CA2175493A1 - Salicylic acid binding protein - Google Patents

Abstract

The present invention relates to catalase, H2O2 and other active oxygen species derived from H2O2 and their role in a plant's immune response.

Description

WO 95/123~1`1 PCTII,'S9~/1262~
} 21 75493 DESCR~PTION
~ALICYL~C ACID 8INDING PROTEIN
CAL FIELD
The invention relates to the fields of biochemistry and molec~ r biology and relates to proteins which are capable of binding ~alicylic acid compounds, methods of isolating same and their use.
The present invention also relates to the cloning of genes for ~alicylic acid bin~i~g proteins.
BAC~Ou~v ~
Perhaps the single best known medication is acetylsalicylic acid--aspirin. Aspirin has long been known to assist in the treatment of pain, swelling and fever. More recently, aspirin has been used to retard blood clotting and lower the risk of heart attacks and strokes.
While the therapeutic value of aspirin has been known for around 100 yeare, the therapeutic value of closely related compounds dates back far longer.
See generally ~J.R. Cutt and D.F. Rless~g, ~Salicylic Acid in Plants - A Changing Perspective~, Pharmaceuticsl Technology, May 1992, pages 26-33, the text of which is hereby incorporated by reference. For example, Hippocrates in the fourth century B.C. is believed to have prescribed the leaves of willow trees for the relief of pain during childbirth. These leaves contain ~alicylic acid (also referred to herein as "SAn), a naturally occurring relative of aspirin. Both can be considered part of a broader family of compounds, naturally occurring and synthetic, known as salicylates. While salicylates have long been known to exist in plants, the role that these compounds have played is only now becoming known. For many years, salicylates were classified as r n~Ary metabolites which played no essential role in functioning of the organism. However, more recently, salicylates are gaining recognition as important factors in a number of important plant functions. An eY~Ancive, but by no 21~5 193 W095l1230~ pcT~lsslll262n means comprehensive list of plant proces6es which are affected by salicylates, and in particular, by the addition of salicylic acid thereto is found in Table 1.
T~blo 1 Procoss ~ffect Flowering +
Thermogenesis Alternative pathway +
Glycolysis +
Rrebs cycle +
Wound response Dis~Ar? resistance +
Ethylene ~iosynthesis Potassium ion absorption Transpiration Stomatal closure Leaf abscission Seed germination Seed germination +
Growth inhibition +
Adventitious root initiation +
Fruit yield +
Somatic embryogenesis Photonastic leaflet movement +
Scotonastic leaflet movement Gene regulation +
+ = induces or enhances - = reduces or inhibits It is interesting to note that salicylic acid has been shown to have effec~s on both the wound response and disease resistance of plants. In this way, plants and animals appear to share some similaritie~.
Although much remains to be learned about a plant's response to wounds, ~jS^Ar?, and the attack by plant pathogens, two specific phenomena have already been observed. First, plants activate a number of a ~local" response~ in their attempt to restrict the spread of pathoqens. This often results in the death WOg~/123~ 2 1 7 5 ~ 9 3 PCT~lS9 I/1262~

of a very limited part of the plant immediately surro~nAing the site of infection. This is called the hypersensitive or local defense response.
In addition to the local defen~e response, many plants respond to infection by activating defenses in uninfected parts of the plant. As a result, the entire plant become6 more resistant to secondary infection. T~is phenomenon, sometimes termed systemic acquired resistance, can persist for some exten~e~
period of time and often confers upon the plant a resistance to unrelated types of pathogens.
It is known that adding salicylic acid to certain plants enhances their resistance to disease.
It has also been shown that such additions of salicylic acid can induce the expression of the genetic material (genes) wit~in plants to produce certain proteins related to disease resistance. Based on this information and the observation that ~alicylic acid is not directly toxic to ~ost pathogens under normal condition~, it is ~elieved that salicylic acid participates in a chain of biochemical events which ends in t~e production of ~is~A~e combating proteins and possibly other factors or compounds. Salicylic acid may therefore be thought of as a signal molecule in the transduction pathway of plant ~ir^Ace resistance or a link in the chain of events leading to a plant's ~immune" response. See Z . Chen and D . F . ~l ~ssig, "Identification of Soluble Salicylic Acid-Binding Protein That May Function in Signal Transduction in the Plant-Disease Response", Proc. Natl. Acad. sci.
USA 88, 8179-8183, (Sept. 1991), the text of which is hereby incorporated by reference. The fact that when salicylic acid is added to plants, a broad number of plant functions are affected sugge~ts that salicylates or related compounds may be effectors or signal compounds along the transduction pathway of a number of pl~nt functions other than just ~ s~ resistance.

217~q93 WO 9~/1230 ~ PCT~S9~112621 The present inventors have identified, purified and characterized a protein which is made by plants. This endogenous protein (endogenous meaning made by the plant) is capable of participating in the S binding of salicylic acid in plants. Therefore, it is believed that the endogenous protein may be a link in the transduction pathway of various plant functions such as, for example, ~ ? resistance. Furthermore, the inventors have cloned and se~l~ce~ a gene from tobacco which encodes this bin~in~ protein.
This discovery is important for a number of reason~. First, the identification of this binding protein, (referred to herein as either "Salicyclic Acid Binding Protein", or "S~3Pn,), is important in gaining a further understanding of the various pathways through which disease resistance, flowering, and o~her normal plant biological processes function. Furthermore, because salicylates such as aspirin and salicylic acid appear to ~ave advantageous properties in both plants and animals with regard to treatment andJor prevention of diseafie, it i6 possible that this di~covery will provide information regarding the identification and characterization of parallel me~hA~isms of, for example, di~ease resistance in both plants and animals.
This could lead to the discovery of new drugs and new forms of treatment for a list of maladies ranging from headaches to high blood pressure. Becau e of the discovery of this binding protein, the cloning of its gene and the growing unders~n~ing of its role in signal transduction, it may be possible to introduce into plants disease resistance mech~isms which might otherwise not be found in that species. It may also be possible to provide enhanced ~ e resistance, as well as other functions to plants which do use salicylic acid as a signal. The cause of world hunger could also be advanced by ~uch discoveries because di~ease resistant crops could be generated with reduced incident of crop failure.

21 7599~
WOg~/123~ PCT~S9~/1262 The present inventors have discovered that the binding protein that they have isolated and purified exhibits a quantitative and qualitative correlation between bi nA i ng activities and the physiological activities of salicylic acid compounds.
In other words, the more biologically active a salicylate is in a plant, the more tightly it will be bound by the native salicylic acid binAi~g protein of the present invention and vice versa. Therefore, assays or tests can be developed and used as a first step in determining whether certain salicylates, either of natural or synthetic origin, might be biologically active and, therefore, agriculturally or pharmaceutically important.
Finally, the inventors have discovered that the identified salicylic acid bi~inq protein has catalase activity which is inhibited by binding.
Inhibition of catalase's H202-scavenging activity would result in an elevated level of H202 and other reactive oxygen species ("ROS"). The previously documented involvement of reactive oxygen species in host defence against microorganisms (Orlandi ~t ~1., Physiol. and Mol. Plant Pathol. ~0: 173 (1992); Schwaclce et al., Planta 187: 136 (1992); Apostol et al., Plant 2S Physiol. 90: 109 (1989); Legendre et al., Plant Physiol. 102: 233 (1993); Baker et al., Plant Physiol. 102: 1341 (1993)) and the discovery that the salicylic acid binding protein is a catalase whose activity is inhibited by SA bin~ing suggest that the role of salicylic acid in defence may be th ough its modulation of t~e abundance of reactive oxygen species via the influencing of plant catalase activity.
8~MMARY OP T~E lNYr~ ON
In accordance with the present invention and in one aspect thereof, there is provided a salicylic acid binding protein having an apparent average native molecular weight of about I80kDa, an apparent Xd of 14p5 ~M for salicylic acid and being capable of WOg~/123~ 2 1 7 5 4 9 3 PCT~IS9~/l262n binding salicylic acid compounds in direct correlation with a salicylic acid compound's physiologic activity, said protein being in purified form.
There is also provided, in accordance with this aspect of the present invention, a protein species having an apparent Rd of 14p5 ~M for salicylic acid and being capable of participating in the binding of salicylic acid compounds in direct correlation with a salicylic acid compound's physiologic activity, said protein species resulting from a ~.oce~s including the steps of providing a plant tissue; homogenizing and buffering the tissue to form a homogenate; filtering and clarifying the homogenate and collecting a first supernatant; chromatographically fractionating the first supernatant in a first anion eY~h~nge column to yield a first eluent; and chromatographically treating the first eluent in a s~con~ gel filtration column to yield a second eluent.
The term ~salicylic acid compound(s)~ as used herein is meant to encompass salicylic acid and benzoic acid analogues thereof. The term includes, but is not limited to, such compounds as 2-hydroxybenzoic acid (salicylic acid~; (acetylsalicylic acid) (aspirin);

2,6-dihydroxybenzoic acid;, 3-hydroxybenzoic acid; 4-hydroxybenzoic acid; 2,3-dihydroxybenzoic acid; 2,4-dihydroxybenzoic acidi and 2,5-dihydroxybenzoic acid.
See ~lso Table 3.
Kd of 14p5 ~H as used herein is the dissociation constant fcr the salicylic acid binding protein. Kd is defined herein as the concentration of salicylic acid needed to occupy 50% of the ~inding sites on the salicylic acid bi n~ inq protein when the binding process reaches its equilibrium. Thus, a smaller value of Kd indicates a stronger interaction between salicylic acid and salicylic acid binding protein and vice versa. Kd is determined by measuring the amounts of salicylic acid bound to salicylic acid binding protein at various concentrations of salicylic 21 7~93 W09~/1230~ PCT~S911l262 acid and then plotting the ratio of bound to free salicylic acid concentrations versus bound salicylic acid concentrations to give a linear plot with a slope of -l/Rd-The phrase "apparent average native molecular weight" indicates the average molecular weight as determined by gel filtration. Altho~lqh these mol~c~lAr weight determinations may not be as precise as those obtained by SDS-PAGE analyses, they do represent a measurement of the mol~c~lAr weight of the native binding protein as opposed to, for example, a denatured species. See Example 3. Further, the term "salicylic acid binding protein" as used herein includes not only the specific protein identified in the immediately pr~o~ing section, i.e., the native or naturally occurring species, but also derivations and analogue6 thereof which are capable of bi~i n~ ~alicylic acid compounds as defined ~erein.
As alluded to pre~iously, calicylic acid appears to be an effector or signal molecule which ic produced in plants. Salicylic acid when present in a plant or, in fact, when added to a plant, can broadly affect a number of plant pr~ce~re~. These processes include inducing the expression of genes which produce anti-pathogenic compounds such as gl~c~nA~es, chitinases, and permatins. The influence of salicylic acid i~, t~erefore, significant with regard to ~i~eAr?
resifitance in plants. As chown in Table 1, other salicylic acid influenced functions include flowering, thermogenesis, and fruit yield.
The ~alicylic acid bin~;n~ protein characterized by the inventors has also been shown to have catalase ~ctivity. Catalases are present in all organisms which grow aerobically ~nd convert the reactive oxygen species H202 to H20 and 2 During the rapid and intense oxidative burst associated with pathogen or elicitor treatment in plants ((Orl~ndi et al., Physiol. and Mol. Plant Pathol. ~.0: 173 (1992);

wo s~/~23n~ 21 7 5 ~ 9 3 PCTA~s~/1262~l Schwacke et al., Planta 187: 136 ~1992); Apostol et al ., Plant Physic~l . 90 : 109 (1989); Legendre et al ., Plant Physiol . 102 : 233 (1993); Baker et al ., Flant Physiol. 102: 1341 (1993) ) ) elevated levels of reactive oxygen species (ROS) are thought to be generated by plasma membrane-localized NAD(P)H oxidases. The inventors have shown that plants appear to utilize ROS
for s~h-equent development of Systemic Acquired Resistance (SAR), and that the mechAnism of ROS
0 generation is unigue. Rather than producing H22 via oxidases, they block breakdown by catalases of H2 2 ~
which is constitutively synthesized as a LY~L ud~ct of several metabolic pathways (e.g. photorespiration and B-oxidation of fatty acids). This difference may reflect the plant's energy consciol~ne~s since the increase of H2 2 associated with SAR G~ throughout the plant where H202 is a Ly~Od~ct while the rapid oxidative burst that requires energy takes place only in a small num~er of cells around the site of 20 infection.
The discovery of a salicylic acid binding protein has a number of important implications with regard to a wide variety of plant functions. Salicylic acid binding protein (SABP), in its native form, may be introduced into a plant by genetic engineering. For example, using standard molecular biological techni ques, the gene( 5) enco~ing the salicylic bin~in~
protein can be genetically engineered into plants to ~odify plant phenotype, response to ~i Fe~?, and other salicylic acid mediated responses. The gene can either be expressed in sense orientation to increase the Ah~ln~nce of the protein or in antis~nre orientation to reduce the level of endogenous calicylic acid bin~ing protein. Thus, the characteristics of salicylic acid bin~ing can be modified in such a way as to influence salicylic acid-infll~n~e~ plant functionc.
The sense or antisense SABP gene can be expressed in different tissues by using tissue or cell-WO 9~/1230~ 2 1 7 5 ~ 9 3 PCT/US9~/1262(1 specific promoters to drive expression and to modify salicylic acid-influenced responses in a tissue or cell-specific manner without affecting tissues in which the selected promoter is not expressed.
The foregoing ~iÇcl~csion was based on plants which have a salicylic acid-inflll~nce~ transduction pathway amongst their si~nAling mechAnicms. But the applicability of the present invention is not limited thereto. It is possible to genetically engineer a salicylic acid transduction pathway including the mechAnisms for producing salicylic acid bin~inq protein and other downstream elements of the ~alicylic acid transduction pathway into a plant. This would provide to the plant specific characteristics not otherwise lS naturally occurring therein. Por example, a foreign gene whose product helps protect a plant against di6ease and whose promoter (naturally or through genetic engineering), contain6 an element which makes it activatable by salicylic acid, could be i--L~o~ ceA
into a plant by standard molec~lAr biological techniques. Simply by applying salicylic~acid to the plant at a~ appropriate time (e.g.. at the first sign of pathogen infestation), the gene would be induced to produce its protective compound. In this way, a complete ~alicylic acid or salicylic acid like transduction pathway can be introduced into a plant.
The discovery of the salicylic acid bin~ing protein of the present invention also allow6 for the development of biochemical assays. These assays can be developed for screening novel biologically or physiologically active analogues of 6alicylic acid which would fall under the general he~;nq of salicylic acid compounds as defined herein. For example, an assay could be developed based on the ability of a 6alicylic acid compound to bind to naturally occurring salicylic acid binding protein or to compete with salicylic acid for binding to tbe protein. Thus, the~e as6ay6 will allow for `testing and comparing the WO 9:~/1230~ 21 7 5 g 9 ~ PCT/US9~/1262~

activities of various salicylic acid compounds. In addition, known catalases could be screened using a similar procedure aimed at identifying compounds which may inhibit catalase activity. Assays of the kind described above will be useful in the identification of new chemical compounds which might modulate the host plant's resistance to disease.
The present inventors have also discovered that the compound 2,6-dichloroisonicotinic acid (INA), like salicylic acid, has the ability to bind SABP/catalase. Like salicyclic acid, INA is known to induce resistance to a variety of pathogens (Metraux et al., In Advanced in Molecular Genetics of Plant-Microbe Interactions 1:4~2, 1991, Kluwer Academic Publishers, Dordrecht; Ward et al., Plant Cell 3:1085, 1991 UknQS et al., Plant Cell 4:645, 1992) and to induce expression of a common set of genes in the 6ystemic acquired resistance respon6e (Ward et al., Plant Cell 3:1085, 1991). The inventors have demonstrated that INA, like salicylic acid, functions in inducing ~ystemic acquired resistance by bin~;ng and inhibiting the catalase activity of SABP/catalase.
Thus, the screening assays referred to above can be directed towards the identification of any compounds which modify catalase activity and these compounds could be analogues of salicyclic acid, analogues of INA
or structurally different compounds with no relation to either salicylic acid or INA, but which exert a cellular effect by modifying catalase activity. A
particularly interesting use of such assays is in the identification of compounds which inhibit catalase to a similar or greater extent than do salicylic acid or INA
and which may have great utility as inducers of systemic acquired resistance for plant pathogen resistance.
In addition to the method for identifying compounds which bind the SABP more ~L.ol.~ly than salicylic acid and which may therefore modify the wo 9~/123n~ 21 7 5 Q 9 J PcrnTss~

function of the SABP and thus modify salicylic acid-based responses, it is also possible to modify the SABP
itself for modified binding of specific compounds including endogenous salicylic acid. Eor example, modification of the coding sequence of the SABP can be undertaken, and the resultant protein expressed in E. coli using techniques well known in the art. The modified SABPC thus generated can be a~sayed in vitro for differential affinity of binding of ~alicylic acid and other compounds; this would be determined by comparison of the compounds to unmodified SABP.
Modified SABP6 which are bound more tightly by salicylic acid and other compounds will be identifiable, as well as modified SABPs to which these same compounds exhibit reduced affinity.
Of course, a salicylic acid-like pathway can be developed including analogues of salicylic acid and a modified complementary bi n~ i ng protein. By so doing, it is possible to render a specific plant's function, such as disease resistance, more efficient. For example, plants may have internal mech~nisms which negatively affect or inactivate 6alicylic acid. This ~ay be done as part of the normal cellular mech~nism to control the amount of salicylic acid available at any given time. The present invention may offer a ~olution. It has been diccovered that a 6alicylic acid-gluco6ide exists in plantc. J. ~alamy, J.
~ennig, and D.F. ~lessig, ~Temperature-dependent induction of salicylic acid and its conjugates during its resistance response to tobacco mosaic virus infection~. Plant Cell ~ (1992) 359-366. This discovery, coupled with observations that several plant hormones are inactivated by conjugation with gluco~e, suggests t~at an internal mechanism which negatively affects or inactivates s~licylic acid exists in plants.
Either the introduction of an analogous pathway into the plant, or the application to the plant of a _alicylic acid analogue which can bind to salicylic wo s~/l23n I PC r~ss~/l262n acid binding protein, but which will not be affected by the plant's inactivation mechAnism could increase the efficiency for induction of a salicylic acid-like response in the plant.
It may also be possible to genetically engineer into a plant a system which parallels the naturally occurring salicylic acid trAnC~ ction pathway but which i6 based on discrete and non-competitively binding analogues. In that way, the normal salicylic acid based cellular functions of a plant will continue undisturbed. However, increases in newly introduced functions can be induced. The plant which contains the complementary salicylic acid binding protein analogue and other downstream me~Anisms n~ - cAry for salicylic acid-induced expression can be in~lcDt~ to express by the application to the plant of the non-competitive salicylic acid compound analogue. The salicylic acid analogue can then bind to the modified salicylic acid binding protein analogue. Plant functions will therefore be influenced by two discrete transduction systems.
It will be clear from the above description of the invention that it is possible to modify certain cellular functions. For example, the insertion of a do~inAnt-negative mutation into the plant genome encoding a SABP which has been modified such that it no longer binds 6alicy1ic acid will result in a SABP which fails to transduce the salicylic acid signal. As the inventors have s~own that the SABP functions in a multimeric complex, then a complex cont~ining both functional and non-functional SABPs will likely not transduce the ~alicylic acid signal. An alternative approach which would provide the same result would be the overexpression of a wild-type gene encoAing a form of catalase which is not inhibited by salicylic acid, but which is capable of assembly with the endogenous catalase-sensitive form(s). In contrast, overexpression of a catalase gene in which the active wog~tl230~ PCT~lS9~11262(~
-13- 2175~93 site has been modified blocking catalase activity, but not the ability of the polypeptides to ass~mble with other subunits may cause the opposite result, i.e., elevated levels of the ROS signal, even in the absence of SA.
Expression of the unmodified SABP in antisense orientation h~h i ~ a ~ y promoter will also result in a failure to transduce the SABP signal, that is salicylic acid bin~in~ protein-based cellular functions can be modified by blocking the production of salicylic acid binding protein. In this technique, the genetically engineered salicylic acid binding protein gene is expressed (transcribed) in the opposite direction relative to the endogenous or normal gene.
This results in the synthesis of anti~ense RNA which is complementary to the sense or normally o~ ing mRNA
which would normally encode salicylic acid binding protein. These two complementary RNAs hybridize or ba6e pair together to form an RNA duplex which is poorly translated into protein or which is destroyed by specific enzymes ~ithin the plant. The end result is that very little salicylic acid binding protein is produced from the endogenous salicylic acid binding protein gene.
A third approach to modify salicylic acid binding protein-based cellular functions utilizes production of large amounts of a salicylic acid bin~ing protein analogue that binds salicylic acid, perhaps even more tightly than the naturally OC~lL ing protein.
The binding protein analogue would have no physiological activity. However, the salicylic acid binding protein analogue could bind all or most of the salicylic acid produced by or added to a plant thereby interrupting the salicylic acid signal. In this way, little or no salicylic acid is available to facilitate salicylic acid induced responr^L, whether or not these responses are in fact mediated by salicylic acid binding protein.

