Pierce's disease (PD) of grapevine (Vitis vinifera) is caused by the bacterium Xylella fastidiosa and is vectored by xylem sap-sucking insects, whereas Grapevine Red Blotch Virus (GRBV) causes Red Blotch Disease and is transmitted in the laboratory by alfalfa leafhopper Spissistilus festinus. The significance of anthocyanin accumulations in distinct tissues of grapevine by these pathogens is unknown, but vector feeding preferences and olfactory cues from host anthocyanins may be important for these disease etiologies. Phosphate, sugar, and UV light are known to regulate anthocyanin accumulation via miR828 and Trans-Acting Small-interfering locus4 (TAS4), specifically in grape by production of phased TAS4a/b/c small-interfering RNAs that are differentially expressed and target MYBA5/6/7 transcription factor transcripts for post-transcriptional slicing and antisense-mediated silencing. To generate materials that can critically test these genes' functions in PD and GRBV disease symptoms, we produced transgenic grape plants targeting TAS4b and MYBA7 using CRISPR/Cas9 technology. We obtained five MYBA7 lines all with bi-allelic editing events and no off-targets detected at genomic loci with homology to the guide sequence. We obtained two independent edited TAS4b lines; one bi-allelic, the other heterozygous while both had fortuitous evidences of bi-allelic TAS4a off-target editing events at the paralogous locus. No visible anthocyanin accumulation phenotypes were observed in regenerated plants, possibly due to the presence of genetically redundant TAS4c and MYBA5/6 loci or absence of inductive environmental stress conditions. The editing events encompass single base insertions and di/trinucleotide deletions of Vvi-TAS4a/b and Vvi-MYBA7 at expected positions 3 nt upstream from the guideRNA proximal adjacent motifs NGG. We also identified evidences of homologous recombinations of TAS4a with TAS4b at the TAS4a off-target in one of the TAS4b lines, resulting in a chimeric locus with a bi-allelic polymorphism, supporting independent recombination events in transgenic plants associated with apparent high Cas9 activities. The lack of obvious visible pigment phenotypes in edited plants precluded pathogen challenge tests of the role of anthocyanins in host PD and GRBV resistance/tolerance mechanisms. Nonetheless, we demonstrate successful genome-editing of non-coding RNA and MYB transcription factor loci which can serve future characterizations of the functions of TAS4a/b/c and MYBA7 in developmental, physiological, and environmental biotic/abiotic stress response pathways important for value-added nutraceutical synthesis and pathogen responses of winegrape.

中文翻译：

---

CRISPR / Cas9介导的葡萄砧木101-14中TAS4和MYBA7基因座的定向诱变。

葡萄皮尔斯氏病（PD）是由细菌Xylella fastidiosa引起的，并由木质部树液吸食性昆虫传播，而葡萄红斑病毒（GRBV）引起红斑病，并在实验室中通过苜蓿叶蝉（Spissistilus）传播费斯蒂纳斯。这些病原体在葡萄的不同组织中积累花色苷的重要性尚不清楚，但宿主的花色苷的载体喂养偏好和嗅觉提示对于这些病因可能很重要。已知磷酸盐，糖和紫外线会通过miR828和反作用小干扰基因座4（TAS4）调节花色苷的积累，特别是在葡萄中，通过产生分阶段表达的TAS4a / b / c小干扰RNA，该RNA差异表达并靶向MYBA5 / 6/7转录因子转录本，以进行转录后切片和反义介导的沉默。为了产生可以严格测试这些基因在PD和GRBV疾病症状中的功能的材料，我们使用CRISPR / Cas9技术生产了针对TAS4b和MYBA7的转基因葡萄植物。我们获得了五个MYBA7品系，所有品系均具有双等位基因编辑事件，并且在与指导序列同源的基因组位点上未检测到脱靶。我们获得了两条独立编辑的TAS4b品系；一个是双等位基因，另一个是杂合子，而两个都在旁系位点有双等位基因TAS4a脱靶编辑事件的偶然证据。在再生植物中未观察到可见的花色苷积累表型，可能是由于存在遗传上多余的TAS4c和MYBA5 / 6基因座或没有诱导性环境胁迫条件。编辑事件包括Vvi-TAS4a / b和Vvi-MYBA7的单个碱基插入和V / MYBA7的二碱基/三核苷酸缺失，位于预期RNA邻近邻近引导基序NGG的3 nt上游。我们还确定了TAS4b系之一中TAS4a与TAS4a脱靶的TAS4a与TAS4b同源重组的证据，从而导致了具有双等位基因多态性的嵌合基因座，支持了具有明显高Cas9活性的转基因植物中的独立重组事件。编辑后的植物缺乏明显的可见色素表型，因此无法进行病原体对花色苷在宿主PD和GRBV抗性/耐受性机制中的作用的挑战性测试。尽管如此，