# Patent

## Title
Compositions and methods for controlling parasitic nematodes

## Abstract

The present invention relates to compositions and methods for controlling nematode infestation of plants or animals. In particular, the present invention provides vectors comprising sequences designed to control nematodes by RNA interference (RNAi) and transgenic plants transformed with such vectors.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. §371 national phase entry of pending International Patent Application No. PCT/US2010/056358, international filing date Nov. 11, 2010, which claims the benefit of U.S. Provisional Application 61/260,248, filed Nov. 11, 2009, the entire contents of which are incorporated herein by reference.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with U.S. Government support under grant number 2004-3560714970 awarded by the United States Department of Agriculture. The Government has certain rights to the invention.


FIELD OF THE INVENTION
The present invention relates to compositions and methods for controlling nematode infestation of plants or animals. In particular, the present invention provides vectors comprising sequences designed to control nematodes by RNA interference (RNAi) and transgenic plants transformed with such vectors.
BACKGROUND OF THE INVENTION
Glycine max (L.) Merr. (soybean) is the second largest crop in the United Sates, with an estimated annual value of 11 billion dollars. Plant parasites such as the Heterodera glycines Ichinohe and Meloidogyne spp. cause significant damage to soybean, with diseased plants exhibiting symptoms ranging from stunting, chlorosis and wilting to enhanced susceptibility to other diseases. Recent estimates of annual production losses by the H. glycines, the most damaging soybean pest, range from $460 million to 818 million for the US alone (Wrather and Koenning 2006). Nematicides, crop rotation and resistant varieties represent the current options for H. glycines management; however, each has serious limitations. Nematicides, including organophosphate and carbamate compounds, are extremely toxic and increase production costs. Crop rotation can require prolonged intervals without a host crop to be effective. Resistant cultivars have a narrow genetic base, while H. glycines populations display broad genetic diversity leading to frequent virulence selection (Dong et al. 1997). Many populations of H. glycines, for instance, are now able to reproduce on soybean cultivars derived from PI88788, the most widely used source of H. glycines resistance in the USA (Mitchum et al. 2007; Hershman et al. 2008). It is therefore imperative that new strategies for H. glycines control be explored to complement existing approaches.
Genetic engineering represents one promising approach to H. glycines management, but improving nematode resistance in plants through this method requires increased knowledge of potential target genes. The search for novel targets for genetically engineered resistance to H. glycines has led to intense study of the secretions of subventral and dorsal esophageal gland cells of the nematode, as they play important roles in the host-parasite interaction. As a result of these studies, genes encoding secreted proteins of H. glycines have been identified, including genes encoding polygalacturonase (Mahalingam et al. 1999) and chorismate mutase (Bekal et al. 2003). Numerous additional putative H. glycines parasitism genes have been identified using microarray analysis (Klink et al. 2007; Ithal et al. 2007; Klink et al. 2009a, b). Although the functions of many of these genes remain to be investigated, Alkharouf et al. (2007) and Klink et al. (2009c) have identified specific genes involved in female development by knocking out these genes' functions in vitro and in vivo, respectively.
RNA interference (RNAi) is a potentially powerful gene-silencing tool for analysis of gene function. The mechanism of RNAi was first identified in the free-living nematode Caenorhabditis elegans, in which the expression of unc22 gene was suppressed via the RNAi pathway (Fire et al. 1998). During this process, long double-stranded RNA is processed into 21-23 nucleotide siRNAs by Dicer, a member of the RNase family (Bernstein et al. 2001). The DCR-2/R2D2 complex binds to siRNAs and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (Liu et al. 2003). This pathway recently has shown promise as the basis of a novel control strategy for plant-parasitic nematodes, with numerous independent studies demonstrating suppression of target nematode populations following soaking nematodes in dsRNA solutions (Urwin et al. 2002; Bakhetia et al. 2005; Huang et al. 2006; Alkharouf et al. 2007) and, more importantly, using in planta transgenic systems expressing dsRNA fragments of nematode genes (Huang et al. 2006; Steeves et al. 2006; Yadav et al. 2006; Sindhu et al. 2009). Yadav et al. (2006) reported that RNAi was induced by using dsRNA fragments of two genes encoding an integrase and a splicing factor in the plant-parasitic nematode M. incognita, leading to protection against nematode infection in tobacco. The expression of root-knot nematode parasitism gene 16D10 dsRNA in transgenic Arabidopsis resulted in resistance against four major root knot nematode species (Huang et al. 2006), while Sindhu et al. (2009) obtained reductions in H. schachtii females ranging from 23 to 64% in transgenic Arabidopsis lines expressing RNAi constructs of four parasitism genes. RNA interference appears to be similarly effective against H. glycines in transformed soybean lines. Steeves et al. (2006) successfully produced transgenic soybean lines using this RNAi strategy targeting a major sperm protein of H. glycines. Bioassay data indicated transgenic plants had up to a 68% reduction in eggs g−1 root tissue. The effects of plant-derived dsRNA molecules appeared to continue into the next generation.
Targets for host-delivered RNAi suppression of plant parasitic nematodes can be selected based on known RNAi effects on corresponding C. elegans genes. Alkharouf et al. (2007), for instance, used bioinformatics to yield 1,508 candidate H. glycines genes whose homologous genes of C. elegans have lethal phenotypes when silenced in C. elegans. They also reported in vitro silencing a conserved ribosomal gene from H. glycines (Hg-rps-23) resulted in dead and dying worms as shown by positive Sytox fluorescence. Klink et al. (2009c) used microarray analysis to demonstrate that 32 of 150 conserved H. glycines homologues of C. elegans genes with lethal phenotypes were induced during feeding site establishment, and subsequently inhibited female development by engineering transgenic soybean plants with tandem inverted repeats of selected homologs.
Novel approaches to SCN management are needed to complement current strategies, and prolong the effectiveness of available resistance genes.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for controlling nematode infestation of plants. In particular, the present invention provides vectors comprising sequences designed to control nematodes by RNA interference (RNAi) and transgenic plants transformed with such vectors.
In some embodiments, the present invention provides transgenic plants comprising a first exogenous nucleic acid sequence having a sense sequence linked to its complementary antisense sequence and encoding a double stranded RNA that inhibits expression of a target pest gene (i.e., an RNAi construct) and a second exogenous sequence encoding at least a portion of said target pest gene. In some embodiments, the first exogenous nucleic acid sequence forms a hairpin structure when expressed. In some embodiments, the second exogenous sequence is expressed as an antisense RNA sequence. In some embodiments, the exogenous sequence is expressed as a sense RNA sequence. In some embodiments, the first exogenous sequence and said second exogenous sequence are present on the same vector. In some embodiments, the transgenic plant is a monocot or eudicot. In some embodiments, the transgenic plant is a crop, cereal, fruit, vegetable, or horticultural plant. In some embodiments, the sense and antisense sequences that form the RNAi construct correspond to a sequence derived from a gene selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1. In some embodiments, the RNAi constructs comprise SEQ ID NOs:60-77, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the target RNA molecule is selected from the group consisting of SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the second exogenous sequence corresponds to a sequence derived from a gene selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1. In some embodiments, the second exogenous sequence corresponds to a sequence derived from a gene selected from the group consisting of SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto.
In some embodiments, the present invention provides systems for controlling a plant pest comprising at least one vector comprising a first exogenous nucleic acid sequence having a sense sequence linked to its complementary antisense sequence and encoding a double stranded RNA that inhibits expression of a target pest gene (i.e., an RNAi construct) and a second exogenous sequence encoding at least a portion of said target pest gene. In some embodiments, the sense and antisense sequences that form the RNAi construct correspond to a sequence derived from a gene selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1. In some embodiments, the RNAi constructs comprise SEQ ID NOs:60-77, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the target RNA molecule is selected from the group consisting of SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the second exogenous sequence corresponds to a sequence derived from a gene selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1. In some embodiments, the second exogenous sequence corresponds to a sequence derived from a gene selected from the group consisting of SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the first exogenous sequence and said second exogenous sequence are present on the same vector. In some embodiments, the first exogenous sequence and the second exogenous sequence are on separate vectors. In some embodiments, the systems comprise vectors with RNAi constructs that target two or more pest target sequences and vectors that express sense or antisense sequences of the two or more pest target sequences. In some embodiments, the RNAi constructs are stacked constructs as described in more detail herein.
In some embodiments, the present invention provides methods of reducing damage due to pests comprising cultivating a transgenic plant comprising the system described above.
In some embodiments, the present invention provides a transgenic plant comprising a nucleic acid sequence having at least two sense sequences from at least two different target pest genes linked to at least two antisense sequences from said at least two different target pest genes so that said nucleic acid sequence encodes a double stranded RNA that inhibits expression of said at least two target pest genes. In some embodiments, the at least two target pest genes are selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1. In some embodiments, the at least two target pest genes are selected from the group consisting of SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto.
In some embodiments, the present invention provides a vector comprising a nucleic acid sequence having at least two sense sequences from at least two different target pest genes linked to at least two antisense sequences from said at least two different target pest genes so that said nucleic acid sequence encodes a double stranded RNA that inhibits expression of said at least two target pest genes. In some embodiments, the vectors comprise additional exogenous sequences encoding at least a portion of said at least two target pest genes. In some embodiments, the at least two target pest genes are selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1. In some embodiments, the at least two target pest genes are selected from the group consisting of SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto.
In some embodiments, the present invention provides methods of reducing damage due to pests comprising: cultivating a transgenic plant comprising the vectors described above.
In some embodiments, the present invention provides a transgenic plant comprising a first exogenous nucleic acid sequence having a sense sequence linked to its complementary antisense sequence and encoding a double stranded RNA that inhibits expression of a target gene, wherein said target gene is selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1. In some embodiments, the target RNA molecule is at least 80%, 85%, 90%, 95% or 99% identical to a Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1 sequence. In some embodiments, the target RNA molecule is selected from the group consisting of SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the sense and antisense strands in the double stranded RNA are linked by a linker sequence. In some embodiments, the linker sequence is a Gus linker.
In some embodiments, the present invention provides vectors comprising a first exogenous nucleic acid sequence having a sense sequence linked to its complementary antisense sequence and encoding a double stranded RNA that inhibits expression of a target gene, wherein said target gene is selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1. In some embodiments, the target RNA molecule is at least 80%, 85%, 90%, 95% or 99% identical to a Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1 sequence. In some embodiments, the target RNA molecule is selected from the group consisting of SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the sense and antisense strands in the double stranded RNA are linked by a linker sequence. In some embodiments, the linker sequence is a Gus linker.
In some embodiments, the present invention provides methods of reducing damage due to pests comprising: cultivating a transgenic plant comprising a vector as described above.
In some embodiments, the present invention provides compositions comprising a part of a transgenic plant as described above. In some embodiments, the plant part is a seed, stem, leave, shoot, root or branch or combinations thereof. In some embodiments, the plant part is provided in an animal feed. In some embodiments, the seed are treated with a coating that reduces fungal infection or enhances germination.



