Integrity of the Post-LRR Domain Is Required for TIR-NB-LRR Function.

Plants trigger appropriate defense responses, notably, through intracellular nucleotide-binding (NB) and leucine-rich repeat (LRR)-containing receptors (NLRs) that detect secreted pathogen effector proteins. In NLR resistance genes, the toll/interleukin-1 receptor (TIR)-NB-LRR proteins (TNLs) are an important subfamily, out of which approximately half the members carry a post-LRR (PL) domain of unknown role. We first investigated the requirement of the PL domain for TNL-mediated immune response by mutating the most conserved amino acids across PL domains of Arabidopsis thaliana TNLs. We identified several amino acids in the PL domain of RPS4, required for its ability to trigger a hypersensitive response to AvrRps4 in a Nicotiana tabacum transient assay. Mutating the corresponding amino acids within the PL domain of the tobacco TNL gene N also affected its function. Consequently, our results indicate that the integrity of the PL domain at conserved positions is crucial for at least two unrelated TNLs. We then tested the PL domain specificity for function by swapping PL domains between the paralogs RPS4 and RPS4B. Our results suggest that the PL domain is involved in their TNL pair specificity, 'off state' stability, and NLR complex activation. Considering genetically paired Arabidopsis TNLs, we finally compared the PL and TIR domains of their sensor and executor sequences, respectively. While TIR and PL domains from executors present complete motifs, sensors showed a lack of conservation with degenerated motifs. We here provide a contribution to the functional analysis of the PL domain in order to decipher its role for TNL function.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Evidence of Sexual Reproduction Events in the Dagger Nematode Xiphinema index in Grapevine Resistance Experiments Under Controlled Conditions.

The dagger nematode Xiphinema index has a major economic impact because of its transmission of Grapevine fanleaf virus to grapevines. This vector nematode, which was introduced into Western countries from the Middle East together with the domesticated grapevine, mostly reproduces by meiotic parthenogenesis, but microsatellite multilocus genotype (MLG) analysis has revealed the occurrence of rare sexual reproduction events in field conditions. In a previous 6-year study under controlled conditions, we evaluated the durability of resistance to X. index in accessions derived from a muscadine resistance source and reference accessions. In this previous study, we used an equal-proportion mixture of four lines (from Spain, Italy, Greece, and Iran) representative of X. index diversity as the inoculum, and we collected random samples in 3-, 4-, 5-, and 6-year-old vines. Here, we genotyped the individuals from these samples using the MLG technique, and we analyzed the changes in line frequency and the occurrence of sexual reproduction events between lines over time. The nematode lines differed in aggressiveness and hybrids between lines were detected at a low, but apparently increasing rate. Hybridization events were recovered in all accessions, regardless of resistance status and propagation type. Finally, our data provide the first evidence of sexual reproduction in the nematode X. index under controlled conditions.

Characterization of genetic determinants of the resistance to phylloxera, Daktulosphaira vitifoliae, and the dagger nematode Xiphinema index from muscadine background.

BACKGROUND: Muscadine (Muscadinia rotundifolia) is known as a resistance source to many pests and diseases in grapevine. The genetics of its resistance to two major grapevine pests, the phylloxera D. vitifoliae and the dagger nematode X. index, vector of the Grapevine fanleaf virus (GFLV), was investigated in a backcross progeny between the F1 resistant hybrid material VRH8771 (Vitis-Muscadinia) derived from the muscadine R source 'NC184-4' and V. vinifera cv. 'Cabernet-Sauvignon' (CS). RESULTS: In this pseudo-testcross, parental maps were constructed using simple-sequence repeats markers and single nucleotide polymorphism markers from a GBS approach. For the VRH8771 map, 2271 SNP and 135 SSR markers were assembled, resulting in 19 linkage groups (LG) and an average distance between markers of 0.98 cM. Phylloxera resistance was assessed by monitoring root nodosity number in an in planta experiment and larval development in a root in vitro assay. Nematode resistance was studied using 10-12 month long tests for the selection of durable resistance and rating criteria based on nematode reproduction factor and gall index. A major QTL for phylloxera larval development, explaining more than 70% of the total variance and co-localizing with a QTL for nodosity number, was identified on LG 7 and designated RDV6. Additional QTLs were detected on LG 3 (RDV7) and LG 10 (RDV8), depending on the in planta or in vitro experiments, suggesting that various loci may influence or modulate nodosity formation and larval development. Using a Bulked Segregant Analysis approach and a proportion test, markers clustered in three regions on LG 9, LG 10 and LG 18 were shown to be associated to the nematode resistant phenotype. QTL analysis confirmed the results and QTLs were thus designated respectively XiR2, XiR3 and XiR4, although a LOD-score below the significant threshold value was obtained for the QTL on LG 18. CONCLUSIONS: Based on a high-resolution linkage map and a segregating grapevine backcross progeny, the first QTLs for resistance to D. vitifoliae and to X. index were identified from a muscadine source. All together these results open the way to the development of marker-assisted selection in grapevine rootstock breeding programs based on muscadine derived resistance to phylloxera and to X. index in order to delay GFLV transmission.

