Susceptibility to South African cassava mosaic virus is Associated with a RING Finger E3 Ubiquitin Ligase

Abstract

BackgroundThe ubiquitylation of proteins is reprogrammed by plant geminiviruses which alter the ubiquitin proteasome system (UPS) to fully infect the host. A RING Finger E3 Ubiquitin Ligase (MeE3L) is located on a major cassava mosaic disease resistance-associated quantitative trait locus. Here, we examine the genetic structure and relative expression of MeE3L (native and gene-edited mutant), and determine how MeE3L affects geminivirus South African cassava mosaic virus (SACMV) DNA accumulation. MethodsCassava protoplasts of model, susceptible and tolerant genotypes were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 hours post-transfection. Relative SACMV load quantitation was determined using DpnI-digested total DNA via qPCR and MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using the 2-ΔΔ method. The MeE3L exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations and for construction of a phylogenetic tree using the Maximum Likelihood method and Tamura-Nei model.ResultsResults show that SACMV DNA accumulation is cassava genotype-dependent. The study also reveals that native and mutant MeE3L is differentially expressed during SACMV infection in protoplasts of susceptible and tolerant cassava landraces. The susceptible cassava landrace encodes a RINGless MeE3L and the MeE3L base sequence is a determinant of cassava’s response to SACMV. Results further show that SACMV silences the MeE3L RING domain in the susceptible and tolerant landraces; and specifically targets the tolerant MeE3L gene homolog for silencing. ConclusionsThese findings suggest that MeE3L is a target of SACMV, contributing to susceptibility in cassava. The MeE3L base sequence is a determinant of cassava’s response to SACMV. The MeE3L RING domain is actively silenced by SACMV and therefore may be essential for host defence against geminiviruses. The study provides further evidence, in addition to existing literature, that plant E3 ligases are exploited by geminiviruses to enhance pathogenicity.