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      Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus


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          Ferulate‐5‐hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate‐5‐hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D‐NMR profiling in KO‐7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.


          The oilseed rape production is unremittingly affected by Sclerotinia sclerotiorum, which causes stem and leaf rot and reduces the production of rapeseed oil. Lignin is an established physical barrier against pathogens, and the different lignin monomer G and S content can have different lignin structures. In this study, four dominantly expressed BnF5H genes were knocked out by CRISPR/Cas9 simultaneously in B. napus, and the f5h mutants were generated. The S‐/G‐lignin composition ratio was decreased compared to that of the wild type (WT) based on the results of Mӓule staining and 2D‐NMR profiling in the KO‐7 line. The resistance to S. sclerotiorum in stems and leaves increased in knock‐out lines. Furthermore, we found that the stem strength of mutants was significantly increased compared to that of the WT. Collectively, for the first time, we demonstrate that knockout of the lignin pathway gene F5H decreases the S/G ratio, improves S. sclerotiorum resistance in B. napus, and increases stem strength.

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          Most cited references66

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          Plants elaborate a vast array of natural products, many of which have evolved to confer selective advantage against microbial attack. Recent advances in molecular technology, aided by the enormous power of large-scale genomics initiatives, are leading to a more complete understanding of the enzymatic machinery that underlies the often complex pathways of plant natural product biosynthesis. Meanwhile, genetic and reverse genetic approaches are providing evidence for the importance of natural products in host defence. Metabolic engineering of natural product pathways is now a feasible strategy for enhancement of plant disease resistance.
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            Formaldehyde stabilization facilitates lignin monomer production during biomass depolymerization

            Practical, high-yield lignin depolymerization methods could greatly increase biorefinery productivity and profitability. However, development of these methods is limited by the presence of interunit carbon-carbon bonds within native lignin, and further by formation of such linkages during lignin extraction. We report that adding formaldehyde during biomass pretreatment produces a soluble lignin fraction that can be converted to guaiacyl and syringyl monomers at near theoretical yields during subsequent hydrogenolysis (47 mole % of Klason lignin for beech and 78 mole % for a high-syringyl transgenic poplar). These yields were three to seven times those obtained without formaldehyde, which prevented lignin condensation by forming 1,3-dioxane structures with lignin side-chain hydroxyl groups. By depolymerizing cellulose, hemicelluloses, and lignin separately, monomer yields were between 76 and 90 mole % for these three major biomass fractions.
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              Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen.

              SUMMARY Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen causing disease in a wide range of plants. This review summarizes current knowledge of mechanisms employed by the fungus to parasitize its host with emphasis on biology, physiology and molecular aspects of pathogenicity. In addition, current tools for research and strategies to combat S. sclerotiorum are discussed. Sclerotinia sclerotiorum (Lib.) de Bary: kingdom Fungi, phylum Ascomycota, class Discomycetes, order Helotiales, family Sclerotiniaceae, genus Sclerotinia. Hyphae are hyaline, septate, branched and multinucleate. Mycelium may appear white to tan in culture and in planta. No asexual conidia are produced. Long-term survival is mediated through the sclerotium; a pigmented, multi-hyphal structure that can remain viable over long periods of time under unfavourable conditions for growth. Sclerotia can germinate to produce mycelia or apothecia depending on environmental conditions. Apothecia produce ascospores, which are the primary means of infection in most host plants. S. sclerotiorum is capable of colonizing over 400 plant species found worldwide. The majority of these species are dicotyledonous, although a number of agriculturally significant monocotyledonous plants are also hosts. Disease symptoms: Leaves usually have water-soaked lesions that expand rapidly and move down the petiole into the stem. Infected stems of some species will first develop dark lesions whereas the initial indication in other hosts is the appearance of water-soaked stem lesions. Lesions usually develop into necrotic tissues that subsequently develop patches of fluffy white mycelium, often with sclerotia, which is the most obvious sign of plants infected with S. sclerotiorum. http://www.whitemoldresearch.com; http://www.broad.mit.edu/annotation/fungi/sclerotinia_sclerotiorum.

                Author and article information

                Plant Cell Environ
                Plant Cell Environ
                Plant, Cell & Environment
                John Wiley and Sons Inc. (Hoboken )
                01 December 2021
                January 2022
                : 45
                : 1 ( doiID: 10.1111/pce.v45.1 )
                : 248-261
                [ 1 ] College of Agronomy and Biotechnology, Academy of Agricultural Sciences Southwest University Chongqing China
                [ 2 ] Department of Biochemistry and the D.O.E. Great Lakes Bioenergy Research Center Wisconsin Energy Institute, University of Wisconsin Madison Wisconsin USA
                Author notes
                [*] [* ] Correspondence

                Liezhao Liu, College of Agronomy and Biotechnology, Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China.

                Email: liezhao@ 123456swu.edu.cn

                Author information
                © 2021 The Authors. Plant, Cell & Environment published by 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.

                : 13 October 2021
                : 09 April 2021
                : 16 October 2021
                Page count
                Figures: 8, Tables: 0, Pages: 14, Words: 10024
                Funded by: National Natural Science Foundation of China , doi 10.13039/501100013314;
                Award ID: 31771830
                Award ID: 31971902
                Award ID: 31371655
                Funded by: Higher Education Discipline Innovation Project , doi 10.13039/501100013314;
                Award ID: B12006
                Funded by: DOE Great Lakes Bioenergy Research Center
                Award ID: DE‐SC0018409
                Original Article
                Original Articles
                Custom metadata
                January 2022
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.1.5 mode:remove_FC converted:09.05.2022

                Plant science & Botany
                brassica napus,ferulate‐5‐hydroxylase,lignin monomer,nmr,sclerotinia sclerotiorum


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