Grapevine Resistance to the Nematode Xiphinema index Is Durable in Muscadine-Derived Plants Obtained from Hardwood Cuttings but Not from In Vitro.

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 Xiphinema index has a high economic impact by transmitting Grapevine fanleaf virus (GFLV) and, to delay GFLV transmission, rootstocks resistant to this vector are being selected, using Muscadinia rotundifolia in particular as an R source. To optimize in fine 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 X. index 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.

New Data Completing the Spectrum of the Ma, RMia, and RMja Genes for Resistance to Root-Knot Nematodes (Meloidogyne spp.) in Prunus.

Root-knot nematodes (RKN) (Meloidogyne spp.) are worldwide pests that affect a considerable number of plants, among which stone fruit (Prunus spp.) are severely attacked. Prevalent RKN species are Meloidogyne arenaria, M. incognita, and M. javanica in stone fruit but the emergent M. ethiopica and M. enterolobii are also reported to challenge perennial crops. In Prunus spp., the complete-spectrum resistance (R) gene Ma from plum and the more restricted-spectrum R genes RMia from peach and RMja from almond completely inhibit nematode multiplication and gall formation of the RKN species that they control. This study aimed to update the resistance spectra of these three major genes by evaluating their activity toward one isolate of the yet-untested RKN species mentioned above. To state whether a given gene controls a particular species, the principle of our experiment was to genotype with appropriate markers a number of individuals segregating for this gene and then to phenotype these individuals. A perfect matching of the genotype and the phenotype of individuals indicates that the gene of interest is active against and, thus, controls the corresponding isolate of this RKN species. Segregating materials used were an Ma F1 plum progeny, an RMia F2 peach progeny, and an RMja F2 almond progeny. In addition to previous data, our results establish a clear spectrum for each of the three genes toward isolates from both the three prevalent species and the two emerging species. Ultimately, our results reveal that (i) Ma controls all of them, (ii) RMja controls all species except M. incognita and M. floridensis, and (iii) RMia controls M. arenaria, M. incognita, and M. ethiopica but not M. javanica or M. enterolobii. Our data should have wide implications for RKN resistance management and breeding and for deciphering the molecular mechanisms of the spectrum of RKN R genes.

TNL genes in peach: insights into the post-LRR domain.

