细菌蛋白Rhp-PSP通过核酸内切酶活性抑制植物病毒增殖

doi:10.1016/j.jia.2024.03.051

摘要/Abstract

摘要： 植物病毒病的发生在全球范围内造成巨大的作物损失。然而，目前没有非常有效的措施来控制植物病毒病。此前，我们鉴定了一种由沼泽红假单胞菌JSC-3b菌株产生的抗病毒蛋白Rhp-PSP。本研究中，我们发现Rhp-PSP的抗病毒活性依赖于其核酸内切酶活性。将129位的精氨酸（R）残基转化为丙氨酸（A），降低了对烟草花叶病毒（TMV）外壳蛋白（CP）RNA的核糖核酸内切酶活性，从而损害了Rhp-PSP的抗病毒活性。进一步的研究表明，突变体Rhp-PSP^R129A不能像野生型那样形成同源三聚体，这说明了四级连接对核糖核酸内酶活性的重要性。Rhp-PSP在本氏烟草中的过表达显著增强了幼苗对TMV的抗性，而Rhp-PSP^R129A过表达则没有，这证实了核糖核酸内切酶活性是Rhp-PSP抗病毒活性的原因。此外，Rhp-PSP喷施在番茄和辣椒叶面上，显著降低了病毒性疾病的发病指数，表明Rhp-PSP在实践中显示出开发抗病毒药物的潜力。

Abstract:

Plant virus causes massive crop losses globally. However, there is currently no effective measure to control plant viral disease. Previously, we identify an antiviral protein Rhp-PSP, produced by the bacterial Rhodopseudomonas palustris strain JSC-3b. In this study, we discover that the antiviral activity of Rhp-PSP relies on its endoribonuclease activity. Converting the arginine (R) residue at position 129 onto alanine (A) abolishs its endoribonuclease activity on coat protein (CP) RNA of tobacco mosaic virus (TMV), consequentially, compromises the antiviral activity of Rhp-PSP. Further investigation demonstrates that, the mutant Rhp-PSP^R129A is unable to form the homotrimer as the wild type, indicating the importance of quaternary junction for the endoribonuclease activity. Overexpression of Rhp-PSP in Nicotiana benthamiana significantly enhances the resistance against TMV of seedlings, while expression of Rhp-PSP^R129Adid not, confirming that endoribonuclease activity is responsible for the antiviral activity of Rhp-PSP. In addition, foliar spray of Rhp-PSP solution on tomato and pepper plants significantly reduces the disease index of viral diseases, indicating that Rhp-PSP shows potential to develop antiviral agent in practice.

Key words: Rhodopseudomonas palustris , Rhp-PSP , tobacco mosaic virus , endoribonuclease , antiviral activity

中图分类号:

','1');return false;" target="_blank"> ">

. 细菌蛋白Rhp-PSP通过核酸内切酶活性抑制植物病毒增殖[J]. Journal of Integrative Agriculture, 2024, 23(6): 1967-1978.

Lijie Chen, Qianze Peng, Xiaohua Du, Weixing Zhang, Ju’e Cheng, Shu’e Sun, Deyong Zhang, Pin Su, Yong Liu.

A bacterial protein Rhp-PSP inhibits plant viral proliferation through endoribonuclease activity [J]. Journal of Integrative Agriculture, 2024, 23(6): 1967-1978.

参考文献

Anantharaman V, Makarova K S, Burroughs A M, Koonin E V, Aravind L. 2013. Comprehensive analysis of the HEPN superfamily: Identification of novel roles in intra-genomic conflicts, defense, pathogenesis and RNA processing. Biology Direct, 8, 15.

Brown B, Wilkins M, Saha R. 2022. Rhodopseudomonas palustris: A biotechnology chassis. Biotechnology Advances, 60, 108001.

Cota-Sánchez J H, Remarchuk K, Ubayasena K. 2006. Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue. Plant Molecular Biology Reporter, 24, 161–167.

Du X, Huang R, Zhang Z, Zhang D, Cheng J, Tian P, Wang Y, Zhai Z, Chen L, Kong X, Liu Y, Su P. 2021. Rhodopseudomonas palustris quorum sensing molecule pC-HSL induces systemic resistance to TMV infection via up-regulation of NbSIPK/NbWIPK expressions in Nicotiana benthamiana. Phytopathology, 111, 500–508.

Duong-Ly K C, Gabelli S B. 2014. Gel filtration chromatography (size exclusion chromatography) of proteins. Methods in Enzymology, 541, 105–114.

