Proteins secreted by root-knot nematodes accumulate in the extracellular compartment during root infection

Root-knot nematodes are biotrophic parasites that invade the root apex of host plants and migrate towards the vascular cylinder where they induce the differentiation of root cells into hypertrophied multinucleated giant cells. Giant cells are part of the permanent feeding site required for nematode development into the adult stage. To date, a repertoire of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection has been identified. However, the precise role of these candidate effectors during root invasion or during giant cell induction and maintenance remains largely unknown. Primarily, the identification of the destination of nematode effectors within plant cell compartment(s) is crucial to decipher their actual functions. We analyzed the fine localization in root tissues of five nematode effectors throughout the migratory and sedentary phases of parasitism using an adapted immunocytochemical method that preserves host and pathogen tissues. We showed that secretion of effectors from the amphids or the oesophageal glands is tightly regulated during the course of infection. The analyzed effectors accumulated in the root tissues along the nematode migratory path and along the cell wall of giant cells, showing the apoplasm as an important destination compartment for these effectors during migration and feeding cell formation.Key words: plant pathogen, effector, immunocytochemistry, root-knot nematode, secretion, plant apoplasm     

Identification of genes involved in Meloidogyne incognita-induced gall formation processes in Arabidopsis thaliana

Root-knot nematodes (RKN; Meloidogyne incognita) are phytoparasitic nematodes that cause significant damage to crop plants worldwide. Recent studies have revealed that RKNs disrupt various physiological processes in host plant cells to induce gall formation. However, little is known about the molecular mechanisms of gall formation induced by nematodes. We have previously found that RNA expression levels of some of genes related to micro-RNA, cell division, membrane traffic, vascular formation, and meristem maintenance system were modified by nematode infection. Here we evaluated these genes importance during nematode infection by using Arabidopsis mutants and/or β-glucronidase (GUS) marker genes, particularly after inoculation with nematodes, to identify the genes involved in successful nematode infection. Our results provide new insights not only for the basic biology of plant–nematode interactions but also to improve nematode control in an agricultural setting.     

Enhanced levels of plant cell cycle inhibitors hamper root-knot nematode-induced feeding site development

Root-knot nematodes (RKN) are highly specialized, obligatory plant parasites. These animals reprogram root cells to form large, multinucleate, and metabolically active feeding cells (giant cells) that provide a continuous nutrient supply during 3–6 weeks of the nematode’s life. The establishment and maintenance of physiologically fully functional giant cells are necessary for the survival of these nematodes. As such, giant cells may be useful targets for applying strategies to reduce damage caused by these nematodes, aiming the reduction of their reproduction. We have recently reported the involvement of cell cycle inhibitors of Arabidopsis, named Kip-Related Proteins (KRPs), on nematode feeding site ontogeny. Our results have demonstrated that this family of cell cycle inhibitors can be envisaged to efficiently disrupt giant cell development, based on previous reports which showed that alterations in KRP concentration levels can induce cell cycle transitions. Herein, we demonstrated that by overexpressing KRP genes, giant cells development is severely compromised as well as nematode reproduction. Thus, control of root-knot nematodes by modulating cell cycle-directed pathways through the enhancement of KRP protein levels may serve as an attractive strategy to limit damage caused by these plant parasites.     

A root-knot nematode-secreted protein is injected into giant cells and targeted to the nuclei

Root-knot nematodes (RKNs) are obligate endoparasites that maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors synthesized in the oesophageal glands and injected into the plant tissue through the syringe-like stylet certainly play a central role in these processes. In a search for nematode effectors, we used comparative genomics on expressed sequence tag (EST) datasets to identify Meloidogyne incognita genes encoding proteins potentially secreted upon the early steps of infection. We identified three genes specifically expressed in the oesophageal glands of parasitic juveniles that encode predicted secreted proteins. One of these genes, Mi-EFF1 is a pioneer gene that has no similarity in databases and a predicted nuclear localization signal. We demonstrate that RKNs secrete Mi-EFF1 within the feeding site and show Mi-EFF1 targeting to the nuclei of the feeding cells. RKNs were previously shown to secrete proteins in the apoplasm of infected tissues. Our results show that nematodes sedentarily established at the feeding site also deliver proteins within plant cells through their stylet. The protein Mi-EFF1 injected within the feeding cells is targeted at the nuclei where it may manipulate nuclear functions of the host cell.     

