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Background  The study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava’s recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the response to South African cassava mosaic virus (SACMV) in cassava protoplasts, with reference to the ubiquitin E3 ligase gene,  MeE3L .Methods  Cassava protoplasts of model, and SACMV-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 h post-transfection. Relative SACMV DNA accumulation was determined via qPCR using  DpnI -digested total DNA,  MeE3L  relative expression was determined via reverse transcriptase qPCR, and results were analysed using one-way ANOVA, Tukey’s HSD test and the 2 −ΔΔCT statistical method. The  MeE3L  exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations using MAFTT and MEGA-X software. Construction of a phylogenetic tree was done using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model.    Results  The differential expression of unedited and mutant  MeE3L  during SACMV infection of model, susceptible and tolerant cassava protoplasts was determined within 7 weeks after commencement of tissue culture. The study also revealed that SACMV DNA accumulation in cassava protoplasts is genotype-dependent and induces multiple mutations in the tolerant landrace  MeE3L  homolog. Notably, the susceptible cassava landrace encodes a RINGless  MeE3L which is silenced by SACMV-induced mutations. SACMV also induces mutations which silence the  MeE3L  RING domain in protoplasts from and tolerant cassava landraces.    Conclusions  This protocol presented here halves the turnaround time for high-throughput screening of genes associated with the host response to SACMV. It provides evidence that a cassava E3 ligase is associated with the response to SACMV and forms a basis for validation of these findings by  in planta  functional and interaction studies.

A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus