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.     

The plant cell inhibitor KRP6 is involved in multinucleation and cytokinesis disruption in giant-feeding cells induced by root-knot nematodes

The plant cell cycle inhibitor gene KRP6 has been investigated in roots infected by plant-parasitic root-knot nematodes (Meloidogyne spp.). Unexpectedly, KRP6 overexpressing lines revealed a distinct role for this specific KRP as an activator of the mitotic cell cycle. This function was confirmed in Arabidopsis thaliana suspension cultures ectopically expressing KRP6. A blockage in the mitotic exit was observed in cell suspensions and in giant cells resulted in the appearance of multi-nucleated cells. KRP6 expression during nematode infection and the similarity in phenotypes among KRP6 overexpressing cell cultures and giant-cell morphology strongly suggest that KRP6 is involved in multinucleation and acytokinesis occurring in giant-cells. Once again nematodes have been shown to manipulate the plant cell cycle machinery in order to promote gall establishment.     

Whole-mount confocal imaging of nuclei in giant feeding cells induced by root-knot nematodes in Arabidopsis

? Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide. ? The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita. ? Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression. ? The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.     

Enhancement of Cylindrocladium crotalariae Root Rot by Meloidogyne arenaria (Race 2) on a Peanut Cultivar Resistant to Both Pathogens

Two populations of Meloidogyne arenaria (race 2, incompatible on peanut) enhanced development of Cylindrocladium black rot (CBR) on CBR-resistant peanut cv. NC 3033 in greenhouse factorial experiments. Nematode populations 256 and 486 (0, 10³, 10⁴ eggs per 15-cm pot) were tested in all combinations with Cylindrocladium crotalariae (0, 0.5, 5, 50 microsclerotia per cm³ of soil). Root-rot index increased in the presence of either population. Positions but not slope values of inoculum density-disease curves were changed by both populations, indicating increased efficiency of microsclerotia when peanuts were grown in the presence of these nematodes. Although little or no reproduction occurred with either nematode population on NC 3033, larvae of 256 and 486 penetrated roots. Meloidogyne arenaria 486 did not induce root galls and was not snccessful in establishing feeding sites. Meloidogyne arenaria 256 produced a few very small eliptical galls and had a range of success in establishing a feeding site, varying from no giant cell development to large giant cell with production of a few eggs.     

Comparative Response of Alfalfa to Pratylenchus penetrans Populations

Four populations of Pratylenchus penetrans did not differ (P > 0.05) in their virulence or reproductive capability on Lahontan alfalfa. There was a negative relationship (r = -0 .7 9 ) between plant survival and nematode inocula densities at 26 ± 3 C in the greenhouse. All plants survived at an inoculum level (Pi) of 1 nematode/cm³ soil, whereas survival rates were 50 to 55% at 20 nematodes/cm³ soil. Alfalfa shoot and root weights were negatively correlated (r = - 0.87; P < 0.05) with nematode inoculum densities. Plant shoot weight reductions ranged from 13 % at Pi 1 nematode/cm³ soil to 69% for Pi 20 nematodes/cm³ soil, whereas root weight reductions ranged from 17% for Pi 1 nematode/cm³ soil to 75% for Pi 20 nematodes/cm³ soil. Maximum and minimum nematode reproduction (Pf/Pi) for the P. penetrans populations were 26.7 and 6.2 for Pi 1 and 20 nematodes/cm³ soil, respectively. There were negative correlations between nematode inoculum densities and plant survival (r = 0.84), and soil temperature and plant survival (r = -0 .7 8 ). Nematode reproduction was positively correlated to root weight (r = 0.89).     

Description of a Unique,Complex Feeding Socket Caused by the Putative Primitive Root-Knot Nematode,Meloidogyne kikuyensis

Meloidogyne kikuyensis produces unique galls that form on one side of the root resembling nitrogen-fixing nodules that are produced on legumes in response to infection by Rhizobium and related bacteria. The gall caused by this root-knot nematode is made up of a complex feeding socket composed of several giant cells that are ramified with xylem vessels extending perpendicular from the vascular cylinder. The anterior portion of the second-stage juvenile, which develops into an adult, plugs into this unique feeding socket. The socket and the surrounding parenchyma together form a gall that is very different in morphology from those typically caused by other species of root-knot nematodes. Even though M. kikuyensis was considered to be a primitive species because of its low chromosome count, the complexity of its feeding site and minor plant damage suggests a more derived systematic position.     