A role for ALF4 during gall and giant cell development in the biotic interaction between Arabidopsis and Meloidogyne spp.

Root‐knot nematodes (RKNs; Meloidogyne spp.) are a major pest for the agriculture worldwide. RKNs induce specialized feeding cells (giant cells, GCs) inside galls which are de novo formed pseudo‐organs in the roots that share similarities with other developmental processes as lateral root (LR) and callus formation or grafting involving new vascular development or pericycle proliferation. Hence, it is pertinent to study the molecular mechanisms directing the plant‐nematode interaction. In this respect, ALF4 is a key gene during LR formation, vascular vessels reconnection in grafting, hormone‐induced callus formation or de novo root organogenesis from leaf explants. Our results show that ALF4 is also induced in galls at early infection stages in an auxin‐independent way. Furthermore, ALF4 activity is necessary for the formation of proper galls and GCs, as the mutant alf4‐1 presents aberrant galls and GCs with severe structural abnormalities leading to a dramatic reduction in the nematode egg production. However, a low‐reproduction rate is maintained, that might be explained by the local auxin maximum build by the nematodes in galls, partially rescuing alf4‐1 phenotype. This would be similar to the partial rescue described for LR formation with exogenous auxins and also agrees with the LR emergence from alf4‐1 galls but not from uninfected roots. In addition, ALF4 is also induced in syncytia formed by cyst nematodes. All these data support a pivotal role for ALF4 during de novo organogenesis processes induced by endoparasitic nematodes, in addition to its role in LR formation, callus development or vessel reconnection during grafting.     

Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins

Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization.The root-knot (Meloidogyne spp.) and cyst (Globodera and Heterodera spp.) nematodes are sedentary endoparasites of the root system in a wide range of plant species. These obligate parasites engage in intricate relationships with their host plants that result in the transformation of normal root cells into specialized feeding sites, which provide the nematodes with all the nutrients required for their development. The initiation and maintenance of functional feeding cells by root-knot nematodes (giant cells) and cyst nematodes (syncytia) seems to be a dynamic process involving active dialogue between the nematodes and their host plants. The nematodes use their stylet, a needle-like apparatus, to deliver effector proteins into the host cells (Williamson and Hussey, 1996; Davis et al., 2004). These effector proteins are mainly synthesized in the nematode esophageal glands, which consist of one dorsal cell and two subventral cells. The activity of these glands is developmentally regulated, with secretions from the two subventral glands being most dynamic during the early stage of infection, consisting of root penetration, migration, and feeding site initiation. Secretions from the single dorsal cell seem to be more active during the sedentary stage of nematode feeding (Hussey and Mims, 1990).Recent progress in the functional characterization of effector proteins from a number of phytonematodes has elucidated diverse mechanisms through which these effectors facilitate the nematode parasitism of host plants. One such mechanism involves depolymerization of the main structural polysaccharide constituents of the plant cell wall by using a diverse collection of extracellular effector proteins (Davis et al., 2011; Wieczorek, 2015). Another mechanism includes the molecular mimicry of host proteins in both form and function (Gheysen and Mitchum, 2011). This strategy could be highly successful when the nematode-secreted effectors imitate host functions to subvert cellular processes in favor of nematodes while escaping the regulation of host cellular processes. Another mechanism of effector action is the modulation of central components of auxin signaling to apparently generate unique patterns of auxin-responsive gene expression, leading to numerous physiological and developmental changes required for feeding site formation and development (Cabrera et al., 2015). In addition, cyst and root-knot nematodes have evolved to efficiently suppress defense responses during their prolonged period of sedentary biotrophic interaction with their hosts. Accordingly, a large number of nematode effectors are engaged in suppressing host immune responses and defense signaling (Hewezi and Baum, 2013; Goverse and Smant, 2014). Finally, there is accumulating evidence that nematode effector proteins target and exploit the host posttranslational machinery to the parasite’s advantage. Posttranslational modifications (PTMs) are tightly controlled and highly specific processes that enable rapid cellular responses to specific stimuli without the requirement of new protein synthesis (Kwon et al., 2006). Phosphorylation, ubiquitination, and histone modifications, among others, have recently been identified as fundamental cellular processes controlling immune signaling pathways (Stulemeijer and Joosten, 2008; Howden and Huitema, 2012; Marino et al., 2012; Salomon and Orth, 2013). This finding underscores the importance of targeting and coopting host posttranslational machinery by pathogen effectors to exert their virulence functions. Here, we review recent progress in the functional characterization of nematode effector proteins and the parasitic strategies that involve modifications of the plant cell wall, molecular mimicry of host factors, alteration of auxin signaling, subversion of defense signaling, and targeting and utilizing the host posttranslational machinery.     

