Philippe Castagnone-Sereno

Root‐knot nematode parasitism and host response: molecular basis of a sophisticated interaction

UNLABELLED SUMMARY Taxonomy: Eukaryota; Metazoa; Nematoda; Chromadorea; order Tylenchida; Tylenchoidea; Heteroderidae; genus Meloidogyne. Physical properties: Microscopic-non-segmented worms. Meloidogyne species can reproduce by apomixis, facultative meiotic parthenogenesis or obligate mitotic parthenogenesis. Obligate biotrophic parasites inducing the re-differentiation of plant cells into specialized feeding cells. Hosts: Meloidogyne spp. can infest more than 3000 plant species including vegetables, fruit trees, cereals and ornamental flowers. SYMPTOMS Root swellings called galls. Alteration of the root vascular system. Disease control: Cultural control, chemical control, resistant cultivars. Agronomic importance: Major threat to agriculture in temperate and tropical regions.

The Genomes of Root-Knot Nematodes

Plant-parasitic nematodes are the most destructive group of plant pathogens worldwide and are extremely challenging to control. The recent completion of two root-knot nematode genomes opens the way for a comparative genomics approach to elucidate the success of these parasites. Sequencing revealed that Meloidogyne hapla, a diploid that reproduces by facultative, meiotic parthenogenesis, encodes approximately 14,200 genes in a compact, 54 Mpb genome. Indeed, this is the smallest metazoan genome completed to date. By contrast, the 86 Mbp Meloidogyne incognita genome encodes approximately 19,200 genes. This species reproduces by obligate mitotic parthenogenesis and exhibits a complex pattern of aneuploidy. The genome includes triplicated regions and contains allelic pairs with exceptionally high degrees of sequence divergence, presumably reflecting adaptations to the strictly asexual reproductive mode. Both root-knot nematode genomes have compacted gene families compared with the free-living nematode Caenorhabditis elegans, and both encode large suites of enzymes that uniquely target the host plant. Acquisition of these genes, apparently via horizontal gene transfer, and their subsequent expansion and diversification point to the evolutionary history of these parasites. It also suggests new routes to their control.

Genetic variability of nematodes: a threat to the durability of plant resistance genes?

Plant resistance is currently the most effective and environmentally safe method to control plant parasitic nematodes (PPNs). Resistance genes generally act against sedentary PPNs by inducing a hypersensitive reaction that prevents the parasite installation and/or reproduction. However, the recent emergence of virulent biotypes able to overcome the plant resistance genes may constitute a severe limitation to this control strategy. In selection experiments conducted under controled environment, the genetic variation, specificity and inheritance of nematode virulence have been demonstrated. Moreover, the occurrence of gene-for-gene interactions has been shown in a few cases. Moleculars markers have been extensively used to investigate the genetic variability of PPNs, but so far, the genomic polymorphisms observed are largely independent of virulence. Such data suggest that, within a species, virulent isolates do not share a common origin, but are probably the result of independent mutational events. To understand the molecular mechanisms responsible for virulence in PPNs, several strategies have been developed, in relation with their mode of reproduction (parthenogenesis versus amphimixis). As an example, recent results obtained in our laboratory on the root-knot nematodes Meloidogyne spp. are presented. On a more general point of view, factors that may induce stable genome variability in PPNs, e.g. Transposition of mobile elements and chromosomal rearrangements (leading to polyploidy, aneuploidy, etc) will also be considered. Advances in knowledge in these areas should have important consequences for the management and durability of natural resistance genes, and for the engineering of new forms of resistance.

High‐resolution DNA fingerprinting of parthenogenetic root‐knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root‐knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria, M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root‐knot nematodes.

Diversity and Evolution of Root-Knot Nematodes, Genus Meloidogyne: New Insights from the Genomic Era

Root-knot nematodes (RKNs) (Meloidogyne spp.) are obligate endoparasites of major worldwide economic importance. They exhibit a wide continuum of variation in their reproductive strategies, ranging from amphimixis to obligatory mitotic parthenogenesis. Molecular phylogenetic studies have highlighted divergence between mitotic and meiotic parthenogenetic RKN species and probable interspecific hybridization as critical steps in their speciation and diversification process. The recent completion of the genomes of two RKNs, Meloidogyne hapla and Meloidogyne incognita, that exhibit striking differences in their mode of reproduction (with and without sex, respectively), their geographic distribution, and their host range has opened the way for deciphering the evolutionary significance of (a)sexual reproduction in these parasites. Accumulating evidence suggests that whole-genome duplication (in M. incognita) and horizontal gene transfers (HGTs) represent major forces that have shaped the genome of current RKN species and may account for the extreme adaptive capacities and parasitic success of these nematodes.

Phylogenetic relationships between amphimictic and parthenogenetic nematodes of the genus Meloidogyne as inferred from repetitive DNA analysis

Plant-parasitic nematodes of the genus Meloidogyne are known to reproduce either by cross-fertilization (amphimixis), facultative meiotic parthenogenesis or obligatory mitotic parthenogenesis. Among them, M. incognita, M. arenaria and M. javanica are obligatory mitotic parthenogenetic species, while M. hapla can reproduce by both cross-fertilization and meiotic parthenogenesis. Phylogenetic relationships in this genus have been investigated by hybridization of BamHI-digested genomic DNAs of 18 geographical isolates belonging to six species with three homologous repeated DNA probes cloned at random from a genomic library of one population of M. incognita. Due to the repetitive nature of the probes, the autoradiograms exhibited extensive restriction fragment length polymorphisms (RFLPs) both between and within nematode species. Genetic distance values estimated from hybridization patterns were analysed by two phylogenetic tree-building distance methods, respectively based on constant (UPGMA) and varying (FITCH) rates of nucleotide substitution, and the resulting dendrograms showed a very similar clustering of species and populations. Comparison of these results with the other sources of phylogenetic data available for this genus, i.e. cytogenetic, isoenzymatic and mitochondrial DNA (mtDNA) data, revealed consistency with all but the mtDNA phylogeny. Due to the maternal inheritance of mtDNA, and the parthenogenetic reproductive mode of these organisms, which excludes any possibility of horizontal transfer, we conclude that nuclear DNA phylogeny should represent a more likely evolutionary history of this particular genus, and that interspecific hybridizations between sexual ancestors may account for the results with mtDNA. Thus the early split off of the mitotically parthenogenetic species cluster and M. hapla confirms the amphimictic ancestral mode of reproduction of root-knot nematodes. Moreover, the existence of polymorphism within each species at the repeated DNA level is discussed in relation to the adaptative evolution of these parthenogenetic species.

Genetic variation inMeloidogyne incognita virulence against the tomatoMi resistance gene: evidence from isofemale line selection studies

Resistance to the parthenogenetic root-knot nematodeMeloidogyne incognita is controlled in tomato by the single dominant geneMi, against which virulent pathotypes are able to develop. Isofemale lines (i.e., families) were established from a natural avirulent isolate ofM. incognita in order to study the genetic variability and inheritance of the nematode virulence. From the progeny of individual females, the production of egg masses on the root system of theMi-resistant tomato ‘Piersol’ was analyzed in artificial selection experiments. A family analysis revealed, after two successive generations, a strongly significant variation between the 63 isofemale lines tested, and the results obtained for the mothers and their daughters were also significantly correlated. These results together clearly demonstrate the existence of a genetic variability and inheritance for this character. In a second experiment, a four-generation selection was performed on 31 other isofemale lines. The results revealed a significant response to selection apparently limited only to the two families able to produce, in first generation, a significant minimal egg-mass number on the resistant cultivar.

The pepper resistance genes Me1 and Me3 induce differential penetration rates and temporal sequences of root cell ultrastructural changes upon nematode infection

Abstract In pepper ( Capsicum annuum L.), the resistance genes Me1 and Me3 have been shown to control the main species of root-knot nematodes, Meloidogyne spp. Here, the reactions to inoculation with Meloidogyne incognita second-stage juveniles (J2) of two resistant doubled haploid pepper lines, HDA149 and HDA330, obtained through in vitro androgenesis and carrying Me3 and Me1 respectively, were compared in time-course experiments. Although they both suppressed nematode reproduction, the two resistance genes induced very different response patterns. First, significantly fewer J2 were able to invade roots of HDA149 compared to HDA330. Second, while ultrastructural changes typical to the hypersensitive reaction (HR) occurred in root cells of HDA149 early after nematode inoculation (i.e. necrosis of cells directly involved in nematode penetration and feeding), the resistance mechanism in HDA330 involved a delayed plant response (i.e. cell senescence and death) which took place after the induction of a number of (imperfect) giant cells by the nematode. These differential responses are discussed in relation to the nematode ability to circumvent or not the resistance gene(s), and it is suggested that such virulence could result from the overcoming by the J2 of the early HR occurring within the epidermis and cortex of the root.

Selection forMeloidogyne incognita virulence against resistance genes from tomato and pepper and specificity of the virulence/resistance determinants

Experiments were designed to analyze the relationships between the root-knot nematodeMeloidogyne incognita and resistant tomato and pepper genotypes. From a natural avirulent isolate, near-isogenic nematode lineages were selected with virulence either against the tomatoMi resistance gene or the pepperMe3 resistance gene. Despite the drastic selection pressure used, nematodes appeared unable to overcome the pepperMe1 gene, therefore suggesting some differences in the resistance conferred byMe1 andMe3 in this species. Nematodes virulent onMi-resistant tomatoes were not able to reproduce onMe1-resistant nor onMe3-resistant peppers, and nematodes virulent onMe3-resistant peppers were not able to reproduce onMi-resistant tomatoes nor onMe1-resistant peppers. These results clearly demonstrate the specificity ofM. incognita virulence against resistance genes from both tomato and pepper, and indirectly suggest that gene-for-gene relationships could occur between these two solanaceous crops and the nematode.

Virulence and molecular diversity of parthenogenetic root-knot nematodes, Meloidogyne spp.

Root-knot nematodes (RKN) are sedentary endoparasites causing severe damage to a wide variety of crops, including tomato. Among them, the parthenogenetic species Meloidogyne arenaria, M. incognita and M. javanica are of particular economic importance. The genetic diversity and relationships of 17 populations belonging to these three major species, either avirulent or virulent against the tomato Mi resistance gene, were investigated in order to determine whether (a)virulence of the nematodes could be related to their molecular fingerprints. Genomic polymorphisms between populations were assessed by using amplified fragment length polymorphism (AFLP) markers, and data were treated by means of a multiple correspondence analysis. A total of 1550 polymorphic amplified DNA fragments were identified and used to compute the relationships between the populations. As expected, the three RKN species were clearly distributed into distinct groups, but combination of data for virulence phenotypes and DNA markers showed that clustering of populations was not associated with their (a)virulence against the tomato Mi resistance gene. Such a lack of correlation indicates that most of the observed DNA polymorphism is independent of virulence, which is presumably under host selection. This result demonstrates that virulent populations do not share a common origin, and strongly suggests that they might have appeared late after the establishment of these clonal lineages, as the result of independent mutational events.