WO9~/1230~ PCT~lS9l/1262~1 -14- 217S~93 Table l shows that salicylic acid causes a variety of plant responses. In some cases (such as disease resistance) salicylic acid induces the response, whereas in other cases it inhibits the S response. The cloning of the SABP gene means that it is now possible to enhance and alter these responses by modifying the salicylic acid in~ e~ pathway. Using t~hniques which reduce the Ah~n~nce of SABP (e.g..
antisense), techniques which modify the bin~;ng of salicylic acid to SABP (using dominant negative mutations or salicylic acid insensitive forms of catalase), and technigues which increase the Ah~nAA~ce of SABP, it will be pos~ible to modify these responses both positively and negatively. In one embodiment, a ~odified SABP could be expressed in a plant. Such modified SABP~ are capable of assembling with endogenous SABPs to form inactive enzymes.
The present invention also has implications with regard to animals including humans. While the medicinal effects of aspirin (and salicylic acid) are well established in animals, there is no evidence that salicylic acid affects gene expression in animals or that animals contain a protein analogous to the salicylic acid binding protein described here. This presents the possibility of developing a new inducible gene expression sy~tem in animals based on salicylic acid and its salicylic acid binding protein. The gene enco~ing the salicylic acid binding protein, under control of an appropriate promoter, could be il-LL~d~ced into animals. Production of the salicylic acid binding protein could make the transformed animal responsive to exogenously applied salicylic acid. For exa~ple, gene could be introduced into the animal that contained a promoter which had cis acting elements which allowed it to respond to its appropriate salicylic acid signal.
Thus in the presence of salicylic acid and its salicylic acid binding protein, it should re6pon~.
Since salicylic acid is nontoxic in most animals and WO9~/l230~ PcT~ss~/l262n -15- 21754~

powerful Ci5 acting salicylic acid responsive elements have been identified, this could be an excellent system for inducing high level expression of foreign genes.
There is precedence for transfer of inducible gene expression systems between very divergent organisms.
For example, the GAL4 system found in yeast has been shown to work in plants and animals. See (J. Ma et al., ~Yeast activators stimulate plant gene expression", Nature, 33~ (1988) 631-633; and H.
Kakidani and H. K ~hn~, ~GALA activate gene expressions in mammalism cells. n Cell 52 (1988) 161 167.) In accordance with another aspect of the present invention, there is provided a bi~i nq species selected from the group consisting of a protein having a molecular weight of 48kDa when mea6ured by SDS-PAGE, and a protein having a molec~ r weight of 150kDa when measured by SDS-PAGE, at least one of which having a Kd of 14p5 ~M for ~alicylic acid and being capable of participating in the bin~in~ of salicylic acid compounds in direct correlation with a 6alicylic acid compound's physiologic activity, said protein being in purified form.
Similarly, in accordance with another aspect of the pre~ent invention, there is provided the protein species resulting from the process just described which al~o includes the ~teps of chromatographically fractionating ~aid ~econd eluent on a third gel filtration column to yield a third eluent; and chromatographically fractionating said third eluent on a fourth immobilized reactive dye column to yield a fourth eluent; and collecting said fourth eluent.
The native salicylic acid bin~i nq protein of the present invention is, as previously describæd, a ~5 protein having an apparent average native molec~ r weight of approximately 180kDa. It i~ this bin~in~
protein which actively binds salicylic acid in nor~al plant systems. However, the native bin~inq protein Wos~/l23(~ pcT~ss1ll262~l -16- 2175~3 need not be a single protein. In fact, the binding protein of the present invention may be composed of smaller proteins which are not "capable of binding salicylic acid~ as individual units. However, at lea~t - 5 one of the smaller proteins is capable of participating in binding salicylic acid. For example, one of the protein species may be capable of actually binding to Ralicylic acid. Other component proteins are capable of participating in binding to the extent that they provide some structural ~ l~ or a particular orientation to the actual bin~ing species.
It is also possible that the smaller proteins exist as a homomeric or heteromeric complex, one or all of which is both capable of bin~i~g to salicylic acid and/or participating in the b;n~ing as previously described.
Another aspect of the present invention is the provision of methods for obtaining, in a more purified form, ~alicylic acid bi~;ng protein. One such method include~ the steps of providing a plant tissue; homogenizing and buffering said tissue to form a homogenate; filtering and clarifying said homogenate and collecting a firs~ supernatant; chromatographically fractionating said first supernatant in a first anion exchange column to yield a first eluent; and chromatographically fractionating said first eluent on a ~econd gel filtration column to yield a second eluent.
The process may also include the steps of chromatographically fra~tionating said ~econd eluent on a third gel filtration column to yield a third eluent;
chromatographically fractionating said third eluent on a fourth cation exchange column to yield a fourth eluent; chromatographically fractionating said fourth eluent on a fifth gel filtration column to yield a fifth eluent; and collecting said fifth eluent.
Alternatively, the process may include the steps of chromatographically fractionating said third Wog~/123~ PCT~IS9~/1262~
-17- 2175~S3 eluent on a fourth immobilized reactive dye column to yield a fourth eluent. The fourth eluent may be further fractionated as desirable.
Another aspect of the present invention is the cloning and seguencing of a gene from tobacco which encodes the binding protein.
BRIEF DE8CRIPTION OP T~ DRAWING8 Preferred embodiments of the present invention will be described in greater detail with reference to the accompanying drawings where:
Figure l is a photograph of an SDS-PAGE gel visualized with Coomassie Blue illustrating protein samples resulting from a five step separation proce~re in accordance with one aspect of the present invention.
Figure 2A is a photograph of an SDS-PAGE gel visualized with Coomassie Blue of protein sample fractions resulting from a four step separation process utilizing an immobilized reactive dye column in accordance with the present invention.
Figure 2B is a salicylic acid bi n~ i ng act~vity profile of the fractions illustrated in Figure 2A.
Figure 3A is a graph of relative binding activity (%) of native salicylic acid binding protein as a function of pH where ( ~ ) represents citrate, ( ) represents phosphate/Na+, and ( O ) represents Tris/Na+.
Figure 3B is a graph of relative binding activity (%) of native salicylic acid binding protein as a fu~ction of ionic strength.
Figure 4 is a graph of the salicylic acid binding activity profile resulting from gel filtration chromatography used to determine the apparent average native molecular weight of the bin~ing protein. ( ~ ) represents total protein eluted and ( ) represents binding activity eluted.
Figure 5 is a graph of the effect on relative bin~ing activity of native salicylic acid binding WO 9~/123(~ t PCr/US9 I/12G2~1 -18- 2175~93 protein as a result of exposure to antioxidants where ( O ) represents beta-mercaptoethanol, ( ) represents dithiothreitol, and ( o ) represents ascorbic acid.
Figure 6A is a representation of the ability of salicylic acid (SA), benzoic acid (BA) and O-coumaric acid (CA) to in~uce PRl expression.
Figure 6B is a graph of the relative ability of salicylic acid ( 0 ), benzoic acid ( ), or 0-coumaric acid ( O ) to compete with a labeled [14C] salicylic acid for bin~;ng with native salicylic acid binding protein.
Figure 7 relates to the purification of salicylic acid bind ing protein from tobacco leaves having an apparent molecular weight of 280kDa by SDS-PAGE.
Figure 8 illustrates the elution profiles of proteins and of salicylic acid binding activity of a partially purified protein mixture on blue-dextran agarose and superose 6 column~.
Figure 9 illustrates the immunoprecipitation of salicyli~ acid binding activity and a 280kDa protein by monoclonal antibodies.
Figure 10 illustrates an immunoblot analysis of salicylic acid binding protein.
Figure 11 illustrates the increase in abundance of H202 in SA or 3AT treated tobacco leaves.
Figure 12 illustrates the induction of protein synthesis.
Figure 13A illustrates an immunoblot analysis of PR1 expression based upon catalase activity from leaf tissue.
Figure ~3B illustrates an immunoblot analysis of PR1 expression based upon catalase activity from flower tissue.
Figure 14 illustrates an immunoblot analysis of PRl expression based upon catalase activity from root tissue.

WO 9~/123W PCT/VS9~/1262~
217549~
Figure 15 is a graph of relative catalase enzyme activity levels of both transgenic and untransformed lines verses the size of TMV-induced lesions.
Figure 16 is a dose r~cpon-? analysis of SA
( ), ( ~ ) and INA ( o ).
B~8T ~OD~ or CARRYING OVT T~ INVENTION
The salicylic acid bi n~ i ng protein of the present invention can be isolated from plant tissue by the use of a unique combination of separation steps.
The steps involved include providing a plant tissue, homogenizing and buffering the plant tissue to form a homogenate, filtering the homogenate and collecting the first supernatant following centrifugation.
Thereafter, the first supernatant is chromatographically fractionated on a first anion exchange column. The first eluent from the first fractionation step is again chromatographically fractionated on a second gel filtration column.
Plant tissue as used herein refers to any plant ti~sue from any member of the plant kingdom which contain~ salicylic acid bin~ing protein. As shown in Table 2, a wide variety of plants have shown salicylic acid binding activity indicating the presence of salicylic acid binding protein. These include, without limitation:
Sable 2 Plant Acti~it~
Tobacco +
Cucumber +
Tomato +
Maize +
Soybean +
Arabidopsis Other plants which are expected to exhibit such activity include: c~--h~, guava, papaya, o~l palm, rubber, canola, sunflower, rye, beans, ginger, lotus, bamboo, potato, rice, peanut, barley, ~alt, wheat, Wos~/123~l PCT~Ss~/l262~
-20- 217549~

alfalfa, oats, eggplant, squash, onion, broccoli, sugar cane, sugar beets, beets, apples, oranges, grapefruit, pear, plum, peach, pineapple, grape, rose, carnation, dai~y, tulip, douglas fir, cedar, white pine, scotch pine, spruce, peas, cotton, flax and coffee.
By the term plant tissue, it is also understood that any part of a plant might be used, including the stem, flower, leaf, trunk, root, seed or any su~parts thereof. Homogenization of plant tissues can be accomplished with a polytron homogenizer or tissue blender in a homogenization buffer, whose components are specified below.
Buffering can be accomplished using any known buffer. Of course, it is preferred to use buffers which are biochemically compatible. Salicylic acid bi~ proteins have been found to bind in a pH-dependent manner with an optimum pH ranging from between about 5.5 to about 6. 5 . See Figure 3A. The pH
of the homogenized plant tissue should be maintained in a range of between about 5 to about 9 and preferably in a range of between about 5.5 to about 7. Suitable bufferc include tris buffers, rhoc~hAte buffers, citrate buffers and combinations thereof. The buffer may also contain magnesium chloride, glycerol, phenyl-methylsulfonyl fluoride and polyvinylpol~L~olidone.
The buffered homogenate is then filtered toremove cellular debris such that a substantially liquid supernatant is formed. Any method of separation can be used in this regard. However, filtering through four layers of cheesecloth followed by centrifugation for 40 minutes at 40,000g has been shown to be effective.
Thereafter, the supernatant, termed the first supernatant, is collected and fractionated on an anion exchanqe column, termed the firct anion eYc~Ange ~5 column. One type of anion exchange column useful in accordance with the present invention is a diethyl A~ i no ethyl (~EAE)-cellulose column which has been equilibrated with the same buffer used to form the WO 9~/1230~ PCT/11S9~/1262~
-21- 2175~93 homogenate minus polyvinylpolypyrrolidone. The supernatant is loaded onto the column and the column is washed extensively with the buffer. Salicylic acid bi~Ai~g protein is eluted from the column by the use of the same buffer containing a linear gradient of a salt such as potassium chloride. Fractions contAini ng the highest level of salicylic acid bi~A i ~g activity (hereafter referred to as peak fractions) are then pooled for subsequent steps. Other types of anion eY~h~ge columns useful in accordance with the present invention include aminoethyl (AE) and quaternary aminoethyl (QAE) columns, and the like. Other salts such as sodium chloride, sodium acetate, potassium acetate, ammonium chloride, and ~mmonium acetate could be used for elution.
The first eluent, which results from chromatographically fractionating the fir~t supernatant on the fir6t anion exchange column is then further chromatographically fractionated on a se~onA gel filtration column to yield a seCo~A eluent. The term Usecond", as used in conjunction with the gel filtration column is meant to indicate that this is the second column employed, not that a first gel filtration column was used in a prior step. In a preferred embodiment, the fractions with highest bi~Ai~g activity (peak fraction) which had been pooled as the first eluent are concentrated via N2-aid¢d filtration concentrator and are loaded onto a sephacryl 5-300 gel filtration column which is equilibrated with the buffer previously used. The samples are eluted in the same buffer and the fractions are then a~sayed for both protein content and salicylic acid bi n~ i n~ activity.
other gel filtration columns useful for the Fame purposes include Sephadex G-200, Seph~rose 6B/Sepharose CL-6B and Superose 6HR 10/30. In addition to further fractionating the sample, the ~^~o~ gel filtration column also provides an G~Gl L~.ity to obtain the apparent average native molec~ r weight of WO9~11230J pcT~Tss~ll262~

the salicylic acid binding protein. The pooled peak fractions from the first anion exchange column were loaded onto the second gel filtration column and eluted at a flow rate of 40 milliliters per hour at 4C.
Fractions were assayed for salicylic acid binding activity using, for example, 15 ~M of [14C] salicylic acid. The elution volumes of six molec~ mass stAn~Ards, as well as the bed volume and the void volume of the column were determined under the same conditions and a calibration curve was obtAine~. For a more detailed discussion of molecular mass estimation, see Z. Chen and D.F. Rless~g, "Identification of a soluble salicyclic acid-bin~ing protein that may function in signal trAn~ tion in the plant ~i~S~re resistance response", Proc. Natl. Ac~d. sci.
USA, 88, 80, (Sept 1991). See also Example 3. The resulting species was found to have an apparent average native molecular weight of approximately 180kDa.
Figure 4 illustrates the results, both in terms of total protein elu~ed from the gel filtration column and binding activity, i.e. alicylic acid b;nAin~
protein. As Figure 4 aptly illustrates, it is possible to obtain good separation and, therefore, purification of the salicylic acid binding protein by the methods described above. It was also shown that there are two peaks of binding activity: fractions 33-49 and having an apparent average native molec~ r weight of 650kDa and a sec~nd peak eluting in fractions 45-49, and having an apparent nverage native molecular weight of 180kDa. It is believed that the 650kDa species is an aggregate of the 180kDa salicylic acid binding protein of the present invention. In prior reported work, speciflcally Z. Chen ~nd D.F. ~lessig cited i~ediately above, the 650kDa species was isolated to the exclusion of the 180kDa salicylic acid binAi~
protein. The discrepancy in the results illustrates t~e importance of the various separation protocols used. In the Chen and Rlessig articles, ammonium WO95/1230~ pcTnTss~ll262n ` -23- 21754~3 sulfate precipitation was used instead of an anion eYchAn~e column separation as used herein.
The binding protein also exhibited a Kd of 14p5Kd ~. Rd f 14p5~M values were determined by Scatchard analysis with tl4C~ salicylic acid concentrations ranging from 2 to 70 ~. Scatchard analysis of Kd is done by measuring the amounts of salicylic acid bound to the calicylic acid bi nA in~
proteins at various concentrations of salicylic acid and then plotting the r~tio of bound to free salicylic acid versus the concentration of bound salicylic acid to yield a linear Scatchard plot with a slope of minus l/Kd.
The 180kDa native bin~in7 protein isolated was tested for binding affinity and specificity. A
competitive assay was established using salicylic acid binding protein fractionated from plants as previously described. As shown in Table 3, [l4C] salicylic acid (20 ~M) was assayed in the preC~nc~ of 40 ~ (2X) or 200 ~M (lOX) of an unlabeled competitor.
Table 3 Competitor Biological Inhibition (%) activity 2X lOX
Salicylic Acid Compounds 2-Hydroxybenzoic acid (SA) ++ 50 9l 2,6-Dihydroxybenzoic acid ++ 51 9l Acetylsalicylic acid~ ++ 25 57 Benzoic acid + 14 25 2,3-Dihydroxybenzoic acid** + l lO

3-Hydroxybenzoic acid - -2 0

4-Hydroxybenzoic acid - 1 -2 2,4-Dihydroxybenzoic acid - -l -2 2,5-Dihydroxybenzoic acid - 1 -l 2,3,4-Trihydroxybenzoic acid - 1 2 2,4,6-Trihydroxybenzoic acid - -l 3 3,4,6-Trihydroxybenzoic acid - 1 -l 3-Aminosalicylic acid - l 5 4-Aminosalicylic acid - O -l WO 95tl230~ PCT/~IS9~11262 -24- 2 1 7 5 g 9

5-Aminosalicylic acid - 1 -2 Thiosalicylic acid - -5 2-Chlorobenzoic acid - 0 2 2-Ethoxybenzoic acid - -3 2 Catechol - 0 Activity of acetylsalicylic acid is probably due to conversion to salicylic acid, the latter of which is both biologically active and capable of binding.
~ Low level of physiologic activity found in some assays and correspondingly low binding activity.
n I +~ indicates a high level of biological activity.
"+" indicates a lower level of biological activity.
"+" indicates low, but detectable biological activity in some a6say~.
n_n indicates no ~iological activity.
Biological activity was based upon values reported in Ab~d et al., ~The effect of benzoic acid derivatives on Nicotiana Tobacum growth in relation to P~-bl productionn, Anti~iral Pes . 9, (1988), 315-327;
L.C. Van Ooon, ~The induction of pathogenesis-related proteins by pathogens and specific chemicals", Neth . J.
Plant. Pathol., 89, (1983), 265-273; R.F. White, "Acetylsalicylic acid (Aspirin) induces resistance to tobacco mosaic v3.rUS in tobacco", Virology, g9, (1979) 410-412; R.F. W~ite et al., ~The chemical induction of PR-(b) proteins and resistance to TMV
infection in tobacco", Antivir. Res., 6, (1986), 177 185; ~.M. Doherty et al ., ~The wound response of tomato plants can be inhibited by aspirin and related ~ydroxybenzoic acids", Physiol. Mol. Plant Pathol ., 3S (1988) 377-384.
As shown in Table 3, only biologically active salicylic acid compounds (salicylic acid, 2,6-dihydroxybenzoic acid, and aspirin and benzoic acid) were able to effectively compete with ~he [14C]
salicylic acid for bin~ing salicylic acid binding protein in accordance with the present invention.

wos~/1230~ pcT~ss1ll262n Salicylic acid which has not been radioactively labeled showed an incidence of inhibition of approximately 50%
when the binding assay involved a mixture containing a l:2 ratio of labeled to unlabeled salicylic acid.
When the amount of l~nl ~h~led salicylic acid used was ten times that of the radioactive species, as expected, the percent inhibition became approximately 90%.
Similar behavior was observed from 2,6-dihydroxybenzoic acid which is about as biologically active as salicylic acid.
2,3-dihydroxybenzoic acid has a very low level of binding. It has a correspondingly low level of biological activity. Aspirin is aleo capable of binding and has biological activity. Tts activity and binding are greater than 2,3-dihydroxybenzoic acid but lower than salicylic acid. In some assays aspirin has lower biological activity than salicylic acid or 2,6-dihydroxybenzoic acid (J. Raskin et ~1. "Regulation of heat production in the inflore~cenc~s of an Arum lily by endogenous salicylic acid~, Proc. Natl. Acad. Sci.
USA 86 (1989) 2214-2218) and accordingly is less capable of competing with salicylic acid for binding.
As shown in Table 4 aspirin is not actually bound by the salicylic acid binding protein. Rather it is readily hydrolyzed (cleaved) either spontaneously or enzymatically to produce ~alicylic acid. The level of biological activity and binding is therefore attributable to the salicylic acid released by hydrolysis. Benzoic acid as discussed herein is both somewhat biologically active and capable of bin~i~g.
~ABL~ 4 Binding f rl4C] salicylic acid, ECarboxyl l4CJ acetylsalicylic acid and [Acetyl -l4C]
acetylsalicylic acid by Partial Purified Salicylic Acid Binding Protein Binding was assayed with 3 mg/ml of the partially purified salicylic acid binding protein and the listed concentration of each ligand with the same specific radioactivity (9.9 Cilmol). Values were WO9~11230~ PCT~sg~/1262n -26- 2175~33 obtained 'rom three independent binding assays and are reported ~ith the sample (n=3) stanàard deviations.