Comparative proteomic analysis of the response of fibrous roots of nematode-resistant and -sensitive sweet potato cultivars to root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

As a major root-knot nematode (RKN), Meloidogyne incognita causes serious losses in the yield of sweet potato (Ipomoea batatas L.). To successfully colonize the host plant, RKNs elicit changes of dramatic physiological and morphological features in the plants. The expression of several genes is regulated as the nematode establishes its feeding site. Therefore, in this study, we analyzed the proteomes in the fibrous roots of sweet potato plants by an infection of RKN to understand the effect of the infection on the plant root regions. This study revealed differences in proteomes of the RKN-resistant sweet potato cultivar Juhwangmi and RKN-sensitive cultivar Yulmi. During plant growth, Juhwangmi plants were shown to be more resistant to M. incognita than Yulmi plants. No M. incognita egg formation was observed in Juhwangmi plants, whereas 587 egg masses were formed in Yulmi plants. Differentially expressed 64 spots were confirmed by proteomic analysis using 2-D gel electrophoresis with three spots up-regulated in the two cultivars during RKN infection. Of these 64 protein spots, 20 were identified as belonging to such different functional categories as the defense response, cell structure, and energy metabolism. This study provides insight into the molecular and biochemical mechanics of the defense response and metabolism of sweet potato plant during nematode invasion. We anticipate that this study will also provide a molecular basis for useful crop breeding and the development of nematode-tolerant plants.     

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called ''giant cells'' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28°C.Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.     

An insight into critical endocycle genes for plant-parasitic nematode feeding sites establishment

Root-knot and cyst nematodes are biotrophic parasites that invade the root apex of host plants and migrate toward the vascular cylinder where they cause the differentiation of root cells into galls (or root-knots) containing hypertrophied multinucleated giant-feeding cells, or syncytia, respectively. The precise molecular mechanisms that drive the formation of such unique nematode feeding sites are still far-off from being completely understood. The diverse gene expression changes occurring within the host cells suggest that both types of plant-parasitic nematodes modulate a variety of plant processes. Induction and repression of genes belonging to the host cell cycle control machinery have shown to be essential to drive the formation of such specialized nematode feeding cells. We demonstrate that nematodes usurp key components regulating the endocycle in their favor. This is illustrated by the involvement of anaphase-promoting complex (APC) genes (CCS52A and CCS52B), the endocycle repressor DP-E2F-like (E2F/DEL1) gene and the ROOT HAIRLESS 1 PROTEIN (RHL1), which is part of a multiprotein complex of the toposiomerase VI, in the proper formation of nematode feeding sites. Altering the expression of these genes in Arabidopsis plants by down- or overexpressing strategies strongly influences the extent of endoreduplication in both types of nematode feeding site leading to a disturbance of the nematode’s life cycle and reproduction.     