Inheritance of Resistance to Pratylenchus penetrans in Alfalfa

Fifty-two alfalfa (Medicago sativa L.) clones, randomly selected from the cultivar Baker and the experimental line MNGRN-4, were evaluated for resistance (based on nematode reproduction) to Pratylenchus penetrans in growth chamber tests (25 C). Twenty-five clones, representing the range of nematodes and eggs per plant, were selected and retested. Four moderately resistant and two susceptible alfalfa clones were identified. Inheritance of resistance to P. penetrans was studied in these six clones using a diallel mating design. The S₁, Fl, and reciprocal progenies differed for numbers of nematodes and eggs per g dry root and for shoot and root weights (P < 0.05). Resistance, measured as numbers of nematodes in roots, was correlated between parental clones and their S₁ families (r = 0.94), parental clones and their half-sib families (r = 0.81), and S₁ and half-sib families (r = 0.88). General combining ability (GCA) effects were significant for nematode resistance traits. Both GCA and specific combining ability (SCA) effects were significant for plant size traits, but SCA was more important than GCA in predicting progeny plant size. Reciprocal effects were significant for both nematode resistance and plant size traits, which may slow selection progress in long-term selection programs. However, the GCA effects are large enough that breeding procedures that capitalize on additive effects should be effective in developing alfalfa cultivars with resistance to P. penetrans.     

Expression and Regulation of the Arabidopsis thaliana Cel1 Endo 1,4 �� Glucanase Gene During Compatible Plant-Nematode Interactions

The root-knot nematode Meloidogyne incognita is an obligate endoparasite of plant roots and stimulates elaborate modifications of selected root vascular cells to form giant cells for feeding. An Arabidopsis thaliana endoglucanase (Atcel1) promoter is activated in giant cells that were formed in Atcel1::UidA transgenic tobacco and Arabidopsis plants. Activity of the full-length Atcel1 promoter was detected in root and shoot elongation zones and in the lateral root primordia. Different 5’ and internal deletions of regions of the 1,673 bp Atcel1 promoter were each fused to the UidA reporter gene and transformed in tobacco, and roots of the transformants were inoculated with M. incognita to assay for GUS expression in giant cells and noninfected plant tissues. Comparison of the Atcel1 promoter deletion constructs showed that the region between −1,673 and −1,171 (fragment 1) was essential for Atcel1 promoter activity in giant cells and roots. Fragment 1 alone, however, was not sufficient for Atcel1 expression in giant cells or roots, suggesting that cis-acting elements in fragment 1 may function in consort with other elements within the Atcel1 promoter. Root-knot nematodes and giant cells developed normally within roots of Arabidopsis that expressed a functional antisense construct to Atcel1, suggesting that a functional redundancy in endoglucanase activity may represent another level of regulatory control of cell wall-modifying activity within nematode feeding cells.     

Aggressiveness and Damage Potential of Central American and Caribbean Populations of Radopholus spp. in Banana

Monoxenic cultures of burrowing nematode populations extracted from banana roots from Belize, Guatemala, Honduras, and Costa Rica were established on carrot discs. Cultures of Radopholus spp. were also obtained from Florida, Puerto Rico, Dominican Republic, and Ivory Coast. The aggressiveness (defined as reproductive fitness and root necrosis) of these populations was evaluated by inoculating banana plants (Musa AAA, cv. Grande Naine) with 200 nematodes/plant. Banana plants produced by tissue culture were grown in 0.4-liter styrofoam cups, containing a 1:1 mix of a coarse and a fine sand, at ca. 27 °C and 80% RH. Banana plants were acclimated and allowed to grow for 4 weeks prior to inoculation. Plant height, fresh shoot and root weights, root necrosis, and nematode population densities were determined 8 weeks after inoculation. Burrowing-nematode populations varied in aggressiveness, and their reproductive fitness was generally related to damage reported in the field. Plant height and fresh shoot and root weight did not reflect damage caused by nematodes under our experimental conditions. Necrosis of primary roots was closely related to the reproductive fitness of the nematode populations. Variation in aggressiveness among nematode populations followed a similar trend in the two susceptible hosts tested, Grande Naine and Pisang mas. All nematode populations had a low reproductive factor (Rf ≤2.5) in the resistant host except for the Ivory Coast population which had a moderate reproductive factor (Rf ≤ 5) on Pisang Jari Buaya. This is the first report of a burrowing nematode population parasitizing this important source of resistance to R. similis.     

