Abstract Powdery mildew (PM), caused by the fungal pathogen Erysiphe necator, is one of the most destructive diseases of grapevine (Vitis vinifera and other Vitis spp.). Resistance to PM is an important goal for cultivar improvement, and understanding the underlying molecular mechanisms conditioning resistance is critical. Here, we report that transgenic expression of the WRKY transcription factor gene VqWRKY31 from the PM-resistant species Vitis quinquangularis conferred resistance to PM in V. vinifera through promoting salicylic acid signaling and specific metabolite synthesis. VqWRKY31 belongs to the WRKY IIb subfamily, and expression of the VqWRKY31 gene was induced in response to E. necator inoculation. Transgenic V. vinifera plants expressing VqWRKY31 were substantially less susceptible to E. necator infection, and this was associated with increased levels of salicylic acid and reactive oxygen species. Correlation analysis of transcriptomic and metabolomic data revealed that VqWRKY31 promoted expression of genes in metabolic pathways and the accumulation of many disease resistance-related metabolites, including stilbenes, flavonoids, and proanthocyanidins. In addition, results indicated that VqWRKY31 can directly bind to the promoters of two structural genes in stilbene synthesis, STS9 and STS48, and activate their expression. Based on our results, we propose a model where VqWRKY31 enhances grapevine PM resistance through activation of salicylic acid defense signaling and promotion of specific disease resistance-related metabolite synthesis. These findings can be directly exploited for molecular breeding strategies to produce PM-resistant grapevine germplasm.

/pdf/overexpression-of-vqwrky31-enhances-powdery-mildew-ip0uolwg.pdf

Overexpression of VqWRKY31 enhances powdery mildew resistance in grapevine by promoting salicylic acid signaling and specific metabolite synthesis

Abstract Anthocyanins are plant secondary metabolites that have a variety of biological functions, including pigmentation. The accumulation of anthocyanins is regulated by both transcriptional activators and repressors. Studies have shown that bZIP family members act primarily as positive regulators of anthocyanin biosynthesis, but there are few reports of negative regulation. Here, we report that a grapevine (Vitis vinifera) bZIP gene from group K, VvbZIP36, acts as a negative regulator of anthocyanin biosynthesis. Knocking out one allele of VvbZIP36 in grapevine with CRISPR/Cas9 promoted anthocyanin accumulation. Correlation analysis of transcriptome and metabolome data showed that a range of anthocyanin biosynthesis genes were activated in VvbZIP36 mutant plants relative to the wild type, resulting in the accumulation of related metabolites, including naringenin chalcone, naringenin, dihydroflavonols, and cyanidin-3-O-glucoside. The synthesis of stilbenes (α-viniferin), lignans, and some flavonols (including quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, and kaempferol-7-O-rhamnoside) was significantly inhibited, and several genes linked to their metabolism were downregulated in the mutant plants. In summary, our results demonstrate that VvbZIP36 is a negative regulator of anthocyanin biosynthesis that plays a role in balancing the synthesis of stilbenes (α-viniferin), lignans, flavonols, and anthocyanins.

/pdf/crispr-cas9-mediated-mutagenesis-of-vvbzip36-promotes-359cwktq.pdf

CRISPR/Cas9-mediated mutagenesis of VvbZIP36 promotes anthocyanin accumulation in grapevine (Vitis vinifera)

Advances in genomics and genome editing for breeding next generation of fruit and nut crops

A multitude of diverse breeding goals need to be combined in a new cultivar, which always forces to compromise. The biggest challenge grapevine breeders face is the extraordinarily complex trait of wine quality, which is the all-pervasive and most debated characteristic. Since the 1920s, Germany runs continuous grapevine breeding programmes. This continuity was the key to success and lead to various new cultivars on the market, so called PIWIs. Initially, introduced pests and diseases such as phylloxera, powdery and downy mildew were the driving forces for breeding. However, preconceptions about the wine quality of new resistant selections impeded the market introduction. These preconceptions are still echoing today and may be the reason in large parts of the viticultural community for: (1) ignoring substantial breeding progress, and (2) sticking to successful markets of well-known varietal wines or blends (e.g. Chardonnay, Cabernet Sauvignon, Riesling). New is the need to improve viticulture´s sustainability and to adapt to changing environmental conditions. Climate change with its extreme weather will impose the need for a change in cultivars in many wine growing regions. Therefore, a paradigm shift is knocking on the door: new varieties (PIWIs) versus traditional varieties for climate adapted and sustainable viticulture. However, it will be slow process and viticulture is politically well advised to pave the way to variety innovation. In contrast to the widely available PIWIs, competitive cultivars created by means of new breeding technologies (NBT, e.g. through CRISPR/Cas) are still decades from introduction to the market. 

/pdf/a-cool-climate-perspective-on-grapevine-breeding-climate-dhfnb118.pdf

A cool climate perspective on grapevine breeding: climate change and sustainability are driving forces for changing varieties in a traditional market

Abstract During the last decade, targeted genome-editing technologies, especially clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) technologies, have permitted efficient targeting of genomes, thereby modifying these genomes to offer tremendous opportunities for deciphering gene function and engineering beneficial traits in many biological systems. As a powerful genome-editing tool, the CRISPR/Cas systems, combined with the development of next-generation sequencing and many other high-throughput techniques, have thus been quickly developed into a high-throughput engineering strategy in animals and plants. Therefore, here, we review recent advances in using high-throughput genome-editing technologies in animals and plants, such as the high-throughput design of targeted guide RNA (gRNA), construction of large-scale pooled gRNA, and high-throughput genome-editing libraries, high-throughput detection of editing events, and high-throughput supervision of genome-editing products. Moreover, we outline perspectives for future applications, ranging from medication using gene therapy to crop improvement using high-throughput genome-editing technologies. 

OUP accepted manuscript

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

/pdf/whole-genome-sequencing-reveals-rare-off-target-mutations-in-505iwk21rz.pdf

Hongsong Wu

Papers

Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine