# Patent

## Title
Plant root-specific nematode resistance

## Abstract

The invention provides expression vectors comprising root-specific promoters in operative association with polynucleotides that are down-regulated in syncytia of nematode-infected plants, for use in methods of producing transgenic plants with increased resistance to nematode infestation. The invention also provides nematode-resistant transgenic plants and seeds comprising such expression vectors.

## Background
This application is a national stage application under 35 U.S.C. §371 of PCT/EP2009/066062, filed Nov. 30, 2009, which claims the priority benefit of U.S. provisional patent application Ser. No. 61/201,471, filed Dec. 11, 2008. The entire contents of each of the above-identified applications are incorporated herein by reference.


FIELD OF THE INVENTION
The invention relates to enhancement of agricultural productivity through use of nematode-resistant transgenic plants and seeds, and methods of making such plants and seeds.
BACKGROUND OF THE INVENTION
Nematodes are microscopic roundworms that feed on the roots, leaves and stems of more than 2,000 row crops, vegetables, fruits, and ornamental plants, causing an estimated $100 billion crop loss worldwide. A variety of parasitic nematode species infect crop plants, including root-knot nematodes (RKN), cyst- and lesion-forming nematodes. Root-knot nematodes, which are characterized by causing root gall formation at feeding sites, have a relatively broad host range and are therefore parasitic on a large number of crop species. The cyst- and lesion-forming nematode species have a more limited host range, but still cause considerable losses in susceptible crops.
Parasitic nematodes are present throughout the United States, with the greatest concentrations occurring in the warm, humid regions of the South and West and in sandy soils. Soybean cyst nematode (Heterodera glycines), the most serious pest of soybean plants, was first discovered in the United States in North Carolina in 1954. Some areas are so heavily infested by soybean cyst nematode (SCN) that soybean production is no longer economically possible without control measures. Although soybean is the major economic crop attacked by SCN, SCN parasitizes some fifty hosts in total, including field crops, vegetables, ornamentals, and weeds.
Signs of nematode damage include stunting and yellowing of leaves, and wilting of the plants during hot periods. Nematode infestation, however, can cause significant yield losses without any obvious above-ground disease symptoms. The primary causes of yield reduction are due to underground root damage. Roots infected by SCN are dwarfed or stunted. Nematode infestation also can decrease the number of nitrogen-fixing nodules on the roots, and may make the roots more susceptible to attacks by other soil-borne plant nematodes.
The nematode life cycle has three major stages: egg, juvenile, and adult. The life cycle varies between species of nematodes. The life cycle of SCN is similar to the life cycles of other plant parasitic nematodes. The SCN life cycle can usually be completed in 24 to 30 days under optimum conditions, whereas other species can take as long as a year, or longer, to complete the life cycle. When temperature and moisture levels become favorable in the spring, worm-shaped juveniles hatch from eggs in the soil. Only nematodes in the juvenile developmental stage are capable of infecting soybean roots.
After penetrating soybean roots, SCN juveniles move through the root until they contact vascular tissue, at which time they stop migrating and begin to feed. With a stylet, the nematode injects secretions that modify certain root cells and transform them into specialized feeding sites. The root cells are morphologically transformed into large multinucleate syncytia (or giant cells in the case of RKN), which are used as a source of nutrients for the nematodes. The actively feeding nematodes thus steal essential nutrients from the plant resulting in yield loss. As female nematodes feed, they swell and eventually become so large that their bodies break through the root tissue and are exposed on the surface of the root.
After a period of feeding, male SCN migrate out of the root into the soil and fertilize the enlarged adult females. The males then die, while the females remain attached to the root system and continue to feed. The eggs in the swollen females begin developing, initially in a mass or egg sac outside the body, and then later within the nematode body cavity. Eventually the entire adult female body cavity is filled with eggs, and the nematode dies. It is the egg-filled body of the dead female that is referred to as the cyst. Cysts eventually dislodge and are found free in the soil. The walls of the cyst become very tough, providing excellent protection for the approximately 200 to 400 eggs contained within. SCN eggs survive within the cyst until proper hatching conditions occur. Although many of the eggs may hatch within the first year, many also will survive within the protective cysts for several years.