DESCRIPTION OF THE FIGURES
FIG. 1. T-DNA region of vectors pIG121Hm and pANDA35HK. a pIG121Hm. Right border (RB), left border (LB), Hygromycin resistance gene (HPT), Neomycin phosphotransferase gene (NPTII), intron-containing GUS gene (GUS INT), promoter nopaline synthase (PNos), promoter 35 s of CaMV (35S), terminator nopaline synthase (T) (Hiei et al. 1994). b pANDA35HK. Left border (LB), right border (RB), Kanamycin resistance gene, driven by NOS promoter (NPTII), Hygromycin resistance gene, driven by 35S promoter (HPT), Nos terminator (T). Fib-1 or Y25 fragments were achieved by PCR. Sense and antisense gene fragments were inserted into pANDA35 HK by Gateway cloning strategy.
FIG. 2. Generation of transformed roots by A. rhizogenes (R1000). a Four days after ‘Jack’ seeds were placed on 1⁄2 MS medium for germination. Bar 1 cm. b Growth of Jack seeds 4 days after injection with R1000 containing pIG121Hm vector (GUS gene included) in the hypocotyls. The tap roots were excised before injection. Arrow shows the injection site of Jack cotyledon. Bar 1 cm. c Selection in MS medium
with Kanamycin (200 mg/L). Bar 1 cm. d Morphological aspect of branched transgenic roots (a) and roots transformed with R1000 lacking vectors (b) 3 weeks after injection. Bar 2 cm. e Chimeric plants transferred to soil. Bar 5 cm. f An example of b-glucuronidase (GUS) histochemical assay of transgenic roots transformed with pIG121Hm. Although most roots are transgenic, there are a few that are not (i.e. escapes). Arrow one non-transgenic root. Bar 2 cm.
FIG. 3. GFP expression in transgenic roots. a-c roots viewed under fluorescence stereomicroscope Leica MZ FLIII with the GFP filter. d-f same roots as a, b and c (respectively) viewed under normal light. a, b, d and e are from seedlings transformed with R1000 containing pBINmGFP5-ER vector. c and f are from a seedling transformed with only R1000 (no binary vector present). Note that as in the case of GUS staining in FIG. 2, most, but not all roots appear to be transgenic in a and b (arrows the non transgenic roots). Bar 5 mm.
FIG. 4. PCR Results of Fib-1 and Y25 genes and ColE on gene amplification in transformed roots. a The amplification results of Fib-1 gene. GUS-F1 (within Gus linker, located close to site b) and Fib-R were used for amplifying Fib-1 gene. 100 bp ladder (M), PCR results from genomic DNAs of six independent transgenic hairy roots transformed with pANDA35HK: Fib-1 (1-6) vector pANDA35HK: Fib-1(P). b The amplification results of Y25 gene. GUS-F1 and Y25-R were used for amplifying Y25 gene. PCR results from genomic DNAs of four independent transgenic hairy roots transformed with pANDA35HK: Y25 (1-4), vector pANDA35HK: Y25 (P), negative control 1, PCR results from genomic DNAs of soybean plants transformed with only R1000 (N1), negative control 2 (N2), PCR results from genomic DNAs of non-transgenic soybean roots.
FIG. 5 Southern blot analysis of composite hairy roots. All the genomic DNAs were digested by SacI. a six independent hairy roots transformed with pANDA35HK: Fib-1 (1-6). b four independent transgenic hairy roots transformed with pANDA35HK: Y25 (1-4). pANDA35HK vector (50 pg) (P), negative control, soybean plants transformed with only R1000 (N). HPT gene digested from pUC119 using BamHI was used as template for synthesizing probe.
FIG. 6. PCR analysis of Cpn-1, Y25 and Prp-17 genes and ColE on replication site. a The amplification results of Cpn-1 gene. GUS-F1 (located close to 5′ end of Gus linker) and Cpn reverse were used for amplifying Cpn-1 gene antisense fragment; GUS-R1 (located close to 3′ end of Gus linker) and Cpn reverse were used for amplifying Cpn-1 gene sense fragment. M 1 kb ladder for Cpn-1 gene and 100 bp ladder for on gene, C1-C4 PCR results from genomic DNAs of roots of four independent transgenic composite plants transformed with pANDA35HK: Cpn-1, P vector pANDA35HK:Cpn-1. b The amplification results of Y25 gene. GUS-F1 and Y25 reverse, GUS-R1 and Y25 reverse were used for amplifying Y25 gene antisense and sense fragments, separately. Y1-Y4 PCR results from genomic DNAs of roots of four independent composite plants transformed with pANDA35HK: Y25, P vector pANDA35HK: Y25. c The amplification results of Prp-17 gene. GUS-F1 and Prp-17 reverse, GUS-R1 and Prp-17 reverse were used for amplifying Prp-17 gene antisense and sense fragments, separately. P1-P4 PCR results from genomic DNAs of roots of four independent transgenic composite plants transformed with pANDA35HK: Prp-17, P vector pANDA35HK: Prp-17. N negative control, PCR results from genomic DNAs of roots of soybean plants transformed with only K599 containing no binary vectors.
FIG. 7. Southern blot analysis of Cpn-1, Y25 and Prp-17 transformed roots. Genomic DNAs from roots of independent composite plants digested with SacI and probed with BamHI-digested HPT gene fragment. M 1 kb marker, P undigested pANDA35HK vector (50 pg). N negative control soybean plants transformed with only K599 (no binary vector), C1-C4, Y1-Y4, and P1-P4 DNAs from roots of composite plants transformed with pANDA35HK:Cpn-1, pANDA35HK: Y25, and pANDA35HK:Prp-17, respectively.
FIG. 8. RT-PCR analysis of Cpn-1, Y25 and Prp-17 transformed roots. RibF and RibR were used for amplification of Rib gene (Ribosomal-521, CF921751); GusF and GusR were used for amplification of Gus linker fragment. Cpn-1 forward and Cpn-1 reverse, Y25 forward and Y25 reverse, Prp-17 forward and Prp-17 reverse were used for amplifying Cpn-1, Y25 and Prp-17 genes, separately. C1-C4 roots of four independent composite plants transformed with pANDA35HK:Cpn-1, Y1-Y4 roots of four independent composite plants transformed with pANDA35HK: Y25, P1-P4 roots of four independent transgenic composite plants transformed with pANDA35HK: Prp-17. N negative control, soybean roots transformed with only K599. GOI gene of interest.
FIG. 9. siRNAs detection in Cpn-1, Y25 and Prp-17 transformed roots. C1 roots from one composite plant transformed with A. rhizogenes K599 containing pANDA35HK:Cpn-1, Y1 roots from one composite plant transformed with A. rhizogenes K599 containing pANDA35HK:Y25, P1 roots from one composite plant transformed with A. rhizogenes K599 containing pANDA35HK:Prp-17, N roots of soybean plants transformed with only K599. 408 bp Cpn-1, 294 bp Y25 and 289 bp Prp-17 were used for the synthesis of probes separately, and hybridized with C1, Y1 and P1 in turn. As negative control (N) had no signals with either of three different probes, hybridization result was only shown with 294-bp Y25 fragment synthesized probe.
FIG. 10. Comparison of H. glycines resistance in Cpn-1 RNAi transgenic composite plants and controls 5 weeks after inoculation. a Transgenic Cpn-1 RNAi plant with significantly less cysts development on roots. b Composite plants transformed with empty vector pANDA35HK showing numerous cysts on roots. Arrows indicate cysts on the roots. Bar 1 mm.
FIG. 11. Real-time RT-PCR results showing the down-regulation of target H. glycines genes Y25 and Prp-17 mRNA transcript levels by host-induced RNAi. Y25 and Prp-17 are two different H. glycines samples obtained from Y25 and Prp-17 RNAi composite plants of 5 weeks after H. glycines inoculation. Nematodes feeding on composite plants inoculated with K599 containing no binary vector were used as negative controls. The H. glycines beta-actin (AF318603) gene was used as an endogenous reference for normalization between samples. Data were provided as fold reduction in expression levels (2-DDCT)±standard deviation of the mean based on duplicate qRT-PCR assays of three independent experiments. The relative expression of non-target (NT) gene remain unchanged in each experiment. *P<0.05.
FIG. 12. Partial nucleic acid sequence for Y25 of Heterodera glycines (HM369132) (SEQ ID NO:1).
FIG. 13. Partial nucleic acid sequence for Arx-3 of Heterodera glycines (CD748919) (SEQ ID NO:2).
FIG. 14. Partial nucleic acid sequence for Cpn-1 of Heterodera glycines (GU074018) (SEQ ID NO:3)
FIG. 15. Full length nucleic acid sequence for Prp-17 of Heterodera glycines (AF113915) (SEQ ID NO:4).
FIG. 16. Partial nucleic acid sequence for Rnr-1 of Heterodera glycines (CD749124) (SEQ ID NO:5).
FIG. 17. Partial nucleic acid sequence for Fib-1 of Heterodera glycines (CB279515) (SEQ ID NO:6).
FIG. 18. Partial nucleic acid sequence for Asb-1 of Heterodera glycines (BI396626) (SEQ ID NO:7).
FIG. 19. Partial nucleic acid sequence for Rpt-1 of Heterodera glycines (CB376265) (SEQ ID NO:8).
FIG. 20. Partial nucleic acid sequence for F-55 of Heterodera glycines (CA940536) (SEQ ID NO:9).
FIG. 21. Partial nucleic acid sequence for Eat-3 of Heterodera glycines (CB375718) (SEQ ID NO:10).
FIG. 22. Partial nucleic acid sequence for Vap-2 of Heterodera glycines (AY033601) (SEQ ID NO:11).
FIG. 23. Partial nucleic acid sequence for Y25 of Pratylenchus neglectus (SEQ ID NO:12).
FIG. 24. Partial nucleic acid sequence for Rnr-1 of Pratylenchus neglectus (SEQ ID NO:13).
FIG. 25. Partial nucleic acid sequence for Y25 of Radopholus similis (SEQ ID NO:14).
FIG. 26. Partial nucleic acid sequence for Rnr-1 of Radopholus similis (SEQ ID NO:15).
FIG. 27. Partial nucleic acid sequence for Rnr-1 of Rotylenchulus reniformis (SEQ ID NO:16).
FIG. 28. Schematic depiction of stacked RNAi constructs.
FIG. 29. Partial nucleic acid sequence for Cdk-1 (J12), H. glycines, (SEQ ID NO: 51).
FIG. 30. Partial nucleic acid sequence for Fzy-1 (J13), H. glycines, (SEQ ID NO: 52).
FIG. 31. Partial nucleic acid sequence for Tba-2 (J14), H. glycines, (SEQ ID NO: 53).
FIG. 32. Partial nucleic acid sequence for Arx-1 (J15), H. glycines, (SEQ ID NO: 54).
FIG. 33. Partial nucleic acid sequence for Tbb-2 (J17), H. glycines, (SEQ ID NO: 55).
FIG. 34. Partial nucleic acid sequence for Unc-26 (J20), H. glycines, (SEQ ID NO: 56).
FIG. 35. Partial nucleic acid sequence for Prp-4 (J21), H. glycines, (SEQ ID NO: 57).
FIG. 36. Partial nucleic acid sequence for Pfn-1 (J23), H. glycines, (SEQ ID NO: 58).
FIG. 37. Partial nucleic acid sequence for Vbh-1 (J7), H. glycines, (SEQ ID NO: 59).
FIG. 38. RNAi construct sequence (containing underlined antisense fragment of Y25, shaded Gus linker and sense fragment of Y25) (SEQ ID NO:108).
FIG. 39. RNAi construct sequence (containing underlined antisense fragment of Arx-3, shaded Gus linker and sense fragment of Arx-3) (SEQ ID NO:60).
FIG. 40. RNAi construct sequence (containing underlined antisense fragment of Cpn-1, shaded Gus linker and sense fragment of Cpn-1) (SEQ ID NO:61).
FIG. 41. RNAi construct sequence (containing underlined antisense fragment of Prp-17, shaded Gus linker and sense fragment of Prp-17) (SEQ ID NO:62).
FIG. 42. RNAi construct sequence (containing underlined antisense fragment of Rnr-1, shaded Gus linker and sense fragment of Rnr-1) (SEQ ID NO:63).
FIG. 43. RNAi construct sequence (containing underlined antisense fragment of Fib-1, shaded Gus linker and sense fragment of Fib-1) (SEQ ID NO:64).
FIG. 44. RNAi construct sequence (containing underlined antisense fragment of Rpt-1, shaded Gus linker and sense fragment of Rpt-1) (SEQ ID NO:65).
FIG. 45. RNAi construct sequence (containing underlined sense fragment of F-55, shaded Gus linker and antisense fragment of F-55) (SEQ ID NO:66).
FIG. 46. RNAi construct sequence (containing underlined sense fragment of Eat-3, shaded Gus linker and antisense fragment of Eat-3) (SEQ ID NO:67).
FIG. 47. RNAi construct sequence (containing underlined sense fragment of Vap-2, shaded Gus linker and antisense fragment of Vap-2) (SEQ ID NO:68).
FIG. 48. RNAi construct sequence (containing underlined sense fragment of Asb-1, shaded Gus linker and antisense fragment of Asb-1) (SEQ ID NO:69).
FIG. 49. RNAi construct sequence, Y25 of Pratylenchus neglectus, (containing underlined sense fragment of Y25, shaded Gus linker and antisense fragment of Y25) (SEQ ID NO:70).
FIG. 50. RNAi construct sequence, Rnr-1 of Pratylenchus neglectus, (containing underlined sense fragment of Rnr-1, shaded Gus linker and antisense fragment of Rnr-1) (SEQ ID NO:71).
FIG. 51. RNAi construct sequence, Y25 of Radopholus similis, (containing underlined sense fragment of Y25, shaded Gus linker and antisense fragment of Y25) (SEQ ID NO:72).
FIG. 52. RNAi construct sequence, Rnr-1 of Radopholus similis, (containing underlined sense fragment of Rnr-1, shaded Gus linker and antisense fragment of Rnr-1) (SEQ ID NO:73).
FIG. 53. RNAi construct sequence, Rnr-1 of Rotylenchulus reniformis, (containing underlined sense fragment of Rnr-1, shaded Gus linker and antisense fragment of Rnr-1) (SEQ ID NO:74).
FIG. 54. RNAi construct sequence of Y25-Prp-17 (containing underlined sense fragment of Y25-Prp-17, shaded Gus linker and antisense fragment of Y25-Prp-17) (SEQ ID NO:75).
FIG. 55. RNAi construct sequence of Y25-Cpn-1 (containing underlined sense fragment of Y25-Cpn-1, shaded Gus linker and antisense fragment of Y25-Cpn-1) (SEQ ID NO:76).
FIG. 56. RNAi construct sequence of Cpn-Rnr-1 (containing underlined antisense fragment of Cpn-Rnr-1, shaded Gus linker and sense fragment of Cpn-Rnr-1) (SEQ ID NO:77).
FIG. 57. Schematic depiction of vectors for overexpression of target sequence with target siRNA.
FIG. 58. Y25 gene fragment used for over-expressing in pPTN289 vector (SEQ ID NO:78).
FIGS. 59A and 59B. Sequence comparison of Hg25 and HgRnr-1 genes among three different Heterodera glycines populations and Radopholus similis, root 1. The Y25 fragments (291 bp) and Rnr-1 (251 bp) from H. glycines (Hg) race 1, race 3, race 4 and Radopholus similis (RS) (SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, respectively; consensus sequences SEQ ID NO:87 and SEQ ID NO:88, respectively, and Lesion Rnr-1 sequences SEQ ID NO:109 and 110, respectively), root lesion nematode Pratylenchus neglectus (lesion) were amplified using specific primers by PCR method. PCR products were cloned into pGEM-T easy vector, and sequenced by T7 primer. DNAman software was performed to compare the sequence difference.
FIG. 60. Actual sequence used for cdk-1 RNAi>J12 (exons) sequenced from cDNA (SEQ ID NO:89).
FIG. 61. Sequence of pANDA35HK/cdk-1 J12 RNAi construct sequence (containing underlined antisense and sense fragment of cdk-1 and shaded Gus linker) (SEQ ID NO:90).
FIG. 62. Actual sequence used for Fzy-1 RNAi>J13 (with introns) sequenced from gDNA (SEQ ID NO:91).
FIG. 63. Sequence of pANDA35HK/fzy-1 J13 (containing underlined antisense and sense fragment of fzy-1 and shaded Gus linker) (SEQ ID NO:92).
FIG. 64. Actual sequence used for tba-2 RNAi>J14 (with introns) sequenced from gDNA (SEQ ID NO:93).
FIG. 65. Sequence of pANDA35HK/tba-2 J14 (containing underlined antisense and sense fragment of tba-1 and shaded Gus linker) (SEQ ID NO:94).
FIG. 66. Sequence used for arx-1 RNAi>J15 (with introns) sequenced from gDNA (SEQ ID NO:95).
FIG. 67. Sequence of pANDA35HK/arx-1 J15 (containing underlined antisense and sense fragment of arx-1 and shaded Gus linker) (SEQ ID NO:96).
FIG. 68. Actual sequence used for tbb-2 RNAi>J17 (exon) from cDNA (SEQ ID NO:97).
FIG. 69. Sequence of pANDA35HK/tbb-2 J17 (containing underlined antisense and sense fragment of tbb-2 and shaded Gus linker) (SEQ ID NO:98).
FIG. 70. Sequence used for unc-26 RNAi>J20 (with introns) from gDNA (SEQ ID NO:99).
FIG. 71. Sequence in pANDA35HK/unc-26 J20 (containing underlined antisense and sense fragment of unc-26 and shaded Gus linker) (SEQ ID NO:100).
FIG. 72. Sequence used for prp-4 RNAi>J21 (exon) from cDNA (SEQ ID NO:101).
FIG. 73. Sequence in pANDA35HK/prp-4 J21 (containing underlined antisense and sense fragment of prp-4 and shaded Gus linker) (SEQ ID NO:102).
FIG. 74. Sequence used for pfn-1 RNAi>J23 (exon) from cDNA (SEQ ID NO:103).
FIG. 75. Sequence in pANDA35HK/pfn-1 J23 (containing underlined antisense and sense fragment of pfn-1 and shaded Gus linker) (SEQ ID NO:104).
FIG. 76. Sequence used for vbh-1 RNAi>J7 (with introns) from gDNA (SEQ ID NO:105).
FIG. 77. Sequence in pANDA35HK/vbh-1 J7 (containing underlined antisense and sense fragment of vbh-1 and shaded Gus linker) (SEQ ID NO:106).



