Parasitism proteins in nematode–plant interactions

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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.

Introduction

The root-knot nematodes and cyst nematodes are devastating pathogens of major crops worldwide and remarkable examples of evolution for a parasitic lifestyle [1]. These microscopic worms completely penetrate the zone of elongation in roots of host plants as motile infective second-stage juveniles (J2) and induce dramatic changes in selected root vascular cells (Figure 1) to form elaborate feeding cells (summarized in reference [2]) to permanently supply nutrients for the nematodes to swell, become sedentary, and develop into reproductive adults. The complex changes in nematode feeding cell morphology are accompanied by dramatic changes in gene expression in the affected root cells (reviewed in reference [3]), and most recently, microarray analyses of excised nematode infection sites [4, 5, 6, 7] and feeding cells isolated by laser-capture microdissection [8••] have provided increased resolution of the alterations in plant gene expression throughout nematode feeding cell development.

Since feeding cell formation is localized around the head of the nematode, and conversely, so are defense responses in the presence of resistance genes similar to those identified for other pathogens [9], ‘effector’ molecules must be active at this interface. With the exception of an unidentified factor exuded from J2 of root-knot nematodes that can stimulate Nod factor pathways in legumes [10] and a putative avirulence protein secreted from the amphids (chemosensory openings in the head) of root-knot nematodes [11], the majority of candidate effector molecules identified are proteins encoded by nematode parasitism genes that are secreted through the nematode stylet (protrusible hollow mouth spear) into plant tissues (reviewed in reference [12, 13]). Nematode parasitism proteins originate from three enlarged esophageal gland secretory cells [14], one dorsal and two subventral (Figure 2). Although the phytonematode gland cells function as true animal secretory cells [14] with Golgi-derived membrane-bound secretory granules and subsequent exocytosis of granule contents through specialized valves into the esophageal lumen, parasitism protein secretion via the nematode stylet may be likened to type-III secretion systems of plant-pathogenic bacteria with correspondingly similar effector molecule delivery to recipient host cells via a pilus [15]. Interestingly, the adaptation of enlarged esophageal secretory cells is also present in nematode parasites of vertebrates (reviewed in reference [16]) but absent in non-parasitic nematodes like the microbial-feeding biological model Caenorhabditis elegans. Changes in the content and activity of plant-parasitic nematode esophageal gland cells occur throughout the parasitic cycle (reviewed in reference [17]), with strong activity in the subventral gland cells during nematode penetration and migration in roots and a transition to dominance in dorsal gland cell activity as feeding cells are formed and maintained throughout the sedentary nematode life stages (Figure 2). Antibody probes have confirmed the secretion of nematode esophageal gland contents into plant tissues, and video-enhanced microscopy has demonstrated that secretions from the esophageal gland cells are released following insertion of the nematode stylet through the plant cell wall (Figure 3) and that subsequent ingestion of cytoplasm from feeding cells via the stylet is cyclic (reviewed in references [12, 13, 17]). In each feeding cycle, a small opening carefully formed between the nematode stylet orifice and the host cell plasmalemma retains the integrity of the feeding cell that is essential for the sedentary lifestyle of the obligate parasite (Figure 3). Furthermore, a ‘feeding tube’ is formed by the nematode within the host cell cytoplasm just before each ingestion phase to function as a putative molecular sieve for the stylet orifice (reviewed in reference [17]). The increased metabolic activity of the feeding cells and solute transport via the root vasculature maintain a continual supply of nutrients to sustain the growth and reproduction of the nematode.