A nematode can move through the soil only a few inches per year on its own power. However, nematode infestation can spread substantial distances in a variety of ways. Anything that can move infested soil is capable of spreading the infestation, including farm machinery, vehicles and tools, wind, water, animals, and farm workers. Seed sized particles of soil often contaminate harvested seed. Consequently, nematode infestation can be spread when contaminated seed from infested fields is planted in non-infested fields. There is even evidence that certain nematode species can be spread by birds. Only some of these causes can be prevented.
Traditional practices for managing nematode infestation include: maintaining proper soil nutrients and soil pH levels in nematode-infested land; controlling other plant diseases, as well as insect and weed pests; using sanitation practices such as plowing, planting, and cultivating of nematode-infested fields only after working non-infested fields; cleaning equipment thoroughly with high pressure water or steam after working in infested fields; not using seed grown on infested land for planting non-infested fields unless the seed has been properly cleaned; rotating infested fields and alternating host crops with non-host crops; using nematicides; and planting resistant plant varieties.
Methods have been proposed for the genetic transformation of plants in order to confer increased resistance to plant parasitic nematodes. For example, a number of approaches involve transformation of plants with double-stranded RNA capable of inhibiting essential nematode genes. Other agricultural biotechnology approaches propose to over-express genes that encode proteins that are toxic to nematodes. U.S. Pat. Nos. 5,589,622 and 5,824,876 are directed to the identification of plant genes expressed specifically in or adjacent to the feeding site of the plant after attachment by the nematode.
US 2009/0089896 discloses a promoter of an Mtn21-like gene which is induced in syncytia of SCN-infected soybean. WO 2008/077892 discloses a promoter of a peroxidase-like gene which is induced in syncytia of SCN-infected soybean. WO 2008/071726 discloses a promoter of a trehalose-6-phosphate phosphatase-like gen which is induced in syncytia of SCN-infected soybean. WO 2008/095887 discloses a promoter of an Mtn3-like gene which is induced in syncytia of SCN-infected soybean. WO 2008/095888 discloses the promoter of an At5g12170-like gene which is induced in syncytia of SON-infected soybean.
A number of patent publications prophetically disclose and generically claim transgenic plants comprising any one or more of thousands of plant genes and having improved agronomic characteristics. Examples of such publications include US2004/0031072, US2006/0107345, US2004/0034888, US2004/0019927, US2004/0045049, US2004/0019927, US2006/0272060, WO2005/5112608, US2006/0150283, and US2007/0214517. Pathogen resistance, including nematode resistance, is disclosed as one potential improved agronomic characteristic of the transgenic plants described in these publications. However, none of these publications specifically associate any disclosed gene with improved nematode resistance in transgenic plants containing the gene.
Serine-Arginine rich (SR-rich) proteins are key regulators of plant gene expression, with various gene family members contributing to constitutive splicing of RNA, nuclear export, maintenance of mRNA stability and protein translation. SR proteins are also involved in alternative RNA splicing, where they bind specific RNA sequences and guide the formation of spliceosome complexes at weak splicing sites SR rich gene families are moderately populated in plants, with diverse sub-groups falling into approximately five motif-based categories.
The Avr9-elicited 111B-like gene is a transcription factor with sequence homology to 111B ACRE (Avr9/Cf-9 rapidly elicited) from Nicotiana tabacum and DREB1A/CBF3 from Arabidopsis. In tobacco the 111B ACRE gene is a pathogenesis-related transcriptional activator that is rapidly induced in lines expressing the Cf-9 resistance gene in response to Avr9 expressed by Cladosporium fulvum, a biotrophic fungus. In other species, CBF3/DREB1 genes are involved in activating abiotic stress response. U.S. Pat. No. 7,345,217 discloses SEQ ID NO:1408, an Avr9-elicited 111B-like gene which is purported to be a homolog of an Arabidopsis thaliana DNA designated G912. U.S. Pat. No. 7,345,217 generically discloses numerous categories of potential utilities for the thousands of genes disclosed therein, and one of those categories is identified as disease resistance, including nematode resistance. However, the only specific utilities proposed in U.S. Pat. No. 7,345,217 for G912 and its homologs are improved tolerances to cold, freezing, drought, and salt stress.
Basic Helix-Loop-Helix (bHLH) and Dehydration Responsive Element Binding (DREB) transcription factors are also key regulatory molecules in plants. The physiological functions of some bHLH genes have been demonstrated experimentally in plants. The R and TT8 genes are known to regulate anthocyanin accumulation in maize and Arabidopsis, and other bHLH genes interact with phytochrome and regulate light response. Other bHLH genes regulate hormone signaling. The physiological role of most plant bHLH genes is unknown, however, and there is little sequence conservation between bHLH gene family members outside of the core bHLH signature domain.