DEFINITIONS
To facilitate an understanding of the present invention, a number of terms and phrases as used herein are defined below:
The term “plant” is used in it broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and photosynthetic green algae. It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc. The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture. The term “plant part” as used herein refers to a plant structure, a plant organ, or a plant tissue.
The term “crop” or “crop plant” is used in its broadest sense. The term includes, but is not limited to, any species of plant or algae edible by humans or used as a feed for animals or used, or consumed by humans, or any plant or algae used in industry or commerce.
The term plant cell “compartments or organelles” is used in its broadest sense. The term includes but is not limited to, the endoplasmic reticulum, Golgi apparatus, trans Golgi network, plastids including chloroplasts, proplastids, and leucoplasts, sarcoplasmic reticulum, glyoxysomes, mitochondrial, chloroplast, and nuclear membranes, and the like.
The term “host cell” refers to any cell capable of replicating and/or transcribing and/or translating a heterologous gene.
The term “heterologous,” when used in reference to DNA sequences or genes, means a DNA sequence encoding a protein, polypeptide, RNA, or a portion of any thereof, whose exact amino acid sequence is not normally found in the host cell, but is introduced by standard gene transfer techniques.
The term “nematode” as used herein refers to worms that are members of the phylum Nemata.
The term “RNA interference” or “RNAi” refers to the silencing or decreasing of gene expression by iRNA or siRNAs. It is the process of sequence-specific, posttranscriptional gene silencing in animals and plants, initiated by iRNA that is homologous in its duplex region to the sequence of the silenced gene. The gene may be endogenous or exogenous to the organism, present integrated into a chromosome or present in a transfection vector that is not integrated into the genome. The expression of the gene is either completely or partially inhibited. RNAi may also be considered to inhibit the function of a target RNA; the function of the target RNA may be complete or partial.
The term “interfering RNA (iRNA)” refers to a double stranded RNA molecule that mediates RNA interference (RNAi). At least one strand of the duplex or double-stranded region of a siRNA is substantially homologous to or substantially complementary to a target RNA molecule. The strand complementary to a target RNA molecule is the “antisense strand;” the strand homologous to the target RNA molecule is the “sense strand,” and is also complementary to the RNAi antisense strand. RNAi may also contain additional sequences; non-limiting examples of such sequences include linking sequences, or loops, as well as stem and other folded structures.
siRNAs generally comprise a duplex, or double-stranded region, of about 18-25 nucleotides long; often siRNAs contain from about two to four unpaired nucleotides at the 3′ end of each strand. At least one strand of the duplex or double-stranded region of a siRNA is substantially homologous to or substantially complementary to a target RNA molecule. The strand complementary to a target RNA molecule is the “antisense strand;” the strand homologous to the target RNA molecule is the “sense strand,” and is also complementary to the siRNA antisense strand. siRNAs may also contain additional sequences; non-limiting examples of such sequences include linking sequences, or loops, as well as stem and other folded structures. siRNAs appear to function as key intermediaries in triggering RNA interference in invertebrates and in vertebrates, and in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants.
The term “target RNA molecule” refers to an RNA molecule to which at least one strand of the short double-stranded region of an siRNA is homologous or complementary. Typically, when such homology or complementary is about 100%, the siRNA is able to silence or inhibit expression of the target RNA molecule. Although it is believed that processed mRNA is a target of siRNA, the present invention is not limited to any particular hypothesis, and such hypotheses are not necessary to practice the present invention. Thus, it is contemplated that other RNA molecules may also be targets of siRNA. Such targets include unprocessed mRNA, ribosomal RNA, and viral RNA genomes.
As used herein, the term “loop sequence” refers to a nucleic acid sequence that is placed between two nucleic sequences that are complementary to each other and which forms a loops when the complementary nucleic acid sequences hybridize to one another.
The term “nematode target RNA” as used herein refers to an RNA that is expressed in a nematode.
The term “double stranded nematode RNA sequence” refers to an iRNA that is specific for a nematode target RNA.
The term “inhibits the proliferation of nematodes” refers to a reduction in nematode parasitism of a host organism. A variety of assays may be used to measure proliferation, including, but not limited to measuring the number of roots cysts that develop in plants exposed to nematodes.
As used herein, the term “orally active to prevent the proliferation of nematodes” refers to a double stranded nematode RNA sequence that inhibits the proliferation of nematodes when orally ingested by the nematodes.
The terms “protein” and “polypeptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.
As used herein, “amino acid sequence” refers to an amino acid sequence of a protein molecule. “Amino acid sequence” and like terms, such as “polypeptide” or “protein,” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. Furthermore, an “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein.
The term “portion” when used in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino sequence minus one amino acid.
The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or a polypeptide or its precursor (e.g., proinsulin). A functional polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained. The term “portion” when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, “a nucleotide comprising at least a portion of a gene” may comprise fragments of the gene or the entire gene.
The term “gene” also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5′ of the coding region and which are present on the mRNA are referred to as 5′ non-translated sequences. The sequences which are located 3′ or downstream of the coding region and which are present on the mRNA are referred to as 3′ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene which are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5′ and 3′ end of the sequences that are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5′ or 3′ to the non-translated sequences present on the mRNA transcript). The 5′ flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3′ flanking region may contain sequences that direct the termination of transcription, posttranscriptional cleavage and polyadenylation.
The term “heterologous gene” refers to a gene encoding a factor that is not in its natural environment (i.e., has been altered by the hand of man). For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous genes may comprise plant gene sequences that comprise cDNA forms of a plant gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous genes are distinguished from endogenous plant genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
The term “homology” when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). “Sequence identity” refers to a measure of relatedness between two or more nucleic acids, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues that are identical and in the same relative positions in their respective larger sequences. Calculations of identity may be performed by algorithms contained within computer programs such as “GAP” (Genetics Computer Group, Madison, Wis.) and “ALIGN” (DNAStar, Madison, Wis.). A partially complementary sequence is one that at least partially inhibits (or competes with) a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term “substantially homologous.” The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a sequence that is completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target. In a preferred embodiment, a homolog has a greater than 60% sequence identity, and more preferable greater than 75% sequence identity, and still more preferably greater than 90% sequence identity, with a reference sequence.
When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term “substantially homologous” refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described infra.
The term “gene expression” refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein, through “translation” of mRNA. Gene expression can be regulated at many stages in the process. “Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called “activators” and “repressors,” respectively.
The terms “in operable combination”, “in operable order” and “operably linked” refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
The term “regulatory element” refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237, 1987). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect, mammalian and plant cells. Promoter and enhancer elements have also been isolated from viruses and analogous control elements, such as promoters, are also found in prokaryotes. The selection of a particular promoter and enhancer depends on the cell type used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review, see Voss, et al., Trends Biochem. Sci., 11:287, 1986; and Maniatis, et al., supra 1987).
The terms “promoter element,” “promoter,” or “promoter sequence” as used herein, refer to a DNA sequence that is located at the 5′ end (i.e. precedes) the protein coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.
Promoters may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seed tissue) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., leave tissue). Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected. The term “cell type specific” as applied to a promoter refers to a promoter which is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining. Briefly, tissue sections are embedded in paraffin, and paraffin sections are reacted with a primary antibody that is specific for the polypeptide product encoded by the nucleotide sequence of interest whose expression is controlled by the promoter. A labeled (e.g., peroxidase conjugated) secondary antibody that is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy.
Promoters may be constitutive or regulatable. The term “constitutive” when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a transgene in substantially any cell and any tissue. Exemplary constitutive plant promoters include, but are not limited to SD Cauliflower Mosaic Virus (CaMV SD; see e.g., U.S. Pat. No. 5,352,605, incorporated herein by reference), mannopine synthase, octopine synthase (ocs), superpromoter (see e.g., WO 95/14098), and ubi3 (see e.g., Garbarino and Belknap (1994) Plant Mol. Biol. 24:119-127) promoters. Such promoters have been used successfully to direct the expression of heterologous nucleic acid sequences in transformed plant tissue.
In contrast, a “regulatable” promoter is one which is capable of directing a level of transcription of an operably linked nuclei acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.) which is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus.
The enhancer and/or promoter may be “endogenous” or “exogenous” or “heterologous.” An “endogenous” enhancer or promoter is one that is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer or promoter is one that is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of the gene is directed by the linked enhancer or promoter. For example, an endogenous promoter in operable combination with a first gene can be isolated, removed, and placed in operable combination with a second gene, thereby making it a “heterologous promoter” in operable combination with the second gene. A variety of such combinations are contemplated (e.g., the first and second genes can be from the same species, or from different species.
The term “vector” refers to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term “vehicle” is sometimes used interchangeably with “vector.”
The terms “expression vector” or “expression cassette” refer to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
The terms “transfection”, “transformation”, “transfected” and “transformed” are used interchangeably and refer to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, glass beads, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, biolistics (i.e., particle bombardment) and the like.
The terms “infecting” and “infection” when used with a bacterium refer to co-incubation of a target biological sample, (e.g., cell, tissue, etc.) with the bacterium under conditions such that nucleic acid sequences contained within the bacterium are introduced into one or more cells of the target biological sample.
The term “Agrobacterium” refers to a soil-borne, Gram-negative, rod-shaped phytopathogenic bacterium which causes crown gall. The term “Agrobacterium” includes, but is not limited to, the strains Agrobacterium tumefaciens, (which typically causes crown gall in infected plants), and Agrobacterium rhizogens (which causes hairy root disease in infected host plants). Infection of a plant cell with Agrobacterium generally results in the production of opines (e.g., nopaline, agropine, octopine etc.) by the infected cell. Thus, Agrobacterium strains which cause production of nopaline (e.g., strain LBA4301, C58, A208, GV3101) are referred to as “nopaline-type” Agrobacteria; Agrobacterium strains which cause production of octopine (e.g., strain LBA4404, AchS, B6) are referred to as “octopine-type” Agrobacteria; and Agrobacterium strains which cause production of agropine (e.g., strain EHA105, EHA101, A281) are referred to as “agropine-type” Agrobacteria. 
The terms “bombarding, “bombardment,” and “biolistic bombardment” refer to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample. Methods for biolistic bombardment are known in the art (e.g., U.S. Pat. No. 5,584,807, the contents of which are incorporated herein by reference), and are commercially available (e.g., the helium gas-driven microprojectile accelerator (PDS-1000/He, BioRad).
The term “microwounding” when made in reference to plant tissue refers to the introduction of microscopic wounds in that tissue. Microwounding may be achieved by, for example, particle bombardment as described herein.
The term “transgenic” when used in reference to a plant or fruit or seed (i.e., a “transgenic plant” or “transgenic fruit” or a “transgenic seed”) refers to a plant or fruit or seed that contains at least one heterologous gene in one or more of its cells. The term “transgenic plant material” refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous gene in one or more of its cells.
The terms “transformants” or “transformed cells” include the primary transformed cell and cultures derived from that cell without regard to the number of transfers. All progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of transformants.
The term “antisense” refers to a deoxyribonucleotide sequence whose sequence of deoxyribonucleotide residues is in reverse 5′ to 3′ orientation in relation to the sequence of deoxyribonucleotide residues in a sense strand of a DNA duplex. A “sense strand” of a DNA duplex refers to a strand in a DNA duplex that is transcribed by a cell in its natural state into a “sense mRNA.” Thus an “antisense” sequence is a sequence having the same sequence as the non-coding strand in a DNA duplex. The term “antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript or mRNA. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. In addition, as used herein, antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression. “Ribozyme” refers to a catalytic RNA and includes sequence-specific endoribonucleases. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein.
The term “overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. The term “cosuppression” refers to the expression of a foreign gene that has substantial homology to an endogenous gene resulting in the suppression of expression of both the foreign and the endogenous gene. The term “altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
The term “recombinant” when made in reference to a nucleic acid molecule refers to a nucleic acid molecule that is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term “recombinant” when made in reference to a protein or a polypeptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.
The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature.
The term “purified” refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated. An “isolated nucleic acid sequence” is therefore a purified nucleic acid sequence. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. The term “purified” or “to purify” also refer to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percent of polypeptide of interest in the sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.
The term “sample” is used in its broadest sense. In one sense it can refer to a plant cell or tissue. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.
DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for controlling nematode infestation of plants or animals. In particular, the present invention provides vectors comprising sequences designed to control nematodes by RNA interference (RNAi) and transgenic plants transformed with such vectors. The compositions and methods of the present invention can be used to inhibit the growth and reproduction of a number of nematodes species, including, but not limited to plant parasitic nematodes and nematodes in the following genera: Acontylus, Criconemella, Pseudhalenchus, Afenestrata, Cucullitylenchus, Lelenchus, Psilenchus, Aglenchus, Cryphodera, Pterotylenchus, Allotylenchus, Cynipanguina, Macrotrophurus, Punctodera, Amplimerlinius, Malenchus, Anguina, Discocriconemella, Meloidodera, Radopholus, Antarctenchus, Ditylenchus, Meloidoderita, Rhizonema, Antarctylus, Dolichodera, Meloidogyne, Rotylenchulus, Aorolaimus, Dolichodorus, Meloinema, Rotylenchus, Aphasmatylenchus, Duotylenchus, Merlinius, Apratylenchoides, Miculenchus, Sarisodera, Atalodera, Ecphyadophora, Mitranema, Sauertylenchus, Atetylenchus, Ecphyadophoroides, Morulaimus, Scutellonema, Atylenchus, Epicharinema, Mukazia, Senegalonema, Eutylenchus, Sphaeronema, Bakernema, Nacobbodera, Subanguina, Basiria, Filenchus, Nacobbus, Sychnotylenchus, Basirienchus, Nagelus, Basiroides, Bellodera, Geocenamus, Neodolichodorus, Thada, Belonolaimus, Globodera, Neopsilenchus, Thecavermiculatus, Blandicephalanema, Gracilacus, Neothada, Trichotylenchus, Boleodorus, Gracilancea, Nothocriconemoides, Triversus, Brachydorus, Trophonema, Bursadera, Halenchus, Ogma, Trophotylenchulus, Helicotylenchus, Paraphelenchus, Trophurus, Cacopaurus, Hemicriconemoides, Pararotylenchus, Tylenchocriconema, Cactodera, Hemicycliophora, Paratrophurus, Tylenchorhynchus, Caloosia, Heterodera, Paratylenchus, Tylenchulus, Cambellenchus, Hirschmanniella, Pateracephalanema, Tylenchus, Carphodorus, Hoplolaimus, Pleurotylenchus, Tylodorus, Cephalenchus, Hoplotylus, Polenchus, Clavilenchus, Coslenchus, Hylonema, Pratylenchoides, Verutus, Criconema, Pratylenchus, Zygotylenchus; and animal parasitic nematodes of the following genera: Trichuris, Acylostoma, Necator, Strongyloides, Toxocara, Baylisacaris, Trichinella, Draccunculus, Filarioidea, Onchocerca, Loa, Dirofilaria, and Anisakis. 
I. RNAi Systems, Constructs and Vectors
RNAi refers to the introduction of homologous double stranded RNA (dsRNA) to target a specific gene product, resulting in post transcriptional silencing of that gene. This phenomena was first reported in Caenorhabditis elegans by Guo and Kemphues (Par-1, A gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed, 1995, Cell, 81 (4) 611-620) and subsequently Fire et al. (Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, 1998, Nature 391: 806-811) discovered that it is the presence of dsRNA, formed from the annealing of sense and antisense strands present in the in vitro RNA preps, that is responsible for producing the interfering activity.
The present invention contemplates the use of RNA interference (RNAi) to downregulate the expression of genes needed for pest viability and reproduction, thus reducing pest infestation of plants. In both plants and animals, RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, although the protein components of this activity are unknown. However, the 22-nucleotide RNA sequences are homologous to the target gene that is being suppressed. Thus, the 22-nucleotide sequences appear to serve as guide sequences to instruct a multicomponent nuclease, RISC, to destroy the specific mRNAs.
Carthew has reported (Curr. Opin. Cell Biol. 13(2):244-248 (2001) that eukaryotes silence gene expression in the presence of dsRNA homologous to the silenced gene. Biochemical reactions that recapitulate this phenomenon generate RNA fragments of 21 to 23 nucleotides from the double-stranded RNA. These stably associate with an RNA endonuclease, and probably serve as a discriminator to select mRNAs. Once selected, mRNAs are cleaved at sites 21 to 23 nucleotides apart.
In preferred embodiments, the dsRNA used to initiate RNAi, may be isolated from native source or produced by known means, e.g., transcribed from DNA. The promoters and vectors described in more detail below are suitable for producing dsRNA. RNA is synthesized either in vivo or in vitro. In some embodiments, endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. In other embodiments, the RNA is provided transcription from a transgene in vivo or an expression construct. In some embodiments, the RNA strands are polyadenylated; in other embodiments, the RNA strands are capable of being translated into a polypeptide by a cell's translational apparatus. In still other embodiments, the RNA is chemically or enzymatically synthesized by manual or automated reactions. In further embodiments, the RNA is synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6). If synthesized chemically or by in vitro enzymatic synthesis, the RNA may be purified prior to introduction into the cell. For example, RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the RNA may be used with no or a minimum of purification to avoid losses due to sample processing. In some embodiments, the RNA is dried for storage or dissolved in an aqueous solution. In other embodiments, the solution contains buffers or salts to promote annealing, and/or stabilization of the duplex strands.
In some embodiments, the dsRNA is transcribed from the vectors as two separate stands. In other embodiments, the two strands of DNA used to form the dsRNA may belong to the same or two different duplexes in which they each form with a DNA strand of at least partially complementary sequence. When the dsRNA is thus-produced, the DNA sequence to be transcribed is flanked by two promoters, one controlling the transcription of one of the strands, and the other that of the complementary strand. These two promoters may be identical or different. In some embodiments, a DNA duplex provided at each end with a promoter sequence can directly generate RNAs of defined length, and which can join in pairs to form a dsRNA. See, e.g., U.S. Pat. No. 5,795,715, incorporated herein by reference. RNA duplex formation may be initiated either inside or outside the cell.
Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. RNA molecules containing a nucleotide sequence identical to a portion of the target gene are preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Thus, sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript. The length of the identical nucleotide sequences may be at least 25, 50, 100, 200, 300 or 400 bases.
There is no upper limit on the length of the dsRNA that can be used. For example, the dsRNA can range from about 21 base pairs (bp) of the gene to the full length of the gene or more. In one embodiment, the dsRNA used in the methods of the present invention is about 1000 bp in length. In another embodiment, the dsRNA is about 500 bp in length. In yet another embodiment, the dsRNA is about 22 bp up to about 100, 200, 300, 0r 400 bp in length. In preferred embodiments, the isolated RNAs of the present invention mediate degradation of the target RNA.
The double stranded RNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi for the target RNA. In one embodiment, the present invention relates to RNA molecules of varying lengths that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi cleavage of the target mRNA. In a particular embodiment, the RNA molecules of the present invention comprise a 3′ hydroxyl group. In some embodiments, the amount of target RNA (mRNA) is reduced in the cells of the target organism (e.g., H. glycines) exposed to target specific double stranded RNA as compared to target organisms that have not been exposed to target specific double stranded RNA.
In some embodiments, the present invention provides systems that enhance the effectiveness of RNAi constructs. In some embodiments, the systems comprise vectors that express an RNAi construct for a target RNA molecule, such as a target pest RNA molecule, and a sense or antisense sequence corresponding to at least a portion of the target RNA molecule. Exemplary vectors for use in this system are depicted in FIG. 57. In some embodiments, the sense or antisense sequence corresponds to the sequence used in the RNAi construct. The sense or antisense sequence can either correspond to the full length target sequence or to a portion of the target sequence. In some embodiments, the sense or antisense sequence is at least as long as the target sequence chosen for the RNAi construct. In some embodiments, these two sequences are encoded on the same vector, while in other embodiments, the RNAi construct and sense or antisense sequences are encoded on separate vectors. In some embodiments, the sense or antisense sequence is operably linked to a promoter that allows overexpression of the sense or antisense sequence. In some embodiments, the promoter is a constitutive plant promoter. Suitable vectors for expression of the RNAi construct and sense or antisense sequence are described in more detail below and in the Examples.
The present invention is not limited to any mechanism of action. Indeed, an understanding of the mechanism of action is not necessary to practice the present invention. Nevertheless, it is contemplated that the RNAi effect is enhanced by overexpression of either part or the full target RNA sequence (in either sense or antisense orientation) at least including the fragment used for the RNAi construct hairpin together with the RNAi hairpin in the same or separate vectors. This results in up regulation of the amount of siRNA both in terms of concentration and number of individual molecule species in the transgenic plant. This in turn increases the amount and number of siRNA species the parasitic nematode, insect or pathogenic fungus will ingest/absorb so that it will give rise to an increased level of plant protection for plants.
In some embodiments the target RNA molecule used in this system is an RNA molecule of an organism that is a plant pest. Examples of such plant pests include, but are not limited to, parasites such nematodes and insects as well as pathogenic fungi. In some preferred embodiments, the pest is a nematode.
In some embodiments, the present invention provides RNAi constructs for a target RNA molecule, vectors that express the RNAi constructs and transgenic plants comprising the RNAi construct/vectors. In some embodiments, the target RNA molecule is selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, and Asb-1. The target RNA molecule may be derived from nematodes of various species, including, but not limited to Heterodera glycines, Meloidogyne incognita, M. javanic, M hapla, Globedera spp., Pratylenchus neglectus and P. thornei, Radopholus similis, and Rotylenchulus reniformis. In some embodiments, the target RNA molecule is at least 80%, 85%, 90%, 95%, or 99% identical to Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1 gene sequences. As described above, the RNAi construct corresponding to the target RNA molecule may be varying lengths. In preferred embodiments, the RNAi construct comprising a first exogenous nucleic acid sequence having a sense sequence linked to its complementary antisense sequence and encoding a double stranded RNA that inhibits expression of a target RNA molecule, wherein said target gene is selected from the group consisting of genes at least 80%, 85%, 90%, 95%, or 99% identical to Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-1. In some embodiments, the target RNA molecule is selected from the group consisting of SEQ ID NOs:1-7, 12-22, and 51-59 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. As shown in Example 3, use of RNAi constructs corresponding to the genes results in at least 33% reduction of nematode eggs (e.g., H. glycines eggs) per gram of roots in a hairy root assay. RNAi constructs to other target RNA molecule may be used in conjunction with the currently described systems and RNAi constructs. Exemplary additional target RNA molecules are described, for example, in U.S. Pat. No. 7,803,984, incorporated herein by reference in its entirety.







TABLE 1 







Exemplary RNAi Target Sequences










SEQ ID



Gene
NO:
Sequence





Y25
17
GCAGCCCGACAAGACAATTATGTTCACGCAGC




TGTCCACACGCGTGTCAGAAAACGTGACGGAC




ACAAATTTGTTTGATCTTTCGCTTTCCCAAGC




GCTTGGTACTGCACCCAAAACGACCAAATACA




CCTTTGCCAGCTCCAAACTGGGAAAAGTGATT




CAGTTAGCCGGCTTTTCGGATCCCGTCTATGC




CGAGGCGTACGTCAACGTCAACCAATATGACA




TTGTATTGGACGTACTCGTGGTCAACCAGACT




AGCGACACCTTGCAAAATTTGTCATTGGAACT




CTC


 


Arx-3
18
GTTTCATGTTCGCACGACAAAAATGCGTTTGT




GTGGACATGGGAGGCTGAAAAGAACAGTTGGA




AACCGGAGATGGTGGTCGTTCGGATAAACAGA




GCTGCCACCTGTGTCAAATGGTCGCCCAACGA




AAACAAATTCGCCGTTGGGACGGGTGCGCGTT




TAGTCGCTATTTGTTATTACGAACGTGAAAAT




GACTGGTGGGTAGCGAAGCAAATCAAAAAGCT




GATTCGTTCCACGGTCACCTCATTGAATTGGC




ATCCCAACAACATTTTGTTGGCTGTTGGCGCC




TGTGACTTCAAAACGCGTGTTTTTTCTGCTTA




TGTGAAAGAGATTGACGAAAAACCAACGCCAA




ACCCTTGGGGGACCAAAATGCCATTGGGAGAA




TTGC


 


Prp-17
19
CAATCGAATTGTCCTTTTCCAAATCGTCGATG




ACAAGTTGCGATTCGCTCGTAAAAAGGCCTTC




CGTGGTCACAATACAGCAGGGTACGCCTGCTC




AACTGATTTTTCGCCAGAGATGAGTTTTCTCG




CTTCCGGTGATGCGGACGGTAAAATCACAATG




TGGGACTGGCGCACACACAAAATTGTCTCCAC




ATGGAAGGCACATGATAATGTGTGCATTTCAA




CACTGTGGCATCCGCACGAGAAATCGCGGATG




ATTTCTTGCGGATGGGACAATGTAATCAAAAT




G


 


Rnr-1
20
CAAGAAATCAAAGACCTGTACAAGACCGTGTG




GGAAATACCGCAGAAGGACATTTTGAAAATGG




CCGCCGATCGCGCCGCTTTCATTGACCAAAGC




CAATCCCTTAACATTCACATAGCGCAGCCGAA




CTATGCTAAACTGAGCTCCATGCACTTTTACG




CCTGGTCATTGGGACTTAAAACCGGGATGTAT




TACCTGCGCACTCGTCCGGCTGTCGATGCTGT




TCAGTTCACTGTGGACAAAATGGCCCT


 


Fib-1
21
CCGAAGACGGAAGAGACAACGAATGGCAGCAA




CGGTGTCTCAGCACCCACCGCCGCGGCTGCCG




GCCCGGCACCGCCAGTCGAGTACCGGGTGTGG




AACCCGTTCCGCTCCAAACTGGCCGCCGCCGT




CATGGCCGGCATTGAGGACACGCACATTTACC




CGGG


 


Asb-1
22
TCCACACGGCTTTTCGTTATAAATTTGAAAAG




TACGTATACAAAGTTACCCGTGAACGATTTGG




CAAAATGAAGGCTTACATTGACAATGAATTGA




AGGAAGCCATCGAGTTCCGCAAGACTTCAAAG




GAGCAGGCCGACTCGTTGAAAGCAGTGCATGA




AAACTTTCCCACAATTTTCCAAGAGAATTTGG




CGCTGCAACTTGAAGCGACCTACCGAAAAAAT




GTGGACTACGCCTGGCAAGAGATGAAGCGTCG




GTTGGATTACCTGCAGGAAGTGCAAGCAATCA




AAGACCGAT









In some embodiments, the RNAi construct of the present invention comprise a first exogenous nucleic acid sequence having a sense sequence linked to its complementary antisense sequence and encoding a double stranded RNA that inhibits expression of a target RNA molecule. In some embodiments, the RNAi constructs comprise SEQ ID NOs:60-77, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. These RNAi constructs comprise sense and antisense sequence corresponding to a portion of the target pest sequence joined by a linker so that a double stranded siRNA is formed upon expression of the construct. In some embodiments, the target RNA molecule is at least 80%, 85%, 90%, 95% or 99% identical to a Cpn-1, Prp-17, Y25, Rnr-1, Arx-3, Fib-1, Asb-1, Cdk-1, Fzy-1, Tba-2, Arx-1, Tbb-2, Unc-26, Prp-4, Pfn-1, and Vbh-sequence. In some embodiments, the target RNA molecule is selected from the group consisting of SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the sense and antisense strands in the double stranded RNA are linked by a linker sequence. In some embodiments, the linker sequence is a Gus linker. In some embodiments, where a sense or antisense sequence corresponding to the target sequence is co-expressed with the RNAi construct, the sense or antisense sequence may be selected from sequences corresponding to SEQ ID NOs:1-7, 12-22, 51-59, 89, 91, 93, 95, 97, 99, 101, 103, and 105 and sequences at least 80%, 85%, 90%, 95%, or 99% identical thereto, or portions thereof, so long as the co-expressed sequence corresponds to the sequence contained within the RNAi construct.
The sequences described above and RNAi constructs comprising the sequences are described in Table 1 and FIGS. 12-26, 29-58, and 60-67.
In some embodiments, RNAi constructs of the present invention are “stacked constructs.” In some embodiments, stacked RNAi constructs comprise a nucleic acid sequence having at least two sense sequences from at least two different target pest genes linked to at least two complementary antisense sequences from said at least two different target pest genes so that said nucleic acid sequence encodes a double stranded RNA that inhibits expression of said at least two target pest genes. In some embodiments, the sense and antisense strands in the double stranded RNA are linked by a linker sequence. In some embodiments, the linker sequence is a Gus linker. In some embodiments, the stacked RNAi constructs comprise sense and antisense sequences from 2, 3, 4, 5, 6, 7, 8, 9, or 10 target RNA molecules. The sense and antisense sequences are preferably operably linked so that upon expression, a double stranded RNAi constructs is formed. Exemplary stacked constructs are depicted in FIG. 28. Sequences for stacked RNAi constructs are exemplified by SEQ ID NOs:75-77. The stacked RNAi constructs may comprise sense and antisense sequence to a variety of target RNA molecules. Exemplary target RNA molecules include, but are not limited to, those RNA molecules described in detail above. Suitable vectors are described in detail below.
II. Transgenic Plants
In some embodiments, the present invention provides transgenic plants that express the RNAi constructs and systems described above. It is contemplated that pests (e.g., nematodes) feeding on the transgenic plants ingest the dsRNA molecules, which in turn decrease the abundance of target RNA within the pest species. This results in decreased pest infestation and decreased plant damage.
A heterologous gene encoding an RNAi gene of the present invention, which includes variants of the RNAi gene, includes any suitable sequence that encodes a double stranded molecule specific for a pest target RNA molecule. Preferably, the heterologous gene is provided within an expression vector such that transformation with the vector results in expression of the double stranded RNA molecule; suitable vectors are described below. As described above, in some embodiments, the vectors further comprise a sequence encoding a sense or antisense RNA that is expressed in addition to the RNAi construct.
In yet other embodiments of the present invention, a transgenic plant comprises a heterologous gene encoding a RNAi gene of the present invention operably linked to an inducible promoter, and is grown either in the presence of the an inducing agent, or is grown and then exposed to an inducing agent. In still other embodiments of the present invention, a transgenic plant comprises a heterologous gene encoding an RNAi gene of the present invention and/or a sense or antisense sequence operably linked to a promoter which is either tissue specific or developmentally specific, and is grown to the point at which the tissue is developed or the developmental stage at which the developmentally-specific promoter is activated. Such promoters include seed and root specific promoters. In still other embodiments of the present invention, the transgenic plant comprises an RNAi gene of the present invention and/or a sense or antisense sequence operably linked to constitutive promoter. In further embodiments, the transgenic plants of the present invention express at least one double stranded RNA molecule at a level sufficient to reduce the proliferation of nematodes as compared to the proliferation of nematodes observed in a nontransgenic plant. In some embodiments, the transgenic plants of the invention coexpress at least one sense or antisense RNA molecule at a level sufficient to enhance the reduction the proliferation of a pest as compared to the proliferation of pests observed in a nontransgenic plant
1. Plants
The methods of the present invention are not limited to any particular plant. Indeed, a variety of plants are contemplated, including but not limited to soybean, wheat, oats, milo, sorghum, cotton, tomato, potato, tobacco, pepper, rice, corn, barley, Brassica, pine, and commercial cultivars can be transformed with heterologous genes. In cases where that is not possible, non-commercial cultivars of plants can be transformed, and the trait for expression of the RNAi gene of the present invention moved to commercial cultivars by breeding techniques well-known in the art.
2. Vectors
The methods of the present invention contemplate the use of at least one heterologous gene encoding an RNAi gene and/or an antisense or sense sequence. Heterologous genes intended for expression in plants are first assembled in expression cassettes comprising a promoter. Methods which are well known to those skilled in the art may be used to construct expression vectors containing a heterologous gene and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are widely described in the art (See e.g., Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.).
In general, these vectors comprise a nucleic acid sequence of the invention encoding an RNAi gene and/or an antisense or sense sequence (as described above) operably linked to a promoter and other regulatory sequences (e.g., enhancers, polyadenylation signals, etc.) required for expression in a plant. In some embodiments, the RNAi gene and/or an antisense or sense sequence are arranged in a bicistronic expression unit. In other embodiments, the RNAi gene and/or an antisense or sense sequence are operably linked to separate promoters.
Promoters include but are not limited to constitutive promoters, tissue-, organ-, and developmentally-specific promoters, and inducible promoters. Examples of promoters include but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine amino peptidase (“LAP,” Chao et al. (1999) Plant Physiol 120: 979-992); a chemically-inducible promoter from tobacco, Pathogenesis-Related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); a heat shock promoter (U.S. Pat. No. 5,187,267); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); and seed-specific promoters, such as those for seed storage proteins (e.g., phaseolin, napin, oleosin, and a promoter for soybean beta conglycin (Beachy et al. (1985) EMBO J. 4: 3047-3053)). In some preferred embodiments, the promoter is a phaseolin promoter. All references cited herein are incorporated in their entirety.
The expression cassettes may further comprise any sequences required for expression of mRNA. Such sequences include, but are not limited to transcription terminators, enhancers such as introns, viral sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
A variety of transcriptional terminators are available for use in expression of sequences using the promoters of the present invention. Transcriptional terminators are responsible for the termination of transcription beyond the transcript and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants include, but are not limited to, the CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminator (See e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet., 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).
In addition, in some embodiments, constructs for expression of the gene of interest include one or more of sequences found to enhance gene expression from within the transcriptional unit. These sequences can be used in conjunction with the nucleic acid sequence of interest to increase expression in plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells (Calais et al. (1987) Genes Develop. 1: 1183). Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
In some embodiments of the present invention, the construct for expression of the nucleic acid sequence of interest also includes a regulator such as a nuclear localization signal (Calderone et al. (1984) Cell 39:499; Lassoer et al. (1991) Plant Molecular Biology 17:229), a plant translational consensus sequence (Joshi (1987) Nucleic Acids Research 15:6643), an intron (Luehrsen and Walbot (1991) Mol. Gen. Genet. 225:81), and the like, operably linked to the nucleic acid sequence encoding the RNAi gene and/or an antisense or sense sequence.
In preparing a construct comprising a nucleic acid sequence encoding a RNAi gene of the present invention, various DNA fragments can be manipulated, so as to provide for the DNA sequences in the desired orientation (e.g., sense or antisense) orientation. For example, adapters or linkers can be employed to join the DNA fragments or other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, or the like is preferably employed, where insertions, deletions or substitutions (e.g., transitions and transversions) are involved.
Numerous transformation vectors are available for plant transformation. The selection of a 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 are preferred. Selection markers used routinely in transformation include the nptII gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra (1982) Gene 19: 259; Bevan et al. (1983) Nature 304:184), the bar gene which confers resistance to the herbicide phosphinothricin (White et al. (1990) Nucl Acids Res. 18:1062; Spencer et al. (1990) Theor. Appl. Genet. 79:625), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann (1984) Mol. Cell. Biol. 4:2929), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al. (1983) EMBO J., 2:1099).
In some preferred embodiments, the vector is adapted for use in an Agrobacterium mediated transfection process (See e.g., U.S. Pat. Nos. 5,981,839; 6,051,757; 5,981,840; 5,824,877; and 4,940,838; all of which are incorporated herein by reference). Construction of recombinant Ti and Ri plasmids in general follows methods typically used with the more common bacterial vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with the native plasmids and sometimes constructed from foreign sequences. These may include but are not limited to structural genes for antibiotic resistance as selection genes.
There are two systems of recombinant Ti and Ri plasmid vector systems now in use. The first system is called the “cointegrate” system. In this system, the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the pMLJ1 shuttle vector and the non-oncogenic Ti plasmid pGV3850. The second system is called the “binary” system in which two plasmids are used; the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector and the non-oncogenic Ti plasmid PAL4404. Some of these vectors are commercially available.
In other embodiments of the invention, the nucleic acid sequence of interest is targeted to a particular locus on the plant genome. Site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, for example, homologous recombination using Agrobacterium-derived sequences. Generally, plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described (U.S. Pat. No. 5,501,967). One of skill in the art knows that homologous recombination may be achieved using targeting vectors which contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known.
In some embodiments of the present invention the nucleic acid sequence of interest is introduced directly into a plant. One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is a modified version of the plasmid pCIB246, with a CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator (WO 93/07278).
3. Transformation Techniques
Once a nucleic acid sequence encoding an RNAi gene and/or an antisense or sense sequence of the present invention is operatively linked to an appropriate promoter(s) and inserted into a suitable vector for the particular transformation technique utilized (e.g., one of the vectors described above), the recombinant DNA described above can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant targeted for transformation. In some embodiments, the vector is maintained episomally. In other embodiments, the vector is integrated into the genome.
In some embodiments, the vector is introduced through ballistic particle acceleration using devices (e.g., available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.). (See e.g., U.S. Pat. No. 4,945,050; and McCabe et al. (1988) Biotechnology 6:923). See also, Weissinger et al. (1988) Annual Rev. Genet. 22:421; Sanford et al. (1987) Particulate Science and Technology, 5:27 (onion); Svab et al. (1990) Proc. Natl. Acad. Sci. USA, 87:8526 (tobacco chloroplast); Christou et al. (1988) Plant Physiol., 87:671 (soybean); McCabe et al. (1988) Bio/Technology 6:923 (soybean); Klein et al. (1988) Proc. Natl. Acad. Sci. USA, 85:4305 (maize); Klein et al. (1988) Bio/Technology, 6:559 (maize); Klein et al. (1988) Plant Physiol., 91:4404 (maize); Fromm et al. (1990) Bio/Technology, 8:833; and Gordon-Kamm et al. (1990) Plant Cell, 2:603 (maize); Koziel et al. (1993) Biotechnology, 11:194 (maize); Hill et al. (1995) Euphytica, 85:119 and Koziel et al. (1996) Annals of the New York Academy of Sciences 792:164; Shimamoto et al. (1989) Nature 338: 274 (rice); Christou et al. (1991) Biotechnology, 9:957 (rice); Datta et al. (1990) Bio/Technology 8:736 (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al. (1993) Biotechnology, 11: 1553 (wheat); Weeks et al. (1993) Plant Physiol., 102: 1077 (wheat); Wan et al. (1994) Plant Physiol. 104: 37 (barley); Jahne et al. (1994) Theor. Appl. Genet. 89:525 (barley); Knudsen and Muller (1991) Planta, 185:330 (barley); Umbeck et al. (1987) Bio/Technology 5: 263 (cotton); Casas et al. (1993) Proc. Natl. Acad. Sci. USA 90:11212 (sorghum); Somers et al. (1992) Bio/Technology 10:1589 (oat); Torbert et al. (1995) Plant Cell Reports, 14:635 (oat); Weeks et al. (1993) Plant Physiol., 102:1077 (wheat); Chang et al., WO 94/13822 (wheat) and Nehra et al. (1994) The Plant Journal, 5:285 (wheat).
In other embodiments, direct transformation in the plastid genome is used to introduce the vector into the plant cell (See e.g., U.S. Pat. Nos. 5,451,513; 5,545,817; 5,545,818; PCT application WO 95/16783). The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleic acid encoding the RNA sequences of interest into a suitable target tissue (e.g., using biolistics or protoplast transformation with calcium chloride or PEG). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al. (1990) PNAS, 87:8526; Staub and Maliga, (1992) Plant Cell, 4:39). The presence of cloning sites between these markers allowed creation of a plastid targeting vector introduction of foreign DNA molecules (Staub and Maliga (1993) EMBO J., 12:601). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab and Maliga (1993) PNAS, 90:913). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the present invention. Plants homoplasmic for plastid genomes containing the two nucleic acid sequences separated by a promoter of the present invention are obtained, and are preferentially capable of high expression of the RNAs encoded by the DNA molecule.
In other embodiments, vectors useful in the practice of the present invention are microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (Crossway (1985) Mol. Gen. Genet, 202:179). In still other embodiments, the vector is transferred into the plant cell by using polyethylene glycol (Krens et al. (1982) Nature, 296:72; Crossway et al. (1986) BioTechniques, 4:320); fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley et al. (1982) Proc. Natl. Acad. Sci., USA, 79:1859); protoplast transformation (EP 0 292 435); direct gene transfer (Paszkowski et al. (1984) EMBO J., 3:2717; Hayashimoto et al. (1990) Plant Physiol. 93:857). In still further embodiments, the vector may also be introduced into the plant cells by electroporation (Fromm, et al. (1985) Proc. Natl Acad. Sci. USA 82:5824; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
In addition to direct transformation, in some embodiments, the vectors comprising a nucleic acid sequence encoding a RNAi gene of the present invention are transferred using Agrobacterium-mediated transformation (Hinchee et al. (1988) Biotechnology, 6:915; Ishida et al. (1996) Nature Biotechnology 14:745). Agrobacterium is a representative genus of the gram-negative family Rhizobiaceae. Its species are responsible for plant tumors such as crown gall and hairy root disease. In the dedifferentiated tissue characteristic of the tumors, amino acid derivatives known as opines are produced and catabolized. The bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. Heterologous genetic sequences (e.g., nucleic acid sequences operatively linked to a promoter of the present invention), can be introduced into appropriate plant cells, by means of the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Schell (1987) Science, 237: 1176). Species which are susceptible infection by Agrobacterium may be transformed in vitro. Alternatively, plants may be transformed in vivo, such as by transformation of a whole plant by Agrobacteria infiltration of adult plants, as in a “floral dip” method (Bechtold N, Ellis J, Pelletier G (1993) Cr. Acad. Sci. III-Vie 316: 1194-1199).
4. Regeneration
After selecting for transformed plant material that can express the heterologous gene encoding a RNAi gene of the present invention, whole plants are regenerated. Plant regeneration from cultured protoplasts is described in Evans et al. (1983) Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co. New York); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. III (1986). It is known that many plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables, and monocots (e.g., the plants described above). Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted.
Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate and form mature plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. The reproducibility of regeneration depends on the control of these variables.
5. Generation of Transgenic Lines
Transgenic lines are established from transgenic plants by tissue culture propagation. The presence of nucleic acid sequences encoding a RNAi gene of the present invention (including mutants or variants thereof) may be transferred to related varieties by traditional plant breeding techniques.
These transgenic lines are then utilized for evaluation of oil production and other agronomic traits.
EXPERIMENTAL
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosures which follow, the following abbreviations apply: N (normal); M (molar); mM (millimolar); μM (micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg (micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C. (degrees Centigrade).
Example 1
Rapid in Planta Evaluation of Root Expressed Transgenes in Chimeric Soybean Plants
Production of stable transgenic plants is one factor that limits rapid evaluation of tissue specific transgene expression. To hasten the assessment of transgenes in planta, we evaluated the use of chimeric soybean seedlings expressing transgenic products in roots. Tap roots from four-day old seedlings (cultivars ‘Jack’ and KS4704) were excised and hairy roots were induced from hypocotyls via Agrobacterium rhizogenes-mediated transformation. Inoculated hypocotyls were screened on a MS-based medium containing either 200 mg/L kanamycin or 20 mg/L hygromycin. Beta-glucuronidase (GUS) activity assay indicated that highest GUS expression was observed in hypocotyls exposed to a 4-d pre-inoculation time, a neutral pH (7.0) for the co-cultivation medium. A 170-bp of the Fib-1 gene and 292-bp of the Y25C1A.5 gene fragments, both related to nematode reproduction and fitness, were cloned independently into pANDA35HK vector using a Gateway cloning strategy. The resulting RNAi constructs of the genes fragments were transformed into soybean using the chimeric hairy root system and evaluated for its effect on soybean cyst nematode (Heterodera glycines) fecundity. Confirmation of transformation was attained by polymerase chain reaction and Southern-blot analysis, and some potential for suppression of H. glycines reproduction was detected for the two constructs. This method takes on average four weeks to produce chimeric plants ready for transgene analysis.
Materials and Methods
Bacterial Strains and Vectors.
Agrobacterium rhizogenes strains R1000 and K599 were used in the transformation experiments. The binary vectors include pIG121Hm (Hiei et al. 1994), pBINmGFP5-ER (obtained from MRC laboratory of Molecular Biology, UK) and pANDA35HK (a kind gift from Ko Shimamoto, Japan). All the three vectors contain the nptII gene that confers resistance to kanamycin, and additionally pIG121Hm and pANDA35HK include hygromycin resistance gene driven by cauliflower mosaic virus (CaMV) 35S promoter. In addition, pIG121Hm contains gus gene with an intron driven by CaMV35S promoter; pBINmGFP5-ER harbors a modified version of GFP with enhanced fluorescence, also driven by CaMV35S promoter; pANDA35HK has a RNA interference cassette under the control of CaMV35S promoter (FIG. 1).
Cloning of Fib-1 and Y25C1A.5 (Y25) Genes from Soybean Cyst Nematode and RNA Interference Constructs.
The sequences of Fib-1 gene and Y25 gene from C. elegans were obtained from http://www.wormbase.org, and the corresponding clones were T01C3.7.1 and 2D377. The homologous Fib-1 (CB279515) and Y25 (CB824330) sequences from H. glycines were identified from datasets in http://www.nematode.net. Specific primers Fib-F (5′-CCGAAGACGGAAGAGACAAC-3′) (SEQ ID NO:23) and Fib-R (5′-ACCCGGGTAAATGTGCGTGTC-3′) (SEQ ID NO:24) for amplifying Fib-1 gene and Y25-F (5′-GCAGCCCGACAAGACAAT-3′) (SEQ ID NO:25) and Y25-R (5′-TGAGAGTTCCAATGACAAAT-3′) (SEQ ID NO:26) for amplifying Y25 gene were designed based on the gene's sequence. Using H. glycines genomic DNA as template, 170-bp and 292-bp fragments of Fib-1 and Y25 genes, respectively were obtained via polymerase chain reaction (PCR). PCR products were independently ligated into pGEM-T easy vector (Promega) and subsequently subcloned into pENTR4 vectors by EcoRI restriction sites. The pENTR4 vector carrying Fib-1 gene or the Y25 fragment was recombined with pANDA35HK vector using the LR clonase enzyme mix (Cat No. 11791-019, Invitrogen) to create the RNA interference expression vectors (FIG. 1). The Fib-1 or Y25 gene was in complementary orientation, separated by 930-bp GUS linker fragment, and driven by CaMV35S promoter. The RNAi construct was introduced into Agrobacterium rhizogenes strains R1000 and K599 bp the freezing-thaw method (Chen et al. 1994).
Hairy Roots Induction and Production of Chimeric Plants.
Seeds of soybean varieties Jack and KS4704 were obtained from the soybean breeding program at Kansas State University. Seeds were put into plates and surface sterilized using chlorine gas (3.3 mL concentrated 12 M HCl added into 100 mL 5.25% chlorine bleach) overnight. After that, the plates were wrapped with plastic bag for later use. Sterilized soybean seeds were cultured in the 12 MS liquid medium [1⁄2 MS (Murashige and Skoog 1962) basal nutrient salts subtracting 5 mM NH4− and 20 mM NO3−, B5 (Gamborg et al. 1968) vitamins, 1.90 g/L MES and 0.5% sucrose (pH 5.7)] at 25° C. for germination. To optimize the physiological conditions of explants, 3, 4, 5 and 6 days of pre-inoculation were compared on the 1⁄2 MS liquid medium. R1000 and K599 carrying different vectors were cultured on dishes with 50 mg/L kanamycin and/or 50 mg/L hygromycin B for 24 hr at 25° C. R1000 and K599 were scraped off the dishes and resolved in 0.5 mL 12 PS buffer (Vieweg et al. 2004) with 0.3-0.6 OD600nm. Soybean seedlings were inoculated by an injection for three times parallel to the vascular bundle of hypocotyl, similar to Vicia hirsuta by Quandt et al. (1993). After inoculation, soybean seedlings were co-cultivated in 1⁄2 MS liquid medium that was adjusted with different pH values (5.0, 5.7, 7.0).
After three days co-cultivation, the roots together with cotyledons were transferred onto selection medium (MS medium added with kanamycin or hygromycin). In the selection medium, different concentrations of kanamycin (50, 100, 200, 300 mg/L) and hygromycin (5, 10, 15, 20, 25 mg/L) were in contrast, respectively. About three or four weeks later, as transgenic roots were produced, the chimeric seedlings were transplanted into soil and grown at 22° C. (16 h light/8 h dark) (FIG. 2). It takes on average four weeks to finish the complete process. Roots were harvested for preparing DNA isolation. To examine different parameters on the transformation efficiency, GUS assay was done for the chimeric seedlings transformed with pIG121Hm. Experiments were performed with three replicates on 50 seedlings each per treatment. Means of percentage of roots covered with blue sectors were calculated.
PCR of Transgenic Roots.
Hairy root tissues (about 100 mg) were harvested and genomic DNA was extracted according to the methods of Delloporta et al. (1983). PCR analysis was done with Hybaid limited PCR express thermal cycler (Thermo Hybaid, Fraklin, Mass., USA). Specific primers oriR (5′-GCTTCCTCGCTCACTGACTC-3′) (SEQ ID NO:107) and oriF (5′-GGAGAAAGGCGGACAGGAAT-3′) (SEQ ID NO:27) were used to amplify the origene (expected 346 bp). Gus-F1 (5′-CACGTAAGTCCGCATCTTCA-3′) (SEQ ID NO:28) situated within Gus linker sequence was paired with Fib-R (5′-ACCCGGGTAAATGTGCGTGTC-3′) (SEQ ID NO:29) or Y25-R (5′-TGAGAGTTCCAATGACAAAT-3′) (SEQ ID NO:30) to identify the presence of Fib-1 or Y25 gene, respectively. The PCR amplification was carried out for 30 cycles. The initial denaturation reaction was done at 94° C. for 8 min. Each cycle was performed with denaturation (94° C., 1 min), annealing (56° C., 45 s) and extension (72° C., 50 s). PCR mixture consisted of 50-100 ng of plant DNA, 5 μl of 10 9 Taq buffer, 2.0 μl of 2.5 mM dNTP, 0.25 μl of Taq DNA polymerase (5 U μl-1, New England Biolabs Inc), 1 μl of 10 pmol each pair of primers in a final volume of 50 μl. The PCR products were separated by electrophoresis on 1.0% agarose gels in 1× TAE buffer and were observed by fluorescence with UV light after staining with ethidium bromide.
Visual Observation of GFP and Histochemical GUS Assay.
Imaging of GFP was performed using Leica MZIII fluorescence stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany). The microscope was equipped with a fluorescence module containing a Leica 106Z lamp housing 50 or 100 W high-pressure mercury vapor burners and two GFP filter sets (470/40 nm). The GUS activity of hairy roots transformed with pIG121 Hm was visualized with a staining solution containing 1 mM 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-gluc) as reported by Jefferson (1987). All the materials were incubated overnight at 37° C. in plastic plates.
Southern Blot Analysis.
Genomic DNAs of hairy roots transformed with R1000 containing pANDA35HK: Fib-1 were isolated using the method referred to Murray and Thompson (1980). DNA was digested with SacI, an enzyme which linearizes pANDA35HK: Fib-1 between attR and Nos terminator. Fifteen microgram DNA from roots of each sample was digested over-night, and gel electrophoresed together with positive control vector pANDA35HK: Fib-1 (50 pg) and genomic DNA from a nontransformed plant. The template for probe was obtained by digestion of pHygr in pUC119 using BamHI, and the probe was synthesized with a-32P [dCTP] labeled using DNA polymerase I large (Klenow) fragment (Fisher scientific, USA). Gel electrophoresis, DNA blotting and hybridization were following Sambrook et al. (1989).
Bioassay.
To explore suppression of nematode reproduction in transformed roots, chimeric plants expressing Fib-1 or Y25 RNAi were transplanted from tissue culture into D40 Deepots (Stuewe and Sons Inc., Corvallis, Oreg.) with 450 mL soil infested with 4,500 eggs 100 cm-3 of soil of a HG Type 2.7 population of H. glycines. Soybean plants inoculated with only R1000 containing no vectors were used as negative controls. Eleven negative controls were put in the same medium without kanamycin added for tissue culture as plants transformed with Fib-1. Transplanted soybeans were acclimated to lower humidity conditions and grown in the SCN-infested soil at 26° C. day/24° C. night with a 16-h photoperiod and a light intensity of 360 lmol photons m-2 s-1. After 5 weeks, roots from each plant were washed with a high-pressure water spray to dislodge cysts. Dislodged cysts were collected onto a 100-lm-pore sieve and enumerated under a dissecting microscope. Cysts from each sample were ruptured by mechanical grinding to release eggs and second-stage juveniles (J2) (Niblack et al. 1993). Eggs and J2 were collected onto a 25-lm-pore sieve and enumerated under a compound microscope at 409 magnification. The root mass from which cysts were collected was dried for 5 days at 60° C. and the dry weight was recorded.
Statistical Analysis.
All data were subjected to analysis of variance using the GLM procedure in SAS (SAS Institute, Cary, N.C.). Numbers of cysts and eggs g-1 root were log 10-transformed prior to analysis to reduce heterogeneity of variances. Means were separated using the PDIFF option in SAS.
Results.
Establishment of Soybean Hairy Roots Transgenic System.
In this study, we developed in planta chimeric system in soybean to evaluate root-expressed transgenes. Chimeric soybean plants were obtained by the induction of transgenic hairy roots on non-transformed shoots after inoculation with Agrobacterium rhizogenes. The majority of the root organogenesis was localized at the bacterial inoculation sites. There are 1-3 roots produced from one inoculation site on average. These roots exhibited the hairy root phenotype whereas roots induced at other locations on the hypocotyls did not possess the hairy root phenotype. Under antibiotic selection the lateral roots became brown and necrotic (FIG. 2) similar to hypocotyls transformed with A. rhizogenes R1000 lacking a binary plasmid (FIG. 2D, b).
Although the majority of roots were transgenic, not all the roots formed on the hypocotyls were transgenic as demonstrated by the GUS assay or GFP observation (FIGS. 2f, 3). A few adventitious roots forming from the injection sites were observed in most experiments using GFP and GUS as reporter genes and NPTII as the selectable marker. However, these escapes were not significant as 90±2.5% SE of the roots regenerated were transgenic. For the hairy roots transformed with A. rhizogenes R1000 containing pANDA35HK: Fib-1, different antibiotics concentrations were tried on the selection medium. Experiments indicated that 200 mg/L kanamycin, or 20 mg/L hygromycin could be used for the selection of soybean culture. Higher concentrations of kanamycin (more than 200 mg/L) or hygromycin (more than 20 mg/L) resulted in a significant inhibition of hairy root formation.
Analysis of Transformation Parameters.
Two A. rhizogenes strains R1000 and K599 were tested for the regeneration of transformed roots. Both strains were effective at inducing the formation of regenerated hairy roots however A. rhizogenes R1000 was more efficient than K599 in producing transgenic roots as indicated by the number of seedlings producing branched roots exhibiting GUS activity (Table 2). GUS expression was also used to compare transformation competency of two soybean cultivars (“Jack” and KS4704). Transformation efficiency was observed to be higher in “Jack” than KS4704. Preinoculation time in 1⁄2 MS medium was important for the expression of GUS in soybean seedlings (Table 3). Preinoculation time of 4 days gave the highest expression (23.3%), compared to 3 days (17.7%), 5 days (18.5%) and 6 days (16.7%). A neutral pH of 7.0 for the co-culture medium showed highest GUS expression as compared to a pH 5.0 or pH 5.7 (Table 4).