Evolution of Parasitism in Insect-transmitted Plant Nematodes

Nematode-insect associations have evolved many times in the phylum Nematoda, but these lineages involve plant parasitism only in the Secernentean orders Aphelenchida and Tylenchida. In the Aphelenchida (Aphelenchoidoidea), Bursaphelenchus xylophilus (Pine wood nematode), B. cocophilus (Red ring or Coconut palm nematode) (Parasitaphelenchidae), and the many potential host-specific species of Schistonchus (fig nematodes) (Aphelenchoididae) nematode-insect interactions probably evolved independently from dauer-forming, mycophagous ancestors that were phoretically transmitted to breeding sites of their insect hosts in plants. Mycophagy probably gave rise to facultative or obligate plant-parasitism because of opportunities due to insect host switches or peculiarities in host behavior. In the Tylenchida, there is one significant radiation of insect-associated plant parasites involving Fergusobia nematodes (Fergusobiinae: Neotylenchidae) and Fergusonina (Fergusoninidae) flies as mutualists that gall myrtaceous plant buds or leaves. These dicyclic nematodes have different phases that are parasitic in either the insect or the plant hosts. The evolutionary origin of this association is unclear.     

A Plant Health Care Program for Brambles in the Pacific Northwest

Pratylenchus and Xiphinema species have been associated with decline and mortality of brambles (Rubus species) in the Pacific Northwest of the United States. These nematodes cause direct feeding damage and (or) transmit viruses that result in poor fruit quality and plant decline. A nematode management program has been developed by the author to minimize chemical use and nematode-induced damage while optimizing fruit production. Nematode management is an integral part of a total plant health care program in which foliar and soil pests, plant stresses, and fertility are managed.     

Problems and Strategies Associated with Long-term Use of Nematode Resistant Cultivars

Plant-parasitic nematodes are obligate parasites, and planting cultivars that are highly resistant to these organisms places extensive selection pressure on the target species and affects nontarget nematodes as well. Problems encountered with long-term planting of cultivars resistant to nematodes include shifts in nematode races or species and the occurrence of multiple species of nematodes within the same field. These problems can be alleviated to some extent when crop management is used to lessen the selection pressure for change on the nematode populations. Race shifts within populations and possibly shifts between nematode species can be delayed by rotating susceptible cultivars and nonhost crops with resistant cultivars. Nematicides in conjunction with resistant cultivars may be used to limit damage by multiple species of nematodes. Some cultivars have resistance to multiple species of nematodes, but greatly increased research effort is needed in this area. More intensive plant breeding effort will be required to make nematode resistant cultivars competitive in quality and yield with more productive, susceptible cultivars.     

In Vitro Culture and Feeding Behavior of Belonolaimus longicaudatus on Excised Zea mays Roots

A greenhouse population of the sting nematode, Belonolaimus longicaudatus, obtained from an infested golf course in California''s Coachella Valley, was surface-decontaminated and cuhured on excised roots of Zea mays supported by Gamborg''s B5 medium. At 26-27 °C the females laid eggs, and newly emerged juveniles of the second generation completed three molts within 29 days after egg deposition. Sixty days after inoculation with 60 females and 40 males, an average of 529 nematodes and 83 eggs were recovered from the culture. The feeding process consisted of probing, stylet penetration, ingestion, and stylet retraction. Feeding seemed to be necessary before egg deposition or molting occurred. The sting nematode was observed feeding exclusively as an ectoparasite and preferably at the region of cell division and elongation. Vigorous feeding by many nematodes usually caused discoloration of root tips and termination of growth.     

