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      SUMO and SUMO-Conjugating Enzyme E2 UBC9 Are Involved in White Spot Syndrome Virus Infection in Fenneropenaeus chinensis

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          Abstract

          In previous work, small ubiquitin-like modifier (SUMO) in hemocytes of Chinese shrimp Fenneropenaeus chinensis was found to be up-regulated post-white spot syndrome virus (WSSV) infection using proteomic approach. However, the role of SUMO in viral infection is still unclear. In the present work, full length cDNAs of SUMO ( FcSUMO) and SUMO-conjugating enzyme E2 UBC9 ( FcUBC9) were cloned from F. chinensis using rapid amplification of cDNA ends approach. The open reading frame (ORF) of FcSUMO encoded a 93 amino acids peptide with the predicted molecular weight (M.W) of 10.55 kDa, and the UBC9 ORF encoded a 160 amino acids peptide with the predicted M.W of 18.35 kDa. By quantitative real-time RT-PCR, higher mRNA transcription levels of FcSUMO and FcUBC9 were detected in hemocytes and ovary of F. chinensis, and the two genes were significantly up-regulated post WSSV infection. Subsequently, the recombinant proteins of FcSUMO and FcUBC9 were expressed in Escherichia coli BL21 (DE3), and employed as immunogens for the production of polyclonal antibody (PAb). Indirect immunofluorescence assay revealed that the FcSUMO and UBC9 proteins were mainly located in the hemocytes nuclei. By western blotting, a 13.5 kDa protein and a 18.7 kDa protein in hemocytes were recognized by the PAb against SUMO or UBC9 respectively. Furthermore, gene silencing of FcSUMO and FcUBC9 were performed using RNA interference, and the results showed that the number of WSSV copies and the viral gene expressions were inhibited by knockdown of either SUMO or UBC9, and the mortalities of shrimp were also reduced. These results indicated that FcSUMO and FcUBC9 played important roles in WSSV infection.

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          Protein modification by SUMO.

          Small ubiquitin-related modifier (SUMO) family proteins function by becoming covalently attached to other proteins as post-translational modifications. SUMO modifies many proteins that participate in diverse cellular processes, including transcriptional regulation, nuclear transport, maintenance of genome integrity, and signal transduction. Reversible attachment of SUMO is controlled by an enzyme pathway that is analogous to the ubiquitin pathway. The functional consequences of SUMO attachment vary greatly from substrate to substrate, and in many cases are not understood at the molecular level. Frequently SUMO alters interactions of substrates with other proteins or with DNA, but SUMO can also act by blocking ubiquitin attachment sites. An unusual feature of SUMO modification is that, for most substrates, only a small fraction of the substrate is sumoylated at any given time. This review discusses our current understanding of how SUMO conjugation is controlled, as well as the roles of SUMO in a number of biological processes.
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            SUMO--nonclassical ubiquitin.

            SUMO (small ubiquitin-related modifier) is the best-characterized member of a growing family of ubiquitin-related proteins. It resembles ubiquitin in its structure, its ability to be ligated to other proteins, as well as in the mechanism of ligation. However, in contrast to ubiquitination-often the first step on a one-way road to protein degradation-SUMOlation does not seem to mark proteins for degradation. In fact, SUMO may even function as an antagonist of ubiquitin in the degradation of selected proteins. While most SUMO targets are still at large, available data provide compelling evidence for a role of SUMO in the regulation of protein-protein interactions and/or subcellular localization.
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              Salicylic acid-mediated innate immunity in Arabidopsis is regulated by SIZ1 SUMO E3 ligase.

              Reversible modifications of target proteins by small ubiquitin-like modifier (SUMO) proteins are involved in many cellular processes in yeast and animals. Yet little is known about the function of sumoylation in plants. Here, we show that the SIZ1 gene, which encodes an Arabidopsis SUMO E3 ligase, regulates innate immunity. Mutant siz1 plants exhibit constitutive systemic-acquired resistance (SAR) characterized by elevated accumulation of salicylic acid (SA), increased expression of pathogenesis-related (PR) genes, and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Transfer of the NahG gene to siz1 plants results in reversal of these phenotypes back to wild-type. Analyses of the double mutants, npr1 siz1, pad4 siz1 and ndr1 siz1 revealed that SIZ1 controls SA signalling. SIZ1 interacts epistatically with PAD4 to regulate PR expression and disease resistance. Consistent with these observations, siz1 plants exhibited enhanced resistance to Pst DC3000 expressing avrRps4, a bacterial avirulence determinant that responds to the EDS1/PAD4-dependent TIR-NBS-type R gene. In contrast, siz1 plants were not resistant to Pst DC3000 expressing avrRpm1, a bacterial avirulence determinant that responds to the NDR1-dependent CC-NBS-type R gene. Jasmonic acid (JA)-induced PDF1.2 expression and susceptibility to Botrytis cinerea were unaltered in siz1 plants. Taken together, these results demonstrate that SIZ1 is required for SA and PAD4-mediated R gene signalling, which in turn confers innate immunity in Arabidopsis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                29 February 2016
                2016
                : 11
                : 2
                : e0150324
                Affiliations
                [1 ]Laboratory of Pathology and Immunology of Aquatic Animals, Ocean University of China, Yushan road 5, Qingdao, 266003, PR China
                [2 ]Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, No. 1 Wenhai Road, Aoshanwei Town, Jimo, Qingdao, 266071, China
                Shanghai Ocean University, CHINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: XQT WBZ. Performed the experiments: WL. Analyzed the data: WL XQT. Contributed reagents/materials/analysis tools: JX XZS. Wrote the paper: WL XQT.

                Article
                PONE-D-15-48527
                10.1371/journal.pone.0150324
                4771164
                26927328
                c3278d87-bfe5-496a-804f-755afb2708fa
                © 2016 Tang et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 20 November 2015
                : 11 February 2016
                Page count
                Figures: 5, Tables: 1, Pages: 13
                Funding
                This work was funded by the National Basic Research Program of China (grant 2012CB114405), Key Technology R&D Program of China (grant 2012BAD17B01), Taishan Scholar Program of Shandong Province, and Special Fund for Agro-scientific Research in the Public Interest (grant 201103034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Biochemistry
                Proteins
                Post-translational modification
                SUMOylation
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Blood Cells
                White Blood Cells
                Hemocytes
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Immune Cells
                White Blood Cells
                Hemocytes
                Biology and Life Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Hemocytes
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