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

A muscadine locus confers resistance to predominant species of grapevine root-knot nematodes (Meloidogyne spp.) including virulent populations

Root-knot nematodes (RKNs) Meloidogyne spp. are extremely polyphagous pests and four species severely affect grapevines throughout the world: M. arenaria, M. incognita, M. javanica and M. ethiopica. Californian populations of M. arenaria and M. incognita are reported to be virulent to widely used rootstocks and to the rootstock ‘Harmony’ in particular. Breeding RKNs-resistant grape rootstocks is a promising alternative to highly toxic nematicides. Muscadine (Vitis rotundifolia syn. Muscadinia rotundifolia) is a resistance (R) source with undercharacterised genetics. To this end, we used a segregating progeny between the RKN-resistant Vitis x Muscadinia accession ‘VRH8771’ from the muscadine source ‘NC184-4’ and the RKN-susceptible V. vinifera cv. Cabernet-Sauvignon. We first phenotyped its resistance to isolates of the i) M. arenaria, ii) M. incognita and iii) M. javanica species, and then to iv) two mixed Harmony-virulent Californian populations of M. arenaria and M. incognita. Finally, we created an isolate of M. arenaria and M. incognita from these Harmony populations and phenotyped the progeny to each of them [v) and vi)], and to vii) an isolate of M. ethiopica. The resistance phenotype of all the progeny’s individuals was independent of the RKN isolates or populations used. Resistance was mapped in a region of chromosome 18 in VRH8771, supporting the hypothesis that it is conferred by a single gene with an unprecedented wide spectrum in grapevine, including Harmony-virulent isolates. This dominant gene, referred to as MsppR1, is linked to the telomeric QTL XiR4 for X. index resistance from the same source. Additionally, plant mortality data showed that MsppR1-resistant material expressed a high-level resistance to the Harmony-virulent isolates. Our results are a first step towards the development of marker-assisted breeding using SSR and SNP markers for resistance to RKNs in accession VRH8771. © 2023, International Viticulture and Enology Society. All rights reserved

Xiphinema index-resistant grapevine materials derived from muscadine are also resistant to a population of X. diversicaudatum

Grapevine is severely affected by two major nepoviruses that cause grapevine degeneration: the grapevine fanleaf virus (GFLV) and the arabis mosaic virus (ArMV), specifically transmitted by the dagger nematodes Xiphinema index and X. diversicaudatum, respectively. While natural resistance to X. index has been shown to be a promising alternative for controlling X. index and GFLV transmission, the resistance interaction between X. diversicaudatum and grapevine has not yet been documented. In the present study, we evaluated the host suitability to X. diversicaudatum in materials previously characterised for their resistance to X. index. Two X. index-resistant accessions VRH8771 (F1 hybrid) and Nemadex Alain Bouquet (BC1 hybrid) derived from muscadine, together with the X. index-susceptible reference accession V. vinifera cv. Cabernet-Sauvignon and the X. index-resistant reference accession V. riparia ‘10128’, were challenged with a X. diversicaudatum population obtained from woody host plants and a reference isolate of X. index. The reproduction factors of X. diversicaudatum and its numbers per gram of roots paralleled those of X. index, showing a resistance interaction to the population of the former species and suggesting that resistance determinants to both nematode vectors might be the same or linked. Nevertheless, these two criteria illustrated a poorer host suitability of grapevine materials to this X. diversicaudatum population than to X. index

BMC Plant Biol

Combining innovation in vineyard management andgenetic diversity for a sustainable European viticultur

Le locus de résistance Ma des Prunus vis-à-vis des nématodes à galles : Originalité structurale et évolution dans la famille des NBS-LRRs chez les plantes

