What determines host specificity in hyperspecialized plant parasitic nematodes?

BACKGROUND: In hyperspecialized parasites, the ability to grow on a particular host relies on specific virulence factors called effectors. These excreted proteins are involved in the molecular mechanisms of parasitism and distinguish virulent pathogens from non-virulent related species. The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida are major plant-parasitic nematodes developing on numerous solanaceous species including potato. Their close relatives, G. tabacum and G. mexicana are stimulated by potato root diffusate but unable to establish a feeding site on this plant host. RESULTS: RNA sequencing was used to characterize transcriptomic differences among these four Globodera species and to identify genes associated with host specificity. We identified seven transcripts that were unique to PCN species, including a protein involved in ubiquitination. We also found 545 genes that were differentially expressed between PCN and non-PCN species, including 78 genes coding for effector proteins, which represent more than a 6-fold enrichment compared to the whole transcriptome. Gene polymorphism analysis identified 359 homozygous non-synonymous variants showing a strong evidence for selection in PCN species. CONCLUSIONS: Overall, we demonstrated that the determinant of host specificity resides in the regulation of essential effector gene expression that could be under the control of a single or of very few regulatory genes. Such genes are therefore promising targets for the development of novel and more sustainable resistances against potato cyst nematodes.

Exploring the causes of small effective population sizes in cyst nematodes using artificial Globodera pallida populations.

The effective size of a population is the size of an ideal population which would undergo genetic drift at the same rate as the real population. The balance between selection and genetic drift depends on the effective population size ( Ne), rather than the real numbers of individuals in the population ( N). The objectives of the present study were to estimate Ne in the potato cyst nematode Globodera pallida and to explore the causes of a low Ne/ N ratio in cyst nematodes using artificial populations. Using a temporal analysis of 24 independent populations, the median Ne was 58 individuals (min Ne = 25 and max Ne = 228). Ne is commonly lower than N but in the case of cyst nematodes, the Ne/ N ratio was extremely low. Using artificial populations showed that this low ratio did not result from migration, selection and overlapping generations, but could be explain by the fact that G. pallida populations deviate in structure from the assumptions of the ideal population by having unequal sex ratios, high levels of inbreeding and a high variance in family sizes. The consequences of a low Ne, resulting in a strong intensity of genetic drift, could be important for their control because G. pallida populations will have a low capacity to adapt to changing environments.

The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence.

BACKGROUND: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. RESULTS: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. CONCLUSIONS: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.

Heterozygote deficits in cyst plant-parasitic nematodes: possible causes and consequences.

Deviations of genotypic frequencies from Hardy-Weinberg equilibrium (HWE) expectations could reveal important aspects of the biology of populations. Deviations from HWE due to heterozygote deficits have been recorded for three plant-parasitic nematode species. However, it has never been determined whether the observed deficits were due (i) to the presence of null alleles, (ii) to a high level of consanguinity and/or (iii) to a Wahlund effect. The aim of the present work was, while taking into the possible confounding effect of null alleles, to disentangle consanguinity and Wahlund effect in natural populations of those three economically important cyst nematodes using microsatellite markers: Globodera pallida, G. tabacum and Heterodera schachtii, pests of potato, tobacco and sugar beet, respectively. The results show a consistent pattern of heterozygote deficiency in the three nematode species sampled at the spatial scale of the host plant. We demonstrate that the prevalence of null alleles is weak and that heterozygote deficits do not have a single origin. Our results suggested that it is restricted dispersal that leads to heterozygote deficits through both consanguinity and substructure, which effects can be linked to soil movement, cyst density, and the number of generations per year. We discuss potential implications for the durability of plant resistances that are used to protect crops against parasites in which mating between relatives occur. While consanguineous mating leads to homozygosity at all loci, including loci governing avirulence/virulence, which favours the expression of virulence when recessive, the Wahlund effect is expected to have no particular effect on the adaptation of nematodes to resistances.

