BR112015024341B1 - EXPRESSION CASSETTE FOR INDUCTION OF RESISTANCE TO MULTIPLE NEMATOID SPECIES IN PLANTS, METHODS AND PLANTS THAT USE IT - Google Patents

Abstract

EXPRESSION CASSETTE FOR INDUCTION OF RESISTANCE TO MULTIPLE NEMATOID SPECIES IN PLANTS, METHODS AND PLANTS THAT USE IT. The present invention provides plant expression cassettes capable of silencing genes from multiple nematode species that feed on said plants conferring resistance to said nematode species. More specifically, the expression cassettes of the present invention are capable of expressing the dsRNA molecules in target tissues of nematode infestation. Also provided are vectors comprising said expression cassette, methods for producing a plant resistant to multiple nematode species as well as for controlling nematodes in a crop, plants resistant to multiple nematode species, their seeds and products produced from material extracted from said plants.

Description

field of invention

[1] The present invention relates to the control of nematodes in plants. More specifically, the present invention relates to the control of multiple nematode species by gene silencing through the expression of an RNA molecule in the plant capable of forming a double-stranded RNA comprising a fragment of the plague target gene sequence.

Fundamentals of the Invention

[2] Phytonematodes are among the main factors responsible for the drop in yields of several crops, particularly soybean, especially in tropical and subtropical regions.

[3] The control of nematodes in scale crops such as soybean is designed to integrate several methods and be cost effective. In general, the phytopathological principles of exclusion are considered (avoid infestation of unaffected areas by species or new breeds, on the property or in a larger geographic region); eradication (crop rotation with non-host summer and winter species); regulation (modification of the environment and plant nutrition); and immunization (use of cultivars resistant to certain species or races). The current strategies for nematode control are: use of nematicides, crop rotation, natural genetic resistance and genetic engineering.

[4] Nematicides have been widely used to control parasitic nematodes of sedentary and migratory plants, but these compounds are often associated with harmful effects on the environment.

[5] Crop rotation, an effective cultural practice for many biotic stresses, is also an important strategy for managing plant parasitic nematodes, although in many cases its effectiveness is limited.

[6] The management of phytonematodes is highly dependent on host plant resistance obtained by traditional breeding methods, often derived from a limited genetic base. Conventional breeding, however, has some limitations, such as the genetic link between genes of interest and undesirable genes and interspecific incompatibility.

[7] Genetic engineering is an extremely useful tool as it allows the identification of genes of interest, manipulation of these genes, the construction and introduction of a single gene of interest directly into elite cultivars and the selection of plants that have this gene. The gene to be introduced can come from the same species or from other species, thus allowing the breaking of barriers imposed by sexual incompatibility between different species, in addition to eliminating the effect of unwanted gene links.

[8] The gene silencing strategy, by interference mediated by double-stranded RNA, offers good results in the control of nematodes (MCCARTER, J. Molecular approaches toward resistance to plant parasitic nematodes. In: (Ed.). Plant Cell Monographs v. 15, 2008. p.239-267.). Such a strategy is based on the transformation of plants for the expression of double-stranded RNA (dsRNA) comprising a fragment of the specific sequence of target genes of the phytonematode. Target genes of interest are genes essential to the nematode or genes involved in parasitism, migration, formation or maintenance of the feeding site. During the cycle of nematodes, they ingest the cytoplasmic content of the giant cells of infected roots, causing the absorption of dsRNA, which can result in the silencing of the corresponding gene. Depending on the function of the silenced gene, several dysfunctions can be generated in the phytonematode by silencing and/or reducing the expression of specific genes, so that the infection can be aborted.

[9] RNA-mediated interference (RNAi) refers to the specific reduction of gene expression through the use of RNA molecules of complementary sequence. This phenomenon, first reported in Caenorhabditis elegans by GUO & KEMPHUES (GUO, S. & KEMPHUES, K. J. par-l, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed Cell, v. 81, no. 4, pp. 611-620, 1995) and subsequently demonstrated by FIRE et al. (FIRE, A.; XU, S.; MONTGOMERY, M. K.; KOSTAS, S. A.; DRIVER, S. E. & MELLO, C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, v. 391, n. 6669, p. 806-11, Feb 19 1998), who found that the presence of double-stranded RNA (dsRNA), formed from the annealing of sense and antisense strands present in in vitro RNA preparations, is responsible for gene silencing.

[10] Subsequently, BERNSTEIN et al. (BERNSTEIN, E.; CAUDY, A. A.; HAMMOND, S. M. & HANNON, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, v. 409, n. 6818, p. 363-6, Jan 18 2001) , indicated that DICER (“dsRNA-specific RNase III-type endonuclease”), a type III ribonuclease enzyme (RNase III) was responsible for processing dsRNA into sequences of ~21 nucleotides (nt). The DCR2/R2D2 complex binds to these small molecules of interfering RNA (siRNA) (LIU, Q.; RAND, T. A.; KALIDAS, S.; DU, F.; KIM, H. E.; SMITH, D. P. & WANG, X. R2D2, a bridge between the initiation and effector steps of the DrosophiJa RNAi pathway. Science, v. 301, n. 5641, p. 1921-5, Sep 26 2003), the siRNA is incorporated into a complex called RISC (RNA Induced Silencing) Complex), which then determines the degradation of any RNA molecules that have a complementary sequence (HAMMOND, S. M.; BERNSTEIN, E.; BEACH, D. & HANNON, G. J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, v. 404, no. 6775, p. 293-6, Mar 16 2000). This gene silencing phenomenon occurs in several eukaryotic organisms, including nematodes and higher plants (BERNSTEIN, E.; CAUDY, A. A.; HAMMOND, S. M. & HANNON, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature , v. 409, no. 6818, p. 363-6, Jan 18 2001).