Dirigent-like proteins belong to a large, diverse gene family found in all major land plant groups analyzed to date. Dirigent-encoding genes cluster into 5 phylogenetic subfamilies, Dir-A through Dir-E. The Dir-A subfamily has been shown, in conjunction with phenolic oxidases, to direct the stereospecific assembly of lignins (cell wall components) and lignans (plant antioxidants and defense compounds) in a range of plant species. Expression of PsDIR1, a Dir-A gene from Pisum sativa, confers resistance to multiple fungal pathogens in transgenic canola. Dir-A subfamily genes are induced by a wide variety of stresses, such as mechanical wounding, herbivory and fungal infection. The specific biochemical functions of genes from subgroups Dir-B, Dir-C, Dir-D and Dir-E (Dir-like) proteins are not as well characterized, although genes from the Dir-C subfamily were shown to be induced by jasmonic acid treatment, salicylic acid and feeding by avirulent Hessian fly larvae.
To date, no genetically modified plant comprising a transgene capable of conferring nematode resistance has been deregulated in any country. Accordingly, a need continues to exist to identify safe and effective compositions and methods for controlling plant parasitic nematodes using agricultural biotechnology.
SUMMARY OF THE INVENTION
The present inventors have discovered that expression of a transgene comprising a polynucleotide encoding Serine-Arginine-rich protein, AVR9-elicited—111B-like protein, a bHLH protein, or a Dirigent-like protein in roots can render soybean plants resistant to SCN infection. Accordingly, the present invention provides transgenic plants and seeds, and methods to overcome, or at least alleviate, nematode infestation of valuable agricultural crops.
In one embodiment, the invention provides an isolated expression vector comprising a root-specific promoter in operative association with a polynucleotide selected from the group consisting of: a) a polynucleotide encoding a Serine-arginine rich protein; b) a polynucleotide encoding an AVR9-elicited—111B-like protein; c) a polynucleotide encoding a basic Helix-Loop-Helix protein; and d) a polynucleotide encoding a dirigent-like protein.
In another embodiment, the invention provides a method of making a nematode-resistant transgenic plant, the method comprising the steps of: a) providing a recombinant expression vector comprising a root-specific promoter in operative association with a polynucleotide selected from the group consisting of: i) a polynucleotide encoding a serine-arginine rich protein; ii) a polynucleotide encoding an AVR9-elicited—111B-like protein; iii) a polynucleotide encoding a basic Helix-Loop-Helix protein; and iv) a polynucleotide encoding a dirigent-like protein; b) transforming a plant cell with the recombinant expression vector; c) regenerating transgenic plants from the transformed plant cell; and d) selecting transgenic plants which demonstrate increased resistance to plant parasitic nematode infection when compared to wild type plants which do not comprise the recombinant expression vector.
In yet another embodiment, the invention provides a nematode-resistant transgenic plant comprising a recombinant expression vector comprising a root-specific promoter in operative association with a polynucleotide selected from the group consisting of: a) a polynucleotide encoding a serine-arginine rich protein; b) a polynucleotide encoding an AVR9-elicited—111B-like protein; c) a polynucleotide encoding a basic Helix-Loop-Helix protein; and d) a polynucleotide encoding a dirigent-like protein.
In another embodiment, the invention provides a seed which is true breeding for a transgene comprising a recombinant expression vector comprising a root-specific promoter in operative association with a polynucleotide selected from the group consisting of: a) a polynucleotide encoding a serine-arginine rich protein; b) a polynucleotide encoding an AVR9-elicited—111B-like protein; c) a polynucleotide encoding a basic Helix-Loop-Helix protein; and d) a polynucleotide encoding a dirigent-like protein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a-1b show the table of SEQ ID NOs assigned to corresponding genes and promoters. SEQ ID NOs 1, 37, 39 and 49 correspond to full length G. max nucleotide sequences for polynucleotides encoding Serine/Arginine-rich protein (SEQ ID NO:1), Avr9-elicited 111b protein (SEQ ID NO:37), bHLH protein (SEQ ID NO:39) and Dirigent-like protein (SEQ ID NO:49), respectively. Syncytia-induced promoter sequences are given in SEQ ID NO:57 (TPP-like promoter from A. thaliana), SEQ ID NO:58 (MtN3-like promoter from G. max) and SEQ ID NO:59 (promoter from locus At5g12170 of A. thaliana). The constitutive ubiquitin promoter designated PcUbi4-2, from P. crispum is given in SEQ ID NO:60.
FIGS. 2a-2c show an amino acid alignment of exemplary Serine/Arginine-rich proteins performed using Vector NTI software suite v10.3.0 (gap opening penalty=10, gap extension penalty=0.05, gap separation penalty=8).
FIG. 3 shows an amino acid alignment of exemplary basic-helix-loop-helix proteins performed using Vector NTI software suite v10.3.0 (gap opening penalty=10, gap extension penalty=0.05, gap separation penalty=8).
FIG. 4 shows an amino acid alignment of exemplary Dirigent-like proteins performed using Vector NTI software suite v10.3.0 (gap opening penalty=10, gap extension penalty=0.05, gap separation penalty=8).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention may be understood more readily by reference to the following detailed description and the examples included herein. Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. As used herein, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a cell” can mean that at least one cell can be used.
As used herein, the word “or” means any one member of a particular list and also includes any combination of members of that list.
As defined herein, a “transgenic plant” is a plant that has been altered using recombinant DNA technology to contain an isolated nucleic acid which would otherwise not be present in the plant. As used herein, the term “plant” includes a whole plant, plant cells, and plant parts. Plant parts include, but are not limited to, stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores, and the like. The transgenic plant of the invention may be male sterile or male fertile, and may further include transgenes other than those that comprise the isolated polynucleotides described herein.
As defined herein, the term “nucleic acid” and “polynucleotide” are interchangeable and refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. An “isolated” nucleic acid molecule is one that is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid (i.e., sequences encoding other polypeptides). For example, a cloned nucleic acid is considered isolated. A nucleic acid is also considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into a cell by transformation. Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. While it may optionally encompass untranslated sequence located at both the 3′ and 5′ ends of the coding region of a gene, it may be preferable to remove the sequences which naturally flank the coding region in its naturally occurring replicon.
The term “gene” is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of consecutive amino acid residues.
The terms “operably linked” and “in operative association with” are interchangeable and as used herein refer to the association of isolated polynucleotides on a single nucleic acid fragment so that the function of one isolated polynucleotide is affected by the other isolated polynucleotide. For example, a regulatory DNA is said to be “operably linked to” a DNA that expresses an RNA or encodes a polypeptide if the two DNAs are situated such that the regulatory DNA affects the expression of the coding DNA.
The term “promoter” as used herein refers to a DNA sequence which, when ligated to a nucleotide sequence of interest, is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is typically, though not necessarily, located 5′ (e.g., upstream) of a nucleotide of interest (e.g., proximal to the transcriptional start site of a structural gene) whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription.
The term “transcription regulatory element” as used herein refers to a polynucleotide that is capable of regulating the transcription of an operably linked polynucleotide. It includes, but not limited to, promoters, enhancers, introns, 5′ UTRs, and 3′ UTRs.
As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. A vector can be a binary vector or a T-DNA that comprises the left border and the right border and may include a gene of interest in between. The term “expression vector” is interchangeable with the term “transgene” as used herein and means a vector capable of directing expression of a particular nucleotide in an appropriate host cell. The expression of the nucleotide can be over-expression. An expression vector comprises a regulatory nucleic acid element operably linked to a nucleic acid of interest, which is—optionally—operably linked to a termination signal and/or other regulatory element.
The term “homologs” as used herein refers to a gene related to a second gene by descent from a common ancestral DNA sequence. The term “homologs” may apply to the relationship between genes separated by the event of speciation (e.g., orthologs) or to the relationship between genes separated by the event of genetic duplication (e.g., paralogs).
As used herein, the term “orthologs” refers to genes from different species, but that have evolved from a common ancestral gene by speciation. Orthologs retain the same function in the course of evolution. Orthologs encode proteins having the same or similar functions. As used herein, the term “paralogs” refers to genes that are related by duplication within a genome. Paralogs usually have different functions or new functions, but these functions may be related.
The term “conserved region” or “conserved domain” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. The “conserved region” can be identified, for example, from the multiple sequence alignment using the Clustal W algorithm.
The term “cell” or “plant cell” as used herein refers to single cell, and also includes a population of cells. The population may be a pure population comprising one cell type. Likewise, the population may comprise more than one cell type. A plant cell within the meaning of the invention may be isolated (e.g., in suspension culture) or comprised in a plant tissue, plant organ or plant at any developmental stage.
The term “true breeding” as used herein refers to a variety of plant for a particular trait if it is genetically homozygous for that trait to the extent that, when the true-breeding variety is self-pollinated, a significant amount of independent segregation of the trait among the progeny is not observed.