~14~] radioact iYity ~ound ~dp~t~Oo~l) Assay condition wit~ protein wi~ut p,oteln SO ~ [ ~cetyl _ 1~] 96~10 10~~9 acetylsalicylic acid 50 ~ [c~--boxyl-14C] 397+25 112+10 acetylsalicylic acid 50 ~M [14~] sa~icylic acid 989l78 89+7 As further support for the proposition that salicylic acid binding activity is capable of binding both quantitatlvely and qualitatively in terms of the biological activity of the bound substance, binding assays were conduc~ed using two proposed salicylic acid precursors, benzoic acid and O-coumaric acid. Benzoic acid has been shown to induce plant disease resistance only in concentrations higher than salicylic acid (R.F.
White, "Acetylsalicylic acid (Aspirin) induces resistance to tobacco mosaic virus in tobacco virology 99 (1579) 410-412) and as shown in Figure 6A, benzoic acià was able to induce the expression of PR1 genes in tabacco. But higher concentrations (approximately ten- fold) of benzoic acid than salicylic acid are required to induce similar levels of PRl gene expression. This alone suggests that benzoic acid has a reduced biological activity compared to salicylic acid. O-coumaric acid did not induce PR1 gene expression at concentrations of up to 10 mM, indicating that 0-coumaric acid is biologically inactive.

SUBSrl~UrE SHEET (RULE 26) WOg~/1230~ PCT~S9~/126~n -27- 2 1 7 5 ~ 9 3 In a competitive binding assay, as shown in Figure 4B, benzoic acid inhibited [l4C] salicylic acid binding about 20% as well as unlabeled salicylic acid.
This is consistent with the fact that higher concentrations of benzoic acid than salicylic acid were required to induce similar levels of PRl expression.
O-coumaric acid was unable to compete for binding to salicylic acid binding protein. ~his is consistent with the fact that O-coumaric acid has no known biological activity. Thus, there is ,a- quantitative, as well as a qualitative, correlation between biological or physiological activity and binding activity.
The salicylic acid binding activity of the native salicylic acid binding protein in accordance with the present invention appears to be independent of salt concen~ration. As shown in Figure 3B, increasing KCl concentration to l.0 M did not significantly reduce the binding activity thereof.
As shown in Figure 5, the activity of the salicylic acid binding protein is affected by reducing agents beta-mercaptoethanol and dithiothreitol. On the other hand, the antioxidant ascorbic acid had little inhibitory effect on salicylic acid binding protein.
This was measured in buffers containing 20 mM
citrate, 5 mM MgCl and 10% glycerol pH 6.5 in addition to the indicated concentrations of antioxidant.
It is possible to further purify the native salicylic acid binding protein of the present invention. This is accomplished by subjecting the second eluent to subsequent chromatographic steps. For example, chromatographically fractionating the fractions containing the highest levels (peak) of s~licylic acid binding activity from second eluent can be accomplished using a third gel filtration column.
This will yield a third eluent. one type of third gel filtration column which may be -used to chromatographically fraction the second eluent is an FPLC superose 6 column. FPLC, which stands for fast S~JBSmU~E SHEFr (RlJLE 26) wos~/1230~ PcT~ss~ll262(~
-28- 2175~93 protein liquid chromatography, has found widespread application in protein purification because of its fast speed and high resolution. In accordance with the present invention, peak fractions from the second eluent of the prior gel filtration can be loaded onto an FPLC superose column which has been equilibrated with the buffer previously used. The samples can then be eluted with the same buffer, and the collected fractions can then be assayed again for both protein content and salicylic acid binding activity.
Thereafter, the peak fractions from third eluent is pooled and further fractionated chromatographically by the use of a fourth cation exchange column. A cation exchange column in accordance with the present invention can be, for example, a heparin sepharose column. In this case, peak fractions from the third element can be loaded onto a heparin sepharose column which is equilibrated with the ~uffer previously used. The column is washed extensively with the same buffer and is then eluted with the buffer containing a linear gradient of 0-lM KCl. ~he fractions are then again assayed for both protein and salicylic acid binding activity.
Alternative approaches useful for the same purpose may include chromatography with, for example, affinity chromatography with immobilized nucleotide ligand (e.g.
adenine 2, 5 diphosphate).
An additional separation step may be employed using a fifth gel filtration column. This column may be, for example, an FPLC superose 6 column a~
previously used in the third gel filtration column.
The chromatographic conditions used in that instant are the same as previously described. Alternative gel filtration columns include those previously described with regard to the second and third separation steps.
After the fifth separation step, the fifth eluent is collected and analyzed using SDS-PAGE
procedures.

SlJ8SrlTUrE SHEET (F~JLE 26) wos~tl23~ PcT~s9~ll262n SDS-PAGE, which stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is an excellen~ method to identify and monitor proteins during purification and to access the homogeneity of purified fractions. In addition, SDS-PAGE is routinely used for the estimation of protein's subunit molecular weights and for determining the subunit composition of purified proteins. SDS-polyacrylamide gel is prepared and run according to U.K. Laemmli, Nature (London) 10(1970), 680.
As a result of the SDS-PAGE analysis, three significant proteins were identified. One of the proteins identified had an apparent molecular weight of approximately 50kDa. Other proteins had an apparent 15molecular weight of 34kDa, and 38kDa respectively. See Figure 1 which is a SDS-PAGE gel as prepared by the procedures of Example 7. Lane 1 is the crude extract (180 ~g of total protein loaded), lane 2 is pooled peak fractions of the DEAE-cellulose eluent (100 ~g of total 20protein loaded), lane 3 is pooled peak fractions of the sephacryl 5-300 eluent ~20 ~g of total protein loaded), lane 4 is the superose 6 HR 10130 eluent (20 ~g of total protein loaded), lane 5 is pooled peak fractions of the heparin-sepharose eluent (20 ~g of total protein 25loaded), and lane 6 is a second sepharose 6 HR 10/30 eluent (10 ~y of total protein loaded).
A preferred alternative separation method as exemplified in Example 9 eliminates the fourth cation exchange column in fa~or of an immo~i~ized reactive dye 30column such as, a blue-dextran column. A higher concentration of binding activity, i.e. a higher concentration of protein exhibiting the ability to bind salicylic acid compounds per mg of protein was realized using this methodology. SDS-PAGE analysis of fractions 35eluted from the blue-dextran column indicate that at least two prominent proteins are found in the fractions with highest salicylic acid ~inding activity. One protein identified had an apparent molecular weight of SUBS~ITUTE SHEE~ LE 26) WOss/l2301 PCT~S9~11262(\

approximately l~0kDa. The second prominent protein had an apparent molecular weight of 48kDa. See Figure 2A
which is an SDS-PAGE gel prepared by the procedure shown in Example 7 of the fourth column purification step of the four step purification protocol described above using a blue-dextran immobilized reactive dye column. See also Figure 2B which shows the salicylic acid binding protein activity profile of the eluent from the blue-dextran column.
Isolation of the gene encoding salicylic acid binding protein can be achieved using standard molecular biology techniques known to those of ordinary skill in the fields of biochemistry and molecular biology. One technique calls for the use of high affinity, mono specific polyclonal or monoclonal antibodies generated against salicylic acid binding protein. These antibodies can be used to screen Agtl expression libraries from, for example, tobacco. This cDNA library and these antibody screening techniques have previously been used to isolate members of two families of PR genes from it (PRla-c, J.R. Cutt e~
al., Virolo~y "Disease response to tobacco mosaic virus in transgenic tobacco plants that constitutively express the pathogenesis- related protein PRlb" 17~
(1989) 89-97; PR2c, Cote et al., "The pathogenesis-related acidic B-l,3-glucanase genes of tobacco are regulated by both stress and development signals" ~ol.
Plant -Microbe Inte~actions ~ (1991) 173-181).
A library was made from TMV-infected tobacco plants. To rule out the unlikely possibility that TMV
infection might repress salicylic acid binding protein gene expression leading to the absence of its cDNA
clone in the infected plant library, it has been shown that TMV infection neither represses nor induces salicylic acid binding activity. Additional libraries are available from Eric Lam at the Waksman Institute and Stratagene Cat. No. 936002.

SUBSmUrE SHEET (RULE 26) WO95/l230~ PCT~TS9~/1262n An alternative to the use of antibodies to screen expression libraries is the use of mixed oligonucleotide probes (based on the protein sequence) as primers f or polymerase chain reaction (PCR) generation of a cDNA probe (eg. J.A. Cassill et al., "Isolation of Drosophila genes encoding G protein-coupled receptor kinases", Proc. Natl. Acad. Sci.
USA 88: (1991) 11067-11070). A partial amino-terminal sequence determination can be made directly on the salicylic acid binding protein purified to homogeneity, providing the N-terminus is not blocked. If the N-t is blocked, salicylic acid binding protein will be cleaved with a sequence specific protease (eg. trypsin), the peptides separated by HPLC, and then one of the larger and best separated peptides sequenced.
Another approach is applicable where the salicylic acid binding protein is only partially pure but the protein responsible for binding salicylic acid identified (perhaps using anti salicylic acid binding protein antibodies or by affinity labeling), then the mixture can be fur~er fractionated by SDS-PAGE. The fractionated polypeptides will be transferred to polyvinylidene difluoride (PVDF) membranes, visualized by staining with coomassie brilliant blue, and the region of membrane containing salicylic acid binding protein subjected to N-t gas-phase sequencing (T.E.
Rennedy et al., "Se~uencing proteins from acrylamide gels" Nature 336: (1988) 499-500). Because of the excellent sensitivity of gas-phase sequencing (10-100 pmoles), obtaining sufficient quantities of salicylic acid bindin~ protein wil~ not be a problem (note salicylic acid binding protein is relatively abundant at 5 pmoles/~g of soluble protein). Sequencing can be conducted by the Protein Microchemistry Laboratory, a Network Service Laboratory of the N. J. CABM. See generally t~e procedures discussed in V. Cleghon and D.F. Klessig, ~Characterization of the adenovirus DNA-binding protein's nucleic acid binding region by SUBSrllUTE SIIEE~ (F~ 26) wos~ll230~ PCT~S9~/1262~
-32- 2175~93 partial proteoloysis and photochemical crosslinking", J. Biol. Chem., Sept. 1992.
Based on the amino acid sequence, two sets of mixed (degenerate) primers (18-26 bases in length that correspond to the amino acid sequence) can be made corresponding to the N-t(5') and reverse complement of the most C-t sequenced portion (3') of the polypeptide/protein. Each primer will have a restriction enzyme recognition site (EcoRI or SalI
for 5' primer and Hind Ill for 3' primer) plus a GGGC
clamp or extension (for efficient cleavage of the restriction site) for efficient, directional cloning into a pUC based sequencing vector such as pUC118~119. Either reverse transcribed RNA from tobacco or the cDNA from the Agtll libraries could be used as the PCR templates. The previously described cDNA libraries can be employed using any one of a number of recently pu~lished protocols (eg. S.M.
Beverly, "Enzymatic Amplification of DNA by the Polymerase Chain Reaction", In Current Protocols in Molecular Biology, Wiley and Sons, New York, (1991) pp. 15.4.1-15.4.6; S.J. Scharf, "Cloning with PCR"
In PCR Protocols, Academic Press, New York, (1990) pp. 84-91; G.H. Keller, "Probe and Target Amplification Systems" In DNA Probes, Macmillan Publishers, United Kingdom, (19B9) pp. 215-231; J.A. Cassill et al., "Isolation of Drosophila genes enco~ing G protein-coupled receptor kinases" Proc. Natl. Acad. Sci.
USA 88: (1991) 11067-11070).
The PCR products can be fractionated on acrylamide gels, and those, whose products correspond in length to the distance between the ends of the primers (60-180 bases depending on size of the sequenced region of the polypeptide), will be cloned.
Individual clones will be sequenced, and any clone which matches the amino acid sequence will be used as a probe to screen the cDNA library using the standard closing procedure described in detail in the laboratory SUBSmUrE SHEET p~ULE 2B) WO95~1~0~ PCT~lS9~/1262n -33- 2175~3 manual "Molecular Cloning" Second Edition 3. Sambrook, ~.F. Fritsch and T. Maniatis 1989 Cold Spring Harbor Laboratory Press, the text of which is hereby incorporated by reference. Eventually a genomic library which is co~ercially available from Clontech Cat. No. FL1070d will be employed.
To increase the chances of identifying a PCR
clone which matches the amino acid sequence a larger population of clones can be screened with an oligonucleotide probe corresponding to amino acid's internal to the seguenced segment of the polypeptide.
A strategy of nested primers (Mullis and Faloona, "Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction" Methods in ~nzymology 155 (1987) 335-350) can be used to increase the specificity of amplification. After amplifying a region of DNA
with one set of primers, amplification can be continued with a second pair of primers that are closer together (providing the sequenced region of the polypeptide is sufficiently long). Any nonspecific amplification products directed by the first primer set are very unlikely to serve as a template for the second primer set.
The cDNA and/or genomic clones isolated as described above has been sequenced using the dideoxynucleotide-based sequenase version 2 DNA
sequencing kit fro~ U.S. Biochemical. cDNA or genomic clones encoding the salicylic acid binding protein can be transferred to plants using a Ti based binary vector and utilizing the 35S promoter of Cauliflower Mosaic Virus for expression. A number of such vectors are available including pGA643. Using standard procedure for transformation of plant cells via cocultivation of Agrobacterium tumefaciens contalning the binary vector with plant (tobacco) protoplasts, transgenic plants will be constructed. See G. An et al., "Binary vectors" In Plant Molecular Biology Manual Editors S.B.
Geluh, R.A. Schilperoort, D.P.S. Verma Kluwer, Academic SUBSrlllJ~E StlEEI (F~LE 26) WO95/1230~ PCT~Ss~/l262n Publishers 1988 pg. A3/1-A3/19). Other promoters such as the l9S and nos could be used to control expression.
Other DNA transformation procedures can be used to obtain transformed plants such as the "biolistics" or particle gum procedures known to the field. These procedures are particularly suitable for the transformation of monocotyledonous species. See I .
Potrykus "Gene transfer to Plants: Assessment of Published Approaches and Results" Ann. ~ev. Plant Physiol. Plant Mol. Bid., 42 (1991) 205-225.
The inventors have shown that the cloned SABP
gene sequence has high homology to catalase and also possesses catalase activity. The involvement of reactive oxygen species in host defence against microorganisms and the discovery that the SABP is a catalase suggest that the role of salicylic acid in defence may in fact be through its modulation of the abundance of reactive oxygen species via its influencing the activity of plant catalases. In the presence of salicylic acid the catalase activity of the highly purified SABP was inhibited by 80% (table 6). A
similar level of ir~ibition of catalase activity by SA
(-70%) was also observed with crude extracts;
inhibition appeared to be reversible since the catalase activity could be largely recovered by extensive dialysis. 2,6dihydroxybenzoic acid and acetylsalicylic acid, both of whic~ are active inducers of PR genes and resistance, were effective inhibitors of the catalase activity of SABP. Quantitatively 2,6-dihydroxybenzoic acid was somewhat stronger and acetylsalicylic acid was weaker than SA for inhibition of catalase. 2,3-dihydroxybenzoic acid, which has only weak biological activity, was a poor inhibitor while five structurally similar but biologically inactive analogues were ineffective in ir~ibiting the catalase activity.
Moreover, the analogues~ effectiveness in inhibiting SABP's catalase activity correlated with their ability to compete with [l4C]SA for binding to SABP, indicating SU~ITUTE SIIEET (RLIl 26) wos~l23o1 pcT~s9~ll26zn ~17549`3 that binding of SA and its analogues to SABP was responsible for the inhibition of the catalase activity.
The inventors have also shown that INA, a further compound capable of inducing the systemic acquired resistance response in plants and expression of the same SAR genes as salicylic acid, operates by inhibiting SABP/catalase. INA does not, however, operate through salicylic acid as it does not induce an increase in the abundance of salicylic acid. Rather, it operates directly by inhibiting catalase activity.
The inventors have found that the physiological mode of action of INA is different to most abiotic inducers of SAR gene expression ~e.g. polyacrylic acid, thiamine-HCl, ~ l-aminobutyric acid, and barium chloride) which they show to act through salicylic acid (Table 7).
Using a tobacco cell suspension culture and an oxygen electrode to measure in vivo catalase activity by determining the rate of H202-dependent 2 production immediately after the addition of H202 it was possible to demonstrate that S~ and INA have similar dose response curves for catalase inhibition in vivo (Fig. 16).