[11] Researchers have been able to add RNA synthesized in vitro directly into cells to achieve a silencing of gene expression. For example, KLINK & WOLNIAK (KLINK, V. P. & WOLNIAK, S. M. Centrin is necessary for the formation of the motile apparatus in spermatids of Marsilea. MoI Biol Cell, v. 12, n. 3, p. 761-76, Mar 2001) were able to silence centrin (centrin) mRNA using dsRNA synthesized in vitro, and for knockout effects, they demonstrated that dsRNA is at least ten times more effective than sense or antisense RNA separately. To achieve control of plant parasitic nematodes using the gene silencing strategy, the RNAi mechanism is partially executed by the plant and partially by the nematode. Plants express dsRNA from nematode genes, which are processed by DICER and generate siRNA. When nematodes feed on these plants, both dsRNA and siRNA are ingested.

[12] As in the plant, nematodes digest dsRNA into siRNA using their DICER complex. Ingested siRNAs, or processed by the nematode itself, bind to RISC to induce the degradation of specific mRNA in the nematode. SiRNAs are amplified in nematodes by RNA-dependent RNA polymerase (RDRP) (CHAPMAN, E. J. & CARRINGTON, J. C. Specialization and evolution of endogenous small RNA pathways. Nature Reviews Genetics, v. 8, n. 11, p. 884- 96, Nov 2007; ZAMORE, P. D. &.HALEY, B. Ribo-gnome: the big world of small RNAs. Science, v. 309, n. 5740, p. 1519-24, Sep 2 2005), where mRNA serves template for the synthesis of more specific dsRNA, increasing the effect of gene silencing.

[13] This siRNA-mediated silencing is highly sequence-specific. For example, TuschI et al. demonstrated that even a single base mismatch between the siRNA and the target mRNA influences gene silencing (ELBASHIR, S. M.; MARTINEZ, J.; PATKANI-OWSKA, A.; LENDECKEL, W. & TUSCHL. T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. The EMBO Journal, v. 20, n. 23, p. 6877-88, Dec 3 2001). Although gene silencing is highly specific, it is possible to silence homologous gene families that share conserved sequences (MIKI, D.; ITOH, R. & SHI-MAMOTO, K. RNA silencing of single and multi pie members in a gene family of rice Plant physiology, v. 138, no. 4, p. 1903-13, Aug 2005) or with the chimeric construct of several target genes (ALLEN, R.S.; MILLGATE, A.G.; CHITTY, J.A.;THISLETON, J.; MILLER , J. A.; FIST, A. J.; GERLACH, W. L. & LARKIN, P. J. RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticulin in opium poppy. Nature Biotechnology, v. 22, n. 12, p. 155966, Dec 2004). It has been documented that the effect of RNAi is not only widespread from cell to cell (FAGARD, M. & VAUCHERET, H. Systemic silencing signal(s). Plant Molecular Biology, v. 43, n. 2-3, p. 285-93, Jun 2000; KEHR, J. & BUHTZ, A. Long distance transport and movement of RNA through the phloem. Journal of Experimental Botany, v. 59, n. 1, p. 85-92, 2008), but also throughout the plant (YOO, B. C.; KRAGLER, F.; VARKONYI-GASIC, E.; HAYWOOD, V.; ARCHER-EVANS, S.; LEE, Y. M.; LOUGH, T. J. & LUCAS, W. J. A systemic small RNA signaling system in plants. Plant Cell, v. 16, no. 8, p. 1979-2000, Aug 2004).

[14] Indirect evidence indicates that RNA silencing moves long distances through the phloem and, at fate, spreads cell to cell through plasmodesmata in recipient tissues (JORGENSEN, R. A. RNA traffics information systemically in plants. Proceedings of the National Academy of Sciences, v. 99, n. 18, p. 11561-3, Sep 3 2002; MLOTSHWA, S.; VOINNET, O.; METTE, M. F.; MATZKE, M.; VAUCHERET, H.; DING, S.W.; PRUSS, G. & VANCE, V. B. RNA silencing and the mobile silencing signal. Plant Cell, v. 14 Suppl, p. S289-301, 2002; VOINNET, O.; VAIN, P.; ANGELL, S. & BAULCOMBE, D. C. Systemic Spread of Sequence-Specific Transgene RNA Degradation in Plants Is Initiated by Localized Introduction of Ectopic Promoterless DNA. Cell, v. 95, n. 2, pp. 177-187, 1998), also HIMBER et al. (HIMBER, C.; DUNOYER, P.; MOISSIARD, G.; RITZENTHALER, C. & VOINNET, O. Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. The EMBO Journal, v. 22, n. 17, pp. 4523-33, Sep 1 2003) demonstrated that the RNAi effect can occur both locally and over long distances. LIMPENS et al. (LIMPENS, E.; RAMOS, J.; FRANKEN, C.; RAZ, V.; COMPAAN, B.; FRANSSEN, H.; BISSELING, T. & GEURTS, R. RNA interference in Agrobacterial rhizogenes transformed roots of Arabidopsis and Medicago truncatula, Journal of experimental botany, v. 55, n. 399, p. 98392, May 2004) also found that the silencing signal was carried systematically from Arabidopsis thaliana roots to the shoot, although the degree of silencing has been limited and highly variable. Although the transformation of plant parasitic nematodes represents an approach for deployment as an RNAi strategy for nematode control, there are no reports of success with this nematode engineering, in part because of its obligatory parasitism nature. In addition to numerous regulatory obstacles to release this transgenic nematode into the environment. In C. elegans, RNAi has been used transiently to silence the expression of almost all genes, inducing different phenotypic effects including lethality (FRASER, A. G.; KAMATH, R. S.; ZIPPERLEN, P.; MARTINEZ-CAMPOS, M.; SOHRMANN, M. & AHRINGER, J. Functional genomic analysis of C. elegans chromosoll1e I by systematic RNA interference. Nature, v. 408, n. 6810, p. 325-30, Nov 16 2000; GONCZY, P.; ECHEVERRI, C.; OE-GEMA, K.; COULSON, A.; JONES, S. J.; COPLEY, R. R.; DUPERON, J.; OE-GEMA, J.; BREHM, M.; CASSIN, E.; HANNAK, E.; KIRKHAM, M. .; PICHLER, S.; FLOHRS, K.; GOESSEN, A.; LEIDEL, S.; ALLEAUME, A. M.; MARTIN, C.; OZLU, N.; BORK, P. & HYMAN, A. A. Functional genolnic analysis of cell division in C. elegans using RNAi of genes on cell rolll0some 111. Nature, v. 408, n. 6810, p. 331-6, Nov 16 2000.; KAMATH, R. S.; FRASER, A. G.; DONG, Y.; POULIN, G. ; DURBIN, R.; GOTTA, M.; KANAPIN, A.; LE BOT, N.; MORENO, S.; SOHRMANN, M.; WELCHMAN, D. P.; ZIPPERLEN, P. & AHRINGER, J. Systematic functional ana lysis of the Caenorhabditis elegans genome using RNAi. Nature, v. 421, no. 6920, p. 231-7, Jan 16 2003; MAEDA, 1.;KO-HARA, V.;VAMAMOTO, M.& SUGIMOTO, A. Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Current biology: CB, v. 11, no. 3, p. 171-176, 2001). RNA-mediated interference can be performed in C. elegans by ingestion of bacteria expressing dsRNA (TIMMONS, L. & FIRE, A. Specific interference by ingested dsRNA. Nature, v. 395, n. 6705, p. 854, Oct 29 1998), by oral absorption of dsRNA from solution (TABARA, H.; GRISHOK, A. & MELLO, C. C. RNAi in C. elegans: soaking in the genome sequence. Science, v. 282, n. 5388, p. 430-1, Oct 161998) Or by microinjection (MELLO, C. C. & CONTE, D., JR. Revealing the world of RNA interference. Nature, v. 431, n. 7006, p. 338-42, Sep 16 2004). Considering plant parasitic nematodes, the ingestion protocol of bacteria expressing dsRNA is not feasible because they feed only on the cytoplasmic content of the feeding site. The protocol initially used for dsRNA administration to phytonematodes was the immersion of nematodes in dsRNA solution to induce ingestion and/or absorption. Since 2006, dsRNA molecules have been produced and offered directly by the genetically modified host plant.

