Rex Allen

Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene

Secreted parasitism proteins encoded by parasitism genes expressed in esophageal gland cells mediate infection and parasitism of plants by root-knot nematodes (RKN). Parasitism gene 16D10 encodes a conserved RKN secretory peptide that stimulates root growth and functions as a ligand for a putative plant transcription factor. We used in vitro and in vivo RNA interference approaches to silence this parasitism gene in RKN and validate that the parasitism gene has an essential function in RKN parasitism of plants. Ingestion of 16D10 dsRNA in vitro silenced the target parasitism gene in RKN and resulted in reduced nematode infectivity. In vivo expression of 16D10 dsRNA in Arabidopsis resulted in resistance effective against the four major RKN species. Because no known natural resistance gene has this wide effective range of RKN resistance, bioengineering crops expressing dsRNA that silence target RKN parasitism genes to disrupt the parasitic process represents a viable and flexible means of developing novel durable RKN-resistant crops and could provide crops with unprecedented broad resistance to RKN.

A Root-Knot Nematode Secretory Peptide Functions as a Ligand for a Plant Transcription Factor

Parasitism genes expressed in the esophageal gland cells of root-knot nematodes encode proteins that are secreted into host root cells to transform the recipient cells into enlarged multinucleate feeding cells called giant-cells. Expression of a root-knot nematode parasitism gene which encodes a novel 13-amino-acid secretory peptide in plant tissues stimulated root growth. Two SCARECROW-like transcription factors of the GRAS protein family were identified as the putative targets for this bioactive nematode peptide in yeast two-hybrid analyses and confirmed by in vitro and in vivo coimmunoprecipitations. This discovery is the first demonstration of a direct interaction of a nematode-secreted parasitism peptide with a plant-regulatory protein, which may represent an early signaling event in the root-knot nematode-host interaction.

Signal Peptide-Selection of cDNA Cloned Directly from the Esophageal Gland Cells of the Soybean Cyst Nematode Heterodera glycines

Secretions from the esophageal gland cells of plant-parasitic nematodes play critical roles in the nematode-parasitic cycle. A novel method to isolate cDNA encoding putative nematode secretory proteins was developed that utilizes mRNA for reverse transcription-polymerase chain reaction derived from microaspiration of the esophageal gland cell contents of parasitic stages of the soybean cyst nematode Heterodera glycines. The resulting H. glycines gland cell cDNA was cloned into the pRK18 vector, and plasmid DNA was transformed into a mutated yeast host for specific selection of cDNA inserts that encode proteins with functional signal peptides. Of the 223 cDNA clones recovered from selection in yeast, 97% of the clones encoded a predicted signal peptide. Fourteen unique cDNA clones hybridized to genomic DNA of H. glycines on Southern blots and, among them, nine cDNA clones encoded putative extracellular proteins, as predicted by PSORT II computer analysis. Four cDNA clones hybridized to transcripts within the dorsal esophageal gland cell of parasitic stages of H. glycines, and in situ hybridization within H. glycines was not detected for eight cDNA clones. The protocol provides a direct means to isolate potential plant-parasitic nematode esophageal gland secretory protein genes.

Molecular characterisation and expression of two venom allergen-like protein genes in Heterodera glycines.

Secretory proteins encoded by genes expressed in the oesophageal gland cells of plant-parasitic nematodes have key roles in nematode parasitism of plants. Two venom allergen-like protein cDNAs (designated hg-vap-1 and hg-vap-2)were isolated from Heterodera glycines gland cell cDNA libraries. Both cDNAs hybridised to genomic DNA of H. glycines in Southern blots. The hg-vap-1 cDNA contained an open reading frame encoding 215 amino acids with the first 25 amino acids being a putative secretion signal. The hg-vap-2 cDNA contained an open reading frame encoding 212 amino acids with the first 19 amino acids being a putative secretion signal. Genes of hg-vap-1 and hg-vap-2 contained four introns, which ranged in size from 44 to 574 bp, and five exons ranging in size from 43 to 279 bp. In situ hybridisation analyses showed that mRNAs of both vap genes accumulated specifically in the subventral gland cells of H. glycines during parasitism. The gland cell-specific expression and presence of predicted secretion signal peptides in both VAPs suggest that these proteins are secreted from the nematode and may play a role in the infection of host plants by this parasite.

