A Coiled-Coil Nucleotide-Binding Domain Leucine-Rich Repeat Receptor Gene MeRPPL1 Plays a Role in the Replication of a Geminivirus in Cassava

Abstract

Disease resistance gene (R gene)-encoded nucleotide-binding leucine-rich repeat proteins (NLRs) are critical players in plant host defence mechanisms because of their role as receptors that recognise pathogen effectors and trigger plant effector-triggered immunity (ETI). This study aimed to determine the putative role of a cassava coiled-coil (CC)-NLR (CNL) gene MeRPPL1 (Manes.12G091600) (single allele) located on chromosome 12 in the tolerance or susceptibility to South African cassava mosaic virus (SACMV), one of the causal agents of cassava mosaic disease (CMD). A transient protoplast system was used to knock down the expression of MeRPPL1 by clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9). The MeRPPL1-targeting CRISPR vectors and/or SACMV DNA A and DNA B infectious clones were used to transfect protoplasts isolated from leaf mesophyll cells from the SACMV-tolerant cassava (Manihot esculenta) cultivar TME3. The CRISPR/Cas9 silencing vector significantly reduced MeRPPL1 expression in protoplasts whether with or without SACMV co-infection. Notably, SACMV DNA A replication was higher in protoplasts with lower MeRPPL1 expression levels than in non-silenced protoplasts. Mutagenesis studies revealed that protoplast co-transfection with CRISPR-MeRPPL1 silencing vector + SACMV and transfection with only SACMV induced nucleotide substitution mutations that led to altered amino acids in the highly conserved MHD motif of the MeRPPL1-translated polypeptide. This may abolish or alter the regulatory role of the MHD motif in controlling R protein activity and could contribute to the increase in SACMV-DNA A accumulation observed in MeRPPL1-silenced protoplasts. The results herein demonstrate for the first time a role for a CNL gene in tolerance to a geminivirus in TME3.