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      • Record: found
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      Function identification of miR482b, a negative regulator during tomato resistance to Phytophthora infestans

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          Abstract

          Tomato is an important horticultural and economic crop cultivated worldwide. As Phytophthora infestans becomes a huge threat to tomato production, it is necessary to study the resistance mechanisms of tomato against P. infestans. Our previous research has found that miR482 might be involved in tomato– P. infestans interaction. In this study, miR482b precursor was cloned from Solanum pimpinellifolium “L3708” and miR482b was shown to decrease in abundance in tomato following P. infestans infection. Compared to wild-type tomato plants, tomato plants that overexpressed miR482b displayed more serious disease symptoms after P. infestans infection, with more necrotic cells, longer lesion diameters, and increased P. infestans abundance. Meanwhile, silencing of miR482b was performed by short tandem target mimic (STTM), resulting in enhancement of tomato resistance to P. infestans. Using miRNA and degradome data sets, NBS–LRR disease-resistance genes targeted by miR482b were validated. Negative correlation between the expression of miR482b and its target genes was found in all miR482b-overexpressing and -silencing tomato plants. Our results provide insight into tomato miR482b involved in the response to P. infestans infection, and demonstrate that miR482b– NBS–LRR is an important component in the network of tomato– P. infestans interaction.

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          Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

          Erika Varkonyi-Gasic, Rongmei Wu, Marion Wood … (2007)
          MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.
          Article 0 comments Cited 394 times – based on 0 reviews     Review now
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            Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome.

            Charles Addo-Quaye, Tifani W. Eshoo, David Bartel … (2008)
            MicroRNAs (miRNAs) regulate the expression of target mRNAs in plants and animals [1]. Plant miRNA targets have been predicted on the basis of their extensive and often conserved complementarity to the miRNAs [2-4], as well as on miRNA overexpression experiments [5]; many of these target predictions have been confirmed by isolation of the products of miRNA-directed cleavage. Here, we present a transcriptome-wide experimental method, called "degradome sequencing," to directly detect cleaved miRNA targets without relying on predictions or overexpression. The 5' ends of polyadenylated, uncapped mRNAs from Arabidopsis were directly sampled, resulting in an empirical snapshot of the degradome. miRNA-mediated-cleavage products were easily discerned from an extensive background of degraded mRNAs, which collectively covered the majority of the annotated transcriptome. Many previously known Arabidopsis miRNA targets were confirmed, and several novel targets were also discovered. Quantification of cleavage fragments revealed that those derived from TAS transcripts, which are unusual in their production of abundant secondary small interfering RNAs (siRNAs), accumulated to very high levels. A subset of secondary siRNAs are also known to direct cleavage of targets in trans[6]; degradome sequencing revealed many cleaved targets of these trans-acting siRNAs (ta-siRNAs). This empirical method is broadly applicable to the discovery and quantification of cleaved targets of small RNAs without a priori predictions.
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              A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs.

              Padubidri V. Shivaprasad, Ho-Ming Chen, Kanu Patel … (2012)
              Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack.
              Article 0 comments Cited 273 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Jun Meng: mengjun@dlut.edu.cn
                Yushi Luan: ysluan@dlut.edu.cn
                Journal
                Journal ID (nlm-ta): Hortic Res
                Journal ID (iso-abbrev): Hortic Res
                Title: Horticulture Research
                Publisher: Nature Publishing Group UK (London )
                ISSN (Electronic): 2052-7276
                Publication date (Electronic): 1 March 2018
                Publication date PMC-release: 1 March 2018
                Publication date Collection: 2018
                Volume: 5
                Electronic Location Identifier: 9
                Affiliations
                [1 ]ISNI 0000 0000 9247 7930, GRID grid.30055.33, School of Life Science and Biotechnology, , Dalian University of Technology, ; Dalian, 116024 China
                [2 ]ISNI 0000 0000 9247 7930, GRID grid.30055.33, School of Computer Science and Technology, , Dalian University of Technology, ; Dalian, 116024 China
                Article
                Publisher ID: 17
                DOI: 10.1038/s41438-018-0017-2
                PMC ID: 5830410
                PubMed ID: 29507733
                SO-VID: 6598357c-8b80-4b7c-8fcb-3411952671b8
                Copyright © © The Author(s) 2018
                License:

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                Date received : 18 September 2017
                Date revision received : 29 October 2017
                Date accepted : 11 January 2018
                Categories
                Subject: Article
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                issue-copyright-statement © The Author(s) 2018

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