Genetic diversity of the golden potato cyst nematode Globodera rostochiensis and determination of the origin of populations in Quebec, Canada.

The golden cyst nematode (Globodera rostochiensis), native to South America, has been introduced in many parts of the world, including Europe and North America. Recently, it was found for the first time in the province of Quebec, Canada in the locality of St. Amable near Montreal. To date, very few studies have examined the population genetics of this pest. Consequently, there is a lack of knowledge about the genetic structure and evolution of this nematode. In this study, twelve new microsatellite markers were developed in order to explore these questions. These markers were used to genotype fifteen populations originating from different regions of the world, including five from Canada. Within populations, the highest genetic diversity was consistently observed in the populations from Bolivia, the postulated region of origin of the golden nematode, and the lowest in populations from British Columbia (Canada) and New York (USA). The two Quebec populations were very similar to each other and to the population found in Newfoundland, but surprisingly, they were significantly different from three other North American populations including those from New York and British Columbia. Based on our results, we conclude that the golden cyst nematode has been introduced in North America at least twice from distinct regions of the world.

Evolution and variability of Solanum RanGAP2, a cofactor in the incompatible interaction between the resistance protein GPA2 and the Globodera pallida effector Gp-RBP-1.

BACKGROUND: The Ran GTPase Activating Protein 2 (RanGAP2) was first described as a regulator of mitosis and nucleocytoplasmic trafficking. It was then found to interact with the Coiled-Coil domain of the Rx and GPA2 resistance proteins, which confer resistance to Potato Virus X (PVX) and potato cyst nematode Globodera pallida, respectively. RanGAP2 is thought to mediate recognition of the avirulence protein GP-RBP-1 by GPA2. However, the Gpa2-induced hypersensitive response appears to be relatively weak and Gpa2 is limited in terms of spectrum of efficiency as it is effective against only two nematode populations. While functional and evolutionary analyses of Gp-Rbp-1 and Gpa2 identified key residues in both the resistance and avirulence proteins that are involved in recognition determination, whether variation in RanGAP2 also plays a role in pathogen recognition has not been investigated. RESULTS: We amplified a total of 147 RanGAP2 sequences from 55 accessions belonging to 18 different di-and tetraploid Solanum species from the section Petota. Among the newly identified sequences, 133 haplotypes were obtained and 19.1% of the nucleotide sites were found to be polymorphic. The observed intra-specific nucleotide diversity ranges from 0.1 to 1.3%. Analysis of the selection pressures acting on RanGAP2 suggests that this gene evolved mainly under purifying selection. Nonetheless, we identified polymorphic positions in the protein sequence at the intra-specific level, which could modulate the activity of RanGAP2. Two polymorphic sites and a three amino-acid deletion in RanGAP2 were found to affect the timing and intensity of the Gpa2-induced hypersensitive response to avirulent GP-RBP-1 variants even though they did not confer any gain of recognition of virulent GP-RBP-1 variants. CONCLUSIONS: Our results highlight how a resistance gene co-factor can manage in terms of evolution both an established role as a cell housekeeping gene and an implication in plant parasite interactions. StRanGAP2 gene appears to evolve under purifying selection. Its variability does not seem to influence the specificity of GPA2 recognition but is able to modulate this activity by enhancing the defence response. It seems therefore that the interaction with the plant resistance protein GPA2 (and/or Rx) rather than with the nematode effector was the major force in the evolution of the RanGAP2 locus in potato. From a mechanistic point of view these results are in accordance with a physical interaction of RanGAP2 with GPA2 and suggest that RBP-1 would rather bind the RanGAP2-GPA2 complex than the RanGAP2 protein alone.

The evolution of the Gp-Rbp-1 gene in Globodera pallida includes multiple selective replacements.