YJBSrl~UTE SH~ET ~E 26) Wo 95/1230~ PCT/llS9~/1262~1 -36- 217~493 ~ o O O

n_ ~ N
_ ~n g ~ OD~

~ ~n X ~

n O ~
~_ + + + + +
. _ C~S

C~S

~ 8 I Q "~
~- ~ o O
" ~, E E ~ s z o O

WO9~/1230~ pcT~s9~ll262n ~37~ 217~93 Furthermore, numerous analogues of INA were tested in this system. A close correlation was observed between the biological activity of the analogues and their ability to bind catalase and inhibit its activity, suggesting that inhibition of catalase was responsible for the biological activity of INA (see Table 8). Using the cell suspension described above, three chlorinated analogues of salicylic acid were identified which were potent inducers of PR-l gene expression and enhanced resistance to TMV infection.
They were also more effective than salicylic acid in inhibiting catalase activity (see Table 8).
Furthermore, the inventors have shown that the abundance of H202 increased in SA- or 3-amino-1,2,4- triazole (3AT; a specific inhibitor of plant and animal catalases) treated tobacco leaves by 50-60% over the control levels observed in water-treated leaves.
By contrast, 3-hydroxybenzoic acid was unable to enhance the in vivo abundance of H202, consistent with its ineffectiveness in both binding SABP and inhibiting catalase activity. Thus, elevated levels of H202, and in turn, enhanced oxidative stress in vi~o, were consistent with inhibition of catalase activity by SA
observed in vitro.
In addition, the injection of H202 or the catalase inhibitor 3AT into tobacco leaves was found to induce the expression of PR-l; the induction of PR-l parallels t~e well documented induction by salicylic acid. PR-l gene expression was also induced by treating tobacco leaves with glycolate (an intermediate in photorespiratlon which serves as a substrate for the generation of H202 by glycolate oY;~ce present in leaves) and with paraquat (a herbicide which can be reduced in vivo and subsequently reoxidized by transfer of its electrons to oxygen to form superoxide anion;
superoxide can be converted either spontaneously or enzymatically by superoxide dismutase to H202). Thus, the SA signal appears to be propagated through H202 S~BSTnnE S~ ~ 2~

wos5ll23o1 pcT~s9~l262n -38- 217~4~

which may act as a secondary messenger, to activate defense-related PR genes.
Because salicylic acid binding protein's role in defense may be through its modulation of the Ah~n~nce of reactive oxygen species, it is likely that the expression of the SABP gene in transgenic plants in antisense orientation so as to reduce the abundance of native SABP may be advantageous. Standard procedures for the production of appropriate constructions and their transformation into transgenic plants have been used for this. Transgenic tobacco plants expressing the SABP/catalase gene of this invention in antisense orientation were found to have reduced abundance of both low molecular weight (LWM) and high molecular weight (HMW) forms of catalase. Although the LMW form was typically eliminated entirely, the HMW form was eliminated to various degrees in different transgenic lines, presumably reflecting its lower homology to the expressed antisense transgene. Analysis of transgenic lines revealed there to be a direct correlation between inhibition of catalase activity and induced expression of PR-1 and enhanced resistance to TMV.
E~AMPLE 1 Plant tissues (200g) (leaves of tobacco) were sliced and then homogenized with a polytron homogenizer in one liter of a homogenization buffer containing 20 mM citrate (pH 6.5), S mM MgC12, 10% glycerol, 30 ~g/ml polyvinylpolypyrrolidone. The homogenate was filtered through four layers of cheesecloth and then centrifuged four minutes at 40,000g at 4C in an Eppendorf microcentrifuge.
E~AMP~ 2 The resulting supernatant from Example 1 was loaded onto a DEAE-Sephacel column (2.5 x 15cm;
purchased from Pharmacia LKB, Piscataway New Jersey) that had been equilibrated with the aforementioned homogenization buffer without polyvinylpolypyrrolidone (Buffer A). The column was washed extensively with SU~ITUI S~IEE~ p~lJLE 26) woss/l~o~ PCT~IS9~/1262~
3g 2175493 Buffer A, and the salicylic acid binding protein was then eluted with 300 ml of Buffer A containing a linear gradient of 0-O.SM KCl. The peak fractions with the highest salicylic acid binding actiYity (peak fractions) were pooled, concentrated with N2-aided filtration concentrator (purchAs~ from Millipore, Bedford, Massachusetts) and loaded onto a Sephacryl S-300 gel filtration column t2.5 x lOOcm; purch~s~
from Pharmacia, Piscataway, New Jersey). The salicylic acid bin~ing protein was eluted with the Buffer A at the flow rate of 40 ml/hr. The peak fractions (8 ml/fraction) from the gel filtration column were then pooled and used for further characterization. In addition to purification, this gel filtration chromatography was used to estimate the molecular weight of the salicylic acid binding protein.
E~P~E 3 Apparent average native molecular weight was determined as follows: the bed volume (Vb) was directly calculated from the column dimensions and the void volume (Vv) was determined using blue-dextran (Pharmacia). The elution volumes (Ve) of the following molecular weight (Mr) standards were also measured:
thyroglobulin (Mr-669kDa); ferritin (Mr-440kDa);
catalase (Mr-230kDa); aldolase (~ -158kDa); albumin (Mr-67kDa) and ovalbumin (Mr 43kDA). A calibration curve was obtained by plotting the Xav value [(Ve~
Vv)/(Vb-Vv)] against the log Mr of each standard. The molecular weight of t~e salicylic acid binding activity was then estimated by fitting its elution volume to the calibration curve using linear regression procedure.

The pooled peak fractions from the first sephacryl S-300 gel filtration column were loaded onto a superose 6 HR10/30 column (which is operated by an FPLC system; purchased from Pharmacia, Piscataway, New Jersey). Because of the large volume of the pooled fractions from the sephacryl 5-300 column, the SUBSTl~U~ SHEET (RULE 26) PCT/US9~/12620 WO 9~/1230-1 -40- 2175~93 fractionation by FPLC on the superose 6 column had to be repeated many times (approximately 20) using 0.5 ml for each injection. Buffer A is used here for both equilibration and elution. The flow rate used is o.35 ml/min. The protein concentration of each fraction (0.35 ml) was monitored by a W detector at 280 nm and the binding activity was determined with spin-column chromatography as described in Example 8.
ESAMP~E 5 Fractions with peak activ~t-y resulting from Example 4 were tAen pooled and loaded onto a heparin-sepharose mini-column (2 ml; in Bio-Rad poly-prep mini column purchased from Pharmacia, Piscataway, New Jersey), which was equilibrated with Buffer A. After loading the sample, the column was extensively washed with t~e Buffer A, and the salicylic acid binding protein was eluted with 30 ml of the Buffer A
containing a linear gradient of 0-lM KCl. The peak fractions was identified by directly assaying for the salicylic acid binding activity using the spin-column exclusion chromatography.
~XAMPLE 6 The peak fractions from Example 5 were then pooled and subjected to another superose 6 HR 10/30 gel filtration column operated by an FPLC system as previously described. Gel filtration was extensively used because most green plant tissue contains very large amounts of soluble ribulose bisphosphate carboxylase/oxygenase which is a major cont~rinAnt during the purification but has a large molecular weight (approximately 550kDa) that facilitates its separation from salicylic acid binding protein by gel filtration. SDS-PAGE was t~en used to identify the significant protein species still present in the peak fractions from the second FPLC gel filtration chromatography.

SU~llUllE SHEET ~RULE 26) WO95/1230~ PCT~lS9~/12C2~
-41- 2175~93 EXAMP~E 7 Polyacrylamide gels (7-15%) was prepared by copolymerization of acrylamide with N,N'-methylenebisacrylamide using stAnAArd protocols.
Samples for SDS-PAGE were prepared by mixing the protein sample with gel loading buffer containing both SDS (1%) and reducing agent B-mercaptoethanol (100 mM) (or 50 mM DTT) to break possible disulfide bonds present in proteins. Gels were run at constant voltage (50V for stacking gel portions and 1OOV for resolving gel), and the proteins on the gel were visualized by either Coomassie blue or silver staining.
E~AMPL~ 8 A key element during the characterization and the purification process is a simple, inexpensive, sensitive and reliable method for determining the salicylic acid binding activity. For this purpose, spin-column exclusion chromatography was used to separate the bound [14C] salicylic acid (excluded) from the bulk free salicylic acid (included). Specifically, a 1.5 ml Eppendorf tube was punctured at the tip with a 20-gauge needle and a small amount of glass wool was added to cover the small hole. The tube was then filled with Bio-Gel P-6DG desalting gel (exclusion limit 6RDa; purchased from Bio-~ad, Melville, New York) which have been swollen in water. An alternative gel is Sephadex G-25 fine (from Pharmacia, Piscataway, New Jersey). Excess liquid in the gel was removed and centrifuged for 5 minutes at full speed in a Dynamic centrifuge (Becton Dickinson). The process was then repeated several ~imes until the gel filled the tube.
After incubation of an aliquot of the extract or column fractions for 2 ~r at 4OC with 5~M-20~uM [14C salicylic acid] (specifically radioactivity 55 Ci/mol; purchased from New England Nuclear), 150 ~l of proteins bin~ing sample was loaded on~o the tube column, which was then rapidly centrifuged for 2 minutes at full speed and the solution of proteins and bound [l4C-salicylic acid]

SUBS~lTllrE SHEET (RlJLE 26) WOg~/l230~ pcT~ss1ll262n -42- ~175493 that was excluded from the gel was collected into another tube that had been placed under the tube column during the centrifugation. One hundred microliters of this solution was then used to determine the amount of bound salicylic acid. With this binding assay, characterization of the salicylic acid binding protein can be carried out very rapidly. For example, to determine the inhibitory effect of various salicylic acid analogues on salicylic acid binding activity, these individual analogues were included in the binding mixture containing both the protein and [14C] salicylic acid during the 2 hr incubation, and resulting binding activity was determined in comparison to the activity in the absence of these analogues. Likewise, to determine the effect of pH and antioxidants on the binding activity the protein sample was either dialyzed against a large volume of buffers with different pH or antioxidants were added to the protein sample to various concentrations and the resulting binding actiYities were then determined with the spin-column method in comparison with the activity determined in the absence of these treatments.
E~AMPLE 9 The procedures used in Examples 1-4 above were repeated and fractions with peak activity resulting from Example 4 were pooled and loaded onto a blue-dextran agarose mini column (2.5 ml in Bio-Rad poly-prep mini column, purchased from Sigma Company, St. Louis, which has been e~uilibrated with buffer A.
After loading the sample, the column was washed with the buffer A and the salicylic acid binding protein was eluted with 30 ml of the buffer A containing a linear gradient of O~1.0 mM ATP and 0-1~ Kcl. The peak fractions can be identified by directly assaying for the salicylic acid binding using the spin-column exclusion chromatography and the proteins of each fraction can be examined by polyacrylamide gel electrophoresis (shown in Figure 2).

YJB5111UTE SHEE~ ~RULE 26~

woss/l~o~ PcT~ss~ll262~

E~AMPLE 10 The level of PRl protein in tobacco leaves was analyzed by immunoblot analysis to establish biological activity. Three leaf discs (1 cm in diameter) were homogenized in 200 microliters of buffer containing 50 mM Tris (pH 8.0), 1 mM EDTA, 12 mM beta-mercaptoethanol and 10 mg/ml phenylmethylsulfonyl fluoride. The homogenate was centrifuged at full speed for 10 minutes in an Eppendorf microcentrifuge. The supernatant was fractionated with 15% polyacrylamide gel under denatured conditions. Protein blotting was done with a monoclonal antibody specific to PR1 protein and protein-antibody complexes were detected with an ECL kit from Amersham.
E~MPLES 11-15 originally~ the inventors believed that tobacco derived salicylic acid binding protein had an apparent average native molecular wei~ht of about 180kDa. They have subsequently shown, however, that in fact the apparent average native molecular weight of tobacco derived salicylic acid binding protein, as determined by gel filtration, is approximately 240kDa. This was confirmed by SDS-PAGE
procedures from which an apparent mol~ lAr weight of 280kDa was determined. The 40kDa difference in molecular weight between the two procedures is insignificant and results from the inherent differences between the two weig~t determination procedures.
The difference between the originally determined 180~Da value of the apparent average native molecular weight and the subsequently determined 240kDa value appears to be based upon the salt concentration used during gel filtration chromatography. When lower salt concentration buffers were used, as exemplified in Examples 2, 4, and 6, it appears that the salicylic acid binding protein has some weak, nonspecific interaction (binding) to the gel matrix. Thi~ slows the movement of the protein through the ~el. When the SUBSrlTU~E SHEEr (RULE 26) Wo95/1230~ PCT~TS9~11262~
-44- 21754~3 salt content of the ~uffer used was elevated by a lOOmM
KCl supplement, as exemplified by Example 11, the nonspecific binding of the protein to the matrix was, apparently, suppressed. This led to a more accurate determination of salicylic acid binding protein's apparent average native molecular weight of approximately 24OkDa.
What follows is the ~iC-llcsion of the modified purification protocol used to obtain the 240kDa molecular weight determination and a discussion of the subsequent characterization steps taken by the inventors.
E~AMPLE 11 Modified Purific~tion Scheme As reported in Examples 1-10, two "low salt"
concentration purification schemes were initially utilized in accordance with the present invention. The first purification scheme employed the methods utilized in Examples 1, 2, 4, 5 and 6, in that order. The second purification scheme employed the tPchn;~ues described in Examples 1, 2, 4 and 9, also in that order. Subsequently, a new purification procedure was developed whic~ roughly corresponded to the use of the separation steps described in Examples 1, 2, and 4, in that order. This purification protocol resulted in the realization of the 240kDa molecular weight determination.
The only modifications to Example 2 was the addition of lOOmM KC~ to Buffer A during gel filtration chromatography on sephacryl S-300. The next step in purification used t~e procedures described in Example 9 with the modification that ~he salicylic acid binding activity was step eluted with Buffer A supplemented with 0.7M KCl. This replaced elution with a linear gradient of O-lOmM ATP and O-lM KCl as previously described. The final purification step corresponding to previous Example 4, was also modified. However, SJBS~l~U~E S) IEET (Rl1LE 26) PCT~Ss~/1262(~

~45~ 2175 193 the only difference in this step involved the use of a Buffer A supplemented with lOOmM KCl.
Specifically, crude homogenate was prepared from leaves of tobacco as described in Example 1 except that lmM EDTA ([ethylene dinitrilo]-tetraacetic acid) was added to the homogenization buffer; all subsequent buffers also contained lmM EDTA. The filtered and clarified (centrifuged) homogenate was then subjected to ion ~Ych~nge chromatography on DEAE-sephacel and then to gel filtration chromatography on sephacryl S-300 as described in Example 2 with the modification that Buffer A was supplemented with lOOmM KCl during gel filtration chromatography.
The pooled peak fractions from the sephacryl S-300 gel filtration column were pooled and concentrated 5 fold with a Whatman ultra filtration apparatus using a YM-30 membrane. The concentrated protein was chromatographed on a blue-dextran agarose column as described in Example 9 except that the salicylic acid binding acti~ity was step eluted with Buffer A supplemented with 0.7M KCl rather than using a linear gradient of 0-lOmM ATP and 0-lM XCl.
The pooled peak fractions from the blue-dextran agarose column were chromatographed on a superose 6 HR 10/30 column as described in Example 4 with the modification that Buffer A was supplemented with lOOmM KCl.
A small sample of the pooled peak fractions for each chromatography step was analyzed by SDS-PAGE
as described in Example 7 and the results are shown in Figure 7. Figure 7A is the SDS-PAGE (7.5-15%) of protein samples from a crude extract (homogenate) and the peak fractions from the four chromatography steps including DEAE sephacel, sephacryl S-300, blue-dextran agarose and superose 6 described in this example. This illustrates the elution profiles of the salicylic acid binding protein. Molecular masses of marker proteins are shown on the left side. The 280kDa protein that SU8Sll~U~E SHEET (RlLE 26) WO9~1123~ PCT~lS9~/1262~
-46- 2175~93 copurified with salicylic acid binding activity is indicated on the right side. The gel was silver stained.
Figure 7B tabularizes the recovery of proteins and salicylic acid binding activity during purification of salicylic acid binding protein. The four chromatoqraphy steps described above resulted in a reduction of total protein by a factor of 1570 and a 250-fold increase in-specific binding activity.
Figure 8 illustrates the elution profiles of proteins and salicylic acid binding acti~ity on blue-dextran agarose (Figures 8A and 8C) and superose 6 (Figures 8B and 8D) columns. Active fractions from the sephacryl S-300 column were pooled and applied to a blue-dextran agarose column. After extensive washing, the bound salicylic acid binding activity was eluted with the loading and washing buffer (Buffer A) containing 0.7 M KCl (Figure 8A). Various flow-through and eluted fractions were subject to SDS-PAGE (7.5-15%) analysis. A 280kDa protein was highly enriched in fractions 72-76 (Figure 8C) which also contained the highest levels of salicylic acid binding activity (Figure 8A). The active fractions from the blue-dextran agarose column were then co~bined and applied to a superose 6 HR 10/30 column usinq a FPLC system (Figure 8B). The eluted fractions were again subjected to SDS-PAGE analysis (Figure 8D). The 280kDa protein was found to co-elute with the salicylic acid binding activity, i.e. fractions 40-44 (Figure 8B). Fraction n-~h~rs from t~e colu~ns are indicated on top of the silver stained gels (Figure 8C and Figure 8D).
A 280kDa protein species copurified with the salicylic acid binding activity (indicated by arrows in Figures 7 and 8).
The result of the modified purification scheme descri~ed herein is a highly purified mixture of a limited number of proteins. As shown in Fig. 7A, under the right hand most column identified by the SU~TUTE SHEET (RULE 26) wo s~/l23n~ Pcrrus9~/l262~
217549~

legend "superose 6", two bands are prevalent. The band at the top indicated by the arrow has an apparent molecular wei~ht of 28OkDa. A second band located below it, having an apparent molec~ r weight of about 150kDa, is also apparent. Figure 8 illustrates that in its final two steps of purification (i.e.
blue-dextran agarose and superose 6 chromatography) the 280kDa protein and the salicylic acid binding activity co-elute. In other words, the fractions with the highest levels of salicylic acid binding activity (eg. fraction 74 in Fig. 8A and fraction 42 in Fig. 8B) also had the highest amounts of the 280kDa protein (Fig. 8C and 8D). In contrast, while 150kDa species also appeared to co-elute with the salicylic acid lS binding activity, the correspondence between levels of binding acti~ity and amounts of lSOkDa protein were not as compelling. Thus, while the two prevalent proteins contained within the purified protein mixture obtained by the practice of Example 11 are a 280kDa and a 150kDa protein, respectively, the inventors suspected that the 280kDa protein is salicylic acid binding protein.
To validate that suspicion, the tests described in Examples 12-15 were performed.
Briefly, the purified protein mixture, including the two prevalent proteins and a number of other less abundant proteins resulting from Example 11, were injected into two mice as described in Example 12.
As with any foreign su~stance i~,L~Gduced into a mammal, the B cells, which form a part of the mouse's immune system, produced antibodies to each of the various proteins in the mixture. Each B cell can produce only a single type of specific antibody. Individual antibodies can specifically bind to the individual proteins.
B cells from the mouse spleen were fused with an established cell line of myeloma cells to produce a hybridoma. The hybridoma retained the characteristics SUBSrlTUrE SHEE~ (RIJLE 26) WO9~/1230~ pcT~Ts9~ll262n of the fused B cells and are capable of producing antibodies.
The progeny of each independent B cell fused with a myeloma cell were grown separately. The antibodies ~called monoclonal antibodies - MAbs) which each fused pair of cells produced and secreted into the culture media was individually tested to determine if the antibody recognized any one of the proteins in the partially purified salicylic acid binding protein mixture used to immunize the mice. The ELISA
procedure, described in Example 13, was used for this test or screen. At that point, the inventors had a partially purified protein mixture and a series of hybridoma which produce antibodies specifically recognizing the proteins in that mixture.
The next step is called immunoprecipitation.
In immunoprecipitation, antibodies produced by different hybridomas were separately incubated (mixed) with the partially purified protein mixture in order to determine which bound (recognized) the salicylic acid binding protein. The antibodies produced by each hybridoma were attached to sepharose beads using protein A. These beads were then systematically placed into samples of the partially purified protein mixture. When binding occurs between an antibody and a protein, the bound protein zlso becomes bound to the bead. The bead, including the bound protein, can then be separated from the mixture by centrifugation. The proteins which remained in the mixture because they were not ~ound by antibodies were then tested for salicylic acid binding activity. If binding activity remains, then the protein removed by reaction with a specific antibody is not the binding protein. ~f, on the other hand, the protein that reacts with the antibody is t~e salicylic acid binding protein, then its removal from the mixture also renders the remaining mixture incapable of binding salicylic acid.

