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\u2014namely VvDMR6.1, VvDMR6.2, VvDLO1, VvDLO2\u2014in 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

A DNA-free editing approach for viticulture sustainability: dual editing of DMR6-1 and DMR6-2 enhances resistance to downy mildew

The sustainability of viticulture hinges on maintaining quality and yield while reducing pesticide use. Promising strides in this direction involve the development of clones with enhanced disease tolerance, particularly through the knockout of plant susceptibility genes. Knocking out of Downy Mildew Resistant 6 (DMR6) led to increased levels of endogenous salicylic acid (SA), a regulator of immunity, resulting in enhanced tolerance to Downy Mildew (DM) and other diseases in various crops. Mutations in both DMR6-1 and DMR6-2 genes were introduced into two grapevine cultivars using CRISPR-Cas9 using two methods. In the first case, transgene delivery mediated by A. tumefaciens was employed, while in the second case, we developed a 'single-cell technology' for gene editing, creating non-transgenic grapevine mutants through the regeneration of protoplasts previously edited with the CRISPR/Cas9 ribonucleoprotein. We tested the susceptibility of single and double mutants to DM through artificial inoculation assays on detached leaves and whole plants. Our findings indicate that a simultaneous mutation in both DMR6-1 and DMR6-2 is needed to significantly enhance resistance to DM, with the double mutant (dmr6-1-dmr6-2) outperforming either single mutant in both cultivars. Elevated levels of endogenous SA were only observed in the double mutant, while single mutation in DMR6-1 or DMR6-2 proved ineffective. Collectively, our data highlight the need for a double knockout to achieve appreciable results against DM-susceptibility. Currenlty, we are adapting the 'single-cell technology' to generate edited vines from various agronomically relevant cultivars. In parallel, we are assessing the performance of plants edited in different susceptibility genes

Breeding for grapevine downy mildew resistance via gene editing

Downy mildew (DM) caused by the oomycete Plasmopara viticola ranks in the top diseases affecting grapevine (Vitis vinifera L.) cultivation and its control requires every year a large use of fungicides. The Farm to Fork strategy newly promoted by the EU aims to accelerate the transition to a sustainable food system and has set very ambitious targets including the reduction by 50% of the use and risk of pesticides by 2030. The introduction of disease-tolerant grapevine varieties or clones clearly represents a step forward to reach this goal. The recent advent of new breeding tools such as genome editing and cis-genesis offers a great opportunity to obtain resistant plants with higher precision and speed than by conventional breeding, either by knocking down susceptibility genes or by introducing known resistance-genes in commercial cultivars. Based on reports in other crops, the family of Downy Mildew Resistant 6 (DMR6) and DMR6-like oxygenases (DLOs) are candidate susceptibility genes for the control of DM resistance in V. vinifera. Deep-sequencing the putative susceptibility genes in 190 genetically diverse grapevine genotypes identified several Single Nucleotide Polymorphisms then screened for their impact on protein structure/function and association with DM resistant genotypes. Gene expression and gene network analysis suggested that grapevine DMR6 and DLO genes have distinct functions, and that VviDMR6-1 is co-regulated with several Pathogenesis-related genes. Based on this evidence, we generated a large collection of DMR6-1 and DMR6-2 single and double knock-out mutants in multiple grapevine cultivars and evaluated their resistance to DM. Phenotypic resistance data upon artificial infection are being collected and will be presented here. In parallel, we also developed a new DNA-free gene editing methodology and obtained non-transgenic and non-chimeric edited grapevine plants regenerated from a single cell

Approaches to mixture risk assessment of PFASs in the European population based on human hazard and biomonitoring data

Per- and polyfluoroalkyl substances (PFASs) are a highly persistent, mobile, and bioaccumulative class of chemicals, of which emissions into the environment result in long-lasting contamination with high probability for causing adverse effects to human health and the environment. Within the European Biomonitoring Initiative HBM4EU, samples and data were collected in a harmonized way from human biomonitoring (HBM) studies in Europe to derive current exposure data across a geographic spread. We performed mixture risk assessments based on recent internal exposure data of PFASs in European teenagers generated in the HBM4EU Aligned Studies (dataset with N = 1957, sampling years 2014-2021). Mixture risk assessments were performed based on three hazard-based approaches: the Hazard Index (HI) approach, the sum value approach as used by the European Food Safety Authority (EFSA) and the Relative Potency Factor (RPF) approach. The HI approach resulted in the highest risk estimates, followed by the RPF approach and the sum value approach. The assessments indicate that PFAS exposure may result in a health risk in a considerable fraction of individuals in the HBM4EU teenager study sample, thereby confirming the conclusion drawn in the recent EFSA scientific opinion. This study underlines that HBM data are of added value in assessing the health risks of aggregate and cumulative exposure to PFASs, as such data are able to reflect exposure from different sources and via different routes.This work was supported by the European Union’s Horizon 2020 research and innovation programme under Grant agreement No 733032 HBM4EU (www.HBM4EU.eu), and received co-funding from the au thors’ organizations. The Norwegian Institute of Public Health (NIPH) has contributed to funding of the Norwegian Environmental Biobank (NEB), and the laboratory measurements have partly been funded by the Research Council of Norway through research projects (275903 and 268465). The PCB cohort (follow-up) received additional funding from the Ministry of Health of the Slovak Republic (program 07B0103).S

Approaches to mixture risk assessment of PFASs in the European population based on human hazard and biomonitoring data

