Antoine Dalmasso

RPE, a plant gene involved in early developmental steps of nematode feeding cells

Sedentary plant‐parasitic nematodes are able to induce the redifferentiation of root cells into multinucleate nematode feeding sites (NFSs). We have isolated by promoter trapping an Arabidopsis thaliana gene that is essential for the early steps of NFS formation induced by the root‐knot nematode Meloidogyne incognita. Its pattern of expression is similar to that of key regulators of the cell cycle, but it is not observed with the cyst nematode. Later in NFS development, this gene is induced by both root‐knot and cyst nematodes. It encodes a protein similar to the D‐ribulose‐5‐phosphate 3‐epimerase (RPE) (EC 5.1.3.1), a key enzyme in the reductive Calvin cycle and the oxidative pentose phosphate pathway (OPPP). Quantitative RT–PCR showed the accumulation of RPE transcripts in potato, as in Arabidopsis NFS. Homozygous rpe plants have a germination mutant phenotype that can be rescued in dwarf plants on sucrose‐supplemented medium. During root development, this gene is expressed in the meristems and initiation sites of lateral roots. These results suggest that the genetic control of NFSs and the first stages of meristem formation share common steps and confirms the previous cytological observations which indicate that root cells undergo metabolic reprogramming when they turn into NFSs.

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.

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.

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.

Cloning and characterization of two satellite DNAs in the low-C-value genome of the nematode Meloidogyne spp.

Two highly reiterated StyI satellite DNAs have been cloned from two nematode species: one from Meloidogyne hapla and another from M. incognita. The monomeric units of these two satellites have a repeat length of 169 and 295 bp, respectively. These StyI repeated element families constitute 5% of the M. hapla and 2.5% of the M. incognita haploid genomes. The A + T content is elevated in both families (i.e., 68% and 77%, respectively). Nucleotide methylation and transcriptional activity are negative. No similarity was found between the two satellites, nor to other known highly repetitive elements. These StyI satellite DNAs are quite homogenous in sequence, showing on average 3% and 3.5% divergence from their respective calculated consensus sequence. An internal subrepeating unit of about 11 bp is observed in the StyI satellite monomer sequences of M. hapla, suggesting that it could have evolved from a shorter sequence. Because of the small size of the Meloidogyne genome (51 Mb) and the abundance of repeated sequences, this genus approaches a limit in terms of coding fraction.

Transmission hereditaire de la resistance aux nematodes Meloidogyne Chitwood (Tylenchida) portee par 2 lignees de Capsicum annuum L. : etude de descendances homozygotes issues d'androgenese

RÉSUMÉ La lignée PM 217 de Capsicum annuum est résistante à Meloidogyne arenaria, M. incognita, M. javanica et M. sp. (Séville). La lignée PM 687 est résistante aux 3 premières espèces. Le parent-hôte opposé à chacune d’elles, « Yolo-Wonder », présente une résistance à M. javanica. Par androgenèse in vitro, on a obtenu des plantes haploïdes à partir des hybrides FI : PM 217 x « Yolo Wonder » et PM 687 x « Yolo-Wonder ». Chaque descendance autodiploïde a été soumise séparément à l’action de 5 populations-clones de Meloidogyne appartenant aux 4 espèces. Il apparaît que PM 217 porte 2 gènes distincts et, vraisemblablement, indépendants, l’un contrôlant la résistance à M. arenaria, M. incognita et M. javanica, l’autre contrôlant la résistance à M. javanica et M. sp. (Séville). PM 687 porte également 2 gènes, l’un actif sur les 3 principales espèces, à l’exception d’une souche de M. arenaria, l’autre contrôlant cette même souche. Ces 2 gènes sont probablement liés. Ces divers gènes sont distincts par leur spectre d’action et/ou leur mode d’expression au niveau des racines. Les gènes à spectre le plus large, portés par chacun des géniteurs résistants, ne sont pas allèles, mais paraissent liés au même chromosome. « Yolo Wonder » porterait un 5e gène à effet plus limité.

Sequences homologous to Tc(s) transposable elements ofCaenorhabditis elegans are widely distributed in the phylum nematoda

SummaryTo have a better understanding of the evolutionary history of mobile elements within the nematodes, we examined the distribution and the conservation of homologues to transposable elements fromCaenorhabditis elegans (Tc1, Tc2, Tc3, Tc4, Tc5, and FB1) in 19 nematode species belonging to the class Secernentea. Our results show that Tc1 elements display a distribution restricted to the family Rhabditidae with poor conservation. The Tc2 and FB1 homologous elements have the same patchy distribution within the Rhabditidae. They were only found inCaenorhabditis and inTeratorhabditis. The Tc3 element is widely distributed among nematode species. Tc3 homologous elements are present in the majority of the Rhabditidae but also in two genera within the family Panagrolaimidae, and inBursaphelenchus, which belongs to the order Aphelenchida. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited toC. elegans. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4, and Tc5 suggests that horizontal transmission may have occurred between reproductively isolated species during their evolutionary history.

Genetic and Molecular Strategies for the Cloning of (A)Virulence Genes in Sedentary Plant-Parasitic Nematodes

In some cases, crop resistance can be overcome by virulent nematode pathotypes, which are able to reproduce on plant carrying a resistance gene. The understanding of the molecular determinants of (a)virulence in the nematode is essential to the development of new and more durable forms of resistance. In this review will be presented the current strategies designed to identify and isolate (a)virulence genes in cyst and root-knot nematodes, in relation with the mode of reproduction of these two groups.