Mining grapevine downy mildew susceptibility genes: a resource for genomics-based breeding and tailored gene editing

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 stresse

Mining downy mildew susceptibility genes: a diversity study in grapevine

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 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 on Arabidopsis, the putative hortologues of genes associated with downy mildew susceptibility in this species, have been discovered also in the grapevine genome. In this work, we deep-resequenced four putative susceptibility genes in 190 highly genetically diverse grapevine genotypes to discover new sources of broad-spectrum recessively inherited resistance. The scouted genes are VvDMR6-1, VvDMR6-2, VvDLO1, VvDLO2 and predicted to be involved in susceptibility to downy mildew. From all identified mutations, 56% were Single Nucleotide Polymorphisms (SNPs) in heterozygosity, while the remaining 44% were homozygous. Regarding the identified mutations with putative impact on gene function, we observed ~4% genotypes mutated in VvDMR6-1 and ~8% mutated in VvDMR6-2, only a handful of genotypes that were mutated in both genes. ~2% and ~7% genotypes showed mutations in VvDLO1 and VvDLO2 respectively, and again a few genotypes resulted mutated in both genes. In particular, 80% of impacting mutations were heterozygous while 20% were homozygous. The current results will inform grapevine genetics and corroborate genomic-assisted breeding programs for resistance to biotic stresses

Grapevine DMR6-1 Is a candidate gene for susceptibility to Downy mildew

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 approac

Emergent Ascomycetes in viticulture: an interdisciplinary overview

The reduction of pesticide usage is a current imperative and the implementation of sustainable viticulture is an urgent necessity. A potential solution, which is being increasingly adopted, is offered by the use of grapevine cultivars resistant to its main pathogenic threats. This, however, has contributed to changes in defense strategies resulting in the occurrence of secondary diseases, which were previously controlled. Concomitantly, the ongoing climate crisis is contributing to destabilizing the increasingly dynamic viticultural context. In this review, we explore the available knowledge on three Ascomycetes which are considered emergent and causal agents of powdery mildew, black rot and anthracnose. We also aim to provide a survey on methods for phenotyping disease symptoms in fields, greenhouse and lab conditions, and for disease control underlying the insurgence of pathogen resistance to fungicide. Thus, we discuss fungal genetic variability, highlighting the usage and development of molecular markers and barcoding, coupled with genome sequencing. Moreover, we extensively report on the current knowledge available on grapevine-ascomycete interactions, as well as the mechanisms developed by the host to counteract the attack. Indeed, to better understand these resistance mechanisms, it is relevant to identify pathogen effectors which are involved in the infection process and how grapevine resistance genes function and impact the downstream cascade. Dealing with such a wealth of information on both pathogens and the host, the horizon is now represented by multidisciplinary approaches, combining traditional and innovative methods of cultivation. This will support the translation from theory to practice, in an attempt to understand biology very deeply and manage the spread of these Ascomycetes

Grapevine DMR6-1 Is a Candidate Gene for Susceptibility to Downy mildew

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

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Grapevine DMR6-1 Is a Candidate Gene for Susceptibility to Downy Mildew

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

Mining Grapevine Downy Mildew Susceptibility Genes: A Resource for Genomics-Based Breeding and Tailored Gene Editing

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

Exploring the role of downy mildew susceptibility genes in Vitis by studying the genetic variation and the in vivo function