Molecular cloning and characterisation of a venom allergen AG5-like cDNA from Meloidogyne incognita

RNA fingerprinting was used to identify RNAs that were expressed in parasitic second-stage juveniles of Meloidogyne incognita, but absent from or reduced in preparasitic second-stage juveniles. A cDNA encoding a putative secretory protein was cloned from a M. incognita second-stage juvenile cDNA library by probing with a 0.5kb fragment derived from fingerprinting that was more strongly expressed in parasitic second-stage juveniles. The cDNA, named Mi-msp-1, contained an open reading frame encoding 231 amino acids, with the first 21 amino acids being a putative secretory signal. In Southern blot analysis the Mi-msp-1 hybridised with genomic DNA from M. incognita, Meloidogyne arenaria, Meloidogyne javanica, but not Meloidogyne hapla, Heterodera glycines or Caenorhabditis elegans. In Northern blot analysis a 1kb transcript was detected in both preparasitic and parasitic second-stage juveniles, but not in adult females of M. incognita. Comparing the predicted amino acid sequence with protein databases revealed significant similarity to the venom allergen antigen 5 family of proteins in hymenoptera insects and homologues found in several other nematode species.

Two chorismate mutase genes from the root-knot nematode Meloidogyne incognita

SUMMARY Parasitism genes encoding secretory proteins expressed in the oesophageal glands of phytoparasitic nematodes play critical roles in nematode invasion of host plants, establishment of feeding sites and suppression of host defences. Two chorismate mutase (CM) genes potentially having a role in one or more of these processes were identified from a Meloidogyne incognita oesophageal gland-cell subtractive cDNA library. These M. incognita enzymes (designated as MI-CM-1 and MI-CM-2) with amino-terminal signal peptides, were significantly similar to chorismate mutases in M. javanica and bacteria. The complementation of an Escherichia coli CM-deficient mutant by the expression of Mi-cm-1 or Mi-cm-2 confirmed their CM activity. In-situ mRNA hybridization showed that the transcripts of Mi-cm-1 and Mi-cm-2 accumulated specifically in the two subventral oesophageal gland cells of M. incognita. RT-PCR analysis confirmed that their transcript abundances were high in the early parasitic juvenile stages, and low (Mi-cm-1) or undetectable (Mi-cm-2) in later parasitic stages of the nematode. Southern blot analysis revealed that these CM genes were members of a small multigene family in Meloidogyne species. The widespread presence of CMs in the specialized sedentary endoparasitic nematode species suggests that this multifunctional enzyme may be a key factor in modulating plant parasitism.

Developmental expression and biochemical properties of a β -1,4- endoglucanase family in the soybean cyst nematode, Heterodera glycines