[15] Root-knot nematodes (NFGs) and cyst-forming nematodes (NCs) are obligate plant root parasites, making host delivery of dsRNA an ideal strategy for silencing nematode genes as well as providing direct evidence of function of these genes. Previous research confirms the feasibility and effectiveness of offering RNAi by the host to control nematodes. YADAV et al. (YADAV, B. C.; VELUTHAMBI, K. & SUBRAMANIAM, K. Host-generated double stranded RNA induces RNAi in plant-parasitic nematodes and protects the host from infection. Molecular and Biochemistry Parasitology, v. 148, n. 2, p. 219 -22, Aug 2006) reported that it induced RNAi using dsRNAs from two genes encoding integrase and mRNA processing factor from M. incognita, leading to protection against nematode infection in tobacco plants.

[16] The dsRNA expression of the parasitism gene 16D10 of NFG in transgenic Arabidopsis plants resulted in resistance against four of the main species of this genus (HUANG, G.;ALLEN, R. S.; DAVIS, E. L.; BAUM, T. J. & HUSSEY, R. S. Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. n. 0027-8424 (Print), 20060927 DCOM- 20061030 2006), while (SINDHU, A. S.; MAIER, T. R. ; MITCHUM, M. G.; HUSSEY, R. S.; DAVIS, E. L. & BAUM, T. J. Effective and specific in planta RNAi in cyst nenlatodes: expression interference of four parasitism genes reduces parasitic success. Journal of Experimental Botany, v. 60, n. 1, p 315-24, 2009) obtained reductions of 23% to 64%, in females of H. schachtii, in transgenic lines of Arabidopsis expressing the dsRNA of four genes of parasitism. Interfering RNA appears to be equally effective against H. glycines in transformed soybean lines. RYAN et al. (RYAN, M. S.; TIM, C. T.; JULIANE, S. E. & HAROLD, N. T. Transgenic soybeans expressing siRNAs specific to a major spern1 protein gene suppress Heterodera glycines reproduction. Functional Plant Biology, v. 33, n. 11, p. 991- 991, 2006) have successfully produced transgenic soybean lines using the RNAi strategy specific to a major protein of H. glycines sperm. Bioassay data indicated transgenic plants with up to 68% reduction in eggs/g of root tissue.

[17] The effects of dsRNA molecules expressed by the plant proved to be continuous, appearing in the next generations. KLINK et al. (KLINK, V. P.; KIM, K. H.; MARTINS, V.; MACDONALD, M. H.; BEARD, H. S.; ALKHA-ROUF, N. W.; LEE, S. K.; PARK, S. C. & MATTHEWS, B. F. A correlation between host-mediated expression of parasite genes as tandem inverted repeats and abrogation of development of female Heterodera glycines cyst formation during infection of Glycine max. Planta, v. 230, n. 1, p. 53-71, Jun 2009) effectively inhibited H. glycines cyst formation using a construct with inverted repeats of several genes. Recently, LI et al. (LI, J.;TODD, T.C.; OAKLEV, T.R.; LEE, J. & TRICK, H.N. Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines Ichinohe. Planta, v. 232, n. 3, p. 775-85, Aug 2010a); LI et al. (LI, J.; TODD, T. C. & TRICK, H. N. Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants. Plant Cell Reports, v. 29, n. 2, p. 113-23, Feb 2010b) successfully suppressed the reproduction of H. glycines in soybean plants by RNAi for three different genes of H. glycines.

[18] Direct molecular evidence of host delivery of RNAi down-regulates the expression of nematode target genes, observed by real-time PCR (RT-qPCR) analysis of nematodes that fed on transgenic roots (LI, J. ;TODD, T. C.; OAKLEV, T. R.; LEE, J. & TRICK, H. N. Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines Ichinohe. Planta, v. 232, n. 3, p. 775-85, Aug 2010a; SINDHU, A. S.; MAIER, T. R.; MITCHUM, M. G.; HUSSEY, R. S.; DAVIS, E. L. & BAUM, T. J. Effective and specific in planta RNAi in cyst nenlatodes: expression interference of four parasitism genes reduces parasitic success. Journal of Experimental Botany , v. 60, no. 1, p. 315-24, 2009). Transgenic plants expressing dsRNAs from nematode genes have shown greater success in NFGs than in NCs. A likely reason could be the ease of ingestion of molecules in NFGs due to their higher size exclusion limit than in NCs.