The term “null segregant” as used herein refers to a progeny (or lines derived from the progeny) of a transgenic plant that does not contain the transgene due to Mendelian segregation.
The term “wild type” as used herein refers to a plant cell, seed, plant component, plant tissue, plant organ, or whole plant that has not been genetically modified or treated in an experimental sense.
The term “control plant” as used herein refers to a plant cell, an explant, seed, plant component, plant tissue, plant organ, or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype or a desirable trait in the transgenic or genetically modified plant. A “control plant” may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of interest that is present in the transgenic or genetically modified plant being evaluated.
A control plant may be a plant of the same line or variety as the transgenic or genetically modified plant being tested, or it may be another line or variety, such as a plant known to have a specific phenotype, characteristic, or known genotype. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transgenic plant herein.
The term “syncytia site” as used herein refers to the feeding site formed in plant roots after nematode infestation. The site is used as a source of nutrients for the nematodes. A syncytium is the feeding site for cyst nematodes and giant cells are the feeding sites of root knot nematodes.
In one embodiment, the invention provides an isolated expression vector comprising a root-specific promoter in operative association with a polynucleotide selected from the group consisting of: a) a polynucleotide encoding a serine-arginine rich protein; b) a polynucleotide encoding an AVR9-elicited—111B-like protein; c) a polynucleotide encoding a basic Helix-Loop-Helix protein; and d) a polynucleotide encoding a dirigent-like protein.
Any root-specific promoter may be employed in the expression vector of the invention. Exemplary root-specific promoters include, without limitation, the promoter derived from corn nicotianamine synthase gene (US 20030131377) and the rice RCC3 promoter (U.S. Ser. No. 11/075,113). Of particular utility in the present invention are root-specific promoters induced in nematode feeding sites (i.e., syncytia). Preferably, the Mtn3-like nematode-inducible promoter disclosed in WO 2008/095887, the nematode-inducible Mtn21-like promoter disclosed in US 2009/0089896, the nematode-inducible peroxidase-like promoter disclosed in WO 2008/077892, the nematode-inducible trehalose-6-phosphate phosphatase-like promoter disclosed in WO 2008/071726 and the nematode-inducible At5g12170-like promoter disclosed in WO 2008/095888 may be employed in the expression vector of the invention nematode-inducible.
Any polynucleotide encoding a serine-arginine rich protein may be employed in the isolated expression vector of the invention. Preferably, the polynucleotide encodes a serine-arginine rich protein selected from the group consisting of a polypeptide comprising amino acids 1 to 253 of SEQ ID NO: 2; a polypeptide comprising amino acids 1 to 249 of SEQ ID NO: 4; a polypeptide comprising amino acids 1 to 247 of SEQ ID NO: 6; a polypeptide comprising amino acids 1 to 249 of SEQ ID NO: 8; a polypeptide comprising amino acids 1 to 249 of SEQ ID NO: 10; a polypeptide comprising amino acids 1 to 245 of SEQ ID NO: 12; a polypeptide comprising amino acids 1 to 240 of SEQ ID NO: 14; a polypeptide comprising amino acids 1 to 261 of SEQ ID NO: 16; a polypeptide comprising amino acids 1 to 280 of SEQ ID NO: 18; a polypeptide comprising amino acids 1 to 248 of SEQ ID NO: 20; a polypeptide comprising amino acids 1 to 252 of SEQ ID NO: 22; a polypeptide comprising amino acids 1 to 265 of SEQ ID NO: 24; a polypeptide comprising amino acids 1 to 263 of SEQ ID NO: 26; a polypeptide comprising amino acids 1 to 220 of SEQ ID NO: 28; a polypeptide comprising amino acids 1 to 220 of SEQ ID NO: 30; a polypeptide comprising amino acids 1 to 263 of SEQ ID NO: 32; a polypeptide comprising amino acids 1 to 218 of SEQ ID NO: 34; and a polypeptide comprising amino acids 1 to 245 of SEQ ID NO: 36. More preferably, the polynucleotide encodes a serine-arginine rich protein comprising amino acids 1 to 253 of SEQ ID NO: 2.
Any polynucleotide encoding an AVR9-elicited—111B protein may be employed in the isolated expression vector of the invention. Preferably, the AVR9-elicited—111B protein comprises amino acids 1 to 226 of SEQ ID NO:38.
Any polynucleotide encoding a basic Helix-Loop-Helix protein may be employed in the isolated expression vector of invention. Preferably, the basic Helix-Loop-Helix protein is selected from the group consisting of a polypeptide comprising amino acids 1 to 231 of SEQ ID NO: 40; a polypeptide comprising amino acids 1 to 226 of SEQ ID NO: 42; a polypeptide comprising amino acids 1 to 232 of SEQ ID NO: 44; a polypeptide comprising amino acids 1 to 233 of SEQ ID NO: 46; and a polypeptide comprising amino acids 1 to 260 of SEQ ID NO: 48. More preferably, the basic Helix-Loop-Helix protein comprises amino acids 1 to 231 of SEQ ID NO: 40.
Any polynucleotide encoding a dirigent-like protein may be employed in the isolated expression vector of the invention. Preferably, the dirigent-like protein is selected from the group consisting of a polypeptide comprising amino acids 1 to 191 of SEQ ID NO: 50; a polypeptide comprising amino acids 1 to 191 of SEQ ID NO: 52; a polypeptide comprising amino acids 1 to 189 of SEQ ID NO: 54; and a polypeptide comprising amino acids 1 to 189 of SEQ ID NO: 56. More preferably, the dirigent-like protein comprises amino acids 1 to 191 of SEQ ID NO: 50.
In another embodiment, the isolated expression vector of the invention is employed in a method of making a nematode-resistant transgenic plant, the method comprising the steps of: a) providing the above-described recombinant expression vector b) transforming a plant cell with the recombinant expression vector; c) regenerating transgenic plants from the transformed plant cell; and d) selecting transgenic plants which demonstrate increased resistance to plant parasitic nematode infection when compared to wild type plants which do not comprise the recombinant expression vector.
A variety of methods for introducing polynucleotides into the genome of plants and for the regeneration of plants from plant tissues or plant cells are known in, for example, Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7, pp. 71-119 (1993); White F F (1993) Vectors for Gene Transfer in Higher Plants; Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and Wu R, Academic Press, 15-38; Jenes B et al. (1993) Techniques for Gene Transfer; Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225; Halford N G, Shewry P R (2000) Br Med Bull 56(1):62-73.
Transformation methods may include direct and indirect methods of transformation. Suitable direct methods include polyethylene glycol induced DNA uptake, liposome-mediated transformation (U.S. Pat. No. 4,536,475), biolistic methods using the gene gun (Fromm M E et al., Bio/Technology. 8(9):833-9, 1990; Gordon-Kamm et al. Plant Cell 2:603, 1990), electroporation, incubation of dry embryos in DNA-comprising solution, and microinjection. In the case of these direct transformation methods, the plasmids used need not meet any particular requirements. Simple plasmids, such as those of the pUC series, pBR322, M13 mp series, pACYC184 and the like can be used. If intact plants are to be regenerated from the transformed cells, an additional selectable marker gene is preferably located on the plasmid. The direct transformation techniques are equally suitable for dicotyledonous and monocotyledonous plants.
Transformation can also be carried out by bacterial infection by means of Agrobacterium (for example EP 0 116 718), viral infection by means of viral vectors (EP 0 067 553; U.S. Pat. No. 4,407,956; WO 95/34668; WO 93/03161) or by means of pollen (EP 0 270 356; WO 85/01856; U.S. Pat. No. 4,684,611). Agrobacterium based transformation techniques (especially for dicotyledonous plants) are well known in the art. The Agrobacterium strain (e.g., Agrobacterium tumefaciens or Agrobacterium rhizogenes) comprises a plasmid (Ti or Ri plasmid) and a T-DNA element which is transferred to the plant following infection with Agrobacterium. The T-DNA (transferred DNA) is integrated into the genome of the plant cell. The T-DNA may be localized on the Ri- or Ti-plasmid or is separately comprised in a so-called binary vector. Methods for the Agrobacterium-mediated transformation are described, for example, in Horsch RB et al. (1985) Science 225:1229. The transformation of plants by Agrobacteria is described in, for example, White F F, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38; Jenes B et al. Techniques for Gene Transfer, Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225.
The nucleotides described herein can be directly transformed into the plastid genome. Plastid expression, in which genes are inserted by homologous recombination into the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit high expression levels. In one embodiment, the nucleotides are inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplasmic for plastid genomes containing the nucleotide sequences are obtained, and are preferentially capable of high expression of the nucleotides. Plastid transformation technology is for example extensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818, and 5,877,462 in WO 95/16783 and WO 97/32977, and in McBride et al. (1994) PNAS 91, 7301-7305.
The method described above produces another embodiment of the invention, a nematode-resistant transgenic plant comprising a recombinant expression vector comprising a root-specific promoter in operative association with a polynucleotide selected from the group consisting of: a) a polynucleotide encoding a serine-arginine rich protein; b) a polynucleotide encoding an AVR9-elicited—111B-like protein; c) a polynucleotide encoding a basic Helix-Loop-Helix protein; and d) a polynucleotide encoding a dirigent-like protein. The transgenic plants of the invention may be used to control infestation of a crop by a plant parasitic nematode.
The invention also provides a method of plant breeding, e.g., to prepare a crossed fertile transgenic plant. The transgenic plants of the invention may be crossed with similar transgenic plants or with transgenic plants lacking the nucleic acids of the invention or with non-transgenic plants, using known methods of plant breeding, to prepare seeds. Further, the transgenic plant of the present invention may comprise, and/or be crossed to another transgenic plant that comprises one or more nucleic acids, thus creating a “stack” of transgenes in the plant and/or its progeny. The seed is then planted to obtain a crossed fertile transgenic plant comprising the expression vector of the invention. The crossed fertile transgenic plant may have the expression vector inherited through a female parent or through a male parent. The second plant may be an inbred plant. The crossed fertile transgenic may be a hybrid.
“Gene stacking” can also be accomplished by transferring two or more genes into the cell nucleus by plant transformation. Multiple genes may be introduced into the cell nucleus during transformation either sequentially or in unison. In accordance with the invention, multiple genes encoding Serine-Arginine-rich, AVR9-elicited—111B-like, bHLH and Dirigent-like proteins can be stacked to provide enhanced nematode resistance. These stacked combinations can be created by any method including but not limited to cross-breeding plants by conventional methods or by genetic transformation. If the traits are stacked by genetic transformation, the Serine-Arginine-rich, AVR9-elicited—111B-like, bHLH and Dirigent-like proteins genes can be combined in any manner. For example if two genes are to be introduced, the two sequences can be contained in separate transformation cassettes or on the same transformation cassette. The expression of the sequences can be driven by the same or different promoters.
The transgenic plants described above produce yet another embodiment of the invention, a seed which is true breeding for a transgene comprising the recombinant expression vector comprising a root-specific promoter in operative association with a polynucleotide selected from the group consisting of: a) a polynucleotide encoding a serine-arginine rich protein; b) a polynucleotide encoding an AVR9-elicited—111B-like protein; c) a polynucleotide encoding a basic Helix-Loop-Helix protein; and d) a polynucleotide encoding a dirigent-like protein. The transgenic seeds of the invention may be used to control infestation of a crop by a plant parasitic nematode.
Crop plants and corresponding parasitic nematodes are listed in Index of Plant Diseases in the United States (U.S. Dept. of Agriculture Handbook No. 165, 1960); Distribution of Plant-Parasitic Nematode Species in North America (Society of Nematologists, 1985); and Fungi on Plants and Plant Products in the United States (American Phytopathological Society, 1989). For example, plant parasitic nematodes that are targeted by the present invention include, without limitation, cyst nematodes and root-knot nematodes. Specific plant parasitic nematodes which are targeted by the present invention include, without limitation, Heterodera glycines, Heterodera schachtii, Heterodera avenae, Heterodera oryzae, Heterodera cajani, Heterodera trifolii, Globodera pallida, G. rostochiensis, or Globodera tabacum, Meloidogyne incognita, M. arenaria, M. hapla, M. javanica, M. naasi, M. exigua, Ditylenchus dipsaci, Ditylenchus angustus, Radopholus similis, Radopholus citrophilus, Helicotylenchus multicinctus, Pratylenchus coffeae, Pratylenchus brachyurus, Pratylenchus vulnus, Paratylenchus curvitatus, Paratylenchus zeae, Rotylenchulus reniformis, Paratrichodorus anemones, Paratrichodorus minor, Paratrichodorus christiei, Anguina tritici, Bidera avenae, Subanguina radicicola, Hoplolaimus seinhorsti, Hoplolaimus Columbus, Hoplolaimus galeatus, Tylenchulus semipenetrans, Hemicycliophora arenaria, Rhadinaphelenchus cocophilus, Belonolaimus longicaudatus, Trichodorus primitivus, Nacobbus aberrans, Aphelenchoides besseyi, Hemicriconemoides kanayaensis, Tylenchorhynchus claytoni, Xiphinema americanum, Cacopaurus pestis, Heterodera zeae, Heterodera filipjevi and the like.
Plants which may be rendered nematode-resistant in accordance with the invention include monocotyledonous plants and dicotyledonous plants. Nematode-resistant plants produced in accordance with the invention include, without limitation, maize, wheat, rice, barley, oat, rye, sorghum, banana, and ryegrass. The plant can be from a genus selected from the group consisting of pea, alfalfa, soybean, carrot, celery, tomato, potato, cotton, tobacco, pepper, oilseed rape, beet, cabbage, cauliflower, broccoli, lettuce A. thaliana, and the like.
The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof.
EXAMPLE 1
Vector Construction
Using a bioinformatics approach, four soybean genes, TA52573—3847 (SEQ ID NO:3), AVR9-elicited—111B (SEQ ID NO:37), GmbHLH—47172355 (SEQ ID NO:39), and GmDirigent—59580836 (SEQ ID NO:49) were identified as being down-regulated in syncytia of SCN-infected soybean roots, as compared to uninfected root tissue. As described herein, the gene designated TA52573—3847 SEQ ID NO:3 encodes a serine-arginine rich protein. The GmSerine-Arginine-rich gene (SEQ ID NO:1) employed in the isolated expression vectors described below encodes a protein having 93% sequence identity to TA52573—3847 (SEQ ID NO:3).
The constitutive ubiquitin promoter from parsley (WO 2003/102198; SEQ ID NO:60, designated PcUbi4) the nematode-inducible MtN3-like promoter from soybean (WO 2008/095887, SEQ ID NO:58), the nematode-inducible TPP-like promoter from Arabidopsis (WO 2008/071726, SEQ ID NO:57) and the constitutive Super Promoter (see U.S. Pat. No. 5,955,646) were used in to make the constructs described in Table 1 below.










