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      Functional analysis of a pathogenesis-related thaumatin-like protein gene TaLr35PR5 from wheat induced by leaf rust fungus

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          Abstract

          Background

          Plants have evolved multifaceted defence mechanisms to resist pathogen infection. Production of the pathogenesis-related (PR) proteins in response to pathogen attack has been implicated in plant disease resistance specialized in systemic-acquired resistance (SAR). Our earlier studies have reported that a full length TaLr35PR5 gene, encoding a protein exhibiting amino acid and structural similarity to a sweet protein thaumatin, was isolated from wheat near-isogenic line TcLr35. The present study aims to understand the function of TaLr35PR5 gene in Lr35-mediated adult resistance to Puccinia triticina.

          Results

          We determined that the TaLr35PR5 protein contained a functional secretion peptide by utilizing the yeast signal sequence trap system. Using a heterologous expression assay on onion epidermal cells we found that TaLr35PR5 protein was secreted into the apoplast of onion cell. Expression of TaLr35PR5 was significantly reduced in BSMV-induced gene silenced wheat plants, and pathology test on these silenced plants revealed that Lr35-mediated resistance phenotype was obviously altered, indicating that Lr35-mediated resistance was compromised.

          Conclusions

          All these findings strongly suggest that TaLr35PR5 is involved in Lr35-mediated adult wheat defense in response to leaf rust attack.

          Electronic supplementary material

          The online version of this article (10.1186/s12870-018-1297-2) contains supplementary material, which is available to authorized users.

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          Barley stripe mosaic virus-induced gene silencing in a monocot plant.

          RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.
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            Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway.

            Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. In VIGS, viruses engineered to carry sequences derived from plant gene transcripts activate the host's sequence-specific RNA degradation system. This mechanism targets the RNAs of the viral genome for degradation, and as the virus contains transcribed plant sequence, homologous host mRNAs are also targeted for destruction. While routinely used in some dicots, no VIGS system was known for monocot plants until the recent report of silencing in barley (Hordeum vulgare) by barley stripe mosaic virus (BSMV). Here, we report development of protocols for use of BSMV to efficiently silence genes in hexaploid wheat (Triticum aestivum). The VIGS system was first optimized in studies silencing phytoene desaturase expression. Next, we used it to assay genes functioning in leaf rust resistance mediated by Lr21, which encodes a nucleotide binding site-leucine-rich repeat class resistance gene product. We demonstrated that infection with BSMV constructs carrying a 150-bp fragment of Lr21 caused conversion of incompatible interactions to compatible, whereas infection with a control construct or one that silences phytoene desaturase had no effect on resistance or susceptibility. Additionally, silencing the RAR1, SGT1, and HSP90 genes, known to be required in many but not all nucleotide binding site-leucine-rich repeat resistance pathways in diverse plant species, resulted in conversion to compatibility, indicating that these genes are essential in Lr21-mediated resistance. These studies indicate that BSMV-VIGS is a powerful tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.
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              Recommendations for naming plant pathogenesis-related proteins

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                Author and article information

                Contributors
                Lisong.ma@anu.edu.au
                ndwanghaiyan@163.com
                ldq@hebau.edu.cn
                Journal
                BMC Plant Biol
                BMC Plant Biol
                BMC Plant Biology
                BioMed Central (London )
                1471-2229
                4 May 2018
                4 May 2018
                2018
                : 18
                : 76
                Affiliations
                [1 ]ISNI 0000 0001 2291 4530, GRID grid.274504.0, Center of Plant Disease and Plant Pests of Hebei Province, College of Plant Protection, , Hebei Agricultural University, ; Baoding, 071001 China
                [2 ]ISNI 0000 0001 2180 7477, GRID grid.1001.0, Division of Plant Science, Research School of Biology, , Australian National University, ; ACT, Acton, 2601 Australia
                [3 ]ISNI 0000 0001 0526 1937, GRID grid.410727.7, Graduate School of Chinese Academy of Agricultural Sciences, ; Beijing, 100081 China
                Article
                1297
                10.1186/s12870-018-1297-2
                5935958
                29728059
                6ec739cc-0c25-4d8f-a630-116ca821feb0
                © The Author(s). 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 26 October 2017
                : 26 April 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31501623
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2018

                Plant science & Botany
                localization,silencing,talr35pr5,thaumatin-like protein,wheat
                Plant science & Botany
                localization, silencing, talr35pr5, thaumatin-like protein, wheat

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