SlJBSrllU~E SHEET (RULE 26) WO 9~i/1230 1 PCl'fl,TS~`111262(~

As described in more detail in Example 14, all of the monoclonal antibodies which removed or precipitated a 280kDa protein from the mixture also removed the salicylic acid binding activity. This clearly indicates that the protein, having an apparent molecular weight of 280kDa as measured by SDS-PAGE, is the salicylic acid binding protein.
E~AMPLE 12 Monoclon~l AntibodY Production The highly purified salicylic acid binding protein preparation obtained after the four chromatography steps of Example 11 was emulsified in an equal volume of Freund complete adjuvant. Aliquots containing about 50 ~g of proteins were injected intramuscularly into each of two female Balb/c mice. The mice were injected two more times (two and five weeks later), each time with an additional 50 ~g of proteins per mouse. The serum was t~en tested six weeks later by ELISA for bindiny to epitopes on the proteins present in the highly purified salicylic acid binding protein preparation as described in Example 13. A final intraperitoneal injection of 50 ~g of protein per mouse was given and the fusion was performed three days later. The mice's spleen cells were fused with a P3X-derived mouse myeloma cell line according to theprocedure of Galfre and Milstein (1981, Methods Enzymol. 73:3-46). The culture media from -1400 independent fused cell lines (hybridoma) were screened by ELISA for activity to the highly purified salicylic acid binding protein preparation.
~AMPLE 13 E~IS~ (e~zYme lin~ed immunosorotion ass~Y) All steps of EL~SA test were performed at room temperature following the procedure described by Harlow and Lane (1988 Antibodies: A Laboratory Manual, Cold Spring Harbor Lab Press, Cold Spring Harbor, New York). Approximately 2 ~g of the highly purified salicylic acid binding protein preparation was added SUBSrmJ~E SHEET (RIJLE 26) wos~ll23o~ PCT~S9~/1262~
_50_ 2175~93 per microtiter plate well in 50 ~l of coating buffer (5omM carbonate, pH 9.6). After incubating the antigen solution in ~he well overnight, the solution was removed and the wells were washed three times with PBST
buffer (lOOmM phosphate, pH 7.5, lOOmM NaCl, 0.05%
Tween 20). The remaining potential protein binding sites of the well were blocked by a 2 hour incubation with 200 ~l of 2% bovine serum albumin (BSA) in P8ST
buffer. The blocking solution was then removed and the wells were washed three times with PBST buffer.
Hybridoma culture media (50 ~l) were added for a 2 hour incubation. The culture media were removed and the wells were washed four times with the BPST buffer.
Fifty ~l of a solution containing anti-mouse antibodies-alkaline phosphatase complexes (Sigma Chemical; diluted l:lOOO in the BPST buffer with 0.2%
BSA) was added per well for a ~ hour incubation, followed by five washes with BPST buffer. Fifty ~l of p-nitrophenyl phosphate (l mg/ml in 10% dietheneamide, pH 9.6) was added to each well. After a suitable degree of color development (about 30 min), 50 ~l of 4N
NaOH was added to each well to terminate the reaction.
E~AMP~E l4 ImmunPre~ipitatio~ of salicYlic Acid Bindinq Protein In s~andard assays, 100-500 ~l of hybridoma culture media were incubated with 40 ~l of protein A-sepharose beads (50% slurry) at 4C for 2 hours. The antibody-protein A-sep~arose complexes were pelleted by centrifugation in a microfuge and were washed three times with RIPA buffer ~l~QmM NaCl, 5 mMEDTA, 1% sodium deoxycholate, o.1% SDS, lOmM Tris, pH 7.4) and one time with a citrate-NP-40 buffer containing 20mM citrate pH 6.5, 5mM MgS04 10% glycerol, 150mM KCl and 0.1%
NP 40. The complexes were then incubated at 4C for 2 hours with lOO ~l of partially purified salicylic acid binding protein obtained after three or four chromatography steps. These antigen-antibody-protein A-sepharose complexes were then pelleted. The SUBSrl~U~ SHEET (F llLE 26) wos5/1~o~ PCT~S9~/1262(~

supernatants were collected and assayed for the amount of salicylic acid binding activity remaining.
As in Figure 9A, increasing amounts of culture media (supernatants) from hybridomas (which S contain monoclonal antibodies - MAbs) 3B6 and IF5 remove the salicylic acid binding activity from the mixture. In contrast, MAb ZA3 did not remove its binding activity.
As shown in Figure 9B, the beads removed from the antigen-antibody mixture by centrifugation were washed three times with lX RIPA before being resuspended in protein sample buffer and subjected to SDS-PAGE (7.5-15~) analysis. Lane 1 - size marker proteins; lane 2 - antigen only (a ~lue dextran agarose fraction); lane 3 - antigen plus MAb 3B6; lane 4 -MAb 3B6 only; lane 5 - antigen plus MAb 2A3. Analysis of the immune complex composition by SDS-PAGE indicated that the 280kDa polypeptide was the only protein precipitated by MAbs 3B6 as illustrated in lane 3. In contrast, MAb 2A3 which did not remove the salicylic acid binding activity from the mixture also did not precipitate the 28OkDa protein, illustrated in lane 5.
It was found that those MAbs which immunoprecipitated the salicylic acid binding activity also immunoprecipitated the 280kDa protein but not the other proteins presen~ in the partially purified preparations. The other major protein in the purest preparation of salicylic acid binding protein was 150kDa protein. It was not immunoprecipitated by the salicylic acid binding protein-specific MAbs.
Moreover, MAbs that recognized and immunoprecipitated the 150kDa protein (but not the 280kDa protein), failed to immunoprecipitate the salicylic acid binding activity. These results strongly argue that the 150kDa protein is not salicylic acid binding protein.

SUBSrlTU~E SH~ET (RULE 26) WO9~/1230J PCT~TS9~/l262~
-52- 2175 19~

E~AMPLE 15 ~mu~oblot AnalY~i~ of 8ali~Ylic Acid Bindi~ Protei~
Four salicylic acid binding protein-specific MAbs (including 3B6 and IF5 described above) were used to detect the salicylic acid binding protein by immunoblot analysis. In this analysis, 50 ~g of freshly prepared tobacco protein homogenates (labeled C
in Figure 10 for crude extract) made in the presence of B mercaptoethanol or 2 ~g of tobacco salicylic acid binding protein partially purified (labeled P in Figure lO) through ~he first three chromatography steps described in Example 11 were size fractionated by SDS-PAGE (7.5-15%). After size fractionation, the proteins were electrophoretically transferred to nitrocellulose filters in a solution of 48mM Tris, 39mM glycine pH 9.0, 20% methanol for 12-16 hrs at 50 volts. The remaining protein binding sites on the nitrocellulose filters were blocked by incubation with a solution of 5% non-fat dried milk in Buffer B (lOOmM phosphate pH 7.5, lOOmM NaCl, 0.1% Tween 20) for 1 hr at room temperature. The filters were then reacted with a 1:100 dilution, in Bu~fer B, of hybridoma culture media containing one of the four salicylic acid binding protein-specific MAbs (3B6, IF5, 2Cll, and 7F10) or one 2~ of three MAbs which do not recognize the salicylic acid binding protein (5A8, 6E10, and PR-l). MAbs 5A8 and 6ElO were obtained from the same fusion from which the salicylic acid binding protein-specific MAb5 were obtained. However, they failed to react with the partially purified salicylic acid binding protein mixture in the ELISA. MAb PR-l specifically recognizes the I6kDa pathogenesis-related proteins PR-l of tobacco; ~hese proteins are not made in uninfected plants suc~ as those used for preparation of the homogenates. After incu~ation at room temperature, the filters were washed three times with Buffer B. The antigen-antibody complexes were detected using a l:lo,ooo dilution of sheep anti-mouse antibodies SlJBSrlll)~E SHEET (RULE 26) wos~ll23o~ pcT~s9~ll262n -53- 217~4~3 conjugated to horseradish peroxidase using the ECL
(Enhanced rhPmintlmine5cence) detection kit from Amersham.
The four salicylic acid binding protein-specific MAbs each recognized (bound to) the 280kDaprotein in the partially purified preparations of salicylic acid binding protein tFigure 10). This is consistent with i) the copurification of the 280kDa protein with the salicylic acid binding activity and ii) the immunoprecipitation of both the salicylic acid binding activity and the 28OkDa protein by each of these MAbs.
In contrast, the MAb's recognize only a 57kDa protein, in freshly made tobacco leaf homogenates prepared as in Example 1 but with the addition of I5mM
B mercaptoethanol to the homogenization buffer.
(Figure 10). B mercaptoethanol is a reducing agent which represses the activity of polyphenol oxidases that can cause crosslinking of proteins. These results indicate that one of the components of salicylic acid binding protein is the 57kDa protein. Since the salicylic acid binding protein's native molecular weight, as determined by gel filtration, is approximately 240kDa in the presence of lOOmM RCl, salicylic acid binding protein appears to be a multimeric complex. The complex may be composed of multiple subunits of only the 57kDa protein or it may contain other proteins in addition to the 57kDa protein. It appears that when the salicylic acid binding protein is prepared in the absence of reducing agents to decrease polyphenol oxidase activity, the subunits of the complex are crosslinked together. As a result these su~units are not dissociated under denaturing conditions such as during SDS-PAGE and thus migrate as a large protein (complex) of 280kDa. The arrows indicate the position of the 280kDa and 57kDa proteins in Figure 10.

~J~SmUrE SHEET (RIJLE 26) wos~/l230~ PCT~S9~/1262~
_54_ 21754~3 Several of the MAbs that bind to the tobacco 57kDa protein and 280kDa complex also recognize a 57kDa protein in fresh homogenates from cucumbers.
The salicylic acid binding activity from cucumber has similar affinity tRd) and specificity as the tobacco salicylic acid ~inding protein. Like the tobacco salicylic acid binding protein, the cucumber salicylic acid binding protein also has a large native molecular weight (approximately 200kDa). Thus, it appears that the cucumber salicylic acid binding protein is also a complex composed of multiple subunits at least one of which is probably the 57kDa protein recognized by the MAbs made against the tobacco salicylic acid binding protein. It should be noted, however, that there may be some minor variation in molecular weight and Kd for salicylic acid binding protein from one plant species to the next. For example, cucumber derived salicylic acid bindiny protein appears to have an apparent average native molecular weight of about 200kDa and a Kd of about 30p 5~M.
The model of salicylic acid binding protein is consistent with ~he inventors' initial observation that salicylic acid ~inding protein had an apparent native molecular weight of 650kDa as determined by gel filtration chromatography of ammonium sulfate precipitated protein. Ammonium sulfate precipitation exacerbate5 the problem of aggregation and in the presence of polyphenol oxidases facilitates crosslinking. When this precipitation step was eliminated, salicylic acid binding protein had an apparent native molecular weight of I80-240kDa. Note that after salicy~ic acid binding protein was fractionated on cellulose and initially chromatographed on sephacryl S-300 under low ionic conditions t20mM
citrate pH 6.5, lOmM MgSo4, 30 ~l/ml PMSF), salicylic acid binding protein eluted with an apparent molecular weight of 180kDa. In contrast, when gel filtration chromatography was done in the presence of modest ionic SUBS~I~UTE SHEE~ LE 26) WO 9~/1230~ Pcr~ss1/l262n strength (lOOmM RCl) salicylic acid binding protein's apparent native molecular weight was determined to be 240kDa. Presumably at low ionic strength salicylic acid binding protein preferentially interacts with the matrix, causing its migration to be slightly retarded and thus its molecular weight to be underestimated.
In summary, several monoc~onal antibodies raised against the highly purified salicylic acid binding protein immunoprecipitated the salicylic acid binding activity and a 280kDa protein. This 280kDa protein also copurified with the salicylic acid binding activity during the various chromatography steps, indicating that it was responsible for bind ing salicylic acid. The 280kDa molecular weight of the denatured salicylic acid binding protein, as determined by SDS-PAGE, was similar to its apparent average native molecular weight of 240kDa, as determined gel filtration in the presence of lOOmM RC1. The salicylic acid binding protein-specific MAbs also recognized the 280kDa species after SDS-PAGE size fractionation and immunoblot analysis of partially purified salicylic acid binding protein. Tn contrast, in parallel size fractionation and immunoblot analysis of freshly prepared plant homogenates (made in the presence of reducing agents to decrease the activity of polyphenol oxidases), the four different salicylic acid binding protein-specific MAbs recognized (bound) only a 57kDa protein. These results strongly suggest that the native salicylic acid binding protein is a heteromeric or homomeric complex containing the 57kDa protein as a subunit. Upon extraction and subsequent purification of salicylic acid binding protein, the subunits of the complex appear to be covalently crosslinked, probably due to the action of polyphenol oxidases. The crosslinked complex remains active. However, the subunits cannot be dissociated even by SDS-PAGE and hence they migrate together as a large molecular weight entity of 280kDa.

SllBSrl~UTE SHEET ~LE 26) wos~/123~ PCT~S9~/1262n -56- 217549~

E~mPle 16 Isolation of a cDN~ clone vhich encodes a prot-in recognisQd by ~Ab8 that ~mmunopr-c~p~tat-8A th~t i~munoDrecipit~te 8A bin~inq acti~ity An aliquot of amplified ~gtll tobacco cDNA library tprepared from mature leaves of N. tabacum cultivar SRl) was absorbed onto E. coli (YlO90) and plated at a density of lO,000 plaques per 150 mm plate containing LB-ampicillin. The bacteriophages were grown at 42C
for 4 hours to induce lysis. Nitrocellulose filters precoated with lO mM isopropyl thio-B-Dgalactoside (IPTG) were overlaid on the plates and inc~1hAted for 2.5 hours at 37C to in~re expression of the cDNA
inserts. After the incubation, the filters were removed and blocked by incubating for l hour at 4C in BPST buffer (lO0 mM phosphate, pH 7.5, lO0 mM NaCl, 0.1% Tween 20) containing 5~ nonfat milk, and washed three times with BPST buffer for a few minutes each.
Blots were probed with diluted hybridoma media (l:lO0) in BPST buffer containing 0.2% BSA for l hour, and briefly washed as above three times with PBST buffer.
The antigen-antibody complexes were detected with a l:lO,000 dilution of sheep antimouse IgG antibodies conjugated to horseradish peroxidase using the ECL
(Enhanced Chemiluminescence) detection kit from Amersham. An immunopositive bacteriophage clone (~CKl) was identified and purified by sequential low-density plating and immunoblotting by following the same procedure described above. Bacteriophages of the purified clone were amplified and DNA was purified by following the standard procedure described in Molecular Cloning: A Laboratory Manual, 2nd Edition, Editors J.
Sambrook, E. F. ~ritsch, and T. Maniatis 1989, Cold Spring Harbor Laboratory Press. The purified bacteriophage DNA was digested with restriction enzyme EcoR I and the cDNA insert (approximately l.9kb) was purified and subcloned into the EcoRI site of the plasmid Bluescript SK II+ generating the plasmid WO9~/1230~ PCT~S9~/1262(~

designed pCKl. pCKl has been deposited with the ATCC
under the Budapest Treaty.
~X~Pl~ ~7 DN~ seouence determinatio~ of the cDN~ insert of DC~l DNA sequencing was perfor~ed by the dideoxynucleotide chain-termination method (Sanger ~t a~., Proc. Natl. Acad. Sci. USA 1977, 74:5463~, using the Sequenase kit from United States Biochemicals and (~_35S)dATP. The complete sequence of the cDNA
insert has been determined for one strand and partially determined for the second strand. Table 5 shows the DNA sequence and predicted amino acid sequence are shown. Sequences corresponding to the amino acid sequences from the tryptic peptides are underlined.

WO9511230~ PCT~'S9~/1262~
-58- 2175~93 DNA and pred~cted amlno acid sequence o~' pC~l ctctaagtttcgaccatcaagcgcatatgattcccctttcttgacaacaaatgctggtgg ~0 90 110 tcctgtctacaacaacgtttcttccttgactgttggacctagagggcctgttcttcttga P V Y N N V S S L ~ V G P R G P V L L E

ggattatcac~taatagagaagctcgcgacttttgatcgtgagcggatacctgagcgtgt D Y H L I E K L A T F D R E R I P E R V

tgttcatgctagaggtgccagtgcaaaaggtttctttgaagtcactcatgatatttctca tcttacctgtgctgattttctccgagcgcctggggttcaaacacctgttatttgccgttt L T C A D F L R A P G V Q T P V I C R F

20ctctactgtcgtccatgagcgtggaagccccgagtcccttagggacattcgtggttttgc S T V V H E R G S P E S L R D I R G F A

tgtcaaattttacaccagagagggtaactttgatctggttggaaacaacgtccccgtctt V K F Y T R E G N F D L V G N N V P V F

ctttaatcgtgatgcaaaatcgttccctgacacgattcgtgcactgaaaccaaatccaaa F N R D A K S F P D T I R A L K P N P K

gtcacacattcaggaatactggaggatccttgatttcttctctttccttccggagagttt 30S H ~ Q E Y w R I L D F F S F L P E S L

gcatacttttgcctgst_~t~cgatgatgtttgtctcccgacagattacagacacatgga H T F A W F F D D V C L P T D Y R H M E

35aggttatggtgttcacg-cta~caattaatcaacaaggctgggaaagcacattatgtgaa G Y G V H ~ Y Q L I N K A G K A H Y V K

gtttcactggaaaccaac~gtggtgtcaagtgcatgtcggaggaagaagctattagggt F H W K P T C G V K C M S E E E A A R V

cggaggtacaaatcatagccacgccaccaaggatctctacgattcgattgctgctggaaa G G T N H S H ~ T K D L Y D S I A A G N

ctatcccgagtggaaactt tta_ccaaattatggacactgaggatgtagacaaattcga 45Y P E h' K L F ' Q 1 M D T E D V D K F D

ctttgatcc-_~tgatgtaaccaagacctggcctgaggatatcttgccattgatgccagt F D P D V ~ K T W P E D I L P L M P V

50tggacgattggtac~taacaggaatatcgataacttc~ttgctgagaacgagcagctcgc G R V L N R N ~ D N F F A E N E Q L A

SUBSmUrE SHEET (F ULE 2B) WO95/1230~ PCT~S9~/1262~1 ~59~ 2175~3 stttaaccctggccatatt~tc~tg~t~ttta~tattcggaggacaagc~tctccagac F N P G H I V P G L Y Y S E D K L L Q T

taggatattcg~tatgctgatactcagaga~accgtattggaccaaactatatgcagct R I F A Y A D T Q R H R ~ G P N Y M Q L

t~ctgt~aa~gctcccaagtgtsctcatcacaataatcaccgcgat~gtgccatgaactt P V N A P K C A H H N N H R D G A M N F
11~0 1170 1190 catgcatcgcgatgaagaggtggattatttgccctcaaggttcgatccttgtcgtcatgc M H R D E E V D Y L P S R F D P C R H A

tgaacagtacccaattccttctcgtgtcttgacaggaaggcgtgaaatgtgtgtcattga E Q Y P I P S R V L T G R R E M C V I E
~5 12~0 1290 1310 gaaagagaacaacttcaagcaggcaggagaaagatacagatcctgggaacctgaca~gca K E N N F K Q A G E R Y R S W E P D R o 1330 ~350 1370 agacagatatgttagcaaatgggttgagcatttatccgat~cacgagtcacttatgagat D R Y V S K W V E H L S D P R V T Y E

acgcagtatatggatatgctccctgtctcaggctgacaagtcttgtggtcagaaggtcgc R S I W I C S L S Q A D ~ S C G Q R V A

tt~tcgt~tcactttaaagcctacaatgtgatgaagactaagatyaaaacactactggga S R L T L K P T M

aaacgtct~aasttg-agtttgaaggagtactaaa~cAag~ gcattacgtttgt~tg ' 1570 1590 1610 tttttgc~ataaagtgta~tstttcgttttatgttctgtttgtaccaaactttgatat~t tgtgtt~a~tatgacacaa.atatgttgcacttgaataaggtacagatgtatgttcaagt actgtgstcatcttc,.tctattttaccttgtttcacactttttaagcttttgtgccaaa attatgtcatacttg_tcattttggtgcttgaagtataccctcaattctataatgcca~t lBlO 1830 1~50 ggtattgtagttttattgacatgttaataagaaagctgctact~tgtcttccgtt~aa EX~MPLE 18 Isolation of a full len~th cDNA for S~BP
The cDNA pCKl does not include the aminoterminal methionine and is therefore not a full-length clone.
From the predicted molecular weight of the polypeptide product (57 kDa) it is assumed that pCKl is near full-~ength. In order to isolate a full-length cDNA copy, a library of tobacco cDNA is plated and screened with radio-labelled pCK1 cDNA
using techniques well known in the art. Clones identified are sequenced and the 5' untranslated region znd initiating methionine of the cDNA are identified.