Root-knot nematodes (RKNs), Meloidogyne spp., are extremely polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on NBS-LRR receptor genes (or NLRs grouping TNL, CNL and RNL subfamilies) that are pivotal factors for control of pests and pathogens. In Prunus spp., the Ma plum TNL gene confers resistance to all RKNs tested, whereas the RMja almond gene displays a more restricted spectrum of resistance (R). Moreover, the Ma predicted protein shows a peculiar TNL structure due to a C-terminal region made of five repeated domains, designated post-LRR domains (PLs). In this context, this thesis work has characterised the originality and the distribution of this uncommon structure among diverse plant proteomes and has revealed the genetic relationship between the Ma and RMja genes.We first studied the frequency, distribution and structural characteristics of TNL genes and PL domains within the peach genome, the reference genome for Rosaceae. The finding of PL domains, which have been identified in two thirds of the 195 TNLs, allowed us to define specific motifs that improve the detection of this poorly known domain in Angiosperms. We found that the PL domain is specific of TNLs and is present in Angiosperm genomes in a proportion similar to the one established for peach. Besides, TNLs displaying multiple PL domains are rare in plants. The five-PL domain pattern is probably unique to Ma and its orthologues and was probably inherited from their common ancestor in the order Rosales. We then investigated the NBS-LRR repertoire of the conifers (Gymnosperms), an ancient taxonomic group, for which the data related to this gene family are unclear. By analysing seven reference transcriptomes, we highlighted a large and diverse NBS-LRR arsenal in conifers but, surprisingly, no PL signatures have been detected. The examination of ancient plant proteomes revealed that only Ginkgo biloba displayed a few PL signatures. Our results suggest that a partial acquisition of the PL domain occurred early in seed plants and was followed by an adaptive expansion in Angiosperms. Additionally, we showed that conifers and Rosaceae have numerous RNLs and TNLs. By enlarging our study to other land plant genomes, we uncovered an average ratio of 1:10 between RNLs and TNLs numbers.We finally carried out a high-resolution mapping of the RMja gene in almond. Using a BAC library, RMja was localised into the Ma resistance cluster and the Ma orthologue is by far the best candidate. The sequence comparison between three orthologous regions of the Ma locus, i.e. plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum) highlighted a unique conserved structure of the Ma orthologues. Our results suggest that the polymorphism contained in the PL-domain repeats might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. In these immune processes for recognition or signalling, other components such as RNLs might be involved. This work paves the way for future comparative and functional approaches aiming to unravel the molecular determinants involved in the resistance to RKNs.Les nématodes à galles, Meloidogyne spp., sont des ravageurs extrêmement polyphages qui, à l’échelle mondiale, occasionnent de graves dommages aux plantes. La résistance génétique spécifique des plantes aux maladies et ravageurs s’appuie principalement sur les gènes de la famille des récepteurs NBS-LRR (ou NLRs), regroupant les TNLs, CNLs et RNLs. Chez les Prunus, le gène Ma du prunier appartient à la sous-famille des TNLs et confère une résistance à toutes les espèces de Meloidogyne testées, alors que le gène RMja de l’amandier exprime un spectre de résistance (R) plus restreint vis-à-vis de ces ravageurs. De plus, la protéine Ma présente une région C-terminale particulière constituée de cinq domaines répétés, désignés domaines post-LRR (PLs). Notre travail de thèse a caractérisé l’originalité et la distribution de cette région à travers de nombreux protéomes de plantes et a identifié la relation génétique entre les gènes Ma et RMja.Nous avons tout d’abord étudié la fréquence, la distribution et les caractéristiques structurales des gènes TNL et des domaines PL dans le génome du pêcher, génome de référence des Rosaceae. Les domaines PL, retrouvés chez les deux tiers des 195 TNLs identifiés, nous ont permis d’établir des signatures améliorant la détection de ce domaine, jusqu’alors peu étudié, dans divers génomes d’Angiospermes. Nous avons pu établir que le domaine PL est spécifique aux TNLs et qu’il est retrouvé dans des proportions similaires à celle établie chez le pêcher. Par ailleurs, les TNLs disposant de domaines PL multiples sont rares chez les plantes étudiées. La structure à cinq domaines répétés est probablement unique à Ma et ses orthologues et a vraisemblablement été héritée de leur ancêtre commun dans l’ordre des Rosales. Nous avons ensuite étudié le répertoire des NBS-LRRs chez les conifères (Gymnospermes), groupe taxonomique ancien, dont les données sur cette famille de gènes étaient parcellaires. En analysant sept transcriptomes de référence, nous avons pu établir que l’arsenal des NBS-LRRs chez les conifères était large et varié mais, étonnamment, qu’aucun domaine PL précédemment défini n’y était présent. L’examen de protéomes de plantes plus anciennes a montré que seul le Ginkgo biloba portait quelques signatures PL. Ces observations suggèrent une acquisition partielle précoce du domaine chez les plantes à graines et une expansion adaptative chez les Angiospermes. En complément, nous avons montré que les conifères, tout comme les Rosaceae, possèdent de nombreux RNLs et TNLs. En étendant notre étude à diverses plantes terrestres, nous avons mis en évidence un rapport moyen de 1:10 reliant les effectifs de RNLs et de TNLs à travers les divers génomes étudiés. Nous avons finalement conduit une cartographie haute résolution du gène RMja chez l’amandier. En nous appuyant sur une banque BAC, RMja a été localisé dans le cluster de résistance Ma et l’orthologue de Ma est de très loin le meilleur candidat. La comparaison de séquence entre les régions orthologues du locus Ma, chez le prunier (spectre R complet), l’amandier (spectre R incomplet) et le pêcher (spectre R nul) a mis en évidence une structure conservée unique des trois orthologues de Ma. Nos résultats suggèrent que le polymorphisme des répétitions du domaine PL sous-tend des interactions différentielles de résistance vis-à-vis des Meloidogyne et un mécanisme d’immunité original chez les plantes pérennes. Dans ces processus immuns de reconnaissance ou de signalisation, d’autres composants tels les RNLs pourraient être impliqués. Notre travail ouvre la voie à des approches comparative et fonctionnelle d’identification des déterminants moléculaires impliqués dans la résistance aux nématodes à galles