TABLE 2







Effect of A. rhizogenes strains and cultivars


on expression of GUS gene in soybean seedlings












No. of seedlings expressing





Gus/Total No. seedlings



A. rhizogenes
inoculated (%)











Strain
Jack
KS4704







R1000
25.4 ± 0.14 a
18.5 ± 0.14 b



K599
18.2 ± 0.14 b
14.8 ± 0.14 c










Transformation efficiency as reflected by Gus expression was assessed 3 weeks after inoculation with A. rhizogenes for both cultivars. Values are given as transformation frequency (%) and are the means±S.E. of three replications on 50 seedlings each. Different letters show significant differences among treatments according to procedure GLM-PDIFF option in SAS








TABLE 3







Effect of pre-inoculation time on the transformation


efficiency of soybean seedlings











Frequency of GUS



Pre-culture
positive seedlings/Total



time (days)
seedlings (%)







3
17.7 ± 0.18 b



4
23.3 ± 0.22 a



5
18.5 ± 0.21 b



6
16.7 ± 0.20 b










Soybean cultivar Jack seeds were put in the 1⁄2 MS medium for preinoculation. The co-culture medium is 1⁄2 MS medium with pH 7.0. A. rhizogenes strains R1000 containing pIG121Hm was used for inoculation. Means±SE followed with the same letters were not significantly different (P=0.05) according to SAS PDIFF option.








TABLE 4







Effect of pH values in the co-culture medium on the


transformation efficiency of soybean seedlings











Frequency of GUS positive



pH value
seedlings/Total seedlings (%)







5.0
15.2 ± 0.15 b



5.7
16.6 ± 0.12 b



7.0
22.2 ± 0.32 a










Soybean cultivar Jack seeds were put in the pre-inoculation medium (1⁄2 MS) for 4 days. A. rhizogenes strains R1000 containing pIG121Hm was used for inoculation. Means±SE followed with the same letters were not significantly different (P=0.05) under the same treatment according to SAS PDIFF option.

The activity of GFP was detected in most of transgenic hairy roots transformed with R1000 harboring binary vector pBINmGFP5-ER (FIG. 3), indicating GFP gene was inserted into hairy roots and had expressed. No GFP activity was observed in the roots transformed with R1000 containing no vectors or non-transgenic control roots. The efficiency of GFP expression in different parameters was similar to that of GUS (data not shown). We observed on average one to three non-transformed roots per seedling.
Molecular Analysis of Putative Roots.
PCR analysis was performed on genomic DNAs of hairy roots to demonstrate the T-DNA transfer. All the putative transgenic roots contained the bands of expected size (389 bp) for the Fib-1 gene, whereas the negative controls (non-transformed roots and roots transformed with only R1000) did not amplify the gene fragment (FIG. 4a). To detect the presence of A. rhizogenes contamination, the origene which situated outside the left and right borders in the pANDA35HK vector was selected for amplification from all the roots above. A 346 bp band corresponding to the ColE expected size was detected in root samples 1 and 2, suggesting that the two samples had A. rhizogenes contamination. All the roots tested were all positive for the amplification of Y25 gene and no Agrobacterium contamination was detected (FIG. 4b). Southern blot analysis was performed to further confirm the integration of T-DNA in soybean hairy roots. Results from the Southern blot correlated well with the results obtained through the PCR analysis (FIG. 5). Hybridization results indicated that three to seven bands were detected in the Fib-1 hairy roots samples from number 3 to 6 and Y25 samples from number 1 to 4. As expected, no signals were found in Fib-1 root samples number 1, 2 and plants transformed with only R1000. The Southern blot results were matched with PCR results, confirming that the PCR results of identifying transgenic roots are reliable.
Bioassay.
To examine the effect of transgenic soybean roots on H. glycines reproduction, a bioassay was performed by exposing hairy roots transformed with pANDA35HK: Fib-1 and pANDA35HK: Y25 to nematode-infested soil. The integration of Fib-1 and Y25 had been confirmed by PCR with genomic DNA of each independent transgenic plant (data not shown). Back-transformed means of log 10-transformed data of cysts g−1 root tissue and eggs g−1 root tissue were summarized in Table 5. Although the Fib-1 RNAi plants displayed a 25% reduction in cyst g−1 root tissue and a 37% reduction in eggs g−1 root tissue compared to negative plants, the effect was variable and not statistically significant. However, in contrast with control, the Y25 RNAi plants demonstrated a 71% reduction in the number of SCN cysts per gram root and a 85% reduction was found in the number of eggs per gram root in Y25 RNAi roots which was significant.







TABLE 5







Comparison of Heterodera glycines reproduction from the bioassay


with negative control and Fib-1, Y25 transgenic soybean roots













No. of
Cysts g−1
Eggs g−1



Treatment
Plants
root
root







Control rootsa
16
401 a
17,660 a



Fib-1 rootsb
10
301 a
11,000 a



Y25C1A.5c
12
115 b
 2,587 b







Values followed by a different letter were significantly different at P ≦ 0.05 according to procedure GLM-PDIFF option in SAS. Values represent back-transformed means of log10-transformed data



aSoybean plants inoculated with only R1000 as negative control



bSoybean plants inoculated with pANDA35HK: Fib-1



cSoybean plants inoculated with pANDA35HK: Y25






Discussion

Chimeric transgenic systems have been successfully used to study nodulation and other biological functions in roots of Medicago truncatula, Arachis hypogaea and Coffea Arabica (Akasaka et al. 1998; Alpizar et al. 2006; Boisson-Dernier et al. 2001). We developed an in planta chimeric soybean system using hairy roots expressing transgenes that can be used to study a variety of high throughput root related topics including resistance to root-parasitic nematodes. In our system, chimeric plants were obtained within four weeks by inducing hairy roots on non-transformed hypocotyls after inoculation with Agrobacterium rhizogenes. Many of the previous studies on nematode parasitism have used in vitro cultures of hairy roots which can have significant limitations for testing transgenes (Remeeus et al. 1998; Kifle et al. 1999; Cho et al. 2000; Hwang et al. 2000; Cai et al. 2003). For example, Cho et al. (2000) demonstrated a highly efficient in vitro hairy root system using kanamycin sulfate as a selection agent. Although Cho et al. (2000) used his system to culture soybean cyst nematodes, the roots could only be tested in vitro. Therefore, this method had the disadvantages that no plants could be regenerated and nematodes were required to be aseptic prior to inoculation onto the hairy roots.
A few reports have used similar composite hairy root systems to study root gene expression. Quandt et al. (1993) and Vieweg et al. (2004) introduced the hairy root transgenic system for Vicia hirsuta and other species; however, there was no selection for transgenic roots and therefore a number of non-transgenic roots were observed. Similarly, Collier et al. (2005) developed ex vitro composite plants free of selection. In GFP studies of composite soybean plants, Collier observed roughly 50% of the roots were not transgenic. The method described in Collier combined a modified transformation protocol of Quandt et al. (1993) with a screening process of transgenic roots in selection medium. Based on GUS assay, more than 90% of the total root population per chimeric plant was transgenic with the current protocol. The percentage of transgenic roots we obtained in the current methodology were significantly higher than the composite plants obtained by Collier et al. (2005), as our chimeric plants were screened in selection medium.
A GUS assay was used for monitoring the effect of different parameters on the transformation efficiency of our system. Although K599 is often used for hairy root transformation in legumes (Cho et al. 2000; Cho et al. 2004; Kereszt et al. 2007; Savka et al. 1990), more hairy roots and higher transgenic efficiency were achieved by using R1000 than K599 in our experiments. A pre-inoculation time of 4 days gave the highest expression compared to 3, 5 and 6 days, perhaps owing to the fact that the mitotic state of target tissue can affect transformation. This is in accordance with previous reports that plant cell conditions can be an important factor for transformation efficiency (Tepfer 1984; Kifle et al. 1999). A pH of 7.0 for the co-culture medium had the best effect on expression, suggesting a more favorable microenvironment for vir induction on the surface of hypocotyls of soybean, although Stachel et al. (1986) reported that vir induction dropped down at pH 6.0 and above. Santare'm et al. (1998) also utilized pH 7.0 in their soybean cotyledon culture medium and obtained good GUS expression.
The transgenic roots produced from individual chimeric plants were pooled for Southern analysis. One to three roots were observed to regenerate from a single inoculation site and PCR analysis of individual roots demonstrated that about 10% of these where non-transgenic roots. Based on these observations the multiple bands detected were most likely the result of multiple independent transformation events rather than one high-copy event. The multiple independent integrations may also give rise to variable expression levels of transgene, increasing variability in bioassays. Moreover, approximately 10% of the regenerated roots were not transgenic according to the GUS assay results. Considering this potential risk, we have put GFP expression cassette into the vector pANDA35HK allowing us to eliminate non-transformed roots more efficiently.
Nonetheless our primary goal with the hairy root assays is to rapidly identify candidate genes that have the greatest impact to suppress nematode growth and thus the greatest potential of being field-deployed. Even with the inherent variability of this technique, it is possible to identify those genes with the highest potential to accomplish our goal. The most promising genes identified from the composite plants will be used to stably transform soybean and then will be further validated for SCN suppression.
The production of chimeric plants with transgenic hairy roots can be used to test transgenes of nematode resistance. The H. glycines Fib-1 gene was chosen for the study because in five separate RNAi experiments of C. elegans Fib-1, either larva lethal or maternal sterile phenotypes were observed (Hanazawa et al. 2001; Kamath et al. 2003; Piano et al. 2002; Simmer et al. 2003; Sonnichsen et al. 2005). Although the H. glycines Fib-1 gene RNAi did not exhibit either of these phenotypes, the bioassay did demonstrate the potential for the suppression of H. glycines cyst development. Fib-1 encodes a fibrillin, a member of a small family of extracellular glycoproteins. Little is known for this family and it is possible that another family member complemented Fib-1's loss of function, resulting in the limited effect observed for this study. In addition, Y25 gene from C. elegans encodes a beta subunit of the coatomer (COPI) complex (Kirchhausen T, 2000; Nickel et al. 2002). Mass RNAi assays of C. elegans Y25 indicated that Y25 is required for fertility, adult viability, osmoregulation, and general health (Kamath et al. 2003; Nickel et al. 2002). RNAi of H. glycines Y25 gene in soybean significantly reduced the cysts and eggs of H. glycines confirming that the gene plays important roles for nematode health and fertility. These results suggest the potential for suppression of H. glycines reproduction exists for RNA interference of the Fib-1 gene, and Y25 RNAi is more promising for nematode control.
Expression data from transgenic roots transformed with RNAi constructions demonstrated siRNA species homologous with the target nematode genes were present in the root samples. We also tried to detect siRNA molecules in the aerial portions of the composite plants, but we were unable to confirm siRNA movement throughout the plant. This is in contrast to Limpens et al. (2004), who reported RNA silencing can spread to the non-transgenic shoots of chimeric plants. Discrepancies between these two studies could simply reflect a lower amount of siRNA produced in the current study that was insufficient to be detected in the aerial portions. Regardless, the use of chimeric plants was shown to be a fast and reliable in planta method for testing transgene efficacy against root pathogens.
In summary, we have established an Agrobacterium rhizogenes-mediated soybean chimeric transgenic system that can be used to rapidly evaluate root-expressed transgenes. Compared to the genetic transformation and recovery of stable transgenic soybean plants, the production of chimeric hairy root plants takes much less time (around one month), is efficient and less labor intensive. This genetic transformation method can be used as a tool for functional genomics in determining factors associated with root biology including root development, tolerances to abiotic stress, and resistance to biotic stresses.
Example 2
Host-Derived Suppression of Nematode Reproductive and Fitness Genes Decreases Fecundity of Heterodera Glycines Ichinohe
To control Heterodera glycines Ichinohe (soybean cyst nematode) in Glycine max (L.) Merr. (soybean), we evaluated the use of producing transgenic soybean seedlings expressing small interfering RNAs (siRNAs) against specific H. glycines genes. Gene fragments of three genes related to nematode reproduction or fitness (Cpn-1, Y25 and Prp-17) were PCR-amplified using specific primers and independently cloned into the pANDA35HK RNAi vector using a Gateway cloning strategy. Soybean roots were transformed with these constructions using a composite plant system. Confirmation of transformation was attained by PCR and Southern blot analysis. Transgene expression was detected using reverse transcription PCR (RT-PCR) and expression of siRNAs was confirmed in transgenic plants using northern blot analysis. Bioassays performed on transgenic composite plants expressing double-stranded RNA fragments of Cpn-1, Y25 and Prp-17 genes resulted in a 95, 81 and 79% reduction for eggs eroot, respectively. Furthermore, we demonstrated a significant reduction in transcript levels of the Y25 and Prp-17 genes of the nematodes feeding on the transgenic roots via real-time RT-PCR whereas the expression of nontarget genes were not affected. The results of this study demonstrate that over-expression of RNA interference constructs of nematode reproduction or fitness-related genes can effectively control H. glycines infection with levels of suppression comparable to conventional resistance.
Materials and Methods
Bacterial Strains, Vectors, and Soybean Seed.
Agrobacterium rhizogenes strain K599 was used in the transformation experiments. The binary vector pANDA35HK was a kind gift from Ko Shimamoto, Japan (Miki and Shimamoto 2004). pANDA35HK has a RNA interference cassette under the control of constitutive 35S cauliflower mosaic virus (CaMV35S) promoter. This vector contains the nptII gene that confers resistance to kanamycin, and a hygromycin phosphotransferase gene (HPT) driven by CaMV35S promoter. Soybean (Glycine max L.) seeds variety-KS4607 were obtained by a kind gift from Dr. William T. Schapaugh, Kansas State University. KS4607 is susceptible to H. glycines HG Type 7 population. Seeds were surface-sterilized using chlorine gas (3.3 mL concentrated HCl added into 100 mL chlorine bleach) in a closed container overnight. Seeds were then placed in the laminar flow hood for 30 min to remove residue chlorine gas and stored in sealed petri plates.
Cloning of Candidate Genes from H. glycines and RNA Interference Constructs.
The selection of target genes from H. glycines was based on following criteria: their sequences should be specific so that RNAi of these genes would not affect function of soybean genes. The homologous Cpn-1 (GU074018), Y25 (CB824330) and Prp-17 (AF113915) from H. glycines were discovered from datasets in nematode.net. Specific primers for amplifying these genes are listed in Table 6. Using nematode cDNAs as template, 408-, 294- and 289-bp fragments of Cpn-1, Y25 and Prp-17 genes were obtained through PCR method, separately. Cloning of the PCR products into pANDA35HK was performed as described in Li et al. (2010). Briefly, the PCR products were first ligated into pGEM-T Easy vector (Promega, Wis., USA) and then subcloned into pENTR4 vectors by EcoRI restriction sites. Subsequently, the pENTR4 vectors carrying three genes were independently recombined with pANDA35HK vectors using LR clonase (Invitrogen, Calif., USA). The resulting RNAi expression vectors (pANDA35HK:Cpn-1, pANDA35HK:Prp-17 and pANDA35HK: Y25) each contained the gene of interest (GOI) fragments in complementary orientation, separated by 930-bp GUS linker fragment, and driven by CaMV35S promoter. The RNAi constructs were introduced into A. rhizogenes strain K599 bp the freeze-thaw method (Chen et al. 1994).