[19] RNA constructs capable of forming dsRNA for the inhibition of nematode genes through RNA interference and consequent induction of resistance to such pests in the plant, as well as DNA constructs capable of expressing such RNA constructs in the plant are available. (EP2330203).

[20] WO2011060151 teaches vectors comprising constructs for inducing resistance to nematodes, including Meloidogyne incognita and Heterodera glycines, encoding double-stranded RNA strands that inhibit the expression of a target plague gene.

[21] WO2006045591 teaches, among others, methods for the production of transgenic plants resistant to nematodes comprising the co-expression in the plant of double-stranded RNA molecules targeting genes of different species, among them Meloidogyne incognita and Heterodera glycines . WO2006045591 further teaches that dsRNA transfer to the plague can be improved through the use of tissue-specific promoters for the parts most accessible to the plague, such as, among others, root-specific promoters or nematode-induced promoters.

[22] The silencing of the Prp-17 splicing factor gene from Hetero dera glycines through the expression of siRNAs in transgenic soybean was effective in inducing pest resistance (Li, J.; Todd, T.C.; Oaklev, T.R.; Lee, J. & Trick, H. N. Host-derived suppression of reproductive nematode and fitness genes decreases fecundity of Heterodera glycines Ichinohe. Planta, v. 232, no. 3, p. 775-85, Aug 2010).

[23] M. incognita splicing factor (FS) dsRNA expression resulted in a 90% reduction in root-established nematodes (M. incognita) (Yadav, B.C.; Veluthambi, K. & Subramaniam, K. Host- generated double stranded RNA induces RNAi in plant-parasitic nematodes and protects the host from infection.

[24] The dsRNA expression of a gene related to the FS of H. glycines reduced the number of females colonizing the roots to less than 20%. (Klink, V.P. & Matthews, B.F. Emerging Approaches to Broaden Resistance of Soybean to Soybean Cyst Nematode as Supported by Gene Expression Studies. Plant Physiol. 2009 November; 151(3): 1017-1022 ).

[25] PI0701172-5 describes a constitutive soy promoter, the soy ubiquitin conjugation protein gene promoter (UceS 8.3).

[26] The UceS8.3 promoter is induced by root invasion by M. incognita (Miranda, V.J. Characterization of the expression of the gene encoding the ubiquitin-conjugating enzyme (E2) in soybean inoculated with Meloidogyne incognita and infested with Anticarsia gemmatalis. 2011. Magister Science. Cell Biology, University of Brasília).

[27] However, in the field of promoting resistance against nematodes via RNA interference, there is a need for constructs capable of expressing dsRNA molecules that silence nematode genes only in target tissues of nematode infestation.

[28] Known interfering RNA expression constructs use constitutive promoters, usually isolated from viruses, such as CaMV 35S for constitutive expression of dsRNA.

[29] There is, therefore, a need for efficient constructs to silence nematode genes for the induction of nematode resistance in cultures capable of expressing dsRNA molecules under the control of a plant-specific promoter.

[30] The differential of the present invention is that it is a dsRNA expression vector that is specific for target genes of more than one nematode species and is regulated by a nematode-induced soybean promoter. The construct containing the target gene is under the control of a soybean promoter, isolated, characterized and patented by our group, and which has strong expression in the plant root (Grossi-de-Sa et al., 2010), especially when these are parasitized by M. incognita (Miranda et al., 2013), specifically at the feeding site.

[31] Initially the UceS8.3 promoter was isolated from soybean plants by TAIL-PCR with oligonucleotides designed for the ubiquitin conjugation factor 2 gene (GmE2). Subsequently, this promoter was used in genetic transformation of a model plant Arabidopsis thaliana for its functional characterization due to the expression of the GUS reporter gene, demonstrating expression in all plant tissues, especially in roots (Grossi-de-Sa et al., 2010). ). Real-time PCR of soybean roots inoculated with M. incognita at 7, 14, 21 and 28 DAI demonstrate 2-6 fold increase in transcriptional expression of the GmE2 gene, regulated by the UceS8.3 promoter (Miranda et al., 2013) . In situ hybridization demonstrated the localization of expression related to UceS8.3 in giant cells and adjacent to the feeding site in the galls.

Summary of the Invention

[32] The present invention comprises plant expression cassettes capable of silencing genes from multiple nematode species that feed on said plants conferring resistance to said nematode species. More specifically, the expression cassettes of the present invention are capable of expressing the dsRNA molecules in target tissues of nematode infestation.

[33] The present invention further provides vectors comprising said expression cassette, methods for producing a plant resistant to multiple nematode species as well as for controlling nematodes in a crop, plants resistant to multiple nematode species, their seeds and products produced from material extracted from said plants.

Description of Figures

[34] Figure 1: Representative scheme of the gene construct used for soybean transformation targeting resistance to Meloidogyne incognita and Heterodera glycines. The black arrow indicates the position of the promoter of the soy ubiquitin conjugation factor UceS8.3, isolated and characterized in LIMPP and protected by Embrapa by patenting. The gene construction includes sequences of the splicing factor target gene from M. incognita and H. glycines, totaling 440 bp in two palindromic positions, that is, reverse and complementary, in order to allow the formation of double stranded RNA. This palindromic region is separated by the intronic region from the RNAi vector, pKannibal, originally isolated from Arabidopsis thaliana. tNOS is the transcriptional terminator used. The red and green arrows indicate the annealing positions of the oligonucleotides used for diagnosis of soybean transformation (Figure 2A). The sequence of the oligonucleotides is in Table 1.

