Thomas J. Baum

Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall–degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies.

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.

NEMATODE PARASITISM GENES

The ability of nematodes to live on plant hosts involves multiple parasitism genes. The most pronounced morphological adaptations of nematodes for plant parasitism include a hollow, protrusible stylet (feeding spear) connected to three enlarged esophageal gland cells that express products that are secreted into plant tissues through the stylet. Reverse genetic and expressed sequence tag (EST) approaches are being used to discover the parasitism genes expressed in nematode esophageal gland cells. Some genes cloned from root-knot (Meloidogyne spp.) and cyst (Heterodera and Globodera spp.) nematodes have homologues reported in genomic analyses of Caenorhabditis elegans and animal-parasitic nematodes. To date, however, the candidate parasitism genes endogenous to the esophageal glands of plant nematodes (such as the ß-1,4-endoglucanases) have their greatest similarity to microbial genes, prompting speculation that genes for plant parasitism by nematodes may have been acquired by horizontal gene transfer.

The Parasitome of the Phytonematode Heterodera glycines

Parasitism genes expressed in the esophageal gland cells of phytonematodes encode secretions that control the complex process of plant parasitism. In the soybean cyst nematode, Heterodera glycines, the parasitome, i.e., the secreted products of parasitism genes, facilitate nematode migration in soybean roots and mediate the modification of root cells into elaborate feeding cells required to support the growth and development of the nematode. With very few exceptions, the identities of these secretions are unknown, and the mechanisms of cyst nematode parasitism, therefore, remain obscure. The most direct and efficient approach for cloning parasitism genes and rapidly advancing our understanding of the molecular interactions during nematode parasitism of plants is to create gland cell-specific cDNA libraries using cytoplasm microaspirated from the esophageal gland cells of various parasitic stages. By combining expressed sequence tag analysis of a gland cell cDNA library with high throughput in situ expression localization of clones encoding secretory proteins, we obtained the first comprehensive parasitome profile for a parasitic nematode. We identified 51 new H. glycines gland-expressed candidate parasitism genes, of which 38 genes constitute completely novel sequences. Individual parasitome members showed distinct gland cell expression patterns throughout the parasitic cycle. The parasitome complexity discovered paints a more elaborate picture of host cellular events under specific control by the nematode parasite than previously hypothesized.

Developmental Transcript Profiling of Cyst Nematode Feeding Cells in Soybean Roots

Cyst nematodes of the genus Heterodera are obligate, sedentary endoparasites that have developed highly evolved relationships with specific host plant species. Successful parasitism involves significant physiological and morphological changes to plant root cells for the formation of specialized feeding cells called syncytia. To better understand the molecular mechanisms that lead to the development of nematode feeding cells, transcript profiling was conducted on developing syncytia induced by the soybean cyst nematode Heterodera glycines in soybean roots by coupling laser capture microdissection with high-density oligonucleotide microarray analysis. This approach has identified pathways that may play intrinsic roles in syncytium induction, formation, and function. Our data suggest interplay among phytohormones that likely regulates synchronized changes in the expression of genes encoding cell-wall-modifying proteins. This process appears to be tightly controlled and coordinately regulated with cell wall rigidification processes that may involve lignification of feeding cell walls. Our data also show local downregulation of jasmonic acid biosynthesis and responses in developing syncytia, which suggest a local suppression of plant defense mechanisms. Moreover, we identified genes encoding putative transcription factors and components of signal transduction pathways that may be important in the regulatory processes governing syncytium formation and function. Our analysis provides a broad mechanistic picture that forms the basis for future hypothesis-driven research to understand cyst nematode parasitism and to develop effective management tools against these pathogens.

Isolation of a cDNA Encoding a β-1,4-endoglucanase in the Root-Knot Nematode Meloidogyne incognita and Expression Analysis During Plant Parasitism

A beta-1,4-endoglucanase encoding cDNA (EGases, E.C. 3.2.1.4), named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of beta-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode beta-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a beta-1,4-endoglucanase devoid of a cellulose-binding domain.