TABLE 6 







Primer sequences used for amplifying target genes








Primer name
Sequences





Cpn-1 forward
5′-CACCAAGGCATCGATCAGGCTGTG-3′ (SEQ ID NO: 31)


 


Cpn-1 reverse
5′-CGCCTTCGATATCCTCCGCCA-3′ (SEQ ID NO: 32)


 


Y25 forward
5′-GCAGCCCGACAAGACAAT-3′ (SEQ ID NO: 33)


 


Y25 reverse
5′-TGAGAGTTCCAATGACAAAT-3′ (SEQ ID NO: 34)


 


Prp-17 forward
5′-CAATCGAATTGTCCTTTTCCA-3′ (SEQ ID NO: 35)


 


Prp-17 reverse
5′-CATTTTGATTACATTGTCCCATC-3′ (SEQ ID NO: 36)









Agrobacterium rhizogenes-Mediated Production of Hairy Roots and composite plants.
K599s carrying pANDA35HK:Cpn-1, pANDA35HK:Prp-17 and pANDA35HK: Y25 were cultured on LB medium with 50 mg/L kanamycin and 50 mg/L hygromycin B for 24 h at room temperature. K599s carrying different constructs were resolved in 0.5 mL 1⁄2 PS buffer with 0.3-0.6 OD600nm. Four-day-old soybean seedlings were inoculated by an injection three times parallel to the vascular bundle of hypocotyl, as described by Li et al. (2010). The formation and selection of composite plants were also accomplished as described by Li et al. (2010).
Polymerase Chain Reaction (PCR) of Transgenic Roots.
Hairy roots tissues from composite plants (about 20 mg) were harvested and genomic DNAs were extracted according to the method of Delloporta et al. (1983). PCR analysis was done with Hybaid limited PCR express thermal cycler (Perkin-Elmer Corporation, CA, USA). Specific primers oriR (5′-GCTTCCTCGCTCACTGACTC-3′) (SEQ ID NO:37) and oriF (5′-GGAGAAAGGCGGACAGGAAT-3′) (SEQ ID NO:38) were used to amplify the origin (ori) replication site (expected 346 bp). Gus-F1 (5′-CACGTAAGTCCGCATCTTCA-3′) (SEQ ID NO:39) and Gus-R1 (5′-GTATCAGTGTGCATGGCTGG-3′) (SEQ ID NO:40) situating within Gus linker was paired with Cpn-1 reverse or Y25 reverse or Prp-17 reverse to identify the presence of sense and antisense fragments of Cpn-1, Y25 and Prp-17 genes, respectively. The PCR amplification was carried out for 32 cycles. The initial denaturation reaction was done at 94° C. for 8 min. Each cycle was performed with denaturation (94° C., 1 min), annealing (56° C., 45 s) and extension (72° C., 50 s). PCR mixture consisted of 50-100 ng of plant DNA, 5 μl of 10× Taq buffer, 2.0 μl of 5 mM dNTP, 0.25 μl of Taq DNA polymerase (5 U μl-1, New England Biolabs Inc, MA, USA), 1 μl of 10 pmol each pair of primers in a final volume of 50 μl. The PCR products were separated by electrophoresis on 1.0% agarose gels in 19 TAE buffer and were observed by fluorescence with UV light after staining with ethidium bromide.
Southern Blot Analysis.
Genomic DNAs of hairy roots from composite plants transformed with K599 containing pANDA35HK:Cpn-1 or pANDA35HK: Y25 or pANDA35HK:Prp-17 or not were isolated using the method referred to Murray and Thompson (1980). DNAs were digested with SacI which only cuts once in the multiple cloning sites located between GOI and Nos terminator in the binary vector pANDA35HK. Fifteen microgram DNA from roots of each sample was digested overnight, and together with positive control pANDA35HK (50 pg) and 1 kb DNA ladder were loaded in each lane. The template for probe synthesis was obtained by digestion of HPT gene in PUC119 using BamHI, and the probe was synthesized with a-32P [dCTP] labeled using DNA polymerase I large (Klenow) fragment (Fisher scientific, PA, USA). Gel electrophoresis, DNA blotting and hybridization were done following Sambrook et al. (1989).
RT-PCR.
Total RNAs from different transgenic roots of composite plants together with roots transformed with only A. rhizogenes strain K599 were isolated using Trizol reagent (Invitrogen, Calif., USA). For RT-PCR, 1 μg of total RNA was reverse transcribed using AMV Reverse Transcriptase (Promega, Wis., USA) following manufacturer's instructions. RT-PCR was performed using gene-specific primers for ribosomal S21 gene (RibF, 5′-CTAAGATGCAGAACGAGGAAGG-3′ (SEQ ID NO:41), and RibR, 5′-GAGAGCAAAAGTGGAGAAATGG-3′ (SEQ ID NO:42)) and GUS linker (GusF, 5′-CATGAAGATGCGGACTTACG-3′(SEQ ID NO:43) and GusR, 5′-ATCCACGCCGTATTCGG-3′(SEQ ID NO:44)). The primers listed in Table 5 were used for the amplification of three different target genes, separately. PCR cycling comprised an initial step at 94° C. for 8 min, followed by 31 cycles at 94° C. for 1 min, 58° C. for 45 s, and 72° C. for 50 s.
Northern Blot Analysis.
Total RNAs from roots of different transgenic plants were isolated using Trizol reagent (Invitrogen, Calif., USA). Low molecular weight (LMW) RNAs were then separated from high molecular weight (HMW) RNAs using Qiagen RNA/DNA kit (Valencia, Calif., USA) according to protocols described by Steeves et al. (2006). The HMW RNAs were transferred onto HybondTM N+ nylon membranes (Amersham Pharmacia Biotech, NJ, USA) using capillary transferring method. The LMW RNAs (12 μg) were separated by electrophoresis on a polyacrylamide gel [including a 6% (w/v) stacking gel and a 15% (w/v) separating gel] containing 7 M urea and 0.59 TBE. RNAs from the gel were transferred onto HybondTM N+ nylon membranes using electric transferring device and the membranes were cross linked using UV cross linking Both HMW and LMW RNA membranes were hybridized overnight at 42° C. in Ultrasensitive Hybridization Buffer (Ambion, Tex., USA). The 408-, 294- and 289-bp fragments of Cpn-1, Y25 and Prp-17 gene fragments were selected as templates for synthesizing probes and probes were labeled radioactively by a random priming technique. After hybridization, membranes were washed in 29 SSC, 0.1% SDS and 0.29 SSC, 0.1% SDS and exposed in a phosphorimage cassette and visualized with the Storm 840 gel imaging system (GE Healthcare, NJ, USA).
Bioassay.
To explore the suppression of nematode reproduction in transformed roots, composite plants expressing the RNAi vectors of Cpn-1, Y25 and Prp-17 were transplanted from tissue culture into D40 Deepots (Stuewe and Sons, Inc., Corvallis, Oreg.) with 4,500 eggs 100 cm-3 of soil of a HG Type 7 (race 3) H. glycines population. The nematode population originated from a naturally infested commercial soybean field in Cherokee Co., KS, and was maintained on a susceptible soybean variety under greenhouse conditions. The universally susceptible soybean cultivar KS4607 was transformed as described above for use in bioassays. Soybean plants inoculated with K599 containing an empty pANDA35HK vector were used as negative controls. Negative controls were put in the same medium for tissue culture as plants transformed with Cpn-1, Y25 and Prp-17. The conditions for growth of composite plants and controls, and bioassay process were described by Steeves et al. (2006).
Data collected from each bioassay were subjected to analysis of variance using the GLM procedure in SAS (SAS Institute, NC, USA). Numbers of cysts g−1 root, eggs
g−1 root and eggs cyst−1 were log 10-transformed before analysis to reduce heterogeneity of variances. Least squares means (LSMEANS) were employed to compare the reproductive potential of H. glycines on transgenic and control plants.
Real-Time RT-PCR of Candidate Genes from Nematodes Feeding on Transgenic Roots.
Composite plants expressing Y25 and Prp-17 RNAi constructs independently were applied with HG Type 7 populations of H. glycines cysts. 5 weeks post-infection, H. glycines cysts were collected from composite plants. Total RNA was isolated from collected nematodes using the protocol described by http://cmgm.stanford.edu/-kimlab/germline/experimental_procedures.htm. Nematodes feeding on composite plants inoculated with K599 containing no binary vectors were used as negative controls.
Real-time RT-PCR was performed to analyze the transcript abundance of nematode genes. Gene-specific primers external to the RNAi target regions were designed as shown in Table 7. RNA was reverse transcribed into cDNA by using AMV kit (Promega, Wis., USA) according to the manufacturer's protocol. Endogenous mRNA levels were measured by qRT-PCR based on the SYBR Green detection system using the ABI Prism 7000 Sequence Detection system and the SYBR Green PCR Kit (Applied Biosystems, CA, USA), according to the manufacturer's instructions. The PCRs were performed in an iCycler (Bio-Rad, Hercules, Calif., USA) using the following program: 95° C. for 5 min, and 40 cycles of 95° C. for 30, 56° C. for 15 s and 72° C. for 30 s.
Following PCR amplification, dissociation curves application (Applied Biosystems CA, USA) to ensure that only a single PCR product was amplified. The dissociation program was 95° C. for 1 min, 55° C. for 1 min followed by a slow ramp from 55° C. to 95° C. The experiment included three biological replications for each gene, and each biological replication was assayed by using three technical replications. H. glycines beta-actin (Genbank accession No. AF318603), a constitutively expressed gene, was used as internal control to normalize gene expression levels. The expression levels of target gene compared to beta-actin control was determined by using 2-DDCT method as described by Livak and Schmittgen (2001). Using the fold change values for the three biological replications within each experiment, t tests were performed for each RNAi construct at each time point.







TABLE 7 







List of primers used in real-time RT-PCR analysis










Name
Sequence
Gene name
Product size





ACT-F
CGGCATGGGACAGA
H. glycines-actin
63



AGGA (SEQ ID NO: 45)




 


ACT-R
CGTCAGAATACCACG





CTTGGA(SEQ ID NO: 46)




 


Prp-F real
CCTACGATACGCTTGG
Prp-17 gene
152



CTATG (SEQ ID NO: 47)




 


Prp-R real
ATTTATCACTCTCTTTCG





CTTCTG (SEQ ID NO: 48)




 


Y25-F real
CGATCACTGAGGACG
Y25 gene
106



ACTTGG (SEQ ID NO: 49)




 


Y25-R real
AACGAAGCACGGCAC





TCTC (SEQ ID NO: 50)









Results

Targeted H. glycines Genes.
Three H. glycines genes, Cpn-1, Y25 and Prp-17, were selected for this study. The genes Y25 and Prp-17 were chosen based on reports of lethal or sterile phenotypes resulting from silencing the homologous C. elegans genes. The H. glycines Y25 gene (CB824330) has 69% similarity with the homologous C. elegans Y25 gene (NM 062040) and codes for a beta subunit of the coatomer (COPI) complex (Kirchhausen 2000; Nickel et al. 2002). The H. glycines Prp-17 gene (AF113915) encodes a pre-mRNA splicing factor, which shares 72% similarity with the C. elegans Prp-17 gene (NM 060450) for the whole ORF (open reading frame). The unique 30 partial H. glycines Prp-17 gene fragment which is different from plant premRNA splicing factor was used for our RNAi constructs. The H. glycines Cpn-1 gene (GU074018) was inadvertently amplified, as we attempted to amplify the H. glycines Eft-1 gene (AW871659) with expected silencing embryonic lethal phenotype. However, the sequence we amplified had  ̃60% identity with a putative 60 KD chaperonin gene from Heterodera schachtii (CD750139) and 83% identity with a Bordetella holmesii strain ATCC 51541 Cpn60 gene (AY123715). There is no information currently available on the silencing effects of this gene.
Composite Plants Induction.
Agrobacterium rhizogenes K599s carrying either pANDA35HK: Cpn-1, pANDA35HK: Y25 or pANDA35HK: Prp-17 binary vector induced composite plants to produce numerous roots growing from the wound sites on the induction medium supplemented with 200 mg/L kanamycin sulfate. The majority of the roots resembled the hairy root phenotype typical of A. rhizogenes induction. Plants inoculated with K599 containing the empty vector pANDA35HK (i.e., no target gene) produced hairy root phenotype as above. Control plants inoculated with K599 containing no binary vectors typically had very poor root development on the selection medium.
PCR Analysis and Southern Blot Hybridization.
PCR was performed on genomic DNAs of roots from separate composite plants to initially screen for plant transformation. Sampled plants were tested for presence of both sense and antisense fragments of the H. glycines genes and for the absence of the on replication site located outside of the T-DNA region of pANDA35HK. As shown in FIG. 7, both the sense and antisense fragments of the H. glycines genes were amplified from all putative transgenic roots that grew rapidly under selection. No amplification of the on replication site was observed from all the tested root samples, suggesting that all the samples were free from A. rhizogenes contamination. For the plants transformed with RNAi constructs of Cpn-1, Y25 and Prp-17 genes, C1-C4, Y1-Y4 and P1-P4 were positive for the amplification of Cpn-1, Y25, and Prp-17 genes, respectively. Southern blot analysis confirmed the integration of T-DNA in soybean composite plants. Consistent with above PCR results, hybridization results indicated that integration was detected in the root samples C1-C4, Y1-Y4 and P1-P4, but not in root samples transformed with only K599 (no binary vector) (FIG. 8).
Northern Blot Analysis of Gene of Interests and RT-PCR Analysis.
To monitor whether RNAi fragments of different GOIs were transcribed, HMW RNAs from roots of 20 independent Y25 RNAi composite plants were isolated and northern blot analysis was performed using probes labeled with 294-bp Y25 gene fragment used for pANDA35HK: Y25 construct. The integral Y25 fragment was not detected for the hybridization, suggesting that the transcripts of Y25 RNAi were potentially processed (data not shown).
RT-PCR was done with primers GusF and GusR towards Gus linker sequence to confirm transcription of the RNAi cassette of Cpn-1, Y25 and Prp-17 genes. Roots of four independent composite plants from each of three constructs along with control roots from plants infected with K599 only (no binary vector) were analyzed. Strong bands were detected in each of putative samples corresponding to the Gus linker transcripts. The appropriate GOIs used for the RNAi constructs were detected in each sample although the intensity of this band was considerably less than that detected for the Gus linker transcripts (FIG. 9).
siRNAs Detection in Transformed Roots.
LMW RNAs were differentially isolated from roots of independent Cpn-1, Y25 and Prp-17 transgenic plants showing positive for RT-PCR analysis of Gus linker, and a northern blot analysis was performed to explore whether RNAi fragments of GOIs were processed into siRNAs. Radioactive-labeled probes were synthesized using 408 bp Cpn-1, 294 bp Y25 and 289 bp Prp-17 sense fragments, respectively. Consistent with the results of RT-PCR on Gus linker, FIG. 10 demonstrates the transcripts were processed into siRNAs ( ̃21-23 nt in length) in transgenic roots transformed with pANDA35HK vectors harboring each GOI.
Bioassays.
Bioassays were performed by inoculating H. glycines on composite plants independently transformed with pANDA35HK:Cpn-1, pANDA35HK: Y25 and pANDA35HK: Prp-17 to detect suppression of H. glycines reproduction on transgenic soybean plants. Transformations of the composite plants were verified by PCR analysis (data not shown). Visual inspections of the roots at the completion of the bioassay indicated a marked difference in cyst numbers observed between negative control and the transgenic roots (FIG. 11). For composite plants transformed with Cpn-1, Y25 and Prp-17 RNAi constructs, mean values of cysts g−1 root tissue were 552, 1,286 and 1,574, mean values of eggs g−1 root tissue were 16,398, 56,104 and 61,432, and mean values of eggs cyst-1 were 55, 48, and 41, respectively (Table 8). In contrast, the control plants transformed with the empty pANDA35HK vector had mean values of 3,364 cysts g−1 root tissue, 298,813 eggs g−1 root tissue and 92 eggs cyst−1. This translates to an 84, 62 and 53% reduction for cysts g−1 root, a 95, 81 and 79% reduction for eggs g root, and a 40, 48 and 55% suppression for eggs cyst−1 for composite plants transformed with Cpn-1, Y25 and Prp-17 RNAi constructs, respectively. Transgenic plants with all three target genes were significantly different (P B 0.05) from negative controls in numbers of cysts g−1 root tissue, while Cpn-1 and Y25 RNAi transgenic plants were significantly different from negative controls for numbers of eggs g−1 root tissue. Y25 and Prp-17 RNAi transgenic plants displayed statistically significant reductions in eggs cyst−1.