SUBSrlTU~E SHEET (RIJLE 26) wos~/l230~ PCT~S9~/1262n -60- 2175~93 E~u~Ple 19 Amino acid seguence of ~ever~1 trYPtic DePtide~ of the ~ABP
Immunoprecipitation with one of the SABP-specific HAbs (MAb 3B6; see Example 14) was used as the final purification step to purify SABP from about 300 mg of highly purified SABP fraction obtained after the four steps of chromatography described in Example 11.
The immunoprecipitated SABP complexed with MAbs was then subject to SDS-PAGE (5-15%) (See Example 7) and SABP band was identified by Coomassie Blue staining.
The band of SABP was excised and submitted for peptide sequencing to W. M. Reck Foundation Biotechnology Laboratory of Yale University. Amino acids composition of an aliquot (10~) of the submitted gel was first analyzed to verify that there was sufficient protein to proceed. Tryptic digestion was then performed in the gel matrix followed by elution and separation of the resulting peptides by high performance liquid chromatography (HPLC). Several peaks from the HPLC
profile potentially suitable for seguencing were further subject to laser desorption mass spec~rophotometer (LDMS) to ensure that each of these peaks indeed contain a peptidP and contained predominantly one peptide. Gasphase sequence analysis was then performed on these peptides. Below is their amino acid sequences (top line) and the corresponding predicted amlno acid sequence from the cDNA insert of pCKl (~otto~ line).
3 0 ~572-1 r c ~ F D L v c ~ ~ ~ P v r S R

llg94-1 1; ~ ~ P D R Q r ~lA - rl r -- -- -- -- D

v r A L l; ~ P ~t wos~/l230~ PCT~ISs~ll262n 217~ 193 The occasional mismatch of protein-derived sequence and cDNA predicted sequence may be due to the different tobacco cultivars used as starting material for the protein purification and cDNA cloning experiments. Alternatively, the discrepancy in several amino acid residues may have resulted from the - - existence of different SABPS isozymes in tobacco.
~x~Dle 20 Cloninq the 8ABP cDN~ usina PCR tech~icu~s and deqenerate oli~onucleotides desicne~ to ~no~n Peptide se~uence The peptide sequences described in example-18 can be used to design degenerate oligonucleotides which can be used as primers in a PCR reaction for the cloning of the SABP cDNA. In addition to degenerate nucleotide sequence, primers include at their 5' end a restriction endonuclease recognition ~ite and a &GGC
clamp or extension for efficient cleavage of the restriction site. Primers are designed in sense and antisense to the known peptide sequence and sense and antisense primer~ are designed with different restriction site extensions to enable easy cloning into an appropriate vector. Typical primers are based on the sequenoe of between 6 and 10 amino acid residues.
For the amino acid sequence U572-1 described in example 18 the following primers would be suitable:
5'-GA(A/G)~G(N)AA(~)ST(C/S)GA(C~ CtT)T(~)GT

5~-~(c~T)tArs/c)A~(G/A)A~(cJA)AA~N)a~(N)GG
N is an abbreviation of all four nucleotides which can alternatively be replaced by inosine. In each case, and as described above, a different restriction enzyme recognition site and GGGC clamp is added at the 5' end to expedite subsequent cloning of the PCRgenerated fragment.
For peptide fraqments N994-1 and D839 similar sense and antisense oligonucleotides can be designed, WOg~/1230~ PCT~S9~/1262n and these can be used in combination with primers designed to other peptide sequences in order to generate PCR fragments of sufficient size for cloning.
Suitable substrates for PCR reactions using the degenerate oligonucleotides described above are reversed transcribed RNA from tobacco and cDNA
libraries of tobacco. Alternatively genomic libraries of tobacco or genomic DNA may be used. PC~ products thus generated are fractionated in acrylamide or agarose gels, cleaved with the appropriate restriction endonucleases and cloned into suitable DNA cloning vectors. Clones found to carry inserts which match the known amino acid sequence are used as probes to screen cDNA and genomic libraries. The t~çhniques described above for PCR and cloning are well known in the art (see "Current Protocols in Molec11lAr Biologyn, Wiley and Sons, New York, (l99l)).
~ Dle 2l Overexpre~on of 8ABP $~ transqenic ~lants The cDNA described in example 18 is expressed at high levels in transgenic plants. The cDNA is cloned into a plant expression cacette h~ i n~ a promoter expressed at high level~ in transgenic plant~
and upstream of a cleavage and polyadenylation signal which is known to function in plants. A preferred promoter i6 the CaMV 35S promoter and a preferred cleavage and polyadenylation signal is the nopaline synthase clea~age and polyadenylation signal. The expression cassette i~ transferred to binary vector (pCGNl54; - Alexander et al., PNAS 90:7327-tl993) for Agrobacterium transformation and a direct gene transfer vector (pCIB3064; Xoziel et ~l., Biotechnology ll:
l94-200) for direct gene transfer. Agrobacterium is particularly suitable for t~e transformation of dicotyledonous species and direct gene transfer i5 particularly suitable for the transformation of monocotyledonous species. These te~hniques are well known in the art and are described in the two above-wog~/l23n~ PCT~S9~11262~
-63- 217519~

mentioned publications. transgenic plants are screened for high-level expression of the SABP transgene by Western analysis using antibodies which recognize SABP.
Alternatively, promoters for the expression of SABP can be selected which have tissue specific expression pattern and would thus localize the increase in SABP to particular cell types.
~AMPLE 22 8AB~ ha~ homoloqY to catalase and catalase ~CtiVitY
The amino acid sequence deduced from the cDNA
sequence of the cloned SABP gene revealed high homology (60-gO%) to catalases of other organisms, with highest similarity to plant catalases. To determine whether or not the SABP had catalase activity, its hydrogen peroxidase degrading activity was measured directly.
Highly purified SABP obtained after four chromatography steps ( Chen et al ., Proc . Natl . Acad . sci . USA, 90: 9533 fl993J J exhibited high specific catalase activity (3000-lO,000 U/mg) and this activity could be specifically immunoprecipitated by SABP-specific hAbs.
The sizes cf the SABP complex (240-280 kDa) and its subunits (57 kDa) are consistent with the structure of known catalases which are composed of four identical or similar subunits of 50-60 kDa.
E~AMPLE 23 In~ibition of 8ABP-a~ociated catalase ~ctivity ~it~ 8A
In th~ presence of SA, the catalase activity of the highly purified SABP was inhibited by 80%
(Table 6). A similar level of inhibition of catalase activity by SA (-70%) was also observed with crude extracts; inhibition appeared to be reversible since the catalase activity could be largely recovered by extensive dialysis. To ASC~C5 the functional relevance of catalase inhibition by SA, several SA analogues, with or without biological activity in inducing plant PR genes and disease resistance, were compared for their ability to inhibit the catalase activity of SABP
(Table 6).

woss/l~o~ pcT~lss~ll262n Catalase activity of SABP was assayed over 3 minutes at room temperature in a 1 ml-mixture containing 20 mM citrate, pH 6.5, 5 mM MgS04, 1 mM
H202, 1 mM SA or its analogues and 500 ng of SABP
purified from four chromatography steps (Chen et al., Proc. Natl . Acad. Sci . USA, 90:9533 (1993) ) . Ali~uots (50 ~1) were removed from the assay mixture at 30 second intervals to assay for amount of H202 remaining using the luminol method as has been described by Warm and Laties, Phytochem . 21: 827 ( 1982 ) except for the following modifications: 50~1 of test solution and 50 ~1 of luminol (0.5 mM in 0.2 N NH4OH, pH 9.5) were added to 0.8 ml of 0.2 N NH4OH, pH 9.5 in a test tube.
The tu~e was placed in the measuring chamber and assay was initiated with automatic injection into the mixture of 100 ~L of 0.5 mM K3Fe(CN)6 in 0.2N NH40H, pH 9.5.
Measurements were integrated over 5 second periods.
H202 was calculated from the standard cur~e constructed with known amounts of H202. Rate constants of SABP
~0 catalase activity in the presence or absence of SA or its analogues were then calculated based on a first-order mechanism.
2,6-dihydroxybenzoic acid and acetylsalicylic acid, both of which are active inducers of PR genes and resistance, were effective inhibitors of the catalase activity o~ SABP. Quantitatively 2,6-dihydroxybenzoic acid was somewhat stronger and acetylsalicylic acid was weaker than SA for inhibition of catalase. 2,3-dihydroxybenzoic acid, which has only weak biological activity, was a poor inhibitor while five structurally similar but biologically inactive analogues were ineffective in inhibiting the catalase activity.
Moreover, the analogues' effectiveness in inhibiting SABP's catalase activity correlated with their ability to compete with [14C] SA for binding to SABP, indicating that binding of SA and its analogues to SABP
was responsible for the inhibition of the catalase activity.

SU8STl~UrE SHEE~ (RULE 26) wo ss/l23n I PCT~IS9~/1262~

-6~- 2175~9~

$~ 6 l~bitl~n ot c-t-~ ctiv~t~ nd r~ Dir~ing o SA~P by SA ra 1~ es I-l~iC-~
SA r~ n~ A~iti~ C~t~ etivit~ t~ b~
2~ ic cid tSA~ ~ ~O S ~9 2 6~D~ c ~ 91,3 ~z ~c~t~ lic cid ~ 53 6 2, 3-Dih, L `~ iC cid 15 3 V
3 ~ ic cid 3 1 t L 1~ i c c i d ~ 2 2, L rJih, . ~ __ic cid - 5 2 --2, S-Dil~ c cid 3 2 ~, Dil~ Ic cid - 5 1 O
0 . ~ bilit~ tO ~ r~-i-t~ or ~ Dr~S~on or to inhibit licitor~ o~ ~1rott-~ ibitors ~UIite, Vlr~oolo~ ~ ~lD tl9r9) ~
Lo~, ~tt" J. Pl~t l-t~. ô~:265 OP~): ~ t ~ nrlvlr~ s. 9:~15 tl9U5) Doh~r~ t ~l, Ph~iol ~ol Pl-nt p~tr~ol 33 3~T tt9U)) - ~r~ r~ (cnen et ~, Proc ll~tl Ac-d SCj US~ 90 ~53~ tlW3 E2~MPLE 24 . Catal~se ~n~ition in ~ivo The abundance of H202 was monitored in leaves following treat~ents with SA, 3-hydroxybenzoic acid (a biologically inactive analogue of SA), and 3-amino-1,2,4-triazole t3AT; a specific inhibitor of plant and animal catalases). T~e abun~nce of H202 increased in SA- or 3AT-treated tobacco leaves by 50-60~ over the .lLlol levels observed in water-treated leaves.
(Fig. 11) By contrast, 3-hydroxybenzoic acid was unable to enhance the in vi~o ~h~n~nce of H202 consistent wi~h its ineffectiveness in both binding SABP and inhibiting catalase activity. Thus, elevated ievels of H202, and in turn, e~h~ce~ oxidative stress in vivo, were consistent wit~ inhibition of catalase activity by SA observed i~ vitro.
PR gene expression is both induced by SA and associated with the development of SAR. Therefore, it was of interest to test whether SA's mechanism of action for the induction of PR genes was via its inhibition of catalases which leads to ~nhAn~e~ H202 levels. To address this question, H202 Ah~ nr~ was artificially raised in tobacco leaves by treating with H202 or compounds which either inhibit the catalase activity, like SA itself, or promote generation of H202 'SUBSrlTUrE SHEFr ~ 2~

PCT~Ss~/l262 woss/~230~

in vlvo. Injection f H202 or the catalase inhibitor 3AT into tobacco leaves induced the expression of PR-l genes. (Fig. 12) PR-l gene expression was also induced by treating tobacco leaves with glycolate (an intermediate in photorespiration which serves as a substrate for the generation of H202 by glycolate oxidase present in leaves) and wit~
paraquat (a herbicide which can be reduced in vivo and subsequently reoxidized by transfer of its electrons to oxygen to form superoxide anion; superoxide can be converted either spontaneously or enzymatically by - superoxide dismutase to H202). Thus, the SA signal appears to be propagated through H202 which may act as a secondary messenger, to activate defense-related PR
genes.
E~AH~LE 25 ExPre~ion of ~ntisense RN~
to t~e SABP qene in tr~n~qenic plants Antisense RNA to the SABP gene is expressed in transgenic plants by the cloning of the cDNA
described in example 18 behind a suitable promoter in antisense orientation. The cDNA is cloned into a plant expression casette behind a pro~oter expressed at high levels in transgenic plants and upstream of a cleavage and polyadenylation signal which is known to function in plants. A preferred promoter is the CaMV 35S
promoter and a preferred cleavage and polyadenylation signal is the nopaline synthase cleavage and polyadenylation signal. The expression cassette is transferred to a binary vector (pCGNl540 - Alexander et al., PNAS 90:7327 (1993)J for Agrobacterium transformation and a direct gene transfer vector (pCIB3064; Koziel et al., Biotechnology ll: 194-200) for direct gene transfer. Agrobacterium is particularly suitable for the transformation of dicotyledonous species and direct gene transfer is particularly suitable for the transformation of monocotyledonous species. These techn~ques are well known in the art SUBSrlTUlE SHEET (RULE 26) WOg~/1230~ PCT~S9~/1262~
-67- 2175~9~

and are described in the two abovementioned publications. Transgenic plants are screened for expression of SABP antisense RNA by Northern analysis and plants which express at high levels are found to have enhanced resistance to plant pathogens.
Alternatively, promoters for the expression of SABP can be selected which have tissue specific expression pattern and would thus localize the effects of antisense expression to particular cell types.
~P~ 26 re~ion of a salicYlic acid in~en~itive catalaYe qene in tr~nsqenic Dlant~
The inventors also have conducted tests which indicate that catalases from non-plant sources such as fungi and animals are inC~ncitive to inhibition by ~alicylic acid. Genes or cDNA copies for several of these catalases have been cloned (e.g. Scandalios, J.G.
et al., P~oc. Natl. Acad. Sci. USA, 77 (1980) 5360 5364; Bell, G.I. et al., Nucleic Acids Res., ~4, (1986), 5561-5562; Hartig, A. et al., Eur.
J . Bi ~chem, ~ 6 0, ( 19 86) 487-490). A gene ~nco~inq one of these catalases can be introduced and expressed in a plant in the sense orientation using a~ G~ iate regulatory sequences (e.g. CaMV 35S promoter and nopaline synthace cleavage and polyadenylation signal) And vectors (e.g. pCGN 1540 or pCIB3064) as described in Example 25.
Catalase is a tetrameric complex of four E~units. Subunits encoded by different members of the catalase gene family in a given plant species form active heteromeric complexes which contain subunits encoded by two or more genes (Ni et al., Biochem J. 269:233, lg90). A heteromeric complex between the endogenous plant catalase subunits and the introduced non-plant catalase subunit may not be able to form, because the subunits are too divergent. In this case the resulting homomeric catalase enroA~ by the introduced animal gene will not be inhibited by wo s~/l23n~ Pcr~ssl/l262n 2175g9'~
salicylic acid and hence the plant should become more sensitive to pathogens. Alternatively, the plant and non-plant encoded catalase subunits may together for~ a complex which is catalytically inactive. This would result in transgenic plants which are more resistant to pathogens. Also a heteromeric enzymatically active complex might be formed. This complex might be insensitive or 6ensitive to salicylic acid. If it is sensitive to SA, its phenotypes would be the same a6 the untransformed parental plant. However, if the heteromeric complex is insensitive, the transgenic plant should become more sensitive to pathogen attack.
E~AMPt~ 27 ~nhibition of c~t~l~s~ bY exvre~sion of a~ti~ense RN~ to t~e 8ABP/~ene in tr~nsae~ic tob~cco Dlant~ activ~te~ PR~ ne e~Dre~sion a~ ~n~n re~i~tance to tob~cco mos~ic viru~ (~M~).
Transgenic tobacco plants were constructed that constitutively express, under control of the CaMV 35S promoter, a cDNA copy of (from clone pCKl, Che~ et ~1 ., Science 262:~883, 1993) one member of the tobacco SABP/catalase gene family. The cDNA was in6erted, relative to the promoter, in An antisense orientation such that the RNA produced wa6 complementary to the mRNA 6ynthesized from the endogenous tobacco catala6e gene6. The cDNA was in6erted into the XbaI and S6tI sites of pRT100 (Topfer et ~1., Nucl. Acids Res. 15:5890, 1987) between the 35S
promoter and polyadenylation ~ignal of CaMV strain Cabb B-D. The resulting pla~mid was designated pCK4. The fragment c~ntaining the 35S promoter, SAE3P/catalase cDNA, and polyadenylation ~ignal sequences was excised from pCK4 with SphI, blunt-ended with Klenow fragment, and cloned into the KpnI ~ite of the Agrobacterium-ba~ed binary vector p~A482. U~ing the leaf disktransformation procedure (~orsch et ~1 ., Science 227:1229, 1984), transgenic Nicotiana tabacum (N.t.) cultivar Xanthi nc were constructed.