[35] Figure 2: (A) Diagnosis of soybean transformation by PCR. The above image was obtained by ethidium bromide stained agarose gel electrophoresis of the PCR product with the oligonucleotides GmFSMiHg-F and GmFSMiHg-R (described in Table 1 and Figure 1). DNA from leaves was isolated and then used in PCR. The numbering corresponds to the third generation individuals (T3), obtained from the successive multiplication of the 4I T0 transformation event. CN: negative control without template DNA. CP: positive control, UceS8.3::GmFSMiHg vector DNA template. (B) Acclimatization of genetically modified soybean plants. After transformation by bioballistics, the seedlings were kept in magenta for 15 days under selection with the herbicide imidazolinone. After growth, the plants were transferred to cups with organic substrate and clay and kept covered with a plastic bag to maintain humidity for a week. Subsequently, the plants were transferred to 20 L plastic bags with the same substrate and kept until they completed their life cycle. T1 seeds from T0 were multiplied in a greenhouse until T2.

[36] Figure 3: Nematological bioassay. T3 plants were challenged with M. incognita to determine nematode resistance induction. (A) Graph of the result of the challenge of GM soybean (4A, 4B, 4C, 4E, 4G and 4I) and non-transformed control plant (BR-16) events. The upper line denotes the number of plants individually tested in the resistance experiment (n) and the percentage of egg reduction obtained (%). The straight line above the bars denotes the standard error of the experiment. The letter above the bar shows a statistically significant difference; (B) graphic with details of the bioassay, without the bar with count of the control plant and with appropriate scale. The statistical analysis in this case did not take into account the control plant.

Detailed Description of the Invention

[37] One embodiment of the present invention is an expression cassette comprising: (i) a plant-functional, nematode-induced promoter; (ii) a sense fragment substantially similar to SEQ ID NO: 2; (iii) a sense fragment substantially similar to SEQ ID NO: 3; (iv) a separator sequence; (v) an antisense fragment substantially similar to SEQ ID NO: 5; (vi) an antisense fragment substantially similar to SEQ ID NO: 6; (vii) a functional in-plant terminator.

[38] Said cassette is capable of expressing dsRNA in plants comprising fragments of nematode genes that silence conserved genes of said nematodes through a mechanism known as RNA interference (RNAi).

[39] In a preferred embodiment of the present invention, the sense and antisense sequences are separated by a spacer sequence. Optionally, the separator region is an intron. An “intron” is a nucleotide sequence that is transcribed and is present in pre-mRNA, but is removed through cleavage and re-ligation of the mRNA within the cell generating a mature mRNA that can be translated into a protein. Examples of introns include, but are not limited to, pdk intron, castor bean catalase intron, cotton Delta 12 denaturase intron, Arabidopsis Delta 12 denaturase, corn ubiquitin intron, SV40 intron, malate synthase gene introns. Preferably the spacer sequence of the present invention is a PDK intron.

[40] “Promoter” refers to the DNA sequence in a gene, usually located upstream of the coding sequence, which controls the expression of the coding sequence promoting recognition by RNA polymerase and other factors required for transcription itself. . In an artificial DNA construct, promoters can also be used to transcribe dsRNA. Promoters may also contain DNA sequences that are involved in binding protein factors which control the effect of transcription initiation in response to physiological or developmental conditions.

[41] In one aspect of the invention, the promoter is a constitutive promoter. In another aspect of the invention, promoter activity is stimulated by external or internal factors such as, but not limited to, hormones, chemical compounds, mechanical impulses, and biotic or abiotic stress conditions. Promoter activity can also be temporally and spatially regulated (eg, tissue-specific promoters and developmentally regulated promoters).

[42] The promoter may contain “enhancer” elements. An "enhancer" is a DNA sequence that can stimulate promoter activity. It may be an innate element of the promoter or a heterologous element inserted to increase the level and/or tissue-specificity of a promoter. “Constitutive promoters” refer to those that drive gene expression in all tissues and at all times. “Tissue-specific” or “development-specific” promoters are those that direct gene expression almost exclusively in specific tissues, such as leaves, roots, stems, flowers, fruits, or seeds, or at specific developmental stages in a tissue, such as at the beginning or end of embryogenesis. The term "expression" refers to the transcription and stable accumulation of dsRNA derived from the nucleic acid fragments of the invention which, in conjunction with the cell's protein production apparatus, results in altered levels of myo-inositol 1-phosphate synthase. "Interference inhibition" refers to the production of dsRNA transcripts capable of preventing expression of the target protein.

[43] In a preferred embodiment of the present invention, said promoter is specific to tissues colonized by nematodes or induced by nematode infestation. Preferably the promoter has the sequence identified as SEQ ID NO: 1.

[44] Still in accordance with a preferred embodiment of the present invention, the sense and antisense sequences of (ii) and (iv) belong to a gene encoding a nematode splicing factor (FS).

[45] In a preferred embodiment of the present invention the sequences of components (ii) and (iv) belong to the nematode species Heterodera glycines and Meloidogyne incognita, respectively, and more preferably the expression cassette contains the following composition: (i) a promoter sequence having a sequence substantially similar to SEQ ID NO: 1 (ii) the first sense sequence having a sequence substantially similar to SEQ ID NO: 2; (iii) the second sense promoter sequence having a sequence substantially similar to SEQ ID NO: 3; (iv) the promoter spacer sequence having a sequence substantially similar to SEQ ID NO: 4; (v) the first antisense promoter sequence having a sequence substantially similar to SEQ ID NO: 5; (vi) the second antisense promoter sequence having a sequence substantially similar to SEQ ID NO: 6; (vii) the promoter terminator sequence having a sequence substantially similar to SEQ ID NO: 7.

[46] In a preferred embodiment of the present invention, the sequence of the expression cassette is substantially similar to SEQ ID NO: 8.

[47] The term “substantially similar” or “substantial similarity” refers to nucleic acid fragments in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate change in gene expression by gene silencing. through, for example, antisense technology, co-suppression or RNA interference (RNAi). Substantially similar nucleic acid fragments of the present invention may also be characterized by the percentage similarity of their nucleotide sequences to the nucleotide sequences of the nucleic acid fragments described herein (SEQ ID NOs 1-8), as determined by common algorithms employed in state of the art. Preferred nucleic acid fragments are those whose nucleotide sequences have at least about 40 or 45% sequence identity, preferably about 50% or 55% sequence identity, more preferably about 60% or 55% sequence identity. 65% sequence identity, more preferably about 70% or 75% sequence identity, most preferably about 80% or 85% sequence identity, most preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when compared to the reference sequence. Sequence alignment and percentage similarity calculation of the present invention were performed using the DNAMAN program for windows (Lynnon Corporation, 2001), using sequences deposited in GenBank, through the integration of the Web browser.

[48] One of the ways to form dsRNA is if the nucleotide sequence of the target gene is present in the DNA molecule in the sense orientation, and a sequence of nucleotides in the antisense orientation, with or without a spacer region between the nucleotide sequences. sense and antisense. The mentioned nucleotide sequences may consist of from about 19nt to 2000nt or even from about 5000 nucleotides or more, each having substantial total sequence similarity of about 40% to 100%. The longer the sequence, the less stringency is required for substantial overall sequence similarity. Fragments containing at least about 19 nucleotides should preferably have about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity as compared to the reference sequence, with the possibility of having about 2 distinct non-contiguous nucleotides. Preferably fragments above 60bp are used, most preferably fragments between 100 to 500bp.

[49] In one aspect of the invention, the dsRNA molecule may comprise one or more regions having substantial sequence similarity to regions with at least about 19 consecutive nucleotides of the sense nucleotides of the target gene, defined as the first region and, one or more regions having substantial sequence similarity to regions of about 19 consecutive nucleotides of the complement of the sense nucleotides of the target gene, defined as the second region, where these regions may have base pairs separating them from one another.

[50] The invention further comprises vectors comprising the cassettes of the present invention as well as methods for producing a plant resistant to multiple nematode species, comprising inserting a cassette of the present invention into a plant cell and regenerating a plant from said cell. vegetable.

[51] The invention further comprises plants resistant to multiple nematode species having a cassette of the present invention integrated into their genome.

[52] “Plants” refers to photosynthetic organisms, both eukaryotes and prokaryotes, where the term “developed plants” refers to eukaryotic plants. The nucleic acids of the invention can be used to provide desired tracts in essentially any plant. Therefore, the invention has use on various plant species, including species of the genera Anacardium, Anona, Arachis, Artocarpus, Asparagus, Atropa, Avena, Brassica, Carica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Hetero-callis, Hordeum, Hyoseyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Passiflora, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Psidium, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea. Preferably the plant of the present invention is a soybean plant.

[53] The invention further comprises seeds from a plant having a cassette of the present invention integrated into its genome as well as products produced from material extracted from a plant having a cassette of the present invention integrated into its genome, such as food products and /or animal feed.

[54] The invention also comprises methods for controlling nematodes in a crop, comprising cultivating a plant having a cassette of the present invention integrated into its genome.

EXAMPLES

[55] The present invention is further defined in the following Examples. It is to be understood that these Examples, while indicating part of the invention, are given by way of illustration only, and therefore do not have any limitation on the scope of the present inventions.

[56] Usual molecular biology techniques such as bacterial transformation and agarose gel electrophoresis of nucleic acids are referred to by common terms to describe them. Details of the practice of these techniques, well known in the art, are described in Sambrook, et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press). Various solutions used in experimental manipulations are referred to by their common names such as “lysis solution”, “SSC”, “SDS”, etc. Compositions of these solutions can be found in the reference Sambrook, et al. (above).

Example 1 - Selection of genes

[57] New target genes for RNAi are selected based on the following criteria: (a) their C. elegans orthologs must be essential genes and/or have lethal phenotypes when silenced or knocked out; (b) their sequences must show high identity only with nematode species; (c) their sequences must be available in the database (Gen-Bank, http://www.ncbi.nlm.nih.gov/nuccore) and (d) To ensure that proposed dsRNAs do not pose a risk to non- -target, the sequences of the fragments of the genes in question used for the synthesis of dsRNA are compared with other sequences available in GenBank, using the BLAST"n tool (version 2.2.15) (ALTSCHUL, S. F.; GISH, W.; MILLER, W.; MYERS, E. W. & LIPMAN, D. J. Basic local alignment search tool. Journal of Molecular Biology, v. 215, no. 3, p. 403-10, Oct 5 1990).

[58] Based on literature reviews, as well as sequences available in the database (GenBank,) an mRNA processing gene (FS) was selected in M. incognita (AW828516) and a gene also involved in mRNA processing ( FS) in H. glycines (AF113915).

Example 2 - Selection of sequences complementary to mRNAs of mRNA processing genes in M. incognita and H. glycines

[59] Once the target genes were selected, their sequences were submitted to the BlockTM RNAi Designer (http: rnaidesigner.invitrogen.com/ rnaiexpress/), to choose the region of each gene with the highest probability of producing efficient siRNAs . Regions containing between 120 and 250 base pairs were chosen.

[60] The gene regions selected in the BlockTM RNAi Designer were submitted to the GenBank™ database by the BLASTn (nucleotide blast) and BLASTp (protein blast) programs, at the NCBI website (http://www.ncbi.nlm.nih. gov/blast/Blast.cgi). in order to identify possible effects of RNAi on plants, hunIall0s, and other non-target organisms.

Example 3 - Oligonucleotides for expression analysis and gene silencing

[61] To assess the efficiency of gene silencing after infestation assays in GM soybean lines, oligonucleotide pairs (primers) were designed for each gene.

[62] The design of the oligonucleotides was performed by the program primer3 v.0.4.0 (http://frodo.wi.mitt.edu/), which suggests the best pairs within the given sequence and for the size of the desired product. . The program also provides important parameters such as Tm (melting temperature), %GC, loop formation and homodimer.

[63] The chosen oligos are described in Table 1 Table 1 - Sequences of oligonucleotides

Example 4 - Obtaining transgenic plants by bioballistics

[64] The shots of tungsten particles with adsorbed DNA were performed in the meristematic region of embryos from seeds of the soybean cultivar BR-16. The co-transformation strategy with the plasmids UceSB.3::GmFSMiHg (for gene silencing of plant nematodes) and pAC321 (herbicide resistance) was used, using the bioballistics protocol (RECH, E. L.; VIANNA, G. R.& ARAGAO, F. J. L. High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nature Protocols, v. 3, n. 3, p. 410-418, 2008.

