Impacting of Root-Knot Nematodes on Tomato: Current Status and Potential Horizons for Its Managing

Root-Knot Nematodes (Meloidogyne spp.) are very serious pathogen on tomato plants among the worldwide. They are widely distributed in soil and causes a highly economical losses for more than 5000 plant species. Therefore, many managements’ strategies are applicable to decrease their effectiveness such as resistant genotypes, soil solarisation and chemical control. Until now, chemical control is the most applied strategy for nematode management. Although nematicides are highly impacted for nematode suppression but environmentally not safety and very toxic. Consequently, several promising studies revealed that root-knot nematode (RKN) can inhibit nematode reproduction based on the susceptibility of their plant host. The plant effectors play a vital role during nematode infection and effect on plant response to nematode requirements. To understand well the relationship between nematode and their host, the molecular and immunolocalization methods illustrated some proteins which are expressed by plant genes involved in plant–nematode interaction. This chapter will focus on the latest status and future perspectives for nematode management

A muscadine locus confers resistance to predominant species of grapevine root-knot nematodes (Meloidogyne spp.) including virulent populations

Root-knot nematodes (RKNs) Meloidogyne spp. are extremely polyphagous pests and four species severely affect grapevines throughout the world: M. arenaria, M. incognita, M. javanica and M. ethiopica. Californian populations of M. arenaria and M. incognita are reported to be virulent to widely used rootstocks and to the rootstock ‘Harmony’ in particular. Breeding RKNs-resistant grape rootstocks is a promising alternative to highly toxic nematicides. Muscadine (Vitis rotundifolia syn. Muscadinia rotundifolia) is a resistance (R) source with undercharacterised genetics. To this end, we used a segregating progeny between the RKN-resistant Vitis x Muscadinia accession ‘VRH8771’ from the muscadine source ‘NC184-4’ and the RKN-susceptible V. vinifera cv. Cabernet-Sauvignon. We first phenotyped its resistance to isolates of the i) M. arenaria, ii) M. incognita and iii) M. javanica species, and then to iv) two mixed Harmony-virulent Californian populations of M. arenaria and M. incognita. Finally, we created an isolate of M. arenaria and M. incognita from these Harmony populations and phenotyped the progeny to each of them [v) and vi)], and to vii) an isolate of M. ethiopica. The resistance phenotype of all the progeny’s individuals was independent of the RKN isolates or populations used. Resistance was mapped in a region of chromosome 18 in VRH8771, supporting the hypothesis that it is conferred by a single gene with an unprecedented wide spectrum in grapevine, including Harmony-virulent isolates. This dominant gene, referred to as MsppR1, is linked to the telomeric QTL XiR4 for X. index resistance from the same source. Additionally, plant mortality data showed that MsppR1-resistant material expressed a high-level resistance to the Harmony-virulent isolates. Our results are a first step towards the development of marker-assisted breeding using SSR and SNP markers for resistance to RKNs in accession VRH8771. © 2023, International Viticulture and Enology Society. All rights reserved

Xiphinema index-resistant grapevine materials derived from muscadine are also resistant to a population of X. diversicaudatum

Grapevine is severely affected by two major nepoviruses that cause grapevine degeneration: the grapevine fanleaf virus (GFLV) and the arabis mosaic virus (ArMV), specifically transmitted by the dagger nematodes Xiphinema index and X. diversicaudatum, respectively. While natural resistance to X. index has been shown to be a promising alternative for controlling X. index and GFLV transmission, the resistance interaction between X. diversicaudatum and grapevine has not yet been documented. In the present study, we evaluated the host suitability to X. diversicaudatum in materials previously characterised for their resistance to X. index. Two X. index-resistant accessions VRH8771 (F1 hybrid) and Nemadex Alain Bouquet (BC1 hybrid) derived from muscadine, together with the X. index-susceptible reference accession V. vinifera cv. Cabernet-Sauvignon and the X. index-resistant reference accession V. riparia ‘10128’, were challenged with a X. diversicaudatum population obtained from woody host plants and a reference isolate of X. index. The reproduction factors of X. diversicaudatum and its numbers per gram of roots paralleled those of X. index, showing a resistance interaction to the population of the former species and suggesting that resistance determinants to both nematode vectors might be the same or linked. Nevertheless, these two criteria illustrated a poorer host suitability of grapevine materials to this X. diversicaudatum population than to X. index

