Mutations in CmVPS41 controlling resistance toCucumber Mosaic Virusdisplay specific subcellular localizations

Resistance to Cucumber mosaic virus (CMV) in melon has been described in several exotic accessions. It is controlled by a recessive resistance gene, cmv1 , which encodes a Vacuolar Protein Sorting 41 (CmVPS41). Cmv1 prevents systemic infection by restricting the virus to the bundle sheath cells, preventing viral phloem entry. CmVPS41 from different resistant accessions carried two causal mutations, either a G85E change, found in Pat-81 and Freeman’s Cucumber, or L348R found in PI161375 , cultivar Songwhan Charmi (SC). The analysis of the subcellular localization of CmVPS41 in N. benthamiana has revealed differential structures in resistant and susceptible accessions. Susceptible accessions showed nuclear and membrane spots and many transvacuolar strands, whereas the resistant accessions showed many intravacuolar invaginations. These specific structures colocalize with late endosomes. Artificial CmVPS41 carrying individual mutations causing resistance in the genetic background of CmVPS41 from the susceptible variety Piel de Sapo (PS), revealed that the structure most correlated with resistance was the absence of transvacuolar strands. Co-expression of CmVPS41 with the viral MPs, the determinant of virulence, did not change these localizations; however, infiltration of CmVPS41 from either SC or PS accessions in CMV-infected N. benthamiana leaves showed a localization pattern closer to each other, with up to 30% cells showing some membrane spots in the CmVPS41SC and fewer transvacuolar strands (from a mean of 4 to 1-2) with CmVPS41PS. Our results suggest that the distribution of CmVPS41PS in late endosomes includes transvacuolar strands that facilitate CMV infection and that CmVPS41 is re-localized during viral infection.