Viticulture is worldly threatened by fungal pathogens. Management of vineyards entails the use of great amounts of fungicides in several applications each year, generating a harmful environmental impact on human health and local biodiversity. In the last decades, resistance (R) loci scouting and deployment have been the main strategy to cope with some of the most aggressive grapevine pathogens: Erysiphe necator, the causal agent of powdery mildew (PM), and Plasmopara viticola, the causal agent of downy mildew (DM). However, R loci-based resistance can be easily overcome by pathogens within a few years from their introgression. Recently, susceptibility (S) genes have been used as a new source of durable and broad-spectrum resistance in many crops and tree species. In grapevine, S genes like VvMLO7 were associated with susceptibility to PM and their role has been studied, also using knock down mutants. On the other hand, S genes associated to susceptibility to DM are not yet available in grapevine. Loss of function mutations in AtDMR6 and AtDLO provide resistance against DM in Arabidopsis, therefore their putative grapevine orthologs VvDMR6.1, VvDMR6.2, VvDLO1 and VvDLO2 are candidate S genes, likely associated to DM in grapevine .In this thesis, we explored the role of the aforementioned grapevine S genes starting from a broad genetic diversity analysis to a functional characterization study on dmr6.1 grapevine plants. Regarding the survey on genetic variability, VvDMR6.1, VvDMR6.2, VvDLO1 and VvDLO2 were investigated in 190 grapevine genotypes belonging to Vitis vinifera spp., wild species, hybrids and the so-called hybrid/wild species, in order to find S genes natural mutants. The scouted genes were deep-sequenced and reads were mapped on PN40024 12× V2 reference genome. A bottleneck analysis was carried out in order to, firstly, identify SNPs (Single Nucleotide Polymorphisms) impacting on the coding sequence, then investigate the potential disrupting role of impacting SNPs on codons and the amino acid sequence and therefore on the protein folding and function. A representative handful of disrupting SNPs were chosen for confirmation by Sanger sequencing. The disrupting impact of amino acid mutations caused by the validated SNPs was then checked on a protein three-dimensional model. DM resistance phenotypic data were collected and compared to the frequency of the reconstructed haplotypes per each gene. Two of them, in VvDMR6.2, were found significantly more represented in DM resistant genotypes. VvMLO7 was sequenced in the 190 grapevine accessions as well, and the resulting data were subjected to the same bottleneck analysis. Once amino acid sequence-disrupting mutations were scouted, we took advantage of the known MLO protein model to identify those mutations that were changing conserved amino acids. Ten mutations were predicted to impact protein function, but no association with phenotypic data was possible since all SNPs were at the heterozygous state. This broad survey provided 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. Functional characterizations of VvDMR6.1, VvDMR6.2, VvDLO1 and VvDLO2 were carried out taking advantage of dmr6.1 grapevine plants previously obtained by the CRISPR/Cas9 genome editing technique. Plants of four different edited lines (each line originated from a separate transformation event) were subjected to P. viticola inoculation assay. Leaves were sampled at 0, 24, 96 hours post-inoculation (hpi) and 8 days post-inoculation (dpi). Samples were collected for several purposes. A gene expression analysis of VvDMR6 and VvDLO genes and pathogenesis-related genes was carried out on 0, 24 and 96 hpi samples. Samples taken at 8 dpi were used for symptom assessment via visual, digital, and histological observation. Since the salicylic acid (SA)-inactive forms 2,5-dihydrobenzoic acid (2,5-DHBA) and 2,3-DHBA are products of S5H (SA-5- hydroxylases) and S3H enzymes, putatively encoded by VvDMR6 and VvDLO, these metabolites were quantified through LC-MS in the same samples collected at 0, 24 and 96 hpi. This overview on the many aspects related to VvDMR6 and VvDLO gene function, made it clear that no uniform effect of VvDMR6.1 knock-out was detectable among different edited lines and further investigations are needed to define the role of the single genes and the relationship among them

Use of the model plant Arabidopsis thaliana to study the role of specific terpenoids in plant-insect interaction

The potential of plant Volatile Organic Compounds (VOCs) in pest control has been recently investigated opening new perspectives for managing pest insects. With the discovery of terpene-synthase gene families and considering the possibility to perform genetic transformation in several plant species, it is now possible to study the role of specific VOCs and their impact on insects behavior in in vivo systems. Given the ease of transformation of Arabidopsis thaliana, the presence in it of a metabolic pathway for the biosynthesis of terpenes, the possibility to use simple detection techniques for volatile compounds, we decided to use Arabidopsis to study the potential role of some specific sesquiterpenes involved in the attraction of grapevine moth: (E)-β-caryophyllene and (E)-β-farnesene. Although we know the volatile blend that attracts the moth consists of many compounds, we started with the creation of transformed plants that emit an individual volatile to study their specific role