WOg~/1230~ PCT~TS9~/1262n Tissues from these antisense transgenic plants and from untransformed control tobacco plants were analyzed to determine the levels of catalase protein and PR-1 protein using immunoblot analysis with a combination of anti-SABP/catalase monoclonal antibodies (3B6) and anti-PR-1 monoclonal antibody (33G1; Fig. 13A and B, and Fig. 14). Leaves of untransformed plants did not express PR-1 genes in the absence of treatment by inducing chemicals such as SA
or infection with TMV. The leaves of untransformed plants contained both a major, hig~er molecular (HMW) form and minor, low molecular weight (LMW) form of the catalase subunits. In leaves of antisense transgenic plant synthesis of the LMW form was essentially eliminated, while synthesis of the major HMW form was inhibited to varying extents in the different transgenic lines. The inventors found that the greater the level of inhibition of the catalase synthesis in the different transgenic lines, the higher the production of PR-1 proteins (Fig. 13A). For example, in the transgenic line where the catalase protein level was only modestly reduced (the third lane under Leaf in Fig. 13A), there was little, if any, production of PR-1 protein. By contrast, in the transgenic line where the catalase protein level was very substantially diminished (the fourth lane under Leaf in Fig. 13A), a high level of PR-1 protein accumulated.
In the sepals and petals of flowers (Fig. 13B) and in roots (Fig. 14) only the LMW form of catalase subunit is produced in untransformed plants.
Its synthesis is effectively inhibited in these organs of the antisense transgenic plants and PR-l gene expression was found to be constitutive.
Reducing catalase protein levels, and hence catalase enzyme activity in the antisense transgenic plants also enhanced resistance to TMV infection. When the relative catalase enzyme activity levels in the leaves of 18 different antisense transgenic lines and SUBSrlTU~E SHEET (RULE 2~) wos~lI23o~ PCT~S9~/1262n the untransformed parental line was grafted versus the size of TMV-induced lesions formed on the various lines at 6 days after inoculation, it was found that there is a good correlation between reduced catalase activity and Pnh~nce~ resistance as measured by a reduction in lesion size tFig. 15).
The results suggest that blocking catalase action leads to induction of defense responses such as PR-l protein synthesis and enhanced ~ ce resistance.
~AMP~E 28 ~ ost abiotic inducers of PR qene exDre~io~ ~n~ acouired resistance act throuoh 8A.
To assess the role of SA in chemically induced PR-1 gene expression, leaves of N.t. cultivar Xanthi nc plants were injected with polyacrylic acid tO.l mM), thiamine-HCl (1 mM), ~-amino butyric acid (50 mM), barium chloride (BaCl, 20 mM), 2,6-dichloro-isonicotinic acid (INA, 1 mM) or water and levels of total SA (free SA and its glucoside) were monitored (Table 7). The SA/SAG valves are given in micrograms per gram fresh weight of leaf tissue and represent averages of values from 3-6 treated plants at 6 days past injection. T~e concentrations of chemicals used strongly induced PR-1 protein accumulation, as determined by immunoblot analysis with a PR-1 specific monoclonal antibody (33G1), and also induced enhanced resistance to TMV, as determined by reduction in lesion size. For determination of reduction in lesion size, longitudinal halves of 2 leaves on 3-6 plants were injection with chemicals, and gix days later the entire leaf was infected with TMV. Twenty lesions were measured on each half of the leaf ~even days post infection; the values given represent the average percent reduction in lesion diameter in the treated leaf half compared to the untreated half. Similar recults were obtained by comparing lesion size to that of control, water-injected plants (data not shown).
Treatment with all of the above chemicals, except INA, W09~ll230~ PCT~Ss~/l262~

resulted in elevated levels of total SA. Neither injection of water or wounding induced PR-1 gene expression, enhanced resistance, or elevated SA levels.
This result argues that many abiotic inducers of PR
gene expression and acquired resistance act by elevating SA levels.
~P~E 29 2,6 Dichloro-i~onicotinic acid, li~e 8A, act~ bY
bin~in~ catalas~ and i~ibiti~q its enzYmatic activit~
10INA treatment induces resistance to a variety of pathogens (M~traux et al., In Advanced in Molecular Genetics of Plant-Microbe Interactions 1:432, 1991, Kluwer Academic Publishers, Dordrecht; W~rd et al., Plant Cell 3:1085, 1991; Uknes et al ., Plant 15Cell 4:645, 1992). It also activates a common set of genes whose expression is induced systemically after local infection of tobacco by TMV. This group of genes i~ also turned on by SA treatment (Ward et al., Plant Cell 3:1085, 1991). INA, however, does not act through SA, as shown in Example 28, Table 7.
In 1984, Rirkman and Gaetani (Proc. Natl.
Acad., Sci. U.S.A. 81:4343) demonstrated that bovine catalase tightly binds nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NADH). However, bi~ing of NADPH or NADH
(NAD(P)H) was ~hown not to be required for or effect catalase's enzymatic activity. Since NAD(P)H and INA
share t~e nicotinic acid moiety, the inventors hypothesized that INA ~lso binds catal_se, but unlike NAD(P)H, this binding would inhibit catalase's ability to convert H202 to H20 and 2 The inventors found that INA inhibited catalase'~ enzymatic activity (Fig. 16 and Table 8).
SA And INA were found to have similar dose response curves for inhibition of catalase in vivo (Fig. 16).
This was determined by adding increasing concentrationc of SA or INA to tobacco ~uspension cells (derived from cultivar Xanthi nc), as described below. The tobacco wo9sll23o~ PCT~S9~/1262~

suspension cell culture was grown in the dark in MS
medium at approximately 22C with agitation (160 rpm).
Cells were maintained by diluting lO-fold every 5 days.
For experimentation, 5 ml of suspension cells obtained 3 days after dilution was transferred to a 25 ml Erlenmeyer flask and agitated (lO0 rpm) at room temperature in the absence or presence of SA or INA
which had been adjusted to pH 5.8. After l hr, l ml of the treated cell suspension was transferred to 7 ml of fresh MS medium containing SA or INA in a lO ml beaker (density was approximately 6 mg cells per ml). Cells were maintained in suspension with a magnetic stir bar.
In vivo catalase activity was determined by measuring, with an oxygen electrode, the rate of H2O2-dependent 2 l~ production immediately after addition of H2O2 to lO mM
final concentration.
To assess whether INA's inhibition of catalase was responsible for its biological activity, various biologically inactive, as well as active, analogues of INA were tested for their ability to bind SABP/catalase and inhibit its enzymatic activity (Table 8). The biological activity o~ the analogues, obtained from CIBA-Geigy Corporation, were determined by measuring their ability at l mM concentration to induce PR-l protein synthesis in tobacco leaf disks during a 24-hour incubation and by their effectiveness in inducing resistance in cUcl~h~r to microbial pathogen infection (Smith ct a~. Physiol. Molec. Plant Pathol. 38:223-235 (l99l). Bin~ing to SABp/catalace was determined by a competition assay with crude extracts prepared from tobacco leaves. Binding of INA, SA and their analogues was measured as percentage inhibition of binding of 14c-labeled SA t20 ~M] in the presence of 1 mM unlabeled INA, SA or their analogues, as described by Chen and Rlessig (Proc. Natl. Acad.
Sci. U.S.A. 88:8179, l99l). Inhibition of catalase activity in tobacco cuspension cells was measured as described above.

WO 9~ 230~ PCT/I,~S94/1262(1 ~73~ 2175493 Table 8: Inhibition by INA, SA and their analogues of catalase activity and [14C] SA binding INA, SA and Biological Inhibitlon (80) analogues activity Catalase [l4C~SA
~cpd. name ornumber) activity binding "~ (INA) + 98 65 (3) ~ 100 69 ~8) + 48 63 ~6 (7) + 79 2~

(6) - 25 .t9 (9) ~ 15 11 \~=~ O

~ (SA) + 84 90 L~ (~ChloroSA) + 95 8B
L
(5-ChtoroSA) + 100 87 (3.5-DichloroSA) + 100 85 o a-~ (3~ I,oA~ A) _ 0 (4 ll~ c.a~ A) -- O O

WO95/1230~ pcT~ss~ll262 As shown in Table 8, there is 2al 7c~o~s9 correlation between biological activity of the analogues and their ability to binding to catalase and inhibit its activity. The exception to this correlation was analogue #7 which was active in the two in vivo assays (PR-l gene induction and inhibition of catalase) but much less active in the in vitro binding assay. A likely explanation is that the aldehyde form of INA (analogue t7) is rather inactive compared to the acidic form (INA) and in vivo the aldehyde form was converted to INA. In sum, the above dose-response analysis (Fig. 16) and pharmacological data suggest that INA and SA share the same mode of action, namely, inhibition of catalase's ability to convert H2O2 to H2O
and 2 In addition, the chlorinated SA analogues 4-chlorosalicylic acid, 5-chlorosalicylic acid and 3,5-dichlorosalicylic acid were found to be potent inducers of PR-l protein synthesis and enhanced resistance to TMV infection of N.t. cv. Xanthi nc plants as measured by reduction in lesion size compared to water-treated control plants. T~ese SA analogues also very effectively inhi~ite~ catalase activity of tobacco suspension cells in vivo and binding of ~4C-labeled SA
to SABP/catalase in vitro.

Oq~ 0~ 0~ OH

30 ,~
~' 4 Comparison of the structure of INA and its active analogues with the structure of SA and its active analogues (Tables 6 and 8) suggests there are several common features of these compounds which allow or facilitate their binding to and inhibition of catalase activity.

SU~lTUTE SHEET (RIJLE 26~

WO9511230~ PCT~S9~/1262/l -75- 217~3 Both contain a six-membered, conjugated ring and a carboxyl group. Substitution of hydrogen atoms by halide groups at the 3,4 and 5 positions of the ring (relative to the carboxyl group) do not interfere with and may enhance binding to SABP/catalase, whereas substitution of hydroxyl groups at the same positions blocks binding. Addition of a hydroxyl group at position 2 of benzoic acid (BA) App~rs to facilitate binding while substitution of a chloro, ethoxyl, or thiol group at this position inhibits binding (Table 2 from Chen et al., Proc. Natl. Acad. Sci.
U.S.A. 90:9533, 1993).
E~AMPL~ 30 Techni~ue~ for t~e Generation of Transqenic Plants The following example describes and reviews t~çhn iques which are well known in the art for the expression of foreign genes in both monocotyledonous and dicotyledonous plants. These ~echniques are suitable for the expression of SABPtcatalase in antisense ~for example) in transgenic plants, and specific experimental details of such an experiment are given in Example 27.
Con~truction of Pla~t Transformatio~ Vectors Numerous transformation vectors are available for plant transformation, and the genes of this invention can be used in conjunction with any such vectors. ~he selection of vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptII gene which confers resistance to kanamycin (Messing &
Vierra, Gene 19:259-268 (1982); Bevan et al., Nature 304:1B4-187 (1983), the bar gene which confers resistance to the herbicide phosphinothricin (White et al ., Nucl Acids Res 18:1062 (1990), Spc~cer et al .
Theor Appl Genet 79:625-631 (1990), the hph gene which Wos~/1230~ PcT~ss~ll262~l -76- 217~9~

confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4:2929-2931), and the dhfr gene, which confers resistance to methatrexate (Fling & Elwell, 1980).
Vectors suitable for Agrobacteri.um transformation typically carry at least one T-DNA
border sequence. These include vectors such as pBINl9 (Bevan, Nucl. Acids Res. (1984) and pCIB200 (EP O 332 ~04).
Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences.
Transformation techniques which do not reply on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. For example, pCIB3064 is a pUC-derived vector suitable for direct gene transfer technique in combination with selection by the herbicide basta (or phosphinothricin). It is described in W0 93/07278 and ~oziel et al. (Biotechnology ll:194-200 (1993)).
~e~uireme~tY for Construction of Plant ~xDression C~ tt~
Gene sequences inten~e~ for expression in transgenic plants are firstly assembled in expression cassettes behind a suitable promoter and upstream of a suitable cleavage and polyadenylation site. These expression cassettes can then be easily transferred to the plant transformation vector~ described above. The manipulations of required sequences in vectors prior to their transfer to plant transformation vectors is according to techniques well known in the art.

217549~
wo sstl23n~ Pcr~uss~/l262n 2175~93 The present invention encompasses the expression of the genes of this invention in sense or antisense orientation under the regulation of any promoter which is expressible in plants, regardless of the origin of the promoter. Furthermore, the invention enComr~-cc~s the use of any plant-expressible promoter in conjunction with any further sequences required or selected for the expression of the AMS gene. Such sequences include, but are not restricted to, cleavage and polyadenylation site~, extr~n~o~ se~n~s to enhance expression (such as introns [e.g. Adh intron 1], viral sequences [e.g. TMV-~].
Promoter ~ele~tion The selection of promoter used in expression cassettes will determine the spatial and temporal expression pattern of the construction in the transgenic plant. Selected promoters may have constitutive activity and these include the CaMV 35S
promoter, the actin promoter (McElroy et al. Plant Cell 2:163-171 (1990); McElroy et al . Mol.
Gen.Genet. ~ 150-160 (1991); ChihhAr et al. Plant Cell Rep 1~:506-509 (1993), and the ubiquitin promoter (Binet et al. Plant Science 79:87-94 (1991), Christensen et al . Plant Molec. Biol. 12:619-632 (1989); Taylor et al. Plant Cell rep. 12:491-495 (1993)). Alternatively they may be wound-induced (Xu et al. Plant Molec. Biol 22:573-588 (1~93), Logemann e~ al . Plant Cell 1:151-158 tl989), Rohrmeier & Lehle, Plant Molec. Biol. 22:78 -792 (1993), Firek et al.
Plant Molec. Biol. ~:129-142 (1993), Warner et al .
Plant J. 3:191-201 (1993) and thus drive the expression of a transgene at the sites of wounding or pathogen infection. Other useful promoters are expressed in specific cell types (such as leaf epidermal cells, ~eosphyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example). Patent Application WO 93/07278, for example, describes the isolation of the maize trpA gene which is 2175~9~
woss/123o~ PCT~lSs~/l262n preferentially expressed in pith cells. Hudspeth ~
Grula (Plant Molec Bio 12:579-589 (1989) have described a promoter derived from the maize gene encoding phosphoenolpyruvate carboxylase (PEPC) with directs expression in a leaf-specific manner. Alternatively, the selected promoter may drive expression of the gene under a light-induced or other tDmrorally-regulated promoter. A further alternative is that the selected promoter be chemically regulated. It will be appreciated that many different promoterC with different expression patterns are available and well known in the art.
Transcription~l Cleavaqe and Polyadenylation 8ites A variety of transcriptional cleavage and polyadenylation sites are available for use in expression cassettes. These are responsible for correct processing (formation) of the 3' end of mRNAs.
Appropriate transcriptional cleavage and polyadenylation sites which are known to function in plants include the CaMV 35S cleavage and polyadenylation sites, the tml cleavage and polyadenylation sites, the nopaline synthase cleavage and polyadenylation sites, the pea rbcS E9 cleavage and polyadenylation sites. These can be used in both monocotyledons and dicotyledons.
~equence~ for t~s ~n~ancement or Re~ulation of E~ressio~
Numerous ~e~uences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.
Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be 217S4!13 WO9~/1230~ PCT~IS9~/1262 particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (CA 7 7 ic et al ., Genes Develop 1:1183-1200 (1987). In the same experimental system, the intron from the maize bronzel gene had a similar effect in enhancing expression (Callis et al., sup~a ) . Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "U-sequencen), Mai2e Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Ga~lie et ~1. Nucl. Acids Res. 1~:8693-8711 (1987); Skuzeski et al. Plant Molec.
Biol. 15; 65-79 (1990)).
~rans~ormation of DicotYledon~
Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and technigues which do not require Agrobacterium . Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG
or electroporation mediated uptake, particle ho~h~rdment-mediated delivery, or microinjection.
Examples of these techniques are described by Paszkowski et al., EMBO J 3:2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199:169-177 (1985), ~eich et a~., Biotechnology 4:1001-1004 (1986), and ~le~n et al., Nature 327: 70-73 (1987). In each case the transformed cells are reqenerated to whole plants using standard techniques known in the art.
Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its hiqh efficiency of transformation and 217549~
wos~/1230~ PcT~ss~ll262 -8~-its broad utility with many different species. The many crop species which are routinely transformable by Agrobacterium include tobacco, tomato, sunflower, cotton, oilseed rape, potato, soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432 (tomato), Wo 87/07299 (Brassica), US 4,795,855 (popular)).
Agrobacteri~m transformation typically involves the transfer of the binary vector carrying the foreign DNA
of interest (e.g. pCIB200 or pCIB2001) to an a~ iate Agro~cterium strain which may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 (Uknes et al. Plant Cell 5:159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomp}ished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain.
Alternatively, the recombinant binary vector can be transferred to Ag~obacterium by DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16:9877 (1988)).
Transformation of the target plant species by recombinant A,a~ob~cterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follo-~s protocols well known in the art.
Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
Trn~form~tion of Mono~otYle~o~
Transformation of most monocotyledonous species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techni~ues and particle horh~rdment into callus tissue.
Transformation can be undertaken with a single DNA
species or multiple DNA species (i.e. co-2175~93 transformation) and both these techniques are suitable for use with this invention. Co-transformation may have the advantage of avoiding complex vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the ~electable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is less than 100%
freguency ~ith which separate DNA ~pecies ~re integrated into the genome (Schocher et al.
Biotechnology 4:1093-1096 (1986)).
Patent Applications EP O 292 435, EP 0 39 225 and Wo g3/07278 describe terhniques for the preparation of callus and protoplasts of maize, transformation of protop~a~ts using PEG or ele~L~6~G~ation, And the regeneration of maize plants from transformed protoplasts. Gordon-~amm et ~1. (Plant Cell ~:603-618 (1990)) and Fromm et al. (Biotechnoloqy ~:833-839 (1990)) have published techniques for transformation of A188-derived maize line using particle bombardment.
Furthermore, application Wo 93/07278 and ~oziel et ~1.
(Biotechnology 11:194-200 (1993) describe tPchnioues for the transformation of elite inbred lines of maize by particle bombardment.
Transformation of rice can also be undertaken by direct gene transfer tec~niquee utilizing protoplasts or particle bombardment. Protoplaet-mediated transformation has been described for Japonic~-types and Indic~-types (Zhange et al., Plant Cell Rep. 7: 379-384 (1988); Shimamoto et al .
Nature 338: 274-277 (1989); Datta et al . Biotechnoloqy 8:736-740 (1990) ) . Both types are al~o routinely transformable using particle bombardment (Christou et al. Biotechnoloqy 9:957-962 (1991) ) .
Patent Application EP 0 332 581 described techniques for the generation, transfor~ation and reqeneration of Pooideae protoplasts. Furthermore, 217519~
wos~/1230~ pcT~Tss~ll26 wheat transformation has been described by Vasil et al.
(Biotechnology 10:667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology l~:1553-1558 (19g3)) and Weeks et al. (Plant Physiol.
102:1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus.
.vub~KIA~ APPLICABlLITY
The present invention is applicable to the pharmaceutical and agricultural industries and to the provision of disease-resistant plants and animals.