TABLE 1





Vector Name 
Promoter
Gene Name
SEQ ID NO: of genes







RBM024
PcUbi4
GmSerine-Arginine-rich
SEQ ID NO: 1


RBM036
MtN3-like
GmSerine-Arginine-rich
SEQ ID NO: 1


RBM019
PcUbi4
AVR9-elicited_111B
SEQ ID NO: 37


RBM031
MtN3-like
AVR9-elicited_111B
SEQ ID NO: 37


RTP1124
Super
AVR9-elicited_111B
SEQ ID NO: 37


RTP1125
TPP-like
AVR9-elicited_111B
SEQ ID NO: 37


RTP1126
PcUbi4
GmbHLH_47172355
SEQ ID NO: 39


RTP1127
MtN3-like
GmbHLH_47172355
SEQ ID NO: 39


RTP1086
PcUbi4
GmDirigent_59580836
SEQ ID NO: 49


RTP1090
MtN3-like
GmDirigent_59580836
SEQ ID NO: 49









The expression vectors also comprised the mutated form of the acetohydroxy acid synthase (AHAS) selection gene described in WO 2008/124495, which confers resistance to the herbicide ARSENAL (Imazapyr, BASF Corporation, Mount Olive, N.J.). The expression of AHAS2 was driven by the parsely ubiquitin promoter (SEQ ID NO:60).
EXAMPLE 2
Nematode Bioassay
A bioassay to assess nematode resistance conferred by the polynucleotides described herein was performed using a rooted plant assay system disclosed in commonly owned copending U.S. Ser. No. 12/001,234. Transgenic roots are generated after transformation with the binary vectors described in Example 1. Multiple transgenic root lines are sub-cultured and inoculated with surface-decontaminated race 3 SCN second stage juveniles (J2) at the level of about 500 J2/well. Four weeks after nematode inoculation, the cyst number in each well is counted. For each transformation construct, the number of cysts per line is calculated to determine the average cyst count and standard error for the construct. The cyst count values for each transformation construct is compared to the cyst count values of an empty vector control tested in parallel to determine if the construct tested results in a reduction in cyst count. Rooted explant cultures transformed with vectors RBM024, RBM036, RBM019, RBM031, RTP1124, RTP1125, RTP1126, RTP1127 and RTP1090 exhibited a general trend of reduced cyst numbers and female index relative to the known susceptible variety, Williams82. Transgenic roots expressing the GmDirigent—59580836 gene regulated by the constitutive PcUbi4 promoter (vector RTP1086) did not show reduced cyst counts relative to control lines. Some root lines constitutively expressing the GmSerine-Arginine-rich gene with the PcUbi4 promoter developed dark brown patches. The localization of dark brown patches varied among transgenic root lines, in some cases being limited to scattered individual cells, or lateral root emergence zones or extending entirely along the length of older roots. Transgenic roots over-expressing the AVR9-elicited—111B gene regulated by the constitutive PcUbi4 promoter (RBM019) or the constitutive Super promoter (RTP1124) developed thicker and shorter roots and reduced numbers of lateral roots relative to control lines.

## Claims

1. A method of making a nematode-resistant transgenic plant, the method comprising the steps of:
a) providing a recombinant expression vector comprising a root-specific promoter in operative association with a polynucleotide encoding an AVR9-elicited—111B-like protein comprising SEQ ID NO:38;
b) transforming a plant cell with the recombinant expression vector;
c) regenerating transgenic plants from the transformed plant cell; and
d) selecting transgenic plants which demonstrate increased resistance to plant parasitic nematode infection when compared to wild type plants which do not comprise the recombinant expression vector.

2. A nematode-resistant transgenic plant comprising a recombinant expression vector comprising a root-specific promoter in operative association with a polynucleotide encoding an AVR9-elicited—111B-like protein comprising SEQ ID NO:38.

3. A seed which is true breeding for a transgene comprising a recombinant expression vector comprising a root-specific promoter in operative association with a polynucleotide encoding an AVR9-elicited—111B-like protein comprising SEQ ID NO:38.

4. An isolated expression vector comprising a root-specific promoter in operative association with a polynucleotide encoding an AVR9-elicited—111B-like protein comprising SEQ ID NO:38.