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Exploring the role of downy mildew susceptibility genes in Vitis by studying the genetic variation and the in vivo function
Get PDFViticulture 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
Past, present and future of genetic strategies to control tolerance to the main fungal and oomycete pathogens in grapevine
No full textThe 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 the use of chemical pesticides. However, such approaches are in conflict with the growing attention on 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 RNA interference (RNAi) technology. Evidence of their effectiveness, as well as potential risks and limitations based on the current legislative situation, are critically discussed
Use of the model plant Arabidopsis thaliana to study the role of specific terpenoids in plant-insect interaction
No full textThe 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
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Mining downy mildew susceptibility genes: a diversity study in grapevine.
Get PDFSeveral 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
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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