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      Proteinaceous effector discovery and characterization in filamentous plant pathogens

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          Abstract

          The complicated interplay of plant–pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens that produce small proteins called effectors to manipulate the host and aid infection/colonization. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterization of the effectors of filamentous plant pathogens and identification of their host targets.

          Abstract

          The chronological progression of methodologies used in effector discovery; from physical isolation and in silico predictions, to functional characterization and host target identification.

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          Most cited references182

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          Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.

          Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.
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            Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.

            Engineered nucleases can be used to induce site-specific double-strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error-prone non-homologous end-joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single-strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease-induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ-mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740-fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I-SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB-inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR-mediated gene targeting systems but also by the combined action of two nickases as DSB-inducing agents excluding off-target effects in homologous genomic regions. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.
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              Opportunities and challenges for transcriptome-wide association studies

              Transcriptome-wide association studies (TWAS) integrate genome-wide association studies (GWAS) and gene expression datasets to identify gene-trait associations. In this Perspective, we explore properties of TWAS as a potential approach to prioritize causal genes at GWAS loci, by using simulations and case studies of literature-curated candidate causal genes for schizophrenia, low-density-lipoprotein cholesterol and Crohn’s disease. We explore risk loci where TWAS accurately prioritizes the likely causal gene as well as loci where TWAS prioritizes multiple genes, some likely to be non-causal, owing to sharing of expression quantitative trait loci (eQTL). TWAS is especially prone to spurious prioritization with expression data from non-trait-related tissues or cell types, owing to substantial cross-cell-type variation in expression levels and eQTL strengths. Nonetheless, TWAS prioritizes candidate causal genes more accurately than simple baselines. We suggest best practices for causal-gene prioritization with TWAS and discuss future opportunities for improvement. Our results showcase the strengths and limitations of using eQTL datasets to determine causal genes at GWAS loci.
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                Author and article information

                Contributors
                kim.hammond-kosack@rothamsted.ac.uk
                Journal
                Mol Plant Pathol
                Mol. Plant Pathol
                10.1111/(ISSN)1364-3703
                MPP
                Molecular Plant Pathology
                John Wiley and Sons Inc. (Hoboken )
                1464-6722
                1364-3703
                07 August 2020
                October 2020
                : 21
                : 10 ( doiID: 10.1002/mpp.v21.10 )
                : 1353-1376
                Affiliations
                [ 1 ] Department of Biointeractions and Crop Protection Rothamsted Research Harpenden UK
                [ 2 ] School of Biosciences University of Nottingham Nottingham UK
                Author notes
                [*] [* ] Correspondence

                Kim E. Hammond‐Kosack, Department of Biointeractions and Crop Protection, Rothamsted Research, Harpenden, AL5 2JQ, UK.

                Email: kim.hammond-kosack@ 123456rothamsted.ac.uk

                Author information
                https://orcid.org/0000-0003-2679-6636
                https://orcid.org/0000-0002-9699-485X
                Article
                MPP12980
                10.1111/mpp.12980
                7488470
                32767620
                e912e4d1-f4f4-4ffe-a06f-08f9f237fa6d
                © 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 20 March 2020
                : 03 June 2020
                : 05 July 2020
                Page count
                Figures: 4, Tables: 2, Pages: 24, Words: 55266
                Funding
                Funded by: Biotechnology and Biological Sciences Research Council , open-funder-registry 10.13039/501100000268;
                Award ID: BB/M008770/1
                Award ID: BB/P016855/1
                Categories
                Review
                Review
                Custom metadata
                2.0
                October 2020
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.9.0 mode:remove_FC converted:14.09.2020

                Plant science & Botany
                bioinformatic effector predictions,effector host–target interactions,effectors,fungal phytopathogens,in planta methodologies,oomycete phytopathogens

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