W O9~11230l 2 t 7 5 4 9 3 PCT~TS9~/1262() ~Qu~ ISSING

~1) GENERAL INFOR~ATION:
(i) APPLICANT:
~A) NAKE: Rutgers University lB) STREET: Old Queens, Somerse~ Street ~C) CITY: New Brusnwi~k (D) STATE: New Jer~ey ~E) COu~: USA
~F) POSTAL CODE (ZIP~: 08903 (G) TELEPHONE:
~H) TE~EFAX:
~A) NA~E: Daniel Rlessig (B) SIREET: 191 Corn~ll Boulev~rd (C) CISY: Bridgcwater (D) STATE: NJ
(E) Cuu~Y: USA
(F) POSTAL CODE (ZIP~: 08807 ~A) NAME: 7h i Yi A~g Chen ~B) STREET: 834 Davidson Road ~CJ C'TY: Piscataway ~D) STATE: NJ
~E) CUUN~K~: USA
~F) POSTAL CODE (ZIP): 08854 (ii) TISLE OF INVENSION: Salicylic Acid BjT~jn~ Protein ~iii) NUMBER OF S~UU~N~ S: 7 (iv~ Cû.I~u.~ RFiUlABLE FORM:
~A) ~EDI~ TYPE: Floppy disk (B) COMP~ER: IB~ PC ~oT~p~tible (C) OPERAmING SYSTE~: PC-DOS/MS-DOS
~D) SOFTW~E: ASCI' Editor (v) C~RRENT APPLICASION DATA:
~A) APPLICASION N~MBE~: US 08/259,535 (P) FIL~NG DATE: 14-JUN-1994 (C) CBASSIFICATION:
(V1 ) PRIOR APPLICATION DATA:
(A) APPLICATION NU~BER: US 08tl46,317 (B) FI~ING DATE: 02-NOV-1993 (V1 ) PRIOR APPLICATION DATA:
~A) APPLICATION N~M3ER: PCT~US93/07202 ~B) FILING DATE: 30-JUL-19g3 ~vi) PRIOR APPLICATION DATA:
~A) APPLICATION NUKBER: US 08/038,132 ~B) FILING DASE: 26-MAR-1993 (V1 ) PRIOR APPL_CATION DATA:
(A) APPLICASION NU~BER: US 07/923,229 (E) FI'.NG DATE: 31-J~L-1992 (2) INFO~MATION FOR SEQ ID NO:1:
( i ) ~f ~U N~ CHARAC~ERIS~ICS:
~A) LENGTH: 1858 base p~irs ~8) TYPE: nucleic Acid ~C) S~R~U~ S: sin~le iD) TOPOLOÇY: line~r SUBSmU~ SltEE~ (RULE 26) W O9~/123~ PCT~'S9~/1262(~

(ii) MOLECUr~E TYPE: cDNA

~ix) FEATURE:
(A) NANE/KEY: CDS
(B) LOCATION: 2..1468 (D) OTHER INFORMATION: /note= DNA sequence as disclosed in Table 5-(xi) ~ u~ DESCRIPTION: SEQ ID NO:1:

Ser Lys Phe Arg Pro Ser Ser Ala Tyr Asp Ser Pro Phe Leu Thr Thr Asn Ala Gly Gly Pro Val Tyr Asn Asn Val Ser Ser Leu Thr Val Gly Pro Arg Gly Pro Val Leu Leu Glu Asp Tyr His Leu Ile Glu Lys Leu Ala Thr Phe Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Arg Gly Ala Ser Ala Lys Gly Phe Phe Glu Val Thr His Asp Ile Ser His Leu Thr Cys Ala Asp Phe Leu Arg Ala Pro Gly Val Gln Thr Pro GTT ATT TGC CGT TTC TC~ ACT GTC GTC CAT GAG CGT GGA AGC CCC GAG 334 Val Ile Cys Arg Phe Ser Thr Val Val His Glu Arg Gly Ser Pro Glu TCC ~ AGG GAC ATT CGT GGT ~TT GCT GTC AAA TTT TAC ACC AGA GAG 382 Ser Leu Arg Asp Ile Arg Gly Phe Ala Val Lys Phe Tyr Thr Arg Glu Giy Asn Phe Asp Leu Val Gly Asn Asn Val Pro Val Phe Phe Asn Arg Asp Ala Lys Ser Phe Pro Asp Thr Ile Arg Ala Leu Lys Pro Asn Pro Lys Ser Hls Ile Gln Glu ~yr Trp Arg Iie Leu Asp Phe Phe Ser Phe Leu Pro Glu SPr Leu His T~ Phe Ala Trp Phe Phe Asp Asp Val Cys Leu Pro Thr Asp Tyr Arg His Met Glu Gly Tyr Gly Val His Ala Tyr Gln Leu Ile Asn Lys Ala Gly Lys Ala His Tyr Val Lys Phe His Trp WO95/l23W 2 1 7 5 4 9 5 PCT~Usg~ll262n Lys Pro Ths Cys Gly Val Lys Cys Met Ser Glu Glu Glu Ala Ile Arg225 230 2~5 GTC GGA GGT AC~ AAT CAT AGC CAC GCC ACC AAG GAT CTC ,-AC GAT TCG 766 Val Gly Gly Th5 Asn H~s Se~ H~s Ala T~s Lys Asp Leu Tyr Asp Ser ATT GCT GCS GGA AAC TAT CCC GAG TGG AAA CTT T~T ATC CAA AT- ATG 814 Ile Ala Ala Gly Asn Tyr Pro Glu Trp Lys Leu Phe Ile Gln Ile ~et GAC AC, GAG GAT GTA GAC AAA TTC GAC TTT GAT CCT CST GAT GTA ACC 262 Asp Ths Glu Asp Val Asp Lys Phe Asp Phe Asp Pro Leu Asp Val Thr 2?5 280 285 AAG ACC TGG CCT GAG GAT ATC TTG CCA TTG ATG CCA GTT G~A CGA TTG 910 Lys Thr Trp Pro Glu Asp Ile Leu Pro Leu Met Pro Val Gly Arg Leu GSA CTT AAC AGG AAT ATC GAT AAC TTC TTT G~T GAG AAC GAG CAG CSC 9 5 a Val Leu Asn Arg Asn ,le Asp Asn Phe Phe Ala Glu Asn Glu Gln Leu GCG T~S AAC CC~ G~C CAT ATT GTC CCT GGT CTT TAC TAT TCG GAG GAC 1006 Ala Phe Asn Pro Gly H~s Ile Val Pro Gly Leu Tyr Tyr Ser Glu Asp AAG CTT CTC CAG ACT AGG ATA -~ GCG TAT GCT GAT ACT CAG AGA CAC 1054 Lys Leu Leu Gln ~h- Arg _le Phe Ala Tyr Ala Asp Thr Gln Arg His CGT A~T GGA CCA AAC -,AT ATG CAG C-T CCT GTT AAT GCT C~C AAG TGT 1102Arg ~le Gly Pro Asn Tyr Met Gln Leu Pro Val Asn Ala Pro Lys Cys GCT CAT CAC AAT AAT CAC CGG GAT G~T GCC ASG AAC TTC ATG CAT CGC 1150 Ala Hls H~s Asn Asn Hls Arg Asp Gly Ala Met Asn Phe Met %is Arg GAT GAA GAG GTG GAT TAT TTG CC~ T-A AGG TTC GAT CCT TGT CGT CAT 1198 ASp Glu Glu Val Asp ~yr Leu P-o Ser Arg Phe Asp Pro Cy5 Arg His 3e5 390 395 GCT GAA CAG ,-AC ~~ ATT CC~ -CT CG. GTC TSG ACA GGA AG~ CGT GAA 1246Ala Glu Gln Tyr P-o ,le Pro Ser Arg Val Leu Thr Gly Arg Arg Glu ~ 405 410 415 ATG TGT GTC ATT GAG ~AA GAG AAC AAC T~C AAG C~G GCA GGA GAA AGA 1294 Me~ Cys Val Ile Glu Lys Glu Asn Asn Phe Lys Gln Aia Gly Glu Arg TAC AGA TCC TGG G~A CCT GAC AGG CAA GAC AGA TAT GST AGC AAA ~GG 1342 ~yr Arg Se~ ~rp Glu Pro Asp Arg Gln ASp Arg Syr Val Ser Lys Trp Val Glu Hls Leu Ser Asp Pr~ A-g Val Thr Tyr Glu Ile Arg Ser Ile TGG ATA TGC TCC CTG T-- CAG G~T GAC AAG TCT TGT GGT CAG AAG GTC 1438Trp lle Cys Ser Leu Ser Gln Ala Asp Lys Ser Cys Gly Gln Lys Val 465 ~70 475 GCT TCT CGT C~C ACT TTA AAG CCT ACA ATG TGATGAAGAC TAAGATGAAA lg88 Ala Ser Arg Teu Thr Leu Lys Pro Thr Met 480 4~5 ACACTACTGG G~AAACGTCT CAAGTTGCAG T~TGAAGGAG TACT~AACCA ~n~AA~G~AT 1548 SUBSrlTUTE SHEET (RULE 26) WO9~/1230J2 1 7 5 4 3 3 PCT~sg~ll262n TAC~ -l~l ATAAAG~TA ~ . SSA ~ ~ T~TGTACC~A 1608 ACSTTGATAT ~ A CSATGACACA ATATATGTTG CA~TGAATA AG~-TACAGAT 1668 GTATGTTC~A GTA~ ~l CA~ -. CTATmmSAC~ ~ACA ~.-i-.-.-,AAG~ ;728 ~ ~CA AAASTAT~TC ASA~1~1C A~ ~C TSGAAGSASA CCC~CAASSC 1788 TASAASGCCA CSGGTA--~T AGT~TSATTG ACAT~TTAAT AAGAAAOCTG CTA~ G 1848 ~-lCC-~r~AA 1858 ~2) lN~ ~TION FOR SEQ ID NO:2 ,?U~N~ C~lARAC~ERlSSICS:
(A) LENGSH: 489 amino acids ~B) TYPE: am~no acid (D) TOPOLOGY 1ine~r ( ii ) MnTFr~F TYP_ protein (xi) ~Uu~N-~ DESCRIPSION SEQ ID NO:2:
Ser Lys Phe Arq Pro Ser Ser Ala Tyr Asp Ser Pro Phe Leu Thr Shr 1 5 10 lS
fin Ala Gly Gly Pro Val Tyr Asn Asn Val Ser Ser Leu Shr Val Gly Pro Arg Gly Pro Val Leu Leu Glu Asp Tyr His Leu Ile Glu Lys Leu Ala Thr Phe Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Arg Gly Ala Ser Ala Lys Gly Phe Phe Glu Val Thr His Asp Ile Ser His eu Thr Cys Ala Asp Phe Leu Arg Ala Pro Gly Val Gln Thr P~o Val le Cys Arg Phe Ser Thr Val Val His Glu Arg Gly Ser Pro Glu Ser Leu Arg Asp Ile Arg Gly Phe Ala Val Lys Phe Tyr Shr Arg Glu Gly llS 120 125 Asn Phe Asp Leu Val Gly Asn Asn Val Pro Val Phe Phe Asn Ary Asp 130 135 1~0 Ala Lys Ser Phe Pro Asp Thr Tle Ars Ala Leu Lys Pro Asn Pro Lys 145 _50 155 150 er His Ile Gln Glu Tyr Trp Arg Ile Leu Asp Phe Phe Ser Phe Leu ro Glu Ser Leu ~s T~r Phe Ala Trp Phe Phe Asp Asp Val Cys Leu 180 185 ~90 Pro Thr Asp Tyr Ars Hls ~et Glu Gly Tyr Gly Val His Ala Tyr Gln Leu Ile Asn Lys Ala Gly Lys Ala His Tyr Val Lys Phe His Trp Lys Pro Thr Cys Gly Val Lys Cys Met Ser Glu Glu Glu Ala lle Arg Val 225 230 235 2~0 SUBSmU~E SHEET (RULE 26) W O9~11230~ 2 1 7 5 4 9 3 PCT~lS9~/1262~
ly Gly Thr Asn His Ser His Ala Thr Lys Asp Leu Tyr Asp Ser Ile 2g5 250 255 la Ala Gly Asn Tyr Pro Glu Trp Lys Leu Phe Ile Gln Ile Met Asp Thr Glu Asp Val Asp Lys Phe Asp Phe Asp Pro Leu Asp Val Thr Lys Thr Trp Pro Glu Asp Ile Leu Pro Leu Met Pro Val Gly Arg Leu Val Leu Asn Arg Asn Ile Asp Asn Phe Phe Ala Glu Asn Glu Gln Leu Ala he Asn Pro Gly His Ile Val Pro Gly Leu Tyr Tyr Ser Glu Asp Lys eu Leu Gln Thr Arg Ile Phe Ala Tyr Ala Asp Thr Gln Arg His Arg Ile Gly Pro Asn Tyr Met Gln Leu Pro Val Asn Ala Pro Lys Cys Ala His His Asn Asn His Arg Asp Gly Ala Met Asn Phe Met His Arg Asp Glu Glu Val Asp Tyr Leu Pro Ser Arg Phe Asp Pro Cys Arg His Ala lu Gln Tyr Pro Ile Pro Ser Arg Val Leu Thr Gly Arg Arg Glu Met ys Val Ile Glu Lys Glu Asn Asn Phe Lys Gln Ala Gly Glu Arg Tyr Arg Ser Trp Glu Pro Asp Arg Gln Asp Arg Tyr Val Ser Lys Trp Val Glu His Leu Ser Asp Pro Arg Val Thr Tyr Glu Ile Arg Ser Ile Trp Ile Cys Ser Leu Ser Gln Ala Asp Lys Ser Cys Gly Gln Lys Val Ala Ser Arg Leu Thr Leu Lys Pro Thr Met (2) INFORMATION FOR SEQ ID NO:3:
u~N~ CHARACTERISTICS:
(A) LENGTH: 15 amlno acids ~B) TYPE: P~ino acid (C) sTRpNnFnNF~s single ~D) TOPOLOGY: linear (ii) ~OLECULE TYPE: pep~ide (ix) FEATURE:
(A~ NAME/KEY: Peptide (B) LOCA~ION: 1..15 (D) O~HER INFORMATION: /note= 'Amino acid sequence of tryptic fragment U572-1-(xi) -~yu~N~E DESCRIPTION: SEQ ID NO:3:
Glu Gly Asn Phe Asp Leu Val Gly Asn Asn Phe Pro Val Phe Phe W 095~1230~ PCTr~S9~11262n l 5 lO 15 (2~ INFO7~MATION FOR SEQ ID NO:4:
u NLr: CHARACTERISTICS:
tA) LENGTH: 9 amino acids ~B) TYPE: amino acid (C) STRANV~VN~:SS: single ~D) TOPOLOGY: linear ~ii) MO7FCU7F TYPE: peptide ~ix) FEAT7JRE:
~A) Nl~ME~KEY: Peptide ~B) LOCATION: l..9 ~D) OTHER INFORMATION: ~noee= Amino acid sequence of tryptic peptide N994-l-~Xi) ~QU N~ DESCRIPTION: SEQ ID NO:4:
Ser Phe Thr Pro Asp Arg Gln Glu Argl 5 ~2) INFORMATION FOR SEQ ID NO:5:
~iJ ~QU~N~: CHARACTERISTICS:
~A) LENGTH: 9 amuno acids ~B) TYPE: amuno acid ~C) ST7~UNDEDNESS: slngle ~D) TOPOLOGY: linear ~ii) MOLEC7JLE TYPE: peptide ~ix) FEATURE:
~A) NAME~KEY: Peptlde ~B) LOCATION: l..9 (D) OTHER INFOP~ATION: tnote= Amino acid sequence of tryptic peptide D839-(xi) S~yu~ DESCP.IPTION: SEQ ID NO:5:
Trp Val Glu Ala Leu Ser Asp Pro Argl 5 (2) INF07~ATION FOR SEQ .D NO:6:
~i) 5~:YU~N~ CHARAGTERISTICS:
(A) LENGTH: 20 base pafrs ~B) TYPE: nuclelc acid ~C) STRANDEDNESS: slngle (D) TOPOLOGY: llnear ~ii) MOLECULE ~YPE: o~her nucleic acid (A) DESCRIPTION: /àesc = oiigonucleotide primer no.l for U572-l tryptlC fragmenl' (xi) ~ U~N~ DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:

WO 9~;/1230~ 2 1 7 5 4 9 ~ Pcr~lsg~/l262() (i) S~QU N~ CHARACTERISTICS:
~A) LENGTH: 17 base palrs (B) TYPE: nucleic acid (C) STR~N~ N~:~S: single (D) TOPOLOGY: linear ii ) M~T.~rU~.F ~YPE: other nucleic acid ~ A) DESCRIPTION: /desc = oligonucleotide primer no 2 for tryptic fragment U572-1-(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:7:
CKDATRAARA ~N~NGG 17

Claims (27)

1. A process for purifying salicylic acid binding protein characterized by the steps of:
providing a plant tissue;
homogenizing and buffering said tissue to form a homogenate;
filtering and clarifying said homogenate and collecting a first supernatant;
chromatographically fractionating said first supernatant in a first anion exchange column to yield a first eluent; and chromatographically fractionating said first eluent on a second gel filtration column to yield a second eluent.

2. The process of claim 2 further characterized by the steps of:
chromatographically fractionating said second eluent on a third gel filtration column to yield a third eluent;
chromatographically fractionating said third eluent on a fourth immobilized reactive dye column to yield a fourth eluent;
and collecting said fourth eluent.

3. A salicylic acid binding protein having an apparent average native molecular weight of about 240kDa, an apparent Kd of 14p5 µM for salicylic acid and being capable of binding salicylic acid compounds in direct correlation with a salicylic acid compound's physiological activity, said protein being partially purified from tobacco.

4. A process for purifying salicylic acid binding protein characterized by the steps of:
providing a plant tissue;
homogenizing and buffering said tissue to form a homogenate;
filtering and clarifying said homogenate and collecting a first supernatant;

chromatographically fractionating said first supernatant in a first anion exchange column to yield a first eluent; and chromatographically fractionating said first eluent on a second gel filtration column to yield a second eluent;
chromatographically fractionating said second eluent on a third immobilized reactive dye column to yield a third eluent;
chromatographically fractionating said third eluent on a fourth gel filtration column to yield a fourth eluent;
and collecting said fourth eluent.

5. Substantially pure salicylic acid binding protein.

6. A DNA sequence which encodes an antisense catalase gene.

7. A method for inhibiting the breakdown of H2O2 in a plant, said method characterized by the step of:
transforming said plant with the DNA sequence of claim 6 wherein said DNA sequence is operably linked to plant control regions and is capable of expression in the plant.

8. A plant transformed with the DNA
sequence of claim 6.

9. A modified catalase which is capable of assembling with endogenous catalase and forming an inactive enzyme.

10. A DNA sequence which encodes the modified catalase of claim 9.

11. A plant transformed with the DNA
sequence of claim 10.

12. A method of enhancing a plant's resistance to disease characterized by the step of:
increasing the level of cellular H2O2 or other active oxygen species derived from H2O2 within said plant's cells from a first concentration to a second concentration.

13. The method of claim 12 characterized in that said first concentration is insufficient to induce significant expression of defense-related genes such as PR-1 genes and characterized in that said second higher concentration is sufficient to induce the expression of said defense-related genes.

14. The method of claim 13 further characterized by the step of adding H2O2 or other active oxygen species to said plant's cells to increase the concentration of H2O2 derived from H2O2.

15. The method of claim 13 further characterized by the step of treating said plant's cells with a compound which will increase cellular production of H2O2 or other active oxygen species derived from H2O2.

16. The method of claim 13 further characterized by the step of reducing the rate at which H2O2 is degraded or scavenged.

17. The method of claim 16 further characterized by the step of adding to said plant's cells a compound which is capable of binding endogenous catalase and inhibiting its enzymatic activity.

18. The method of claim 16 further characterized by the step of inducing said plant's cells to express a compound capable of binding endogenous catalase within said plant cell and inhibiting its enzymatic activity.

19. The method of claim 16 further characterized by the step of inducing the expression of an antisense capped catalase within said plant's cells.

20. The method of claim 16 further characterized by the step of expressing a sense catalase construct which assembles with native protein to form an inactive catalase gene enzyme within said plant's cells.

21. A method of reducing catalase activity in a plant characterized by the step of:
treating said plant with a compound that will inhibit catalase activity in said plant.

22. The method of claim 21 characterized in that said compound binds with said catalase thereby inhibiting its activity.

23. The method of claim 22 characterized in that said compound is not endogenous to said plant.

24. The method of claim 23 characterized in that said compound is INA.

25. The method of claim 22 characterized in that said compound is endogenous to said plant.

26. The method of claim 25 characterized in that said compound is SA.

27. A method of reducing catalase activity in a plant characterized by the step of: transforming said plant with a exogenous DNA sequence which is operably linked to plant control regions, encodes an antisense catalase and which is capable of expression in said plant.