Building a cluster of NLR genes conferring resistance to pests and pathogens: the story of the Vat gene cluster in cucurbits

Abstract

Most molecularly characterized plant resistance genes (R genes) belong to the nucleotide-binding-site-leucine-rich-repeat (NLR) receptor family and are prone to duplication and transposition with high sequence diversity. In this family, the Vat gene in melon is one of the few R genes known for conferring resistance to insect, i.e., Aphis gossypii, but it has been misassembled and/or mispredicted in the whole genomes of Cucurbits. We examined 14 genomic regions (about 400 kb) derived from long-read assemblies spanning Vat-related genes in Cucumis melo, Cucumis sativus, Citrullus lanatus, Benincasa hispida, Cucurbita argyrosperma, and Momordica charantia. We built the phylogeny of those genes. Investigating the paleohistory of the Vat gene cluster, we revealed a step by step process beginning from a common ancestry in cucurbits older than 50 my. We highlighted Vat exclusively in the Cucumis genera, which diverged about 20 my ago. We then focused on melon, evaluating a minimum duplication rate of Vat in 80 wild and cultivated melon lines using generalist primers; our results suggested that duplication started before melon domestication. The phylogeny of 44 Vat-CDS obtained from 21 melon lines revealed gain and loss of leucine-rich-repeat domains along diversification. Altogether, we revealed the high putative recognition scale offered in melon based on a combination of SNPs, number of leucine-rich-repeat domains within each homolog and number of homologs within each cluster that might jointly confer resistance to a large pest and pathogen spectrum. Based on our findings, we propose possible avenues for breeding programs.

Fig. 1. Genes identified after manual annotation for Vat-related sequences and MAKER annotation within M5-M4 regions.

The sequences were obtained from public databases for Cucumis melo DHL92 (https://www.melonomics.net/), Payzawat, Harukei-3, HS, and Cucumis sativus, Citrullus lanatus, Benincasa hispida (http://cucurbitgenomics.org/), and Momordica charantia. In the seven melon lines, three genetic markers, i.e., V432, V681, and M7, and three specific markers, i.e., Z5182, Z5184, and Z5257, were positioned. The colored boxes correspond to the main collinear blocks shown in Fig. S3

Fig. 2. Phylogenetic tree from CDSs of Vat-related sequences (with one R65aa for C. melo) retrieved for cucurbit species.

All sequences except ‘out of cluster’ sequences were retrieved from CDSs of the Vat-related sequences presented in Fig. 1. The MegaX software suite was then used to infer the evolutionary histories. The percentage of trees in which the associated CDSs clustered is shown next to the branches (500 replications). The tree is drawn to scale, with branch lengths measured according to the number of substitutions per site

Fig. 3. Vat homolog diversity in 80 melon accessions.

Percentage assignation of each accession to three genetic clusters. The baseline indicated the geographical origin of the accession. The upper line gives information obtained from amplicons of the LRR2 domain: the number of digits gives an estimate of the number of homologs in the accession, each digit gives the number of R65aa estimated in each homolog. Twenty SSRs distributed over the 12 chromosomes were amplified and their diversity analyzed by Bayesian clustering. The allele size at the eight microsatellite loci and probabilities of assignation at the three genetic clusters are given in the Table S3

Fig. 4. Phylogenetic tree for 44 Vat homologs obtained from 21 melon accessions.

Each CDS was named Accession name_Vat number in the accession (when known) number of R65aa within the CDS. AN for Anso77, DB for Doublon, PZ for Payzawat, and PI for PI 161375. A Schematic representation of the multiple alignments of the 44 CDS. The alignment was independently managed for three blocks—pre-LRR2/LLR2/post LRR2—with the MUSCLE algorithm in the Seaview software package. B The MegaX software suite was then used to infer the evolutionary histories. The percentage of trees in which the associated CDSs clustered is shown next to the branches (500 replications). The tree is drawn to scale, with branch lengths measured according to the number of substitutions per site

Fig. 5. SNP analysis in 39 CDSs of Vat homologs with more than one R65aa.

Typical alignment obtained for the CDS with different numbers of R65aa (alignment pre-LRR2, LRR2, and post-LRR2 independent), the virtual CDSs did not contain any R65aa. i The virtual CDSs were analyzed by FUBAR, a Bayesian approach to infer nonsynonymous (dN) and synonymous (dS) substitution rates on a per-site basis represented by [alpha-beta]. This method assumes that the selection pressure for each site is constant throughout the entire phylogeny. ii The two parts of virtual CDS and 152 R65aa contained in the 39 CDSs were analyzed independently for the distribution of nonsynonymous SNPs, represented by the probability of nonsynonymous SNPs

Fig. 6. Topology of Cucurbitaceae phylogeny retrieved from the Cucurbitaceae tree http://timetree.org/.

The clade in red corresponds to the Benincaseae tribe