BACKGROUND: Plants develop sustainable defence responses to pathogen attacks through resistance (R) genes contributing to effector-triggered immunity (ETI). TIR-NB-LRR genes (TNL genes) constitute a major family of ETI R genes in dicots. The putative functions or roles of the TIR, NB and LRR domains of the proteins they encode (TNLs) are well documented, but TNLs also have a poorly characterised C-terminal region, the function of which is unknown in most cases. We characterised this prevalent stress-response protein family in a perennial plant, using the genome of peach (Prunus persica), the model Prunus species. The first TNL gene from this genus to be cloned, the Ma gene, confers complete-spectrum resistance to root-knot nematodes (RKNs) and encodes a protein with a huge C-terminal region with five duplicated post-LRR (PL) domains. This gene was the cornerstone of this study. RESULTS: We investigated the role of this C-terminal region, by first describing the frequency, distribution and structural characteristics of i) TNL genes and ii) their PL domains in the peach genome, using the v1.0 Sanger sequence together with the v2.0 sequence, which has better genome annotation due to the incorporation of transcriptomic data. We detected 195 predicted TNL genes from the eight peach chromosomes: 85 % of these genes mapped to chromosomes 1, 2, 7 and 8. We reconstructed the putative structure of the predicted exons of all the TNL genes identified, and it was possible to retrieve the PL domains among two thirds of the TNL genes. We used our predicted TNL gene sequences to develop an annotation file for use with the Gbrowse tool in the v2.0 genome. The use of these annotation data made it possible to detect transcribed PL sequences in two Prunus species. We then used consensus sequences defined on the basis of 124 PL domains to design specific motifs, and we found that the use of these motifs significantly increased the numbers of PL domains and correlative TNL genes detected in diverse dicot genomes. Based on PL signatures, we showed that TNL genes with multiple PL domains were rare in peach and the other plants screened. The five-PL domain pattern is probably unique to Ma and its orthologues within Prunus and closely related genera from the Rosaceae and was probably inherited from the common ancestor of these plants in the subfamily Spiraeoideae. CONCLUSIONS: The first physical TNL gene map for Prunus species can be used for the further investigation of R genes in this genus. The PL signature motifs are a complementary tool for the detection of TNL R genes in dicots. The low degree of similarity between PL domains and the neighbouring LRR exons and the specificity of PL signature motifs suggest that PL and LRR domains have different origins, with PL domains being specific to TNL genes, and possibly essential to the functioning of these genes in some cases. Investigations of the role of the oversized Ma PL region, in ligand binding or intramolecular interactions for example, may help to enrich our understanding of NB-LRR-mediated plant immunity to RKNs.

Resistance to root-knot nematodes Meloidogyne spp. in woody plants.

I. 42 II. 43 III. 44 IV. 47 V. 49 VI. 50 VII. 50 VIII. 50 IX. 52 52 References 52 SUMMARY: Root-knot nematodes (RKNs) Meloidogyne spp. cause major damage to cultivated woody plants. Among them, Prunus, grapevine and coffee are the crops most infested by worldwide polyphagous species and species with a more limited distribution and/or narrower host range. The identification and characterization of natural sources of resistance are important steps to develop RKN control strategies. In woody crops, resistant rootstocks genetically different from the scion of agronomical interest may be engineered. We describe herein the interactions between RKNs and different woody crops, and highlight the plant species in which resistance and corresponding resistance (R) genes have been discovered. Even though grapevine and, to a lesser extent, coffee have a history of rootstock selection for RKN resistance, few cases of resistance have been documented. By contrast, in Prunus, R genes with different spectra have been mapped in plums, peach and almond and can be pyramided for durable resistance in interspecific rootstocks. We particularly discuss here the Ma Toll/interleukin-1 receptor-like-nucleotide binding-leucine-rich repeat gene from Myrobalan plum, one of the longest plant R genes cloned to date, due to its unique biological and structural properties. RKN R genes in Prunus will enable us to carry out molecular studies aimed at improving our knowledge of plant immunity in woody plants.

Identification of Meloidogyne Species from the Central Valley of Chile and Interaction with Stone Fruit Rootstocks.

Root-knot nematodes (RKN; Meloidogyne spp.) are predominant polyphagous pests of crops in the Central Valley of Chile. Twenty RKN populations from this region were collected from diverse crops and subsequently identified with both sequence-characterized amplified region and isoenzyme markers. Populations included three RKN species: Meloidogyne ethiopica (75%), M. javanica (15%), and M. arenaria (10%). This is the first report of the high prevalence and wide host range of M. ethiopica in Chile. The host status of three Prunus rootstocks for isolates obtained from the Chilean RKN populations was then evaluated. Rootstocks assessed included the peach rootstock Nemaguard and the plum rootstock Marianna 2624, both previously considered resistant, and the peach rootstock Pomona as a susceptible accession. In the first experiment, rootstocks were inoculated individually in pots with 10,000 second-stage juveniles and eggs of each isolate, and reproduction and galling were evaluated 5 months after inoculation. In the second experiment, the six most aggressive RKN isolates were used in a mixture to evaluate the host response of the same three rootstocks. No RKN were detected on Marianna 2624 in both experiments, which confirmed its immune host status to M. arenaria and M. javanica; this is the first report of immunity to M. ethiopica. Even though Pomona was classified overall as susceptible, the response of this rootstock to the RKN isolates was highly variable and ranged from susceptible to resistant, depending on the RKN isolates. Nemaguard ranged from resistant (to each of the most aggressive isolates and to their mixture) to highly resistant (to M. arenaria isolates). Our results illustrate that Prunus rootstocks express different levels of resistance to RKN species. RKN resistance may be active either at the isolate level (as in Pomona), at the species level (as in Nemaguard toward M. javanica and M. arenaria), or at the genus level (as in Marianna 2624).

