Guozhong Huang

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 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.

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.

The 8D05 Parasitism Gene of Meloidogyne incognita Is Required for Successful Infection of Host Roots

Parasitism genes encode effector proteins that are secreted through the stylet of root-knot nematodes to dramatically modify selected plant cells into giant-cells for feeding. The Mi8D05 parasitism gene previously identified was confirmed to encode a novel protein of 382 amino acids that had only one database homolog identified on contig 2374 within the Meloidogyne hapla genome. Mi8D05 expression peaked in M. incognita parasitic second-stage juveniles within host roots and its encoded protein was limited to the subventral esophageal gland cells that produce proteins secreted from the stylet. Constitutive expression of Mi8D05 in transformed Arabidopsis thaliana plants induced accelerated shoot growth and early flowering but had no visible effects on root growth. Independent lines of transgenic Arabidopsis that expressed a double-stranded RNA complementary to Mi8D05 in host-derived RNA interference (RNAi) tests had up to 90% reduction in infection by M. incognita compared with wild-type control plants, suggesting that Mi8D05 plays a critical role in parasitism by the root-knot nematode. Yeast two-hybrid experiments confirmed the specific interaction of the Mi8D05 protein with plant aquaporin tonoplast intrinsic protein 2 (TIP2) and provided evidence that the Mi8D05 effector may help regulate solute and water transport within giant-cells to promote the parasitic interaction.

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.

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.

Mining Novel Effector Proteins from the Esophageal Gland Cells of Meloidogyne incognita

Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins which M. incognita secretes into its host plants during infection is an important step toward finding new ways to manage this pest. In this study, we have identified the cDNAs for 18 putative effectors (i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants). These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that, in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically upregulated during different stages of the nematodes life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of M. hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors, we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed on, and reproduce on their host plants. Future studies investigating the roles that these proteins play in planta will help mitigate the effects of this damaging pest.

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.