CRISPR/Cas9-mediated targeted mutagenesis of TAS4 and MYBA7 loci in grapevine rootstock 101-14

Abstract

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 TransActing 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

Secondary Metabolites in Xylella fastidiosa–Plant Interaction

“…nicotianae in tobacco [ 99 , 100 ]. In regards to counteracting the effect of Xf , promising examples of genetic engineering techniques include the use of CRISPR/Cas9-mediated targeted mutagenesis of TAS4 (Trans-Acting Small-interfering locus4) and MYBA7 (MYeloBlastosis viral oncogene-like transcription factor) loci, involved in regulation of anthocyanin accumulation in grapevine [ 101 ]. Even if obvious PD symptoms are anthocyanin accumulation in leaves at the scorched periphery and shriveling of berries that impacts fruit quality and yield, the role of anthocyanin accumulations in grapevine tissues by Xf is unknown; however vector feeding preferences and olfactory cues from host anthocyanins may be important for etiologies.…”

“…Even if obvious PD symptoms are anthocyanin accumulation in leaves at the scorched periphery and shriveling of berries that impacts fruit quality and yield, the role of anthocyanin accumulations in grapevine tissues by Xf is unknown; however vector feeding preferences and olfactory cues from host anthocyanins may be important for etiologies. Besides the lack of visible pigment phenotypes in edited plants precluded pathogen challenge tests of the role of anthocyanins in host PD resistance/tolerance mechanisms, the authors demonstrate successful genome editing which can serve future characterizations of the functions of TAS4 and MYBA7 in biotic stress response pathways [ 101 ]. A further example is represented by the expression of the diffusible signal factor (DSF) molecules, a class of widely conserved quorum-sensing signals used by many Gram-negative bacterial pathogens which are also produced by Xf .…”

Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters

“…For instance, the CRISPR/Cas9-mediated targeting of multiple genes that control the time of flowering and plant morphology can accelerate the domestication of wild tomato or directly develop customized tomato for urban agriculture 37,38 . In recent years, an increasing number of reported studies have implemented targeted genome editing in grape, and almost all the studies were performed with one or several sgRNAs targeting single genes [27][28][29][30][31]39 . The simultaneous editing of different genes of interest could improve different traits of grapes, which would be extremely significant for shortening the period of grape breeding.…”

Comprehensive Genome-Wide Exploration of C2H2 Zinc Finger Family in Grapevine (Vitis vinifera L.): Insights into the Roles in the Pollen Development Regulation

“…Since 2016, sites that are appropriate for CRISPR/Cas9-based genome editing identified in the V. vinifera genome are available through a publicly accessible grape CRISPR database [ 87 ]. To date, there are a growing number of reports on the use of CRISPR-Cas9 for genome editing in grapevine [ 88 , 89 , 90 , 91 , 92 , 93 , 94 ], and using it to understand the function of VviZFP genes can be a powerful reverse genetics approach.…”