The plant WEE1 kinase is involved in checkpoint control activation in nematode-induced galls

Galls induced by plant‐parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such background, potential DNA damage in the genome of gall cells is eminent. We questioned if DNA damage checkpoints activation followed by DNA repair occurred, or was eventually circumvented, in nematode‐induced galls. Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1‐knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1 were used to understand mechanisms that might cope with DNA damage in galls. Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted to the accumulation of mitotic defects. As well, WEE1‐knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root‐knot nematodes. Together with data on DNA damaging drugs, we suggest a conserved function for WEE1 controlling a G1/S cell cycle arrest in response to replication defect in galls

Le cytosquelette des plantes (une cible pour les nématodes phytoparasites pendant une interaction compatible)

Root-knot nematode (RKN) Meloidogyne species are one of the most important obligate sedentary endoparasites attacking many plants species. They are competent to modify plant root cells by inducing specialized feeding structures. The genera Meloidogyne is capable to induce abnormal changes in selectes root vascular cells to form complex feeding cells (giant cells) that supply nutrients for the nematodes to enlarge, become sedentary and finally developing into fertile adults. Giant cells are hypertrophied multinucleated acytokinetic cells containing a dense metabolically active cytoplasm filled with proliferating organelles and showing an entirely rearranged actin and microtubular cytoskeleton. Therefore, the plant cytoskeleton might be one of the main targets during nematode infection and its rearrangement seems to be important for the successful completion of the nematode s life cycle. In order to find out which role the cytoskeleton plays during the fast isodiametric growth of feeding cells and which cytoskeleton components are involved in the cytoskeletal remodelling of nematode feeding site (NFS), we investigated the distribution of the microtubular cytoskeleton and its behaviour during giant cell development. Immunocytochemical analysis of tubulins as well as in vivo observation of GFP-decorates microtubules (MT) revealed that severe changes of the cytoskeleton occur during feeding cell development. Our results provide evidence that cortical microtubules are dense and seemingly disordered and that endoplasmic MT s probably undergo partial depolymerisation during giant cell ontogeny. On the other hand, large and multiple malformed spindles and phragmoplasts are seen in these giant feeding cells during (incomplete) mitotic events. The rearrangement of the cytoskeleton seems important for the proper initiation and development of galls,. In order to better understand the cytoskeleton rearrangement during feeding cell development we have initiated studies to investigate the involvement of y-tubulins which are required for MT nucleation at MT organizing centers (MTOCs). Our quantitative-RT-PCR analysis revealed that the transcripts of Arabidopsis y-tubulin genes (TUBG1 and TUBG2) and two y-tubulin complex proteins genes (GCP3 and GCP4) are upregulated in NFS. By using y-tubulin mutant lines, we demonstrated that both Arabidopsis y-Tubulins are needed for proper feeding cell development and nematode maturity. Immunocytochemical analyses of nematode infected Arabidopsis roots illustrated that y-tubulin-GFP are abundantly present in giant cells, localized to giant cell phragmoplasts and are associated whith the cortical and cytosolic MTs. Furthermore, the immunofluorescence of y-tubulins in roots of nematode infected mutant lines indicated that y-tubulin proteins (TUBG1 and TUBG2) localize differently in giant cells : TUBG1 is highly concentrated around the nuclear surface of giant cell whereas TUBG2 is mainly distributed throughout the celle cortex. We have generated transgenic plants expressing the y-tubulin GFP protein and in vivo observations of root apical meristem revealed abundant protein distributed throughout the cytoplasm and along spindles during mitosis. In giant cells y-tubulin was distributed mainly around the nuclei. Overexpression of y-tubulin also induced roots to skew and to adopt a twisted phenotype. Treatment with cytoskeleton drugs (propyzamide and oryzalin) showed that the twisted phenotype disappeared and roots grew traight as in wild type. Finally, immunolocalization carried out at the light and electron microscope level demonstrated that GCP3 colocalized with y-tubulin along the nuclear surface and in the giant cell cortex suggesting the presence of thze y-tubulin ring complex (yTuRC) in giant cells. This work suggests the presence of MTOCs in giant cells that might responsible for novel MT nucleation at the cell cortex, around the nuclei and during mitosis in giant-feeding cells induced by roots-knot nematodes. Therefore, y-tubulins may be play an important role in the control of cytoskeleton remodelling in nematode induced feeding cells.Les nématodes à galles du genre Méloidogyne