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Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine.

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.

SUBMITTER: Pessina S

PROVIDER: S-EPMC4935963 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine.

Pessina Stefano S Lenzi Luisa L Perazzolli Michele M Campa Manuela M Dalla Costa Lorenza L Urso Simona S Valè Giampiero G Salamini Francesco F Velasco Riccardo R Malnoy Mickael M

Horticulture research 20160420

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-gene ...[more]

PMID: 27390621

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Project description:BackgroundPowdery mildew is a major disease that causes great losses in soybean yield and seed quality. Disease-resistant varieties, which are generated by reducing the impact of susceptibility genes through mutation in host plants, would be an effective approach to protect crops from this disease. The Mildew Locus O (MLO) genes are well-known susceptibility genes for powdery mildew in plant. In this study, we utilized the CRISPR/Cas9 system to induce targeted mutations in the soybean GmMLO genes to improve powdery mildew resistance.ResultsA dual-sgRNA CRISPR/Cas9 construct was designed and successfully transferred into the Vietnamese soybean cultivar DT26 through Agrobacterium tumefaciens-mediated transformation. Various mutant forms of the GmMLO genes including biallelic, chimeric and homozygous were found at the T0 generation. The inheritance and segregation of CRISPR/Cas9-induced mutations were confirmed and validated at the T1 and T2 generations. Out of six GmMLO genes in the soybean genome, we obtained the Gmmlo02/Gmmlo19/Gmmlo23 triple and Gmmlo02/Gmmlo19/Gmmlo20/Gmmlo23 quadruple knockout mutants at the T2 generation. When challenged with Erysiphe diffusa, a fungus that causes soybean powdery mildew, all mutant plants showed enhanced resistance to the pathogen, especially the quadruple mutant. The powdery mildew severity in the mutant soybeans was reduced by up to 36.4% compared to wild-type plants. In addition, no pleiotropic effect on soybean growth and development under net-house conditions was observed in the CRISPR/Cas9 mutants.ConclusionsOur results indicate the involvement of GmMLO02, GmMLO19, GmMLO20 and GmMLO23 genes in powdery mildew susceptibility in soybean. Further research should be conducted to investigate the roles of individual tested genes and the involvement of other GmMLO genes in this disease infection mechanism. Importantly, utilizing the CRISPR/Cas9 system successfully created the Gmmlo transgene-free homozygous mutant lines with enhanced resistance to powdery mildew, which could be potential materials for soybean breeding programs.

Project description:Cannabis sativa is increasingly being grown around the world for medicinal, industrial, and recreational purposes. As in all cultivated plants, cannabis is exposed to a wide range of pathogens, including powdery mildew (PM). This fungal disease stresses cannabis plants and reduces flower bud quality, resulting in significant economic losses for licensed producers. The Mildew Locus O (MLO) gene family encodes plant-specific proteins distributed among conserved clades, of which clades IV and V are known to be involved in susceptibility to PM in monocots and dicots, respectively. In several studies, the inactivation of those genes resulted in durable resistance to the disease. In this study, we identified and characterized the MLO gene family members in five different cannabis genomes. Fifteen Cannabis sativa MLO (CsMLO) genes were manually curated in cannabis, with numbers varying between 14, 17, 19, 18, and 18 for CBDRx, Jamaican Lion female, Jamaican Lion male, Purple Kush, and Finola, respectively (when considering paralogs and incomplete genes). Further analysis of the CsMLO genes and their deduced protein sequences revealed that many characteristics of the gene family, such as the presence of seven transmembrane domains, the MLO functional domain, and particular amino acid positions, were present and well conserved. Phylogenetic analysis of the MLO protein sequences from all five cannabis genomes and other plant species indicated seven distinct clades (I through VII), as reported in other crops. Expression analysis revealed that the CsMLOs from clade V, CsMLO1 and CsMLO4, were significantly upregulated following Golovinomyces ambrosiae infection, providing preliminary evidence that they could be involved in PM susceptibility. Finally, the examination of variation within CsMLO1 and CsMLO4 in 32 cannabis cultivars revealed several amino acid changes, which could affect their function. Altogether, cannabis MLO genes were identified and characterized, among which candidates potentially involved in PM susceptibility were noted. The results of this study will lay the foundation for further investigations, such as the functional characterization of clade V MLOs as well as the potential impact of the amino acid changes reported. Those will be useful for breeding purposes in order to develop resistant cultivars.

Project description:BackgroundSpecific members of the plant Mildew Locus O (MLO) protein family act as susceptibility factors towards powdery mildew (PM), a worldwide-spread fungal disease threatening many cultivated species. Previous studies indicated that monocot and dicot MLO susceptibility proteins are phylogenetically divergent.MethodsA bioinformatic approach was followed to study the type of evolution of Angiosperm MLO susceptibility proteins. Transgenic complementation tests were performed for functional analysis.ResultsOur results show that monocot and dicot MLO susceptibility proteins evolved class-specific conservation patterns. Many of them appear to be the result of negative selection and thus are likely to provide an adaptive value. We also tested whether different molecular features between monocot and dicot MLO proteins are specifically required by PM fungal species to cause pathogenesis. To this aim, we transformed a tomato mutant impaired for the endogenous SlMLO1 gene, and therefore resistant to the tomato PM species Oidium neolycopersici, with heterologous MLO susceptibility genes from the monocot barley and the dicot pea. In both cases, we observed restoration of PM symptoms. Finally, through histological observations, we demonstrate that both monocot and dicot susceptibility alleles of the MLO genes predispose to penetration of a non-adapted PM fungal species in plant epidermal cells.ConclusionsWith this study, we provide insights on the evolution and function of MLO genes involved in the interaction with PM fungi. With respect to breeding research, we show that transgenic complementation assays involving phylogenetically distant plant species can be used for the characterization of novel MLO susceptibility genes. Moreover, we provide an overview of MLO protein molecular features predicted to play a major role in PM susceptibility. These represent ideal targets for future approaches of reverse genetics, addressed to the selection of loss-of-function resistant mutants in cultivated species.

Dataset Information

Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine.

Publications

Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine.

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