The Globodera pallida SPRYSEC Gp-Rbp-1 gene encodes a secreted protein which induces effector-triggered immunity (ETI) mediated by the Solanum tuberosum disease resistance gene Gpa2. Nonetheless, it is not known how the Andes orogeny, the richness in Solanum species found along the Cordillera or the introduction of the nematode into Europe have affected the diversity of Gp-Rbp-1 and its recognition by Gpa2. We generated a dataset of 157 highly polymorphic Gp-Rbp-1 sequences and identified three Gp-Rbp-1 evolutionary pathways: the 'Northern Peru', 'Peru clade I/European' and 'Chilean' paths. These may have been shaped by passive dispersion of the nematode and by climatic variations that have influenced the nature and diversity of wild host species. We also confirmed that, by an analysis of the selection pressures acting on Gp-Rbp-1, this gene has evolved under positive/diversifying selection, but differently among the three evolutionary pathways described. Using this extended sequence dataset, we were able to detect eight sites under positive selection. Six sites appear to be of particular interest because of their predicted localization to the extended loops of the B30.2 domain and/or support by several computational methods. The P/S 187 position was previously identified for its effect on the interaction with GPA2. The functional importance of the other five amino acid polymorphisms observed was investigated using Agrobacterium transient transformation assays. None of these new residues, however, appears to be directly involved in Gpa2-mediated plant defence mechanisms. Thus, the P/S polymorphism observed at position 187 remains the sole variation sufficient to explain the recognition of Gp-Rbp-1 by Gpa2.

Identification and functional characterization of effectors in expressed sequence tags from various life cycle stages of the potato cyst nematode Globodera pallida.

In this article, we describe the analysis of over 9000 expressed sequence tags (ESTs) from cDNA libraries obtained from various life cycle stages of Globodera pallida. We have identified over 50 G. pallida effectors from this dataset using bioinformatics analysis, by screening clones in order to identify secreted proteins up-regulated after the onset of parasitism and using in situ hybridization to confirm the expression in pharyngeal gland cells. A substantial gene family encoding G. pallida SPRYSEC proteins has been identified. The expression of these genes is restricted to the dorsal pharyngeal gland cell. Different members of the SPRYSEC family of proteins from G. pallida show different subcellular localization patterns in plants, with some localized to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localization may reflect diverse functional roles for each individual protein or, more likely, variety in the compartmentalization of plant proteins targeted by the nematode. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.

Identification of plant genes regulated in resistant potato Solanum sparsipilum during the early stages of infection by Globodera pallida.

Using a complementary (c)DNA-amplified fragment length polymorphism (AFLP) approach, we investigated differential gene expression linked to resistance mechanisms during the incompatible potato - Globodera pallida interaction. Expression was compared between a resistant and a susceptible potato clone, inoculated or not inoculated with G. pallida. These clones were issued from a cross between the resistant Solanum sparsipilum spl329.18 accession and the susceptible dihaploid S. tuberosum Caspar H3, and carried, respectively, resistant and susceptible alleles at the resistance quantitative trait loci (QTLs). Analysis was done on root fragments picked up at 4 time points, during a period of 6 days after infection, from penetration of the nematode in the root to degradation of the feeding site in resistant plants. A total of 2560 transcript-derived fragments (TDFs) were analyzed, resulting in the detection of 46 TDFs that were up- or downregulated. The number of TDFs that were up- or downregulated increased with time after inoculation. The majority of TDFs were upregulated at only 1 or 2 time points in response to infection. After isolation and sequencing of the TDFs of interest, a subset of 36 sequences were identified, among which 22 matched plant sequences and 2 matched nematode sequences. Some of the TDFs that matched plant genes showed clear homologies to genes involved in cell-cycle regulation, transcription regulation, resistance downstream signalling pathways, and defense mechanisms. Other sequences with homologies to plant genes of unknown function or without any significant similarity to known proteins were also found. Although not exhaustive, these results represent the most extensive list of genes with altered RNA levels after the incompatible G. pallida-potato interaction that has been published to date. The function of these genes could provide insight into resistance or plant defense mechanisms during incompatible potato-cyst nematode interactions.