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Functional and applied aspects of the DOWNY MILDEW RESISTANT 1 and 6 genes in Arabidopsis

Plants are constantly exposed to micro-organisms including pathogens. They are resistant to the vast majority of potential pathogens due to a multilayered defence system. Plant disease susceptibility, on the other hand, is determined by virulence factors from the pathogen, their targets in the host and the successful suppression of plant defences. To gain insight into the molecular mechanisms underlying disease susceptibility, we study the interaction between the oomycete pathogen Hyaloperonospora arabidopsidis, causing downy mildew, and its host Arabidopsis. A forward genetic screen resulted in several downy mildew resistant (dmr) Arabidopsis mutants of which dmr1 and dmr6 were further characterized. Chapter 2 described the cloning of DMR1 that encodes for homoserine kinase (HSK), a key enzyme in the Aspartate pathway. Mutation of HSK in the dmr1 mutant resulted in accumulation of the amino acid homoserine. Exogenous application of homoserine proved to be sufficient to induce resistance against H. arabidopsidis. No known defence-related pathways were associated with the observed resistance, indicating homoserine accumulation triggers a novel form of plant resistance. HSK was further analysed in chapter 3 where a reverse genetics approach (TILLING) resulted in additional hsk alleles. One mutant accumulated homoserine and is resistant to downy mildew infection. Another mutant did not develop beyond the seedling stage suggesting HSK is an essential plant gene. We also identified HSK orthologs for a number of crop species making them candidates for mutagenesis to further explore dmr1-mediated resistance. Chapter 4 described the characterization of the DMR6 gene, coding for a 2-oxoglutarate Fe(II)-dependent oxygenase. Absence of a functional DMR6 gene resulted in resistance that requires a functional salicylic acid pathway. On the other hand, overexpression of DMR6 resulted in enhanced susceptibility towards biotrophic pathogens indicating DMR6 acts as negative regulator of defence. A 3-dimensional model was constructed of the DMR6 protein that facilitated in the identification of important residues in the substrate pocket. Chapter 5 described the family of DMR6-like oxygenases (DLOs) and their role in plant defence. Two Arabidopsis DLOs, identified through phylogenetic analysis, have a similar molecular function as DMR6. Also, we have identified DMR6 orthologs in several crop species that are able to complement the dmr6-mediated resistance. A DMR6/DLO-specific motif is identified that could be used for the identification of crop DMR6 or DLO proteins. The presence of this conserved motif could determine the specificity for a particular substrate that is shared between different species

The Relationship Between Internal and External Dose: Some General Results Based on a Generic Compartmental Model

Abstract Statements on how the internal-to-external-dose (IED) relationship looks like are often based on qualitative toxicokinetic arguments. For example, the recently proposed kinetically derived maximum dose (KMD) states that the IED relationship must have an inflection point, due to saturation of underlying processes like metabolism or absorption. However, such statements lack a solid quantitative foundation. Therefore, we derived expressions for the IED relationship for a number of scenarios based on a generic compartmental model involving saturation. The scenarios included repeated or single dose, and saturable metabolism or saturable absorption. For some of these scenarios, an explicit expression for the IED relationship can be derived, for others only implicit expressions can be established, which need to be evaluated numerically. The results show that saturable processes will lead to an IED relationship that is nonlinear over the whole dose range, ie, it can be approximated by a linear relationship at the lower end, whereas the approximation will become gradually poorer with increasing doses. The finding that saturation does not lead to an inflection point in the IED relationship, as assumed in the KMD, implies that the KMD is not a valid approach for selecting the top dose in toxicological studies. An additional use of our results is that the derived explicit expressions of the IED relationship can be fitted to IED data, and, possibly, for extrapolation outside the observed dose range.</jats:p

Scouting downy and powdery mildew susceptibility genes: a diversity study in Vitis spp.

World viticulture is continually threatened by both known and emerging pathogens. Until now, the investigation on resistance loci/genes has been the main trend to understand the interaction between grapevine (Vitis spp.) and mildew causal agents. Dominantly inherited gene-based resistance has shown to be race-specific in some cases, not to confer total immunity and to be potentially overcome within a few years. Recently, on the footprint of research conducted on Arabidopsis and barley, genes associated to downy (DM) and powdery (PM) mildew susceptibility have been discovered also in the grapevine genome. In the present work, in order to find new sources of broad-spectrum recessively inherited resistance against pathogens five susceptibility genes were re-sequenced (Illumina, 1000X depth) in 96 grapevine accessions including wild, vinifera and hybrid individuals. The scouted genes were VvDMR6-1, VvDMR6-2, VvDLO1, VvDLO2 involved in susceptibility to DM and VvMLO7 associated with susceptibility to PM. These genes were mapped on the reference genome and analysed to identify polymorphisms and haplotypes using dedicated software to study the mutation impact. Preliminary results showed 10 mutations affecting the VvMLO7 protein structure (high-medium impact) dispersed in 75% of accessions; in particular, one Single Nucleotide Polymorphisms (SNPs) led to premature stop codons. Moreover, 70% of the accessions showed a total of 13 SNPs in VvDMR6-1 and 11 in VvDMR6-2 impacting their coding and amino acid sequences. Finally, 12 mutations were detected in the VvDLO1 coding sequence in 37 individuals, whereas the VvDLO2 sequence appeared much more conserved with only one SNP identified in four accessions. These findings will be validated taking advantage of several reliable parentages. Prior haplotype function confirmation, the final results will corroborate genomic-assisted breeding programs for resistance to biotic stresses