Meloidogyne javanica chorismate mutase 1 alters plant cell development

Root-knot nematodes are obligate plant parasites that alter plant cell growth and development by inducing the formation of giant cells for feeding. Nematodes inject secretions from their esophageal glands through their stylet and into plant cells to induce giant cell formation. Meloidogyne javanica chorismate mutase 1 (MjCM-1) is one such esophageal gland protein likely to be secreted from the nematode as giant cells form. MjCM-1 has two domains, an N-terminal chorismate mutase (CM) domain and a C-terminal region of unknown function. It is the N-terminal CM domain of the protein that is the predominant form produced in root-knot nematodes. Transgenic expression of MjCM-1 in soybean hairy roots results in a phenotype of reduced and aborted lateral roots. Histological studies demonstrate the absence of vascular tissue in hairy roots expressing MjCM-1. The phenotype of MjCM-1 expressed at low levels can be rescued by the addition of indole-3-acetic acid (IAA), indicating MjCM-1 overexpression reduces IAA biosynthesis. We propose MjCM-1 lowers IAA by causing a competition for chorismate, resulting in an alteration of chorismate-derived metabolites and, ultimately, in plant cell development. Therefore, we hypothesize that MjCM-1 is involved in allowing nematodes to establish a parasitic relationship with the host plant.     

Physiological and biochemical changes in different sugar beet genotypes infected with root-knot nematode

Ten genotypes of sugar beet plant either monogerm or multigerm seeds were screened under greenhouse conditions for both susceptibility and biochemical reaction to root-knot nematode (RKN) Meloidogyne incognita. All the tested genotypes were susceptible to nematode infection according to the number of root galls and gall indices. All infected genotypes exhibited significant reduction in chlorophyll a, b and carotenoids compared to non-infected ones. The total indole acetic acid and total phenolic compounds contents (mean of both shoot and root) increased significantly in most infected genotypes compared to non-infected genotypes except Disk-01-99 and Monte Rosa as well as LP16 and LP15 genotypes, respectively. Also, total polyamine contents (putrescine, spermidine and spermine) showed significant increases in response to infection with nematodes in all genotypes. The same trend was observed in lipid peroxidation expressed with malondialdehyde content in all tested genotypes. Activities of polyphenol oxidase, peroxidase, superoxide dismutase and catalase enzymes were also induced in most infected genotypes compared with non-infected genotypes. Generally, infection with RKNs induced the appearance of new protein bands at molecular masses 303, 288, 42 and 37?KDa in all infected genotypes. The differentiation in the appearance and/or disappearance of protein bands according to susceptibility to infection reflects the variation between genotypes in defense against infection.     

Influence of Six Varieties of Cynodon on Four Meloidogyne spp.

Two years of giant star grass, Cynodon nlemluensis var. nlemfuensis, in a field plot markedly reduced the incidence of the root-knot nematodes. Tomato planted following the grass showed very little or no root galling and the yield was thrice that of tomato planted on an adjacent field plot previously cropped to tomato. Replicated greenhouse experiments indicated that six varieties of Cynodon were resistant to root-knot nematode but it took up to 6 months of grass growth to appreciably lower the nematode population. The nematodes were eliminated from the soil by all the six grass varieties after 18 months.     

Interrelationships between Root-nodule Bacteria,Plant-parasitic Nematodes and their Leguminous Host

The effect of infection by Meloidogyne javanica and Heterodera trifolii on number, size, structure and efficiency of nodules formed by Rhizobiurn trifolii on white clover roots was investigated. Introduction of nematodes one week before, simultaneously, or one week following inoculation with Rhizobium bacteria did not hinder nodule formation. Nodule size did not differ between nematode-infected and nematode-free plants. Formation of nodules on M. javanica galls and gall formation on the nodules have been reported. The structure of nodular tissues was not disturbed by nematode infection, even though giant cells were formed inside the vascular bundles. The nitrogen-fixation efficiency of nematode-infected nodules was not impaired; however, earlier disintegration of nodules as a result of M. javanica infection ultimately deprived the plants of nitrogenous materials. The drastic reduction of the total-N in H. trifolii-infected plants reflected stunting of the entire plant due to nematode infection. Both nematodes invaded the entire root system, uniformly showing preference for nodules.