[65] The seeds were first sterilized in 70% ethanol for 10 minutes, followed by immersion in 50% hypochlorite for twenty minutes, and then washed three times with distilled water autoclaved in a laminar flow chamber, remaining immersed in distilled water for a period of time. approximately 16 hours.

[66] The seeds were then incised to remove the embryos with the aid of tweezers and sterile scalpels, being stored in a Petri dish with distilled water to prevent desiccation. Then, the leaf primordia were removed with the aid of a magnifying glass to expose the apical meristem region.

[67] Subsequently, the embryos were dried under exposure to the environment on filter paper in a laminar flow chamber, and later placed in 5 cm diameter Petri dishes containing 11 mL of MS medium (Murashige & Skoog 1962), 3 % sucrose and 0.8% phytagel and pH 5.7. Arranged on the line of a 16 mm diameter circle centered on the plate (death zone), with the apical meristem region facing upwards.

[68] The DNA constructs were precipitated onto tungsten microparticles with the aid of CaCl2 and spermidine. The introduction of the gene constructs of interest occurred through the use of the particle accelerator developed in Brazil.

[69] After co-transformation with the genes of interest, the embryos were transferred to plates containing MS medium supplemented with benzylaminopurine (BAP - 5 mg/ml), 3% sucrose, 0.6% agar and pH 5.7, where they remained approximately 18 hours protected from light at 28°C for induction of multi-sprouting. The embryos were then transferred to magenta containing selective media with MS.3% sucrose, 0.15 μM Imazapyr herbicide, 0.8% agar and vitamin B5, pH 5.7, with 9 embryos in each magenta, which were kept in a growth chamber at a temperature of 28° C, with 16 hours of photoperiod and luminosity of 350 μmols.m-2.s-1 and relative humidity above 80% for approximately 45 days.

[70] After this period, two multi-sprouted embryos were transferred per cup containing sand:vermiculite (1:1), autoclaved and moistened with a nutrient solution and covered with a plastic bag for acclimatization. The multi-sprouted embryos were irrigated with nutrient solution every 7 days and kept for another 28 days in an acclimatized chamber, until transfer to the greenhouse.

[71] After this period of 28 days, they were transferred to a greenhouse in pots containing a mixture of soil and sterilized sand (5:3), covered with a plastic bag for seven days for acclimatization, being replaced by a plastic bag with holes for more five days. After this acclimatization period, the plastic bags were removed for the normal development of the plants until the beginning of the molecular analyzes for the identification of positive plants by the PCR technique.

[72] In a period of 3 months, 2946 embryos were transformed by bioballistics, which were selected for resistance to the herbicide Imazapir, whose resistance is conferred by the Ahas gene. The remaining 1495 embryos were acclimatized and transferred to the greenhouse. Of these earlier ones, 673 were able to regenerate and develop. Of these, 350 plants were tested by PCR, resulting in 31 plants (8.85% of the total analyzed) that amplified the transgene (Table 2). Considering all the steps performed, almost 1% of GM PCR-positive plants were obtained in relation to the number of transformed embryos.

[73] The results were confirmed by different PCR amplifications using specific primer pairs (Figure 1) for the UceS8.3::GmFSMiHg construct.

*Porcentagens são sempre em reahção ao número inicial de embriões bombardeados[74] 3150 amplifications were performed, starting from 1050 DNA samples, to characterize the 350 regenerated plants (Table 2). Table 2 - Results obtained by the transformation process

*Percentages are always in relation to the initial number of bombed embryos

Example 5 - PCR Analysis

[75] The selection of transformed plants was performed by PCR analysis. Using the Extract-N-AmpTM Tissue PCR Kit (Sigma-Aldrich, Saint Louis, MO, USA), the DNA from leaves was purified and PCR amplified fragments of the transgenes in the genome of each generated event. Each plant had its DNA extracted independently three times, each starting from different trifoliates, and each DNA sample served as a template for three independent PCRs. Those that amplify the transgene at least once for each DNA extraction, that is, for each trefoil, were considered as PCR-positive plants. Confirmation of the insertion of the UceS8.3::GnlFSMiHg construct was done separately with two pairs of primers that amplify different regions within the construct. The primers InGmFSMiHg-F and InGmFSMiHg-R (SEQ ID NO 9 and SEQ ID NO 10 - Figure 1) amplify a 135 bp fragment in the intron. The primers GmFSMiHg-F and GmFSMiHg-R (SEQ ID NO 11 and SEQ ID NO 12 - Figure 1) amplify a 440 bp fragment in the sense region. Using 400 nM of each primer, PCR amplifications were performed in a Mycycler thermocycler (BioRad), with the program: initial denaturation of 1'30" at 94° C, 35 denaturation cycles of 30" at 94° C, annealing of 30" at 55°C and 45" extension at 72°C, followed by a final 5' extension at 72°C. The PCR products were separated by electrophoresis on a 1.3% agarose gel, stained with ethidium and visualized in a transilluminator.

[76] PCR-positive plants for both amplicons were transferred to 15L pots with soil and kept in a greenhouse.

Example 6 - Bioassay of transformed events with the UceS8.3::GmFSMiHg construct against M. incognita

[77] To investigate the effect of mRNA processing (FS) gene silencing on nematodes, bioassays were performed with inoculation of M.incognita J2 in plants transformed with the construct to be studied.

Culture of M.incognita

[78] The culture of M.incognita, race 1, was maintained on tomato plants (Solanum lycopersicum), of the susceptible variety Santa Cruz and cultivar Kada Gigante. The collection of nematodes in their different stages of development was performed from 28 days after inoculation (DAI).

