Grapevine (Vitis vinifera) is a valuable crop in Europe for both economical and cultural reasons, but highly susceptible to Downy mildew (DM). The generation of resistant vines is of critical importance for a sustainable viticulture and can be achieved either by introgression of resistance genes in susceptible varieties or by mutation of Susceptibility (S) genes, e.g., by gene editing. This second approach offers several advantages: it maintains the genetic identity of cultivars otherwise disrupted by crossing and generally results in a broad-spectrum and durable resistance, but it is hindered by the poor knowledge about S genes in grapevines. Candidate S genes are Downy mildew Resistance 6 (DMR6) and DMR6-Like Oxygenases (DLOs), whose mutations confer resistance to DM in Arabidopsis. In this work, we show that grapevine VviDMR6-1 complements the Arabidopsis dmr6-1 resistant mutant. We studied the expression of grapevine VviDMR6 and VviDLO genes in different organs and in response to the DM causative agent Plasmopara viticola. Through an automated evaluation of causal relationships among genes, we show that VviDMR6-1, VviDMR6-2, and VviDLO1 group into different co-regulatory networks, suggesting distinct functions, and that mostly VviDMR6-1 is connected with pathogenesis-responsive genes. Therefore, VviDMR6-1 represents a good candidate to produce resistant cultivars with a gene-editing approach.

The production of high-quality wines is strictly related to the correct management of the vineyard, which guarantees good yields and grapes with the right characteristics required for subsequent vinification. Winegrowers face a variety of challenges during the grapevine cultivation cycle: the most notorious are fungal and oomycete diseases such as downy mildew, powdery mildew, and gray mold. If not properly addressed, these diseases can irremediably compromise the harvest, with disastrous consequences for the production and wine economy. Conventional defense methods used in the past involved chemical pesticides. However, such approaches are in conflict with the growing attention to environmental sustainability and shifts from the uncontrolled use of chemicals to the use of integrated approaches for crop protection. Improvements in genetic knowledge and the availability of novel biotechnologies have created new scenarios for possibly producing grapes with a reduced, if not almost zero, impact. Here, the main approaches used to protect grapevines from fungal and oomycete diseases are reviewed, starting from conventional breeding, which allowed the establishment of new resistant varieties, followed by biotechnological methods, such as transgenesis, cisgenesis, intragenesis, and genome editing, and ending with more recent perspectives concerning the application of new products based on RNAi technology. Evidence of their effectiveness, as well as potential risks and limitations based on the current legislative situation, are critically discussed.

Several pathogens continuously threaten viticulture worldwide. Until now, the investigation on resistance loci has been the main trend to understand the interaction between grapevine and the mildew causal agents. Dominantly inherited gene-based resistance has shown to be race-specific in some cases, to confer partial immunity, and to be potentially overcome within a few years since its introgression. Recently, on the footprint of research conducted in Arabidopsis, putative genes associated with downy mildew susceptibility have been discovered also in the grapevine genome. In this work, we deep-sequenced four putative susceptibility genes—namely VvDMR6.1, VvDMR6.2, VvDLO1, VvDLO2—in 190 genetically diverse grapevine genotypes to discover new sources of broad-spectrum and recessively inherited resistance. Identified Single Nucleotide Polymorphisms were screened in a bottleneck analysis from the genetic sequence to their impact on protein structure. Fifty-five genotypes showed at least one impacting mutation in one or more of the scouted genes. Haplotypes were inferred for each gene and two of them at the VvDMR6.2 gene were found significantly more represented in downy mildew resistant genotypes. The current results provide a resource for grapevine and plant genetics and could corroborate genomic-assisted breeding programs as well as tailored gene editing approaches for resistance to biotic stresses.

The advent of CRISPR/Cas9 technology has revolutionized genome editing, enabling the attainment of once-unimaginable goals. CRISPR/Cas’s groundbreaking attributes lie in its simplicity, versatility, universality, and independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. It is therefore more and more used for genome modification including the generation of mutants. Beyond such editing scopes, the recent development of novel or modified Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Precisely targeting DNA or RNA sequences, the CRISPR/Cas system has been harnessed in fields as diverse as gene regulation, deepening insights into gene expression, epigenetic changes, genome spatial organization, and chromatin dynamics. Furthermore, it aids in genome imaging and sequencing, as well as effective identification and countering of viral pathogens in plants and animals. All in all, the non-editing aspect of CRISPR/Cas exhibits tremendous potential across diverse domains, including diagnostics, biotechnology, and fundamental research. This article reviews and critically evaluates the primary CRISPR/Cas-based tools developed for plants and animals, underlining their transformative impact.

PIWI, from the German word Pilzwiderstandsfähig, meaning “fungus-resistant”, refers to grapevine cultivars bred for resistance to fungal pathogens such as Erysiphe necator (the causal agent of powdery mildew) and Plasmopara viticola (the causal agent of downy mildew), two major diseases in viticulture. These varieties are typically developed through traditional breeding, often crossbreeding European Vitis vinifera with American or Asian species that carry natural disease resistance. This study investigates the transcriptional profiles of exocarp tissues in mature berries from four PIWI grapevine varieties compared to their elite parental counterparts using RNA-seq analysis. We performed RNA-seq on four PIWI varieties (two red and two white) and their noble parents to identify differential gene expression patterns. Comprehensive analyses, including Differential Gene Expression (DEGs), Gene Set Enrichment Analysis (GSEA), Weighted Gene Co-expression Network Analysis (WGCNA), and tau analysis, revealed distinct gene clusters and individual genes characterizing the transcriptional landscape of PIWI varieties. Differentially expressed genes indicated significant changes in pathways related to organic acid metabolism and membrane transport, potentially contributing to enhanced resilience. WGCNA and k-means clustering highlighted co-expression modules linked to PIWI genotypes and their unique tolerance profiles. Tau analysis identified genes uniquely expressed in specific genotypes, with several already known for their defense roles. These findings offer insights into the molecular mechanisms underlying grapevine resistance and suggest promising avenues for breeding strategies to enhance disease resistance and overall grape quality in viticulture.

The advent of CRISPR/Cas9 technology has revolutionized genome editing, enabling the attainment of once-unimaginable goals. CRISPR/Cas's groundbreaking attributes lie in its simplicity, versatility, universality, and its independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. Beyond its editing prowess, the discovery of novel Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Researchers have harnessed CRISPR/Cas for gene regulation, deepening insights into gene expression and epigenetic changes. Precisely targeting DNA or RNA sequences, CRISPR/Cas effectively identifies and counters pathogens. Furthermore, it aids in genome imaging and sequencing, revealing genome spatial organization and chromatin dynamics. The non-editing applications of CRISPR/Cas exhibit tremendous potential across diverse domains, including diagnostics, biotechnology, and fundamental research. This article reviews and critically evaluates the primary CRISPR/Cas-based tools developed for plants and animals, underlining their transformative impact.