Multiyear evaluation of the durability of the resistance conferred by Ma and RMia genes to Meloidogyne incognita in Prunus under controlled conditions.

Root-knot nematodes (RKNs) (Meloidogyne spp.) are highly polyphagous pests that parasitize Prunus crops in Mediterranean climates. Breeding for RKN-resistant Prunus cultivars, as an alternative to the now-banned use of nematicides, is a real challenge, because the perennial nature of these trees increases the risk of resistance breakdown. The Ma plum resistance (R) gene, with a complete spectrum, and the RMia peach R gene, with a more restricted spectrum, both provide total control of Meloidogyne incognita, the model parthenogenetic species of the genus and the most important RKN in terms of economic losses. We investigated the durability of the resistance to this nematode conferred by these genes, comparing the results obtained with those for the tomato Mi-1 reference gene. In multiyear experiments, we applied a high and continuous nematode inoculum pressure by cultivating nematode-infested susceptible tomato plants with either Prunus accessions carrying Ma or RMia R genes, or with resistant tomato plants carrying the Mi-1 gene. Suitable conditions for Prunus development were achieved by carrying out the studies in a glasshouse, in controlled conditions allowing a short winter leaf fall and dormancy. We first assessed the plum accession 'P.2175', which is heterozygous for the Ma gene, in two successive 2-year evaluations, for resistance to two M. incognita isolates. Whatever the isolate used, no nematodes reproducing on P.2175 were detected, whereas galls and nematodes reproducing on tomato plants carrying Mi-1 were observed. In a second experiment with the most aggressive isolate, interspecific full-sib material (P.2175 × ['Garfi' almond × 'Nemared' peach]), carrying either Ma or RMia (from Nemared) or both (in the heterozygous state) or neither of these genes, was evaluated for 4 years. No virulent nematodes developed on Prunus spp. carrying R genes, whereas galling and virulent individuals were observed on Mi-1-resistant tomato plants. Thus, the resistance to M. incognita conferred by Ma in Prunus material in both a pure-plum and an interspecific genetic background, or by RMia in an interspecific background, appears to be durable, highlighting the value of these two genes for the creation of Prunus rootstock material.

The Ma gene for complete-spectrum resistance to Meloidogyne species in Prunus is a TNL with a huge repeated C-terminal post-LRR region.

Root-knot nematode (RKN) Meloidogyne species are major polyphagous pests of most crops worldwide, and cultivars with durable resistance are urgently needed because of nematicide bans. The Ma gene from the Myrobalan plum (Prunus cerasifera) confers complete-spectrum, heat-stable, and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato (Solanum lycopersicum), the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous state in the P.2175 accession. High-resolution mapping totaling over 3,000 segregants reduced the Ma locus interval to a 32-kb cluster of three Toll/Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat (LRR) genes (TNL1-TNL3), including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma, as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in Agrobacterium rhizogenes-transformed hairy roots and composite plants. The full-length cDNA (2,048 amino acids) of Ma is the longest of all Resistance genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1,088 amino acids) comprising five repeated carboxyl-terminal PL exons with two conserved motifs. The amino-terminal region (213 amino acids) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 amino acids) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains, which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signaling.

Control of Xiphinema index populations by fallow plants under greenhouse and field conditions.