The Ma resistance locus to root-knot nematodes in Prunus : Structural originality and evolution within the NBS-LRR gene family in plants

Les nématodes à galles, Meloidogyne spp., sont des ravageurs extrêmement polyphages qui, à l’échelle mondiale, occasionnent de graves dommages aux plantes. La résistance génétique spécifique des plantes aux maladies et ravageurs s’appuie principalement sur les gènes de la famille des récepteurs NBS-LRR (ou NLRs), regroupant les TNLs, CNLs et RNLs. Chez les Prunus, le gène Ma du prunier appartient à la sous-famille des TNLs et confère une résistance à toutes les espèces de Meloidogyne testées, alors que le gène RMja de l’amandier exprime un spectre de résistance (R) plus restreint vis-à-vis de ces ravageurs. De plus, la protéine Ma présente une région C-terminale particulière constituée de cinq domaines répétés, désignés domaines post-LRR (PLs). Notre travail de thèse a caractérisé l’originalité et la distribution de cette région à travers de nombreux protéomes de plantes et a identifié la relation génétique entre les gènes Ma et RMja.Nous avons tout d’abord étudié la fréquence, la distribution et les caractéristiques structurales des gènes TNL et des domaines PL dans le génome du pêcher, génome de référence des Rosaceae. Les domaines PL, retrouvés chez les deux tiers des 195 TNLs identifiés, nous ont permis d’établir des signatures améliorant la détection de ce domaine, jusqu’alors peu étudié, dans divers génomes d’Angiospermes. Nous avons pu établir que le domaine PL est spécifique aux TNLs et qu’il est retrouvé dans des proportions similaires à celle établie chez le pêcher. Par ailleurs, les TNLs disposant de domaines PL multiples sont rares chez les plantes étudiées. La structure à cinq domaines répétés est probablement unique à Ma et ses orthologues et a vraisemblablement été héritée de leur ancêtre commun dans l’ordre des Rosales. Nous avons ensuite étudié le répertoire des NBS-LRRs chez les conifères (Gymnospermes), groupe taxonomique ancien, dont les données sur cette famille de gènes étaient parcellaires. En analysant sept transcriptomes de référence, nous avons pu établir que l’arsenal des NBS-LRRs chez les conifères était large et varié mais, étonnamment, qu’aucun domaine PL précédemment défini n’y était présent. L’examen de protéomes de plantes plus anciennes a montré que seul le Ginkgo biloba portait quelques signatures PL. Ces observations suggèrent une acquisition partielle précoce du domaine chez les plantes à graines et une expansion adaptative chez les Angiospermes. En complément, nous avons montré que les conifères, tout comme les Rosaceae, possèdent de nombreux RNLs et TNLs. En étendant notre étude à diverses plantes terrestres, nous avons mis en évidence un rapport moyen de 1:10 reliant les effectifs de RNLs et de TNLs à travers les divers génomes étudiés. Nous avons finalement conduit une cartographie haute résolution du gène RMja chez l’amandier. En nous appuyant sur une banque BAC, RMja a été localisé dans le cluster de résistance Ma et l’orthologue de Ma est de très loin le meilleur candidat. La comparaison de séquence entre les régions orthologues du locus Ma, chez le prunier (spectre R complet), l’amandier (spectre R incomplet) et le pêcher (spectre R nul) a mis en évidence une structure conservée unique des trois orthologues de Ma. Nos résultats suggèrent que le polymorphisme des répétitions du domaine PL sous-tend des interactions différentielles de résistance vis-à-vis des Meloidogyne et un mécanisme d’immunité original chez les plantes pérennes. Dans ces processus immuns de reconnaissance ou de signalisation, d’autres composants tels les RNLs pourraient être impliqués. Notre travail ouvre la voie à des approches comparative et fonctionnelle d’identification des déterminants moléculaires impliqués dans la résistance aux nématodes à galles.Root-knot nematodes (RKNs), Meloidogyne spp., are extremely polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on NBS-LRR receptor genes (or NLRs grouping TNL, CNL and RNL subfamilies) that are pivotal factors for control of pests and pathogens. In Prunus spp., the Ma plum TNL gene confers resistance to all RKNs tested, whereas the RMja almond gene displays a more restricted spectrum of resistance (R). Moreover, the Ma predicted protein shows a peculiar TNL structure due to a C-terminal region made of five repeated domains, designated post-LRR domains (PLs). In this context, this thesis work has characterised the originality and the distribution of this uncommon structure among diverse plant proteomes and has revealed the genetic relationship between the Ma and RMja genes.We first studied the frequency, distribution and structural characteristics of TNL genes and PL domains within the peach genome, the reference genome for Rosaceae. The finding of PL domains, which have been identified in two thirds of the 195 TNLs, allowed us to define specific motifs that improve the detection of this poorly known domain in Angiosperms. We found that the PL domain is specific of TNLs and is present in Angiosperm genomes in a proportion similar to the one established for peach. Besides, TNLs displaying multiple PL domains are rare in plants. The five-PL domain pattern is probably unique to Ma and its orthologues and was probably inherited from their common ancestor in the order Rosales. We then investigated the NBS-LRR repertoire of the conifers (Gymnosperms), an ancient taxonomic group, for which the data related to this gene family are unclear. By analysing seven reference transcriptomes, we highlighted a large and diverse NBS-LRR arsenal in conifers but, surprisingly, no PL signatures have been detected. The examination of ancient plant proteomes revealed that only Ginkgo biloba displayed a few PL signatures. Our results suggest that a partial acquisition of the PL domain occurred early in seed plants and was followed by an adaptive expansion in Angiosperms. Additionally, we showed that conifers and Rosaceae have numerous RNLs and TNLs. By enlarging our study to other land plant genomes, we uncovered an average ratio of 1:10 between RNLs and TNLs numbers.We finally carried out a high-resolution mapping of the RMja gene in almond. Using a BAC library, RMja was localised into the Ma resistance cluster and the Ma orthologue is by far the best candidate. The sequence comparison between three orthologous regions of the Ma locus, i.e. plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum) highlighted a unique conserved structure of the Ma orthologues. Our results suggest that the polymorphism contained in the PL-domain repeats might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. In these immune processes for recognition or signalling, other components such as RNLs might be involved. This work paves the way for future comparative and functional approaches aiming to unravel the molecular determinants involved in the resistance to RKNs

Data from: 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

Full list of 103 trimmed sequences of the PL domain.

These 103 Post LRR predicted peptide sequences have been trimmed and used to build the alignement and the phylogenic tree. Fasta format - use text edito

Additional file 7: of TNL genes in peach: insights into the post-LRR domain

TNL-related peptides obtained with BLASTp analysis using MEME motifs of the post-LRR domain in the Plant Genome Database website. Results are shown for Populus trichocarpa, Vitis vinifera, Medicago truncatula, Arabidopsis thaliana, Solanum tuberosum, Glycine max, Oryza sativa, Prunus persica, Sorghum bicolor, and Zea mays. Identified TNL-related sequences, unrelated TNL sequences and unknown sequences are highlighted in green, blue and yellow, respectively. (DOC 138 kb

Additional file 2: of TNL genes in peach: insights into the post-LRR domain

Exon and domain structures of all predicted TNL-related peptides. Exon sizes are shown above the colour shapes reflecting the different domains. (XLSX 115 kb