The obligate endoparasitic nature of the nematode–plant interaction, the lethality of mutant phytonematode phenotypes, the relatively long life cycle, and the technical difficulties of controlled crosses have challenged genetic studies of nematode parasitism (reviewed in references [1, 13]). Effector molecule identification has, thus, focused on analyses of parasitism proteins and the ‘parasitism genes’ that encode them in the nematode esophageal gland secretory cells (reviewed in references [1, 12, 13]). The ability to stimulate secretion of proteins from the stylet of hatched infective J2 of cyst and root-knot nematodes with neuroactive compounds has promoted proteomic analyses, leading to the isolation of several candidate parasitism genes (reviewed in reference [13]) that appear to function in the early stages of plant parasitism. The lion's share of candidate nematode parasitism genes has been identified through analyses of differential nematode gene expression among parasitic life stages using cDNA–AFLP, subtractive hybridizations, and expressed sequence tag (EST) analyses of cDNA libraries constructed from the microaspirated contents of the esophageal gland cells (reviewed in references [12, 13]). Large-scale EST analyses of multiple parasitic nematode species [18] and the available C. elegans genome now allow comparative genomic analyses [19] to assess genes specific to nematodes, parasitic nematode phylogeny, developmental biology, and putative common parasitic mechanisms. Parasitic nematode ESTs have recently been generated and mined to assess the secretome of several plant-parasitic nematode species [20, 21, 22•, 23]. The genome sequences of the root-knot nematodes, Meloidogyne incognita and M. hapla, and the soybean cyst nematode, Heterodera glycines, will become available within the next 1–2 years (P Abad, C Opperman, and K Lambert, respectively, personal communications) aiding in the identification of the complete repertoire of potential secreted parasitism proteins. Assessing the biological functions of nematode parasitism gene products, however, remains a challenge. This is particularly true among the current parasitism gene candidates since the majority of them encode proteins without described database homologs (reviewed in references [12, 13]). Continual improvements in bioinformatics and the functional analyses described below are, however, providing exciting initial analyses of the function of candidate nematode parasitism proteins.

Section snippets

The walls come tumbling down

The discovery of parasitism genes encoding endoglucanases secreted by phytoparasitic cyst nematodes [24] was the first discovery of endogenous cellulases in animals and among the first reports that support current evidence of lateral gene transfer from prokaryotes to eukaryotes [25••]. Parasitism genes among sedentary and migratory phytonematode species have since been identified that encode a battery of cell-wall-modifying proteins including pectinases, galactosidases, xylanases, and expansins

Altered states

A number of nematode parasitism proteins that induce potential direct modifications of recipient host cells for parasitic benefit have been identified that correlate with observed augmentation of host metabolism, cell cycle, cellular development, and defense response [3, 8••]. A recent report demonstrating that some nematode parasitism proteins have nuclear localization signals that are functional in plant cells [30••] supports the tantalizing scenario that some host cell regulation by

Imitation is the sincerest form of parasitism

The discovery of an expansin-like protein in the secretions of the potato cyst nematode [36], Globodera rostochiensis, was the first report of expansins outside of the plant kingdom and a benchmark example of potential molecular mimicry of host molecules by a phytoparasitic nematode. The Hg-SYV46 parasitism gene expressed in the parasitic stages of the soybean cyst nematode [37] was under analysis for function in planta when a bioinformatic analysis confirmed that the C-terminus of HG-SYV46

At the junction of function

Although the challenges of genetic analyses of phytoparasitic nematode genes and lack of a viable transformation system for them remain, several assays are providing significant functional data on the activity and roles of candidate nematode parasitism proteins. As intimated above, expression of nematode parasitism genes in roots or whole plants provides a measure of potential activity of the encoded products directly in plant tissues. Arabidopsis thaliana provides an exceptional model for such

Conclusions

Several approaches have been fruitful to identify multiple candidate parasitism proteins secreted from phytonematodes, and the identification of additional candidates will most certainly increase as more genomic resources for nematodes are generated. Confirmation of the function of candidate nematode parasitism proteins in nematode–plant interactions and advances in molecular in vivo assays within intact host cells remain key to understanding the evolution and mechanisms of parasitism and hold

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgement

This treatise is dedicated to the memory of our friend and colleague Ling Qin.

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