ou root-knot nematodes (RKN) , sont de redoutables endoparasites sédentaires obligatoires de nombreuses espèces végétales. Ils sont capables d engendrer de profondes modifications au niveau des cellules racinaires, en induisant la formation de structures nourricières spécifiques. Ces cellules vasculaires des racines évoluent en cellules nourricières géantes complexes (cellules géantes) qui fournissent les nutriments indispensables à la croissance, la sédentarisation et le développement des nématodes jusqu au stade adulte fertile. Les cellules géantes sont acytokinétiques, multinucléées et hypertrophiées. Leur cytoplasme est dense et présente une intense activité métabolique. Les organites qu il renferme prolifèrent abondamment. L actine et le cytosquelette microtubulaire sont totalement réarrangés. Par conséquent, le cytosquelette de la cellule végétale pourrait être l une des principales cibles d action du nématode au moment de l infection et sa réorganisation semble être une étape majeure pour la réussite du cycle de vie du parasite. Afin de savoir quel rôle joue le cytosquelette lors de la croissance isodiamétrique des cellules nourricières et quels sont les composants du cytosquelette impliqués dans la mise en place des sites d alimentation du nématode ou nematode feeding sites (NFS) , nous avons entrepris l étude de la répartition et du réarrangement des microtubules du cytosquelette tout au long du développement des cellules géantes. L analyse immunocytochimique des tubulines ainsi que l observation in vivo des microtubules (MT) décorées avec le GFP a mis en évidence d importantes modifications du cytosquelette au cours du développement des cellules géantes. Nos résultats montrent clairement que les microtubules corticaux sont denses et apparemment désordonnés et que les MTs endoplasmiques subissent probablement une dépolymérisation partielle au cours de l ontogénèse des cellules géantes. Par ailleurs on observe dans ces cellules de nombreux sites contenant des phragmoplastes non alignés lors d événements mitotiques qui s avèrent également incomplets. Ce réarrangement global du cytosquelette semble important pour l initiation de la formation puis le développement des galles. Afin de mieux comprendre les réarrangements du cytosquelette qui apparaissent lors du développement des cellules nourricières, nous avons entrepris l étude du rôle spécifique des y-tubulines dans la nucléation MT au niveau des centres d organisation MT (MTOCs). L analyse par RT-PCR quantitative a révélé, au niveau des NFS, une surexpression des transcrits des gènes des deux y-tubulines d Arabidopsis (TUBG1 et TUBG2) ainsi que des gènes codant les protéines du complexe y-tubuline (GCP3 et GCP4). En utilisant des lignées mutantes pour les protéines du complexe y-tubuline, nous avons démontré que les deux y-tubulines (TUBG1 et TUBG2) sont nécessaires au bon développement des cellules nourricières et à la maturité des nématodes. Les analyses immunocytochimiques des racines d Arabidopsis infectées par les nématodes ont montré que les y-tubulines GFPs sont abondamment présentes et localisées au niveau des phragmoplastes des cellules géantes et qu elles y sont associées au MTs corticaux et cytosoliques. En outre, l immunofluorescence des y-tubulines de lignées mutantes dans les racines infectées par les nématodes a indiqué que les y-tubulines protéines TUBG1 et TUBG2 n ont pas la même localisation dans les cellules géantes : TUGB1 est fortement concentrée autour de la membrane nucléaire alors que TUBG2 est répartie dans l ensemble du cortex cellulaire. Nous avons généré des plantes transgéniques exprimant la protéine de fusion y-tubuline-GFP et les observations in vivo du méristène apical des racines ont mis en évidence une répartition homogène de la protéine dans tout le cytoplasme, le cortex cellulaire et le long des fuseaux miotiques et des phragmoplastes pendant la mitose. Dans les cellules géantes, la y-tubuline est répartie principalement autour des noyaux avec une répartition régulière en tache. La surexpression de la y-tubuline a également provoqué une croissance incurvée des racines avec apparition d un phénotype en forme de vrille. Le traitement avec des molécules chimiques actives sur le cytosquelette (propyzamide et oryzaline) a entraîné une réversion de ce phénotype vrillé et un retour à une croissance normale des racines, identique au type sauvage. Enfin, l immunolocalisation réalisée en microscopie électronique a démontré que GCP3 est co-localisée avec la y-tubuline autour de la surface nucléaire et dans le cortex de la cellule géante. De ce fait l existence d une complexe de type y-tubulin ring complex (yTuRC) dans les cellules géantes est très vraisemblable. Ce travail suggère que la présence des MTOCs dans les cellules géantes pourrait être responsable d un nouveau type de nucléation MT au niveau du cortex cellulaire, autour des noyaux et pendant la phase de mitose de cellules géantes induites par les nématodes à galles. Par conséquent, les y-tubulines semblent jouer un rôle important dans le contrôle de la restructuration du cytosquelette des cellules géantes induites par les nématodes.NICE-BU Sciences (060882101) / SudocSudocFranceF