Dynamics of the Nuclear Complement of Giant Cells Induced by Meloidogyne incognita

The total numbers of nuclei in giant cells induced by Meloidogyne incognita in pea, lettuce, tomato, and broad bean were determined. Mature giant cells from pea had the most nuclei per giant cell with a mean of 59 ± 23, lettuce had the fewest with 26 ± 16, and tomato and broad bean were intermediate. The rate of increase in numbers of nuclei for all plant species was greatest during the first 7 days after inoculation. No mitotic activity was observed in giant cells associated with adult nematodes. Number of nuclei per giant cell doubled each day during the period of greatest mitotic activity, but number of total chromosomes per giant cell increased 20-fold per day at the same time. The hypothesis is presented that factor(s) responsible for the polyploid, mulfinucleate condition characteristic of giant cells may be different from factor(s) responsible for aneuploid numbers of chromosome per nucleus or for nuclear aberrations such as the presence of linked nuclei.     

Exploiting cell cycle inhibitor genes of the KRP family to control root‐knot nematode induced feeding sites in plants

Cell cycle control in galls provoked by root‐knot nematodes involves the activity of inhibitor genes like the Arabidopsis ICK/KRP members. Ectopic KRP1, KRP2 and KRP4 expression resulted in decreased gall size by inhibiting mitotic activity, whereas KRP6 induces mitosis in galls. Herein, we investigate the role of KRP3, KRP5 and KRP7 during gall development and compared their role with previously studied members of this class of cell cycle inhibitors. Overexpression of KRP3 and KRP7 culminated in undersized giant cells, with KRP3^OE galls presenting peculiar elongated giant cells. Nuclei in KRP3^OE and KRP5^OE lines presented a convoluted and apparently connected phenotype. This appearance may be associated with the punctuated protein nuclear localization driven by specific common motifs. As well, ectopic expression of KRP3^OE and KRP5^OE affected nematode development and offspring. Decreased mitotic activity in galls of KRP3^OE and KRP7^OE lines led to a reduced gall size which presented distinct shapes – from more elongated like in the KRP3^OE line to small rounded like in the KRP7^OE line. Results presented strongly support the idea that induced expression of cell cycle inhibitors such as KRP3 and KRP7 in galls can be envisaged as a conceivable strategy for nematode feeding site control in crop species attacked by phytopathogenic nematodes.     

Activation of a Pollenin Promoter upon Nematode Infection

Three glycine-rich protein genes of Arabidopsis thaliana (Atgrp-6, Atgrp-7, and Atgrp-8) that correspond to putative genes coding for pollenins (AtolnB;2, AtolnB;3, and AtolnB;4, respectively) are expressed predominantly in the anthers and, more specifically, in the tapetum layer. Tapetal cells are responsible for nutrition of developing pollen grains and show some functional similarities to nematode feeding sites (NFS) induced in plant roots by sedentary parasitic nematodes. The aim of this study was to analyze promoter activity of the Atgrp genes in NFS. Transformed Arabidopsis plants containing a promoter-ß-glucuronidase (gus) fusion of the Atgrp-7 gene were inoculated with the root-knot nematode Meloidogyne incognita and the cyst nematode Heterodera schachtii. GUS assays were performed at different time points after infection. Histochemical analysis revealed an up-regulation of Atgrp-7-gus expression 3 days after inoculation in the feeding sites of both nematodes. Maximal Atgrp-7-gus staining levels in NFS were observed 1 week after nematode infection.     

Molecular Analysis of the Interactions between Cyst Nematodes and Their Hosts

In order to complete its life cycle, a cyst nematode must stimulate the production of a specialized syncytial feeding site within host root tissues. This process is characterized by major changes in local root morphology, including enlargement of affected nuclei and nucleoli, cell wall degradation, and proliferation of subcellular organelles. At the molecular level very little is known about the processes involved in this host response, but recent evidence suggests that cyst nematodes are able to regulate specific host genes. The host-parasite model system provided by Arabidopsis thaliana and Heterodera schachtii will be fundamental to our future understanding of the formation of syncytia. Molecular biology now offers us the opportunity to study this complex host-parasite interaction in great detail. A better understanding of the host genes regulated by cyst nematodes and the mechanisms by which this regulation is achieved will facilitate the engineering of crop cultivars that possess novel forms of resistance to these adept parasites.