Farkas A, Nardai G, Csermely P, Tompa P, Friedrich P. 2004. DUK114, the Drosophila orthologue of bovine brain calpain activator protein, is a molecular chaperone. Biochemical Journal, 383, 165–170.

Hemming M L, Selkoe D J. 2005. Amyloid beta-protein is degraded by cellular angiotensin-converting enzyme (ACE) and elevated by an ACE inhibitor. Journal of Biological Chemistry, 280, 37644–37650.

Hull R. 2002. Matthews' Plant Virology: Purification and Composition of Plant Viruses. 4th ed. Beijing Science Press, China. pp. 75–107.

Ji P, Momol M T, Olson S M, Hong J, Pradhanang P, Anith K N, Jones J B. 2005. New tactics for bacterial wilt management on tomatoes in the Southern U.S. Acta Horticulturae, 695, 153–160.

Kim S W, Li Z, Moore P S, Monaghan A P, Chang Y, Nichols M, John B. 2010. A sensitive non-radioactive northern blot method to detect small RNAs. Nucleic Acids Research, 38, e98.

Lambrecht J A, Browne B A, Downs D M. 2010. Members of the YjgF/YER057c/UK114 family of proteins inhibit phosphoribosylamine synthesis in vitro. Journal of Biological Chemistry, 285, 34401–34407.

Lane L C. 1986. Propagation and purification of RNA plant viruses. Methods in Enzymology, 118, 687–696.

Leitner-Dagan Y, Ovadis M, Zuker A, Shklarman E, Ohad I, Tzfira T, Vainstein A. 2006. CHRD, a plant member of the evolutionarily conserved YjgF family, influences photosynthesis and chromoplastogenesis. Planta, 225, 89–102.

Li J G, Cao J, Sun F F, Niu D D, Yan F, Liu H X, Guo J H. 2011. Control of Tobacco mosaic virus by PopW as a result of induced resistance in tobacco under greenhouse and field conditions. Phytopathology, 101, 1202–1208.

Lindbo J A. 2007. TRBO: A high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiology, 145, 1232–1240.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) method. Methods, 25, 402–408.

López-Rosas I, Marchat L A, Olvera B G, Guillen N, Weber C, Hernández de la Cruz O, Ruíz-García E, Astudillo-de la Vega H, López-Camarillo C. 2014. Proteomic analysis identifies endoribouclease EhL-PSP and EhRRP41 exosome protein as novel interactors of EhCAF1 deadenylase. Journal of Proteomics, 111, 59–73.

Ma Y, Li S. 2022. Purification of total RNAs and small RNAs from fruit tree leaf tissues. Methods in Molecular Biology, 2400, 217–224.

Mistiniene E, Pozdniakovaite N, Popendikyte V, Naktinis V. 2005. Structure-based ligand binding sites of protein p14.5, a member of protein family YER057c/YIL051c/YjgF. International Journal of Biological Macromolecules, 37, 61–68.

Miyakawa T, Hatano K I, Lee W C, Kato Y, Sawano Y, Yumoto F, Nagata K, Tanokura M. 2005. Crystallization and preliminary X-ray analysis of the YjgF/YER057c/UK114-family protein ST0811 from Sulfolobus tokodaii strain 7. Acta Crystallographica Section F: Structural Biology Communications, 61, 828–830.

Morishita R, Kawagoshi A, Sawasaki T, Madin K, Ogasawara T, Oka T, Endo Y. 1999. Ribonuclease activity of rat liver perchloric acid-soluble protein, a potent inhibitor of protein synthesis. Journal of Biological Chemistry, 274, 20688–20692.

Nicaise V. 2014. Crop immunity against viruses: Outcomes and future challenges. Frontiers in Plant Science, 5, 660.

Nookongbut P, Kantachote D, Khuong N Q, Tantirungkij M. 2020. The biocontrol potential of acid-resistant Rhodopseudomonas palustris KTSSR54 and its exopolymeric substances against rice fungal pathogens to enhance rice growth and yield. Biological Control (Theory and Application in Pest Management), 150, 104354.

Ó’Fágáin C, Cummins P M, O’Connor B F. 2017. Gel-filtration chromatography. Methods in Molecular Biology, 1485, 15–25.

Ohyama H, Sakai T, Agari Y, Fukui K, Nakagawa N, Shinkai A, Masui R, Kuramitsu S. 2014. The role of ribonucleases in regulating global mRNA levels in the model organism Thermus thermophilus HB8. BMC Genomics, 15, 386.

Oka T, Tsuji H, Noda C, Sakai K, Hong Y M, Suzuki I, Muñoz S, Natori Y. 1995. Isolation and characterization of a novel perchloric acid-soluble protein inhibiting cell-free protein synthesis. Journal of Biological Chemistry, 270, 30060–30067.

Parsons L, Bonander N, Eisenstein E, Gilson M, Kairys V, Orban J. 2003. Solution structure and functional ligand screening of HI0719, a highly conserved protein from bacteria to humans in the YjgF/YER057c/UK114 family. Biochemistry, 42, 80–89.

Pradhanang P M, Ji P, Momol M T, Olson S M, Mayfield J L, Jones J B. 2005. Application of acibenzolar-S-methyl enhances host resistance in tomato against Ralstonia solanacearum. Plant Disease, 89, 989–993.

Pu Y G, Jiang Y L, Ye X D, Ma X X, Guo P C, Lian F M, Teng Y B, Chen Y, Zhou C Z. 2011. Crystal structures and putative interface of Saccharomyces cerevisiae mitochondrial matrix proteins Mmf1 and Mam33. Journal of Structural Biology, 175, 469–474.

Qin Y, Wang J, Wang F, Shen L, Zhou H, Sun H, Hao K, Song L, Zhou Z, Zhang C, Wu Y, Yang J. 2019. Purification and characterization of a secretory alkaline metalloprotease with highly potent antiviral activity from Serratia marcescens Strain S3. Journal of Agricultural and Food Chemistry, 67, 3168–3178.

Sawasaki T, Morishita R, Ozawa A, Ogasawara T, Madin K, Endo Y. 1999. Mechanism of ribosome RNA apurinic site specific lyase. Nucleic Acids Symposium, 42, 257–258.

Sinha S, Rappu P, Lange S C, Mäntsälä P, Zalkin H, Smith J L. 1999. Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein and member of the highly conserved YjgF family. Proceedings of the National Academy of Sciences of the United States of America, 96, 13074–13079.

Song Y, Wilson A J, Zhang X C, Thoms D, Sohrabi R, Song S, Geissmann Q, Liu Y, Walgren L, He S Y, Haney C H. 2021. Feronia restricts Pseudomonas in the rhizosphere microbiome via regulation of reactive oxygen species. Nature Plants, 7, 644–654.

Su P, Feng T, Zhou X, Zhang S, Zhang Y, Cheng J, Luo Y, Peng J, Zhang Z, Lu X, Zhang D, Liu Y. 2015. Isolation of Rhp-PSP, a member of YER057c/YjgF/UK114 protein family with antiviral properties, from the photosynthetic bacterium Rhodopseudomonas palustris strain JSC-3b. Scientific Reports, 5, 16121.

Su P, Tan X, Li C, Zhang D, Cheng J, Zhang S, Zhou X, Yan Q, Peng J, Zhang Z, Liu Y, Lu X. 2017. Photosynthetic bacterium Rhodopseudomonas palustris GJ-22 induces systemic resistance against viruses. Microbial Biotechnology, 10, 612–624.

Thakur K G, Praveena T, Gopal B. 2010. Mycobacterium tuberculosis Rv2704 is a member of the YjgF/YER057c/UK114 family. Proteins, 78, 773–778.

Vorholt J A. 2012. Microbial life in the phyllosphere. Nature Reviews Microbiology, 10, 828–840.

Williams D B, Barry Carter C. 2009. The Transmission Electron Microscope. Springer, USA.

Wu X, Chen Y, Li C, Zhang X, Tan X, Lv L, Liu Y, Zhang D. 2021. GroEL protein from the potential biocontrol agent Rhodopseudomonas palustris enhances resistance to rice blast disease. Pest Management Science, 77, 5445–5453.

Zern M A, Yap S H, Strair R K, Kaysen G A, Shafritz D A. 1984. Effects of chronic renal failure on protein synthesis and albumin messenger ribonucleic acid in rat liver. The Journal of Clinical Investigation, 73, 1167–1174.

Zhang H M, Gao Y, Li M, Chang W R. 2010. Crystal structure of the PSPTO-PSP protein from Pseudomonas syringae pv. tomato str. DC3000 in complex with D-glucose. Biochemical and Biophysical Research Communications, 397, 82–86.

Zhu F, Zhang P, Meng Y F, Xu F, Zhang D W, Cheng J, Lin H H, Xi D H. 2013. Alpha-momorcharin, a RIP produced by bitter melon, enhances defense response in tobacco plants against diverse plant viruses and shows antifungal activity in vitro. Planta Berlin, 237, 77–88.