TABLE 8







Comparison of H. glycines reproduction on composite plants














No of
Cysts g−1
Eggs g−1
Eggs



Treatment
Plants
root
root
cyst−1







Cpn-1 rootsa
32
 552*
16,398*
55 



Y25 rootsb
31
1,286*
56,104*
48*



Prp-17 Rootsc
26
1,574*
61,432 
41*



Control rootsd
35
3,364 
298,813 
92 







Values represent back-transformed means of log10-transformed data



*Significant reduction (P ≦ 0.05)



aSoybean plants inoculated with K599 containing pANDA35HK: Cpn-1



bSoybean plants inoculated with K599 containing pANDA35HK: Y25



cSoybean plants inoculated with K599 containing pANDA35HK: Prp-17



dSoybean plants inoculated with K599 containing pANDA35HK as negative controls






Down-Regulation of Candidate Genes in Nematodes Feeding on Composite Plants Transformed with Different RNAi Constructs.
Total RNAs extracted from nematodes feeding on roots of soybean composite plants at 5 weeks post-inoculation were used in real-time RT-PCR analysis to analyze the transcript abundance of target genes in each RNA sample. Real-time RT-PCR was not performed for nematodes feeding on plants transformed with Cpn-1 RNAi construct as the quality and quantity of isolated RNA was insufficient to perform the analysis.
A 2.41-fold down-regulation of the Y25 transcripts was observed in nematodes feeding on Y25 RNAi transgenic soybean plants, which is equivalent to a 59% reduction in Y25 gene expression compared to the nematodes feeding on composite plants transformed with K599 containing no binary vectors. The expression of Prp-17 as a non-target gene was measured in nematodes feeding on Y25 RNAi transgenic soybean plants and soybean plants transformed with K599 containing no binary vectors. The transcript levels of Prp-17 did not change in these nematodes (FIG. 11).
For nematodes feeding on Prp-17 RNAi transgenic soybean plants, a 4.76-fold down-regulation of the Prp-17 transcripts was observed when compared to the nematodes feeding on composite plants transformed with K599 containing no binary vectors. This value is equivalent to a 79% reduction in Prp-17 gene expression. The Y25 transcript levels were also measured in these two nematode samples as a non-target control and were not altered significantly (FIG. 11). These results confirmed that all of the targeted H. glycines genes displayed specific down-regulation for their mRNA transcripts in the nematodes collected from composite plants transformed with the corresponding RNAi constructs at 5 weeks post-inoculation.
Discussion
The transformation of soybean roots with transcripts of three different H. glycines genes (Cpn-1, Y25 and Prp-17) designed to induce an RNA interference effect in the nematodes significantly suppressed H. glycines reproduction and development. Additionally, the nematodes feeding on Y25 and Prp-17 RNAi transgenic soybean roots displayed significant suppression of Y25 and Prp-17 mRNA transcripts, respectively, as quantified by real-time RTPCR analysis. Target genes were selected using the strategy of Alkharouf et al. (2007), who compared an EST database of H. glycines with the C. elegans genome and found that 1,500 of the more than 8,000 conserved genes had lethal phenotypes in C. elegans. Our results confirmed the validity of this approach for finding gene targets for transgenic resistance to H. glycines in soybean. Klink et al. (2009c) recently refined this strategy by employing an Affymetrix soybean Genechip and microarray analyses to find a subset of those H. glycines genes with similarity to C. elegans genes having mutant lethal phenotypes that were induced during parasitism by the nematodes.
The Y25 gene from C. elegans encodes a beta subunit of the coatomer (COPI) complex (Kirchhausen 2000; Nickel et al. 2002). RNAi assays of C. elegans Y25 indicated that expression of this gene is required for fertility, adult viability, osmoregulation, and general health (Nickel et al. 2002; Kamath et al. 2003). In an earlier study (Li et al. 2010), we demonstrated that chimeric plants of a partially resistant soybean cultivar ‘Jack’ expressing RNAi construct of the Y25 gene had significantly fewer numbers of cysts g root (71% reduction) and eggs g-1 root (85% reduction) compared to non-transgenic plants after transplantation into soil infested with H. glycines HG Type 2.7. In the current study, composite plants of the susceptible soybean cultivar ‘KS4607’ expressing Y25 RNAi displayed similar effects (62 and 81% reductions in numbers of cysts and eggs g−1 root, respectively), when transplanted into soil infested with H. glycines HG type 7. These results corroborate each other, indicating that Y25 RNAi-transformed roots provide consistent levels of resistance against H. glycines. 
The Prp-17 gene product is required for the second step of pre-mRNA splicing, and C. elegans transformed with RNAi of the C. elegans Prp-17 gene displayed a phenotype of embryonic lethal (Fraser et al. 2000; Maeda et al. 2001; Simmer et al. 2003; Sonnichsen et al. 2005). In our study, RNAi of the H. glycines Prp-17 gene resulted in significant reductions in cysts g−1 root tissue (53% reduction) and eggs cyst−1 (79% reduction) in the transgenic soybean plants inoculated with H. glycines, indicating that this gene also plays an important role in H. glycines fitness or reproduction, which supported the important role of Prp-17 gene in C. elegans. As reported in Alkharouf et al. (2007), greater than 95% lethality of pi-J2 was observed for H. glycines soaked in dsRNA solution of a ribosomal gene Hg-rps-23. In addition, inverted repeat constructs of three genes Hg-rps-3a, Hg-rps-4 and Hg-spk-1 involved in different aspects of mRNA metabolism were associated with 81-88% reductions in numbers of H. glycines cysts on transgenic soybean roots (Klink et al. 2009c). Yadav et al. (2006) reported that double-stranded RNA fragments of two genes encoding an integrase and a splicing factor induced RNAi in the plant-parasitic nematode M. incognita and protected tobacco against infection. All of these genes are involved in mRNA metabolism, suggesting that genes involved in mRNA metabolism may be particularly sensitive to RNAi and should, therefore, make good target genes for H. glycines control.
The cloning of H. glycines Cpn-1 fragment was serendipitous. We attempted to amplify H. glycines Eft-1 gene using two specific primers, although the BLAST result of the PCR products we obtained showed the sequence we amplified had no identity with Eft-1 gene, but had a 60% identity with a putative 60 kD chaperonin gene from Heterodera schachtii (CD750139) and an 83% identity with Bordetella holmesii strain ATCC 51541 Cpn60 gene (AY123715). Although no published information on a silencing phenotype was available for this gene, the transgenic composite plants transformed with H. glycines Cpn-1 RNAi construct in our experiments displayed robust resistance against H. glycines. The 84-95% reductions in numbers of cysts and eggs g-1 root on Cpn-1 RNAi-transformed plants are comparable to the level of resistance in commercialized soybean cultivars, which are categorized as moderately resistant or resistant based on reductions ≧70 and 90%, respectively, in numbers of females that develop on the tested cultivars compared to a standard susceptible cultivar (Schmitt and Shannon 1992).
We have screened RNAi constructs of more than ten different H. glycines genes, including the three genes featured in this report, through composite plant bioassay. Plants transformed with the remaining RNAi constructs showed no significant reductions for H. glycines development or reproduction. The target genes were chosen based on the embryonic lethal or adult lethal phenotype of silencing of corresponding C. elegans genes. The nucleic acid sequence similarity between H. glycines and C. elegans of these genes was around 50-80%. It is possible that these genes were not orthologs of the C. elegans genes and therefore do not hold the same functions in H. glycines. Alternatively, there may be functional redundancies within H. glycines genome making these target genes non-essential.
Sindhu et al. (2009) reported that RNAi of four parasitism genes cloned from H. glycines led to reduction of developing females of H. schachtii ranging from 23 to 64%. As stated in the article of Sindhu et al. (2009), at least 90% nucleotide identity was found between those two species H. glycines and H. schachtii, though only a couple of mis-matching nucleotides may reduce the RNAi effects. RNAi is a highly specific method of gene silencing. For example, Tuschl and colleagues demonstrated that even a single base mismatch between a siRNA and its mRNA target prevented gene silencing (Elbashir et al. 2001). The target genes selected for our study (Cpn-1, Y25 and Prp-17) were more effective in controlling H. glycines than the parasitism genes reported in Sindhu et al. (2009), likely because we made specific RNAi constructs using H. glycines genes.
Improving soybean resistance against H. glycines by genetic engineering requires effective genes and soybean transformation. The traditional stable soybean transformation process is very time consuming and labor intensive. Therefore, there is an urgent need for the development of more rapid methods to identify the function and suitability of target genes. Several options available for this purpose have serious limitations. Feeding C. elegans with bacteria expressing H. glycines Cpn-1, Y25 or Prp-17 derived dsRNA fragments would not be likely to produce useful results as H. glycines Y25 or Prp-17 genes only share 69 and 72% similarity with corresponding C. elegans genes, respectively, and H. glycines Cpn-1 gene has even lower homology with C. elegans genes. As demonstrated by Bakhetia et al. (2005), no phenotype was observed when C. elegans was fed with bacteria expressing M. incognita dual oxidase gene, which had 65% nucleotide identity with the corresponding C. elegans gene. Although transformation of plant-parasitic nematodes represents another approach, attempts to do so have been hindered by the obligate nature of parasitism and low recovery rates.
Composite plant transgenic systems consist of a wildtype shoot with transgenic roots induced by Agrobacterium rhizogenes (Hansen et al. 1989). The composite plant systems can produce transgenic material much faster than methods resulting in stable transgenic plants (Collier et al. 2005; Klink et al. 2009c). Recently, Li et al. (2010) demonstrated the value of composite plant transformation for evaluating root-expressed transgenes. In this study, composite soybean plants expressing RNAi constructs were proven to be effective for the rapid identification of target genes, although some variation in results may occur due to the fact that a limited number of roots induced from inoculation sites may escape transformation or because roots derived from one soybean seedling may represent multiple transgenic events. Our goal is to identify the most effective H. glycines genes for targeting H. glycines reproduction or parasite fitness using this transgenic system. After confirming the effectiveness of RNAi constructs of target genes, such as Cpn-1, Y25 and Prp-17, we are producing stable transgenic plants using particle bombardment mediated method. To date, we have obtained several T1 transgenic lines, and homozygous lines of each will be subjected to H. glycines bioassay testing to confirm the results of our composite plant bioassays.
In conclusion, our results provided robust support for the hypothesis that host-delivered dsRNA molecules can be an effective strategy for H. glycines gene suppression and H. glycines control, and the application of RNAi constructs associated with a composite plant transgenic system provides rapid identification of genes involved in nematode resistance. This strategy also could lead to a better understanding of host-parasite interactions and to the control of other pathogens and insect pests.
Example 3
Results of Hairy Root Bioassay for Different Target Sequences
RNAi sequences from the genes listed in Tables 9a and 9b were cloned into the destination vector pANDA35HK by Gateway cloning method as described in detail above. Agrobacterium rhizogenes K599 containing all these RNAi constructs were used for hairy roots transformation, and the composite plants with hairy roots were used for bioassay. RNAi constructs were used for stable transformation mediated by gene gun.







TABLE 9A







Bioassay results of RNAi constructs containing different SCN target genes














Targeted
Genbank



# of Stable



nematode
Accession
RNAi
Hairy
Bioassay Eggs
transgenic


Name
life stage
No.
constructs
roots
reduction
events
















Cpn-1
—
GU074018
Completed
Completed
85%
3


Prp-17
—
AF113915
Completed
Completed
79%
3


Y25
J3
CB824330
Completed
Completed
65%
1


Rnr-1
J4
CD749124
Completed
Completed
54%
2


Arx-3
J4
CD748919
Completed
Completed
46%
—


Fib-1
J3
CB279515
Completed
Completed
37%
3


Asb-1
J2
BI396626
Completed
Completed
33%
1


Vap-2
—
AY033601
Completed
Completed
31%
—


F-55
Egg
CA940536
Completed
Completed
0%
—


Rpt-1
Virgin
CB376265
Completed
Completed
0%
—



female


Eat-3
Virgin
CB375718
Completed
Completed
0%
—



female
















TABLE 9b







Bioassay results of RNAi constructs


containing different SCN target genes














Bioassay
Bioassay



Target
Genbank
Eggs
Cyst


Name
stage
Accession No.
reduction
reduction





cdk-1 (J12)
Juvenile
CB935405
71%
65%


fzy-1 (J13)
Juvenile
CB377841
78%
70%


tba-2 (J14)
Juvenile
CB935405
63%
49%


arx-1 (J15)
Juvenile
CB299515
84%
68%


tbb-2 (J17)
Juvenile
CA939744
61%
50%


unc-26 (J20)
Juvenile
CD748096
56%
47%


prp-4 (J21)
Juvenile
CB935298
60%
54%


pfn-1 (J23)
Juvenile
CB374607
74%
61%


vbh-1 (J7)
Juvenile
CB279252
*
*





* bioassay not performed






Example 4
Testing of Stacked Constructs
Three RNAi stacking constructs have been made: Cpn-Rnr-1, Cpn-Y25, and Y25-Prp-17. Hairy roots bioassay has been performed for some of the constructs. Bioassay results showed that composite plants derived from stacking constructs Y25-Prp17 RNAi showed more reductions for cysts/g root and eggs/g root than plants derived from Y25 RNAi construct, compared with non-transgenic roots. The reductions for cyst/g root are 61% and 34% for Y25-Prp 17 and Y25 separately, and eggs/g root reductions are 75% and 43% for them, separately.
Example 5
Enhancement of siRNA Amount by Over-Expressing Nematode Genes in Stable Transgenic Soybean Plants Transformed with RNAi Constructs of SCN Genes
Stable transgenic plants transformed with RNAi constructs of Y25 (a.k.a. Y25C1A.5). have been obtained and advanced to the T2 generation. Small RNA sequencing using an Illumina Genetic Analyzer showed that soybean dicer-like (DCL) ribonuclease cleaved expressing Y25 dsRNAs formed by nematode inverted repeats into 20 ̃25 nt siRNAs. To enhance the levels of siRNA species of the Y25 target gene we cloned a fragment of the Y25 gene (FIG. 58), which is longer than the fragment used for RNAi construct and expressed the gene fragment with a G. max ubiquitin promoter, into a pPTN289 (See FIG. 57) vector containing bar gene which confers bialaphos resistance. We germinated seeds from homozygous T2 soybean lines transformed with Y25 RNAi construct and transformed this line with the above vector. By enhancing the target signal within the soybean plant we should expect to see an enhanced level of siRNA species within the plant and greater delivery of these molecules to the feeding nematodes.
Example 6
Sequences Conservation of Target Genes within and Among Plant Parasitic Species
Currently, resistance to SCN in soybean are limited to certain populations, that is not all genetic sources of resistance are equally effective to all SCN populations. Additionally current genetic resistances are species specific. For example, the genetic resistance for SCN does not give resistance to root knot nematodes. By using RNAi technology targeting gene sequences, which are conserved among different SCN populations and other nematode parasites, we should be able to provide broader resistance against nematodes. We amplified two target genes Y25 (294 bp) and Rnr-1 (251 bp) from SCN race 1, 3, 4 and Radophlus similis, root lesion nematode Pratylenchus neglectus, and the sequencing results showed 99% similarity for the two genes among all these nematode populations (FIGS. 59A and 59B). We expect the sequences from Meloidogyne spp. to be equally similar. We also would expect that these sequences will provide broad resistance to all nematodes that share sequence homology and if these traits are transferred to other plants such as wheat, tomato pineapple and various other plants that are affected by parasitic nematodes, resistance could be achieved as demonstrated with transgenic soybeans.
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All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, plant biology, biochemistry, or related fields are intended to be within the scope of the following claims.

## Claims

1. A transgenic soybean plant comprising an exogenous nucleic acid sequence forming a hairpin structure when expressed, said exogenous nucleic acid sequence comprising a sense sequence of from about 18 to 25 nucleotides in length from a Heterodera glycines gene selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1 linked to its complementary antisense sequence and encoding a double stranded RNA that inhibits expression of said Heterodera glycines gene when expressed.

2. A system for controlling a plant pest comprising:
at least one vector comprising an exogenous nucleic acid sequence forming a hairpin structure when expressed, said exogenous nucleic acid sequence comprising a sense sequence of from about 18 to 25 nucleotides in length from a Heterodera glycines gene selected from the group consisting of Cpn-1, Prp-17, Y25, Rnr-1 linked to its complementary antisense sequence and encoding a double stranded RNA that inhibits expression of said Heterodera glycines gene.

3. A method of reducing damage due to pests comprising:
cultivating a transgenic plant comprising the system of claim 2.