Yusuke Ban

CRISPR/Cas9-mediated targeted mutagenesis in grape

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

Evaluating Grapevine Cultivars for Resistance to Anthracnose Based on Lesion Number and Length

Anthracnose is one of the most detrimental diseases of grapevines in humid regions. To lessen the risk of crop damage and to reduce fungicide application, resistant cultivars are needed. The resistance of grapevines to anthracnose caused by Elsinoë ampelina (de Bary) Shear was evaluated in two ways: the resistance against ingress was evaluated using the number of lesions and the resistance against colonization was evaluated using the lesion length. First, both forms of resistance in 13 cultivars were evaluated in a detached-leaf assay for three years using greenhouse-grown cuttings. Both lesion number and lesion length revealed a genetic component of the resistance, with greater phenotypic variation among interspecific hybrid cultivars than among Vitis vinifera cultivars. The detached-leaf assay revealed significant differences in lesion length among V. vinifera accessions; Exotic and Perlette showed moderate resistance. Lesion diameters were measured in a fungicide-free vineyard to evaluate the resistance of six cultivars, and significant differences were found among the cultivars. The lesion shape in susceptible cultivars was almost circular, whereas the shape in resistant cultivars was usually irregular. Results showed that evaluations based on the lesion number and length can reliably predict resistance to anthracnose without the need for large field trials and that some V. vinifera cultivars exhibited moderate resistance to this disease.