The dagger nematode Xiphinema index has a high economic impact in vineyards by direct pathogenicity and above all by transmitting the Grapevine fanleaf virus (GFLV). Agrochemicals have been largely employed to restrict the spread of GFLV by reducing X. index populations but are now banned. As an alternative to nematicides, the use of fallow plants between two successive vine crops was assessed. We selected plant species adapted to vineyard soils and exhibiting negative impact on nematodes and we evaluated their antagonistic effect on X. index in greenhouse using artificially infested soil, and in naturally infested vineyard conditions. The screening was conducted with plants belonging to the families Asteraceae (sunflower, marigold, zinnia, and nyjer), Poaceae (sorghum and rye), Fabaceae (white lupin, white melilot, hairy vetch, and alfalfa), Brassicaceae (rapeseed and camelina), and Boraginaceae (phacelia). In the greenhouse controlled assay, white lupin, nyjer, and marigold significantly reduced X. index populations compared with that of bare soil. The vineyard assay, designed to take into account the aggregative pattern of X. index distribution, revealed that marigold and hairy vetch are good candidates as cover crops to reduce X. index populations in vineyard. Moreover, this original experimental design could be applied to manage other soilborne pathogens.

Histological mechanisms of the resistance conferred by the Ma gene against Meloidogyne incognita in Prunus spp.

The Ma gene from Myrobalan plum is a TNL gene that confers a high-level resistance to all root-knot nematodes of major economic importance, including Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii. The nematode behavior in the roots and the corresponding histological mechanisms of the Ma resistance to M. incognita in the resistant (R) accessions of the plum 'P.2175' and the interspecific hybrid P.2175×almond-peach '35', carrying the Ma1 allele (Ma1/ma), were characterized in comparison with the susceptible (S) accessions in the plum 'P.2032' and the interspecific hybrid P.2175×almond-peach '253' (ma/ma). Second-stage juveniles (J2s) were inoculated in micropropagated plantlets grown in soil substrate under controlled conditions at 25°C. Nematodes penetrated both R and S plants preferentially along the apical zone or close to the young lateral buds and moved via similar routes. Then they migrated into the cortex downward in the direction of the apex and turned up in the meristematic apical region to colonize the differentiating stele. In R accessions, motile J2s neither swelled nor developed into J3s, and initiation of feeding sites was never observed. This complete absence of gall symptoms is associated with cell necroses and corresponding hypersensitive-like reaction (HLR) phenotypes occurring either in the stele or in the meristematic apical region or in the cortex. Nematode attacks often disorganized the meristematic apical tissues of R accessions, which induced the development of subterminal lateral roots replacing primary terminal apices and, thus, provided an active resistance reaction to HLR damage.

Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants.

Resistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum × almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed.

Genetic signature of amphimixis allows for the detection and fine scale localization of sexual reproduction events in a mainly parthenogenetic nematode.

Asexuality is an important mode of reproduction in eukaryotic taxa and has a theoretical advantage over sexual reproduction because of the increased ability to propagate genes. Despite this advantage, hidden signs of cryptic sex have been discovered in the genomes of asexual organisms. This has provided an interesting way to address the evolutionary impact of sex in plant and animal populations. However, the identification of rare sexual reproduction events in mainly asexual species has remained a challenging task. We examined the reproductive history in populations of the plant parasitic nematode Xiphinema index by genotyping individuals collected from six grapevine fields using seven microsatellite markers. A high level of linkage disequilibrium and heterozygous excess suggested a clonality rate of 95-100%. However, we also detected rare sexual reproduction events within these highly clonal populations. By combining highly polymorphic markers with an appropriate hierarchical sampling, and using both Bayesian and multivariate analysis with phylogenetic reconstructions, we were able to identify a small number of sexually produced individuals at the overlapping zones between different genetic clusters. This suggested that sexual reproduction was favoured when and where two nematode patches came into contact. Among fields, a high degree of genetic differentiation indicated a low level of gene flow between populations. Rare genotypes that were shared by several populations suggested passive dispersal by human activities, possibly through the introduction of infected plants from nurseries. We conclude that our method can be used to detect and locate sexual events in various predominantly asexual species.

Transmission competency of single-female Xiphinema index lines for Grapevine fanleaf virus.

Grapevine fanleaf virus (GFLV) is vectored specifically from grapevine to grapevine by the ectoparasitic nematode Xiphinema index. Limited information is available on the vector competency of X. index populations from diverse geographical origins. We determined the transmissibility of two GFLV strains showing 4.6% amino acid divergence within their coat protein (e.g., strains F13 and GHu) by seven clonal lines of X. index developed from seven distinct populations from the Mediterranean basin (Cyprus, southern France, Israel, Italy, and Spain), northern France, and California. X. index lines derived from single adult females were produced on fig (Ficus carica) plants to obtain genetically homogenous aviruliferous clones. A comparative reproductive rate analysis on Vitis rupestris du Lot and V. vinifera cv. Cabernet Sauvignon showed significant differences among clones, with the single-female Cyprus line showing the highest rate (30-fold the initial population) and the Spain and California lines showing the lowest rate (10-fold increase), regardless of the grapevine genotype. However, there was no differential vector competency among the seven X. index lines for GFLV strains F13 and GHu. The implications of our findings for the dynamic of GFLV transmission in vineyards and screening of Vitis spp. for resistance to GFLV are discussed.

Phylogeography of the soil-borne vector nematode Xiphinema index highly suggests Eastern origin and dissemination with domesticated grapevine.

The soil-borne nematode Xiphinema index is closely linked to its main host, the grapevine, and presents a major threat to vineyards worldwide due to its ability to transmit Grapevine fanleaf virus (GFLV). The phylogeography of X. index has been studied using mitochondrial and microsatellite markers in samples from most regions of its worldwide distribution to reveal its genetic diversity. We first used the mitochondrial marker CytB and illustrated the low intraspecific divergence of this mainly meiotic parthenogenetic species. To generate a higher polymorphism level, we then concatenated the sequences of CytB and three mitochondrial markers, ATP6, CO1 and ND4, to obtain a 3044-bp fragment. We differentiated two clades, which each contained two well-supported subclades. Samples from the eastern Mediterranean and the Near and Middle East were grouped into three of these subclades, whereas the samples from the western Mediterranean, Europe and the Americas all belonged to the fourth subclade. The highest polymorphism level was found in the samples of one of the Middle and Near East subclades, strongly suggesting that this region contained the native area of the nematode. An east-to-west nematode dissemination hypothesis appeared to match the routes of the domesticated grapevine during Antiquity, presumably mainly dispersed by the Greeks and the Romans. Surprisingly, the samples of the western subclade comprised only two highly similar mitochondrial haplotypes. The first haplotype, from southern Iberian Peninsula, Bordeaux and Provence vineyards, exhibited a high microsatellite polymorphism level that suggests introductions dating from Antiquity. The second haplotype contained a highly predominant microsatellite genotype widespread in distant western countries that may be a consequence of the massive grapevine replanting following the 19th-century phylloxera crisis. Finally, our study enabled us to draw a first scaffold of X. index diversity at the global scale.

The large repertoire of conifer NLR resistance genes includes drought responsive and highly diversified RNLs.

The NLRs or NBS-LRRs (nucleotide-binding, leucine-rich-repeat) form the largest resistance gene family in plants, with lineage-specific contingents of TNL, CNL and RNL subfamilies and a central role in resilience to stress. The origin, evolution and distribution of NLR sequences has been unclear owing in part to the variable size and diversity of the RNL subfamily and a lack of data in Gymnosperms. We developed, searched and annotated transcriptomes assemblies of seven conifers and identified a resource of 3816 expressed NLR sequences. Our analyses encompassed sequences data spanning the major groups of land plants and determinations of NLR transcripts levels in response to drought in white spruce. We showed that conifers have among the most diverse and numerous RNLs in tested land plants. We report an evolutionary swap in the formation of RNLs, which emerged from the fusion of an RPW8 domain to a NB-ARC domain of CNL. We uncovered a quantitative relationship between RNLs and TNLs across all land plants investigated, with an average ratio of 1:10. The conifer RNL repertoire harbours four distinct groups, with two that differ from Angiosperms, one of which contained several upregulated sequences in response to drought while the majority of responsive NLRs are downregulated.

Ma Orthologous Genes in Prunus spp. Shed Light on a Noteworthy NBS-LRR Cluster Conferring Differential Resistance to Root-Knot Nematodes.