SUMMARY The soybean cyst nematode, Heterodera glycines, produces beta-1,4-endoglucanases (cellulases) that are secreted during infection of soybean. The gene structures of three, hg-eng-4, hg-eng-5 and hg-eng-6, of the six beta-1,4-endoglucanase genes, all family 5 glycosyl hydrolases previously identified from H. glycines, are presented here. Furthermore, we present the detailed expression analyses of beta-1,4-endoglucanase genes as well as the biochemical properties of four H. glycines endoglucanase enzymes. Two of the endoglucanases, HG-ENG-5 and HG-ENG-6, differed significantly in their amino acid sequence of the catalytic domains and their gene structure from that of the other four beta-1,4-endoglucanases. Quantitative real-time RT-PCR revealed distinct developmental expression differences among the hg-eng family members during the early stages of parasitism and relatively low expression levels in late parasitic stages, with the exception of the adult male stage for some eng genes. Recombinant HG-ENGs degraded carboxymethylcellulose and optimum enzyme activity ranged from pH 5.5 for HG-ENG-5 to pH 8 for HG-ENG-6. EDTA, Ca(2+), Co(2+), Mg(2+) and Fe(2+) did not affect enzyme activity of any ENG protein, whereas Zn(2+), Cu(2+) and Mn(2+) inhibited enzyme activity from 23% to 73% in some cases. In tests with 12 different polysaccharide substrates, enzyme activity was restricted to beta-1,4 linkages with all ENG proteins tested. Only HG-ENG-5 and HG-ENG-6 had relatively high activity on xylan and slightly degraded microcrystalline cellulose. Together, these data reveal distinct differences in expression and biochemistry of cyst nematode parasitism genes and proteins, respectively, and cast light on the intricate interactions between a parasitic animal and its plant host.

Use of solid-phase subtractive hybridization for the identification of parasitism gene candidates from the root-knot nematode Meloidogyne incognita

SUMMARY A solid-phase subtractive strategy was used to clone parasitism gene candidates (PGCs) expressed in the oesophageal gland cells of Meloidogyne incognita. Nematode intestinal first-strand cDNA was synthesized directly on magnetic beads and used to enrich for gland-specific sequences by high stringency hybridization to gland-cell mRNA. A gland-specific cDNA library was created from the nonhybridizing gland-cell mRNA by long-distance reverse transcription polymerase chain reaction. Subtraction of the gland cDNA library (1000 clones) with previously cloned M. incognita parasitism genes removed 89 cDNA clones and promoted efficient identification of new PGCs. Sequencing of 711 cDNA clones from the subtracted library revealed that deduced protein sequences of 67 cDNAs were preceded by a signal peptide for secretion, a key criterion for parasitism genes. In situ hybridization with probes from the cDNA clones encoding signal peptides showed that seven cDNA clones were specifically expressed in the subventral gland cells and four in the dorsal gland cell of M. incognita. BLASTP analyses revealed the predicted proteins of five cDNAs to be novel sequences. The six PGCs with similarities to known proteins included a pectate lyase, three beta-1,4-endoglucanases and two chorismate mutases. This subtractive protocol provides an efficient and reliable approach for identifying PGCs encoding oesophageal gland cell secretory proteins that may have a role in M. incognita parasitism of plants.

Characterisation and developmental expression of a chitinase gene in Heterodera glycines

A chitinase full-length cDNA (designated Hg-chi-1) was isolated from a Heterodera glycines oesophageal gland cell-specific long-distance PCR cDNA library. The cDNA hybridised to genomic DNA of H. glycines in Southern blots. The Hg-chi-1 cDNA contained an open reading frame encoding 350 amino acids with the first 23 amino acids being a putative signal peptide for secretion. Hg-CHI-1 contained a chitinase 18 family catalytic domain, and chitinolytic activity of recombinant Hg-CHI-1 was confirmed in glycol-chitin substrate gel electrophoresis. In situ mRNA hybridisation analyses showed that transcripts of Hg-chi-1 accumulated specifically in the subventral oesophageal gland cells of parasitic stages of H. glycines, but Hg-chi-1 expression was not detected in eggs or hatched pre-parasitic second-stage juveniles, suggesting that this chitinase does not have a role in egg hatching of H. glycines. The biological function of Hg-CHI-1 in H. glycines remains to be determined.

Microaspiration of Esophageal Gland Cells and cDNA Library Construction for Identifying Parasitism Genes of Plant-Parasitic Nematodes

Identifying parasitism genes encoding proteins secreted from a plant-parasitic nematodes esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Parasitism genes have been cloned by directly microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages of cyst or root-knot nematodes to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. cDNA clones are sequenced and deduced protein sequences with a signal peptide for secretion are identified for high-throughput in situ hybridization to confirm gland-specific expression.