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      Identification of Susceptibility Genes in Castanea sativa and Their Transcription Dynamics following Pathogen Infection

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          Abstract

          Castanea sativa is one of the main multipurpose tree species valued for its timber and nuts. This species is susceptible to two major diseases, ink disease and chestnut blight, caused by Phytophthora spp. and Cryphonectria parasitica, respectively. The loss-of-function mutations of genes required for the onset of pathogenesis, referred to as plant susceptibility (S) genes, are one mechanism of plant resistance against pathogens. On the basis of sequence homology, functional domain identification, and phylogenetic analyses, we report for the first time on the identification of S-genes ( mlo1, dmr6, dnd1, and pmr4) in the Castanea genus. The expression dynamics of S-genes were assessed in C. sativa and C. crenata plants inoculated with P. cinnamomi and C. parasitica. Our results highlighted the upregulation of pmr4 and dmr6 in response to pathogen infection. Pmr4 was strongly expressed at early infection phases of both pathogens in C. sativa, whereas in C. crenata, no significant upregulation was observed. The infection of P. cinnamomi led to a higher increase in the transcript level of dmr6 in C. sativa compared to C. crenata-infected samples. For a better understanding of plant responses, the transcript levels of defense genes gluB and chi3 were also analyzed.

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          Comparative Protein Structure Modeling Using MODELLER.

          Comparative protein structure modeling predicts the three-dimensional structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and how to use the ModBase database of such models, and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. © 2016 by John Wiley & Sons, Inc.
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            Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.

            Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator-like effector nuclease (TALEN) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.
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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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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Plants (Basel)
                Plants (Basel)
                plants
                Plants
                MDPI
                2223-7747
                02 May 2021
                May 2021
                : 10
                : 5
                : 913
                Affiliations
                Dipartimento di Scienze Agrarie, Forestali e Alimentari-DISAFA, Università degli Studi di Torino, Largo Paolo Braccini 2, Grugliasco, 10095 Torino, Italy; vera.pavese@ 123456unito.it (V.P.); paolo.gonthier@ 123456unito.it (P.G.); daniela.marinoni@ 123456unito.it (D.T.M.); emile.cavaletgiorsa@ 123456unito.it (E.C.-G.); roberto.botta@ 123456unito.it (R.B.)
                Author notes
                Author information
                https://orcid.org/0000-0001-7431-844X
                https://orcid.org/0000-0002-7242-8239
                Article
                plants-10-00913
                10.3390/plants10050913
                8147476
                34063239
                82daa2ca-52f6-4762-8d5d-211cc5c34dd4
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 31 March 2021
                : 28 April 2021
                Categories
                Article

                chestnut,susceptibility genes,phytophthora cinnamomi,cryphonectria parasitica

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