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. 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−ΔΔCTstatistical 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. 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 MeE3Lwhich is silenced by SACMV-induced mutations. SACMV also induces mutations which silence the MeE3L RING domain in protoplasts from and tolerant cassava landraces. 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.

中文翻译：

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用于筛选与南非木薯花叶病毒反应相关的基因的木薯原生质体系统

使用现有协议在病毒感染期间研究木薯植物中的瞬时基因表达是费力的，并且可能需要大约 15 周的时间，因为木薯对转化的顽固。原生质体系统与 CRISPR 介导的基因编辑相结合，有望缩短从植物组织培养到候选基因高通量基因表达筛选的周转时间。在这里，我们详细介绍了筛选与木薯原生质体中南非木薯花叶病毒 (SACMV) 反应相关的基因的协议，参考泛素 E3 连接酶基因 MeE3L。使用 PEG4000 介导的转染，用 SACMV 感染性克隆和/或靶向 MeE3L 的 CRISPR 编辑构建体转化模型的木薯原生质体以及 SACMV 易感和耐受基因型。在转染后 24 小时从转化的原生质体中提取 DNA 和 RNA。使用 DpnI 消化的总 DNA 通过 qPCR 确定相对 SACMV DNA 积累，通过逆转录酶 qPCR 确定 MeE3L 相对表达，并使用单向方差分析、Tukey's HSD 检验和 2-ΔΔCT 统计方法分析结果。MeE3L 外显子区在 ABI 3500XL 基因分析仪平台上测序；使用 MAFTT 和 MEGA-X 软件分析序列的突变。系统发育树的构建是使用最大似然法和基于琼斯-泰勒-桑顿 (JTT) 矩阵的模型完成的。在组织培养开始后 7 周内确定了模型、易感和耐受木薯原生质体在 SACMV 感染期间未编辑和突变的 MeE3L 的差异表达。该研究还表明，SACMV DNA 在木薯原生质体中的积累具有基因型依赖性，并在耐受的地方品种 MeE3L 同源物中诱导多个突变。值得注意的是，易感的木薯地方品种编码无环的 MeE3L，它被 SACMV 诱导的突变沉默。SACMV 还诱导突变，使来自和耐受木薯地方品种的原生质体中的 MeE3L RING 结构域沉默。此处介绍的该协议将与宿主对 SACMV 反应相关的基因的高通量筛选的周转时间减半。它提供了木薯 E3 连接酶与 SACMV 反应相关的证据，并为通过植物功能和相互作用研究验证这些发现奠定了基础。