A parasitism gene from a plant-parasitic nematode with function similar to CLAVATA3/ESR (CLE) of Arabidopsis thaliana

SUMMARY The Hg-SYV46 parasitism gene is expressed exclusively in the dorsal oesophageal gland cell of parasitic stages of the soybean cyst nematode, Heterodera glycines, and it encodes a secretory protein that contains a C-terminal motif of the CLAVATA3/ESR-related (CLE) family in Arabidopsis thaliana. In shoot and floral meristems of Arabidopsis, the stem cells secret CLV3, a founding member of the CLE protein family, that activates the CLV1/CLV2 receptor complex and negatively regulates WUSCHEL expression to restrict the size of the stem cell population. Mis-expression of Hg-SYV46 in Arabidopsis (ecotype Columbia-0) under control of the CaMV35S promoter resulted in a wus-like phenotype including premature termination of the shoot apical meristem and the development of flowers lacking the central gynoecium. The wus-like phenotype observed was similar to reports of over-expression of CLV3 and CLE40 in Arabidopsis, as was down-regulation of WUS expression in the shoot apices of 35S::Hg-SYV46/Col-0 plants. Expression of 35S::Hg-SYV46 in a clv3-1 mutant of Arabidopsis was able partially or fully to rescue the mutant phenotype, probably dependent upon localization and level of transgene expression. A short root phenotype, as reported for over-expression of CLV3, CLE40 and CLE19 in roots, was also produced in primary 35S::Hg-SYV46/Col-0 transgenic plants. The results suggest a functional similarity of HG-SYV46 to plant-secreted CLE ligands that may play a role in the differentiation or division of feeding cells induced in plant roots by parasitic nematodes.

A Profile of Putative Parasitism Genes Expressed in the Esophageal Gland Cells of the Root-knot Nematode Meloidogyne incognita

Identifying parasitism genes encoding proteins secreted from a 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. Meloidogyne incognita parasitism genes were cloned by microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. Of 2,452 cDNA clones sequenced, deduced protein sequences of 185 cDNAs had a signal peptide for secretion and, thus, could have a role in root-knot nematode parasitism of plants. High-throughput in situ hybridization with cDNA clones encoding signal peptides resulted in probes of 37 unique clones specifically hybridizing to transcripts accumulating within the subventral (13 clones) or dorsal (24 clones) esophageal gland cells of M. incognita. In BLASTP analyses, 73% of the predicted proteins were novel proteins. Those with similarities to known proteins included a pectate lyase, acid phosphatase, and hypothetical proteins from other organisms. Our cell-specific analysis of genes encoding secretory proteins provided, for the first time, a profile of putative parasitism genes expressed in the M. incognita esophageal gland cells throughout the parasitic cycle.

Parallel Genome-Wide Expression Profiling of Host and Pathogen During Soybean Cyst Nematode Infection of Soybean

Global analysis of gene expression changes in soybean (Glycine max) and Heterodera glycines (soybean cyst nematode [SCN]) during the course of infection in a compatible interaction was performed using the Affymetrix GeneChip soybean genome array. Among 35,611 soybean transcripts monitored, we identified 429 genes that showed statistically significant differential expression between uninfected and nematode-infected root tissues. These included genes encoding enzymes involved in primary metabolism; biosynthesis of phenolic compounds, lignin, and flavonoids; genes related to stress and defense responses; cell wall modification; cellular signaling; and transcriptional regulation. Among 7,431 SCN transcripts monitored, 1,850 genes showed statistically significant differential expression across different stages of nematode parasitism and development. Differentially expressed SCN genes were grouped into nine different clusters based on their expression profiles during parasitism of soybean roots. The patterns of gene expression we observed in SCN suggest coordinated regulation of genes involved in parasitism. Quantitative real-time reverse-transcription polymerase chain reaction confirmed the results of our microarray analysis. The simultaneous genome-wide analysis of gene expression changes in the host and pathogen during a compatible interaction provides new insights into soybean responses to nematode infection and the first profile of transcript abundance changes occurring in the nematode as it infects and establishes a permanent feeding site within a host plant root.

Parasitism proteins in nematode-plant interactions

The current battery of candidate parasitism proteins secreted by nematodes to modify plant tissues for parasitism includes cell-wall-modifying enzymes of potential prokaryotic origin, multiple regulators of host cell cycle and metabolism, proteins that can localize to the plant cell nucleus, potential suppressors of host defense, mimics of plant molecules, and a relatively large cadre of predicted novel nematode parasitism proteins. Phenotypic effects of expressing nematode parasitism proteins in transformed plant tissues, protein-protein interaction assays, and RNA-mediated interference (RNAi) analyses are currently providing exciting evidence of the biological role of candidate nematode secreted parasitism proteins and identifying potential novel means of developing transgenic resistance to nematodes in crops.