Nematology The 8 D 05 Parasitism Gene of Meloidogyne incognita Is Required for Successful Infection of Host Roots

Xue, B. , Hamamouch, N. , Li, C. , Huang, G. , Hussey, R. S. , Baum, T. J., and Davis, E. L. 2013. The 8D05 parasitism gene of Meloidogyne incognita is required for successful infection of host roots. Phytopathology 103:175-181. 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. The ability to infect the roots of over 1,700 different plant species has made the root-knot nematodes (Meloidogyne spp.) one of the most successful and devastating parasites of crops found worldwide (5,11,48). Infective second-stage juveniles (J2) completely penetrate plant roots and migrate intercellularly to the root vascular tissue where they induce the formation of localized feeding sites called giant-cells from immature plant vascular cells (33). The giant-cells become the essential, permanent food source for the invading nematode as it assumes a sedentary lifestyle through growth and successive molts towards the pyroform reproductive adult stage. Morphological and molecular studies of giant-cell ontogeny suggest that root-knot nematodes manipulate fundamental plant cell biology to achieve the phenotype of giantcells in such a wide array of plant species (1,4,20,21,34). The nematodes feed from giant-cells using the hollow, protrusible stylet in their head, and they also use the stylet to secrete effector proteins into plant cells to initiate and maintain the feeding site (13,15,45). The secreted effector proteins are encoded by parasitism genes that are specifically and developmentally expressed in three large secretory gland cells within the nematode esophagus (14,15,31). The two subventral esophageal gland cells are active mainly during root penetration and migration by the nematode, and become less active as the single dorsal gland cell grows in size and activity during feeding site initiation and maintenance throughout the parasitic cycle of the biotroph. Investigations to date have identified multiple parasitism genes in the sedentary root-knot and cyst endoparasitic nematodes through analyses of developmentally expressed genes (13,15,45) and secretome prediction from genomic analyses (2,39). Functional analyses of predicted effector molecules encoded by different phytoparasitic nematode parasitism genes have remained a challenge because the nematodes are microscopic biotrophs embedded within plant roots and they are currently not amenable to molecular transformation. The adoption of the model plant species Arabidopsis thaliana (50) for studies of parasitism gene function has, however, provided a wealth of technical and genetic resources on the host side of the plant–nematode interaction. Constitutive expression of individual nematode parasitism genes in transformed plant roots has been used for initial assessments of expressed effector effects on plant phenotype and susceptibility to nematode infection (18,24,25,28,35,41,42,54,56). The utility of the RNA interference (RNAi) technology developed in Caenorhabditis elegans (19) has also provided a powerful tool to specifically silence target phytonematode parasitism genes (46) through assays that either soak infective J2 (3,10) in solutions of doublestranded RNA (dsRNA) or provide dsRNA complementary to target parasitism genes for nematode ingestion from within transgenic plants (27,41,42,51). Yeast two-hybrid screens using single expressed parasitism proteins as baits and total root proteins (expressed from root cDNA libraries) as prey (24,25,28,35, 41,44) have begun to identify specific interactions between nematode effectors and host proteins that appear to manipulate different cellular processes in plants (21,45). Greater than 40 parasitism genes were identified in the rootknot nematode, Meloidogyne incognita, using esophageal gland cell microaspiration and transcript mining assays (29,30). While a number of those root-knot nematode parasitism genes encoded effector proteins like cell wall-modifying enzymes and a few Corresponding author: E. L. Davis; E-mail address: eric_davis@ncsu.edu http://dx.doi. org/10.1094 / PHYTO-07-12-0173-R © 2013 The American Phytopathological Society