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      Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine

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          Abstract

          Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO ( Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion.

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          An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development

          Background Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents. Results We describe optimization of an RNA isolation procedure that compensates for the low pH found in grape berries and improves the ability of the RNA to precipitate. This procedure was tested on pericarp and seed developmental series, as well as steady-state leaf, root, and flower tissues. Additionally, the expression stability of actin, AP47 (clathrin-associated protein), cyclophilin, EF1-α (elongation factor 1-α), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), MDH (malate dehydrogenase), PP2A (protein phosphatase), SAND, TIP41, α-tubulin, β-tubulin, UBC (ubiquitin conjugating enzyme), UBQ-L40 (ubiquitin L40) and UBQ10 (polyubiquitin) were evaluated on Vitis vinifera cv. Cabernet Sauvignon pericarp using three different statistical approaches. Although several of the genes proved to be relatively stable, no single gene outperformed all other genes in each of the three evaluation methods tested. Furthermore, the effect of using one reference gene versus normalizing to the geometric mean of several genes is presented for the expression of an aquaporin and a sucrose transporter over a developmental series. Conclusion In order to quantify relative transcript abundances accurately using real-time RT-PCR, we recommend that combinations of several genes be used for normalization in grape berry development studies. Our data support GAPDH, actin, EF1-α and SAND as the most relevant reference genes for this purpose.
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            The barley Mlo gene: a novel control element of plant pathogen resistance.

            Mutation-induced recessive alleles (mlo) of the barley Mlo locus confer a leaf lesion phenotype and broad spectrum resistance to the fungal pathogen, Erysiphe graminis f. sp. hordei. The gene has been isolated using a positional cloning approach. Analysis of 11 mutagen-induced mlo alleles revealed mutations leading in each case to alterations of the deduced Mlo wild-type amino acid sequence. Susceptible intragenic recombinants, isolated from mlo heteroallelic crosses, show restored Mlo wild-type sequences. The deduced 60 kDa protein is predicted to be membrane-anchored by at least six membrane-spanning helices. The findings are compatible with a dual negative control function of the Mlo protein in leaf cell death and in the onset of pathogen defense; absence of Mlo primes the responsiveness for the onset of multiple defense functions.
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              Loss of susceptibility as a novel breeding strategy for durable and broad-spectrum resistance

              Recent studies on plant immunity have suggested that a pathogen should suppress induced plant defense in order to infect a plant species, which otherwise would have been a nonhost to the pathogen. For this purpose, pathogens exploit effector molecules to interfere with different layers of plant defense responses. In this review, we summarize the latest findings on plant factors that are activated by pathogen effectors to suppress plant immunity. By looking from a different point of view into host and nonhost resistance, we propose a novel breeding strategy: disabling plant disease susceptibility genes (S-genes) to achieve durable and broad-spectrum resistance.
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                Author and article information

                Journal
                Hortic Res
                Hortic Res
                Horticulture Research
                Nature Publishing Group
                2052-7276
                20 April 2016
                2016
                : 3
                : 16016
                Affiliations
                [1 ] Research and Innovation Centre, Fondazione Edmund Mach , via Edmund Mach 1, 38010 San Michele all’Adige, Italy
                [2 ] Wageningen UR Plant Breeding, Wageningen University and Research Centre , PO Box 386, 6700 AJ Wageningen, The Netherlands
                [3 ] Department of Ecological and Biological Sciences, Università della Tuscia , Via San Camillo de Lellis, Viterbo 01100, Italy
                [4 ] Council for Agricultural Research and Economics (CREA), Rice Research Unit , S.S. 11 per Torino km 25, Vercelli 13100, Italy
                [5 ] Council for Agricultural Research and Economics (CREA), Genomics Research Centre , Via San Protaso, 302, 29017, Fiorenzuola d’Arda, Piacenza, Italy
                Author notes
                []

                SP propagated and acclimated the transgenic lines, carried out the gene expression analysis and the statistical analysis and wrote the major part of the manuscript. LL designed and performed the inoculation experiments and visually assessed the symptoms with powdery and downy mildew, counted the conidia, took the microscopy pictures and revised the manuscript. MP designed the inoculation experiments, contributed to the analysis of severity and gene expression data, and revised the manuscript. MC carried out the grapevine gene transfer, identified false positives and revised the manuscript. LDC carried out the grapevine gene transfer and revised the manuscript. SU designed and built the construct for RNAi and revised the manuscript. GV conceived the project and revised the manuscript. RV conceived the project and revised the manuscript. FS conceived the project and revised the manuscript. MM conceived the project, contributed to data analysis and was the main reviser of the manuscript.

                Article
                hortres201616
                10.1038/hortres.2016.16
                4935963
                27390621
                7ea69560-7dd2-4c7c-b0e9-de76c8fbebcc
                Copyright © 2016 Nanjing Agricultural University

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 29 November 2015
                : 15 March 2016
                : 15 March 2016
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