Root-knot nematodes (RKNs) are considerable polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on the NBS-LRR genes that are pivotal factors for pathogens control. In Prunus spp., the Ma plum and RMja almond genes possess different spectra for resistance to RKNs. While previous works based on the Ma gene allowed to clone it and to decipher its peculiar TIR-NBS-LRR (TNL) structure, we only knew that the RMja gene mapped on the same chromosome as Ma. We carried out a high-resolution mapping using an almond segregating F2 progeny of 1448 seedlings from resistant (R) and susceptible (S) parental accessions, to locate precisely RMja on the peach genome, the reference sequence for Prunus species. We showed that the RMja gene maps in the Ma resistance cluster and that the Ma ortholog is the best candidate for RMja. This co-localization is a crucial step that opens the way to unravel the molecular determinants involved in the resistance to RKNs. Then we sequenced both almond parental NGS genomes and aligned them onto the RKN susceptible reference peach genome. We produced a BAC library of the R parental accession and, from two overlapping BAC clones, we obtained a 336-kb sequence encompassing the RMja candidate region. Thus, we could benefit from three Ma orthologous regions to investigate their sequence polymorphism, respectively, within plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum). We showed that the Ma TNL cluster has evolved orthologs with a unique conserved structure comprised of five repeated post-LRR (PL) domains, which contain most polymorphism. In addition to support the Ma and RMja orthologous relationship, our results suggest that the polymorphism contained in the PL sequences might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. Besides, our study illustrates how PL exon duplications and losses shape TNL structure and give rise to atypical PL domain repeats of yet unknown role.

BRC4Env, a network of Biological Resource Centres for research in environmental and agricultural sciences.

The Biological Resource Centre for the Environment BRC4Env is a network of Biological Resource Centres (BRCs) and collections whose leading objectives are to improve the visibility of genetic and biological resources maintained by its BRCs and collections and to facilitate their use by a large research community, from agriculture research to life sciences and environmental sciences. Its added value relies on sharing skills, harmonizing practices, triggering projects in comparative biology, and ultimately proposing a single-entry portal to facilitate access to documented samples, taking into account the partnership policies of research institutions as well as the legal frame which varies with the biological nature of resources. BRC4Env currently includes three BRCs: the Centre for Soil Genetic Resources of the platform GenoSol, in partnership with the European Conservatory of Soil Samples; the Egg Parasitoids Collection (EP-Coll); and the collection of ichthyological samples, Colisa. BRC4Env is also associated to several biological collections: microbial consortia (entomopathogenic bacteria, freshwater microalgae…), terrestrial arthropods, nematodes (plant parasitic, entomopathogenic, animal parasitic...), and small mammals. The BRCs and collections of BRC4Env are involved in partnership with academic scientists, as well as private companies, in the fields of medicinal mining, biocontrol, sustainable agriculture, and additional sectors. Moreover, the staff of the BRCs is involved in many training courses for students from French licence degree to Ph.D, engineers, as well as ongoing training.

The Transcriptomes of Xiphinema index and Longidorus elongatus Suggest Independent Acquisition of Some Plant Parasitism Genes by Horizontal Gene Transfer in Early-Branching Nematodes.

Nematodes have evolved the ability to parasitize plants on at least four independent occasions, with plant parasites present in Clades 1, 2, 10 and 12 of the phylum. In the case of Clades 10 and 12, horizontal gene transfer of plant cell wall degrading enzymes from bacteria and fungi has been implicated in the evolution of plant parasitism. We have used ribonucleic acid sequencing (RNAseq) to generate reference transcriptomes for two economically important nematode species, Xiphinema index and Longidorus elongatus, representative of two genera within the early-branching Clade 2 of the phylum Nematoda. We used a transcriptome-wide analysis to identify putative horizontal gene transfer events. This represents the first in-depth transcriptome analysis from any plant-parasitic nematode of this clade. For each species, we assembled ~30 million Illumina reads into a reference transcriptome. We identified 62 and 104 transcripts, from X. index and L. elongatus, respectively, that were putatively acquired via horizontal gene transfer. By cross-referencing horizontal gene transfer prediction with a phylum-wide analysis of Pfam domains, we identified Clade 2-specific events. Of these, a GH12 cellulase from X. index was analysed phylogenetically and biochemically, revealing a likely bacterial origin and canonical enzymatic function. Horizontal gene transfer was previously shown to be a phenomenon that has contributed to the evolution of plant parasitism among nematodes. Our findings underline the importance and the extensiveness of this phenomenon in the evolution of plant-parasitic life styles in this speciose and widespread animal phylum.