Egg extraction

[79] Eggs were extracted according to HUSSEY & BARKER 1973 (HUSSEY, R. S. & BARKER, A. A. Comparison methods of collecting inocula of Meloigogyne spp. including a new technique. The Plant Disease Reporter v. 57, p. 1025-1028, 1973). Briefly, the roots were ground in a blender for two minutes, in sodium hypochlorite (NaOCI) 0.5%. Eggs were counted on a Peters slide using an optical light microscope (VRAIN, T. C.A technique for the collection of larvae of Meloidogyne spp. and a comparison of eggs and larvae as inocula. Journal Nematology, v. 9, p. 249 -251, 1977.).

Extraction of pre-parasitic J2

[80] For the collection of juveniles in the J2 stage, the egg suspension was submitted to the Baernlann funnel technique, kept at room temperature, in a container with distilled water to allow the eggs to hatch and subsequently the nematode collection. The collection of hatched J2 was carried out over a week, every two days.

bioassay

[81] In order to evaluate the induction of resistance to M.incognita in GM soybean events, a bioassay was set up in a greenhouse. Second generation progenies (T3), at the 2-3 trifoliate stage, were planted in pots containing 300 mL of soil, which were inoculated with a population of approximately 1,000 J2 of M.incognita 1. Plants of cultivar BR-16 (not transgenic), which does not have resistance to M.incognita race 1, were planted and inoculated serving as a control. The plants remained in a greenhouse for 6 weeks, being irrigated when necessary. Soybean roots were individually processed for egg extraction at 45 days after inoculation (DAI). The roots were individually washed to remove the soil, dried with paper towels, weighed, crushed in a blender with 0.5% NaClO for 2 minutes, washed with a jet of water and the eggs were separated in a 500 mesh sieve.

[82] Considering each plant, the final volume of the suspension with eggs obtained was corrected to 30 mL, and three 1mL aliquots were withdrawn. After egg counting using a microscope and Peters slide, the count was normalized to root mass, determining the number of g-l root eggs.

[83] All data obtained from the bioassay were statistically evaluated using the Generalized Linear Modeling (GLM) procedure using Program R statistical packages (R:A language and environment for statistical computing. 2005. Available at: http:// www.R-project.org ).

[84] Seeds from the transformed events were germinated and the plants that developed were transplanted into pots in a greenhouse. When they reached the 2-3 trifoliate stage, the plants were genotyped by PCR for transgene detection (Figure 2A). The plants that did not present the corresponding fragment were discarded, and only the positive plants were used in the bioassays (Figure 2B).

[85] Each plant was inoculated with approximately 1000 J2 obtained in a hatching system. Each treatment consisted of 6 to 10 replicates. The challenge was completely repeated twice at different times. Approximately six weeks after inoculation, plant roots were individually extracted and processed to determine the number of eggs. root g-l. All bioassay values were relative to the control treatment to normalize the data between the biological replicates and enable statistical analysis. This normalization was necessary because the data referring to the infection of nematodes vary greatly from one experiment to another, which may cause an error in the interpretation of these data. These variations are inherent both in the experiment with nematodes, as it is not known how many actually penetrate the root, and in the RNAi technique, as it is not known how much dsRNA/siRNA the nematode will ingest. For the statistical evaluation of all data, the analysis of generalized linear models (GLM) was used, and after defining the best fit model for each data group (for example: number of eggs, number of J2 hatched, etc. ) an analysis of variance of variance (ANODEV) was performed (Package “car”, program R.) determine whether there are significant differences between treatments.

[86] When evaluating the data regarding soybean events with the UceS8.3::GmFSMiHg construct, the number of eggs per gram of root better fitted the normal distribution model with identity binding function. When comparing the number of g-l root eggs between the control treatment and the transgenic events expressing dsRNA for FS, it was verified that this number was 71% to 91% lower in the transgenic events (Figure 3A), this difference being significant due to the contrast analysis (p<0.05). The transgenic events were not statistically different when compared to each other, except for the GmFSMiHg-4IT3 event (Figure 3B). Therefore, plants transformed with the expression cassette of the present invention therefore show high resistance against nematodes.

Claims (12)

1. An expression cassette comprising: (i) a plant functional promoter; (ii) a sense fragment as described in SEQ ID NO: 2; (iii) a sense fragment as described in SEQ ID NO: 3; (iv)) a separator sequence; (v)) an antisense fragment as described in SEQ ID NO: 5; (vi) an antisense fragment as described in SEQ ID NO: 6; (vii) a functional in-plant terminator. 2. Expression cassette according to claim 1, characterized in that the promoter functional in plants is induced by nematodes 3. Expression cassette according to any one of claims 1 and 2, characterized in that the promoter functional in plants is the sequence described in SEQ ID NO 1. 4. Expression cassette according to claim 1, characterized in that the separator sequence is an intron. 5. Expression cassette according to claim 4, characterized in that the intron is selected from the group consisting of pdk intron, castor bean catalase intron, Delta 12 cotton denaturase intron, Arabidopsis Delta 12 denaturase, corn ubiquitin intron , SV40 intron, malate synthase gene introns. 6. Expression cassette according to claim 5, characterized in that the intron is the sequence described in SEQ ID NO 4. An expression cassette according to any one of claims 1 to 6, wherein the sense and antisense sequences of (ii, iii) and (iv, v) belong to a gene encoding a splicing factor. An expression cassette according to any one of claims 1 to 7, wherein the sequences of (ii, iv) and (iii, v) are from Heterodera glycines and Meloidogyne incognita, respectively. 9. Expression cassette characterized by having a sequence as described in SEQ ID NO 8. A vector, comprising the cassette as defined in any one of claims 1 to 9. A method for producing a plant resistant to multiple nematode species, comprising inserting a cassette as defined in any one of claims 1 to 9 into a plant cell and regenerating a plant from said plant cell. 12. A method for controlling nematodes in a crop, comprising the steps of (a) growing a plant resistant to multiple nematode species that has an expression cassette as defined in any one of claims 1 to 9 integrated into its genome; (b) observe the presence of nematodes in the cultivation perimeter of the plants of (a); and (c) comparing the observed numbers of nematodes in (b) with the presence of nematodes in growing areas of plants that do not have an expression cassette as defined in any one of claims 1 to 9 integrated into their genome.

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