An immunocytochemical procedure for protein localization in various nematode life stages combined with plant tissues using methylacrylate-embedded specimens

Plant-parasitic nematodes possess a large number of proteins that are secreted in planta, allowing them to be successful parasites of plants. The majority of these proteins are synthesized mainly in the nematode subventral and dorsal glands as well as in other organs. To improve the immunovisualization of these proteins, we adapted a methacrylate embedding method for the localization of proteins inside nematode tissues, and extracellularly when secreted in planta or within plant cells. An important advantage is that the method is applicable for all nematode stages: preparasitic as well as parasitic stages, including large mature females. Herein, the method has been successfully applied for the localization of four nematode secreted proteins, such as Mi-MAP-1, Mi-CBM2-bearing proteins, Mi-PEL3, and Mi-6D4. In addition, we could also localize 14-3-3 proteins, as well as two cytoskeletal proteins, by double-immunolabeling on preparasitic juveniles. Superior preservation of nematode and plant morphology, allowed more accurate protein localization as compared with other methods. Besides excellent epitope preservation, dissolution of methacrylate from tissue sections unmasks target proteins and thereby drastically increases antibody access

Phytopathology

Breeding for varieties carrying natural resistance (R) against plant-parasitic nematodes is a promising alternative to nematicide ban. In perennial crops, the long plant-nematode interaction increases the risk for R breaking and R durability is a real challenge. In grapevine, the nematode has a high economic impact by transmitting (GFLV) and, to delay GFLV transmission, rootstocks resistant to this vector are being selected, using in particular as an R source. To optimize this strategy, the durability has been studied under controlled conditions in F1 and BC1 muscadine-derived resistant accessions previously obtained from either hardwood-cutting or in vitro propagation. After inoculation with a mix, in equal proportions, of four lines representative of the diversity, multiplication on plants has been monitored 3 to 6 years. The nematode reproduction factor remained lower than 1 in resistant plants obtained from hardwood cuttings while it increased at values far beyond 1 in resistant plants of in vitro origin. Data for nematode numbers per gram of roots mostly paralleled those obtained for the reproduction factor. The effect of the propagation type on resistance over years was also evaluated for the ratio female/juvenile and the frequency of males. Altogether our results illustrate that the muscadine-derived resistance based on hardwood cuttings is durable. By contrast, in resistant and reference accessions obtained from in vitro, our data suggest that the increased nematode multiplication might be mainly due to the modification of root architecture consecutive to this propagation method

Feeding cells induced by phytoparasitic nematodes require gamma-tubulin ring complex for microtubule reorganization

Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound c-tubulin ring complexes. Here we investigate the requirement of c-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that c-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis c-tubulin (TUBG1 and TUBG2) and two c-tubulin complex proteins genes (GCP3 and GCP4) are upregulated in galls. Electron microscopy demonstrates association of GCP3 and c-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both c-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the c-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts