A mutation in the melon Vacuolar Protein Sorting 41prevents systemic infection of Cucumber mosaic virus

Abstract

In the melon exotic accession PI 161375, the gene cmv1, confers recessive resistance to Cucumber mosaic virus (CMV) strains of subgroup II. cmv1 prevents the systemic infection by restricting the virus to the bundle sheath cells and impeding viral loading to the phloem. Here we report the fine mapping and cloning of cmv1. Screening of an F2 population reduced the cmv1 region to a 132 Kb interval that includes a Vacuolar Protein Sorting 41 gene. CmVPS41 is conserved among plants, animals and yeast and is required for post-Golgi vesicle trafficking towards the vacuole. We have validated CmVPS41 as the gene responsible for the resistance, both by generating CMV susceptible transgenic melon plants, expressing the susceptible allele in the resistant cultivar and by characterizing CmVPS41 TILLING mutants with reduced susceptibility to CMV. Finally, a core collection of 52 melon accessions allowed us to identify a single amino acid substitution (L348R) as the only polymorphism associated with the resistant phenotype. CmVPS41 is the first natural recessive resistance gene found to be involved in viral transport and its cellular function suggests that CMV might use CmVPS41 for its own transport towards the phloem.

Figure 1

Fine mapping of cmv1. (a) Sequential intervals screened during fine mapping. Each horizontal bar represents a group of recombinants inside the region, colors indicate the origin of each region. Red, SC; green, PS; yellow, heterozygous and white, recombination interval. The phenotype of the recombinants is noted at the right. R, S and R/S for resistant, susceptible and segregating F3 plants. (b) Annotated features inside the smallest cmv1 interval.

Figure 2

Structure of CmVPS41 gene. (a) CmVPS41 structure according to annotation on the melon genome. Both genes MELO3C004831 and MELO3C004827 correspond to a single open reading frame. The genomic DNA expands 28.9Kb. (b) CmVPS41 gene structure based on the complete coding sequence. The 19 exons produce a 2,883 bp cDNA. SNPs between PS and SC are detailed below in blue. In red, the amino-acid changes in exons 4, 5 and 13. The red line demarks one of the ends of the cmv1interval.

Figure 3

CMV infection of T0 transgenic plants expressing PS CmVPS41. Representative results 21 days after the inoculation with either CMV-LS or CMV-FNY are shown (a). RT-PCR detection of CMV-LS-infected plants. (b) RT-PCR results after inoculation with CMV-FNY. (c) In vitro acclimatized T0 transgenic plants inoculated with CMV-FNY. PS: non-transgenic, in vitro regenerated susceptible line; SC: non-transgenic, in vitro regenerated resistant line; SC-T0: transgenic SC lines (16, 17 and 38) expressing CmVPS41 from PS under its own promoter. C- negative RT-PCR control. Arrows indicate the 1200 bp band of the size marker.

Figure 4

(a) Amino acid sequence of the CmVPS41 fragment screened in the TILLING population (exons 5 and 6). The sequence was aligned against the corresponding sequence from representative plant species. Detailed below are the nonsynonymous TILLING mutants detected. (b) CMV-LS accumulation in the inoculated D360N TILLING family. Samples were collected after 21dpi. ELISA results from the fourth true leaf from a representative experiment are shown. PS, Piel de Sapo susceptible line; SC12-1-99, introgression line carrying cmv1; CM, CharMono susceptible TILLING parental line; D360N, TILLING family carrying the mutation D360N in CmVPS41. *p-value for the t-test comparing against M3425 lower than 0.05, **lower than 0.01, ***lower than 0,001.