GhWRKY75通过结合W-box TTGAC(C/T)正向调控GhPR6-5b基因表达和棉花黄萎病抗性反应

doi:10.1016/j.jia.2024.05.017

摘要/Abstract

摘要：

黄萎病是棉花生产中的主要真菌病害，严重影响了棉花产量和品质，因此挖掘棉花黄萎病抗性关键基因具有重要的理论意义和生产应用价值。蛋白酶和蛋白酶抑制剂在植物防卫反应中具有重要的作用，然而蛋白酶抑制剂PR6基因家族的功能和调节机制相关研究少有报道。本研究首先对棉花PR6基因家族进行了全基因组鉴定分析，该基因家族属于马铃薯蛋白酶抑制剂I家族，在亚洲棉、雷蒙德氏棉、海岛棉和陆地棉中共鉴定出39个PR6基因，系统发育分析将PR6基因分为4个组；通过黄萎病诱导表达分析和类病变突变体Ghlmm转录组数据分析，挖掘到了黄萎病抗性关键基因GhPR6-5b，并发现利用病毒诱导的基因沉默技术将PR6-5b沉默后，棉花对黄萎病菌V991更加敏感；进一步研究发现GhWRKY75通过结合GhPR6-5b启动子的W-box TTGAC（T/C），正向调节基因表达和棉花黄萎病抗性反应。本研究为棉花抗黄萎病分子机制研究和遗传改良提供了基因资源和理论基础。

Abstract:

Verticillium dahliae is an important fungal pathogen affecting cotton yield and quality. Therefore, the mining of V. dahlia-resistance genes is urgently needed. Proteases and protease inhibitors play crucial roles in plant defense responses. However, the functions and regulatory mechanisms of the protease inhibitor PR6 gene family remain largely unknown. This study provides a comprehensive analysis of the PR6 gene family in the cotton genome. We performed genome-wide identification and functional characterization of the cotton GhPR6 gene family, which belongs to the potato protease inhibitor I family of inhibitors. Thirty-nine PR6s were identified in Gossypium arboreum, G. raimondii, G. barbadense, and G. hirsutum, and they were clustered into four groups. Based on the analysis of pathogen-induced and Ghlmm transcriptome data, GhPR6-5b was identified as the key gene for V. dahliae resistance. Virus-induced gene silencing experiments revealed that cotton was more sensitive to V. dahliae V991 after PR6-5b silencing. The present study established that GhWRKY75 plays an important role in resistance to Verticillium wilt in cotton by positively regulating GhPR6-5b expression by directly binding to the W-box TTGAC(T/C). Our findings established that GhWRKY75 is a potential candidate for improving cotton resistance to V. dahliae, and provide primary information for further investigations and the development of specific strategies to bolster the defense mechanisms of cotton against V. dahliae.

Key words: cotton , proteinase inhibitors , WRKY transcription factor , Verticillium wilt

Qichao Chai, Meina Zheng, Yanli Li, Mingwei Gao, Yongcui Wang, Xiuli Wang, Chao Zhang, Hui Jiang, Ying Chen, Jiabao Wang, Junsheng Zhao. GhWRKY75通过结合W-box TTGAC(C/T)正向调控GhPR6-5b基因表达和棉花黄萎病抗性反应[J]. Journal of Integrative Agriculture, 2024, 23(10): 3343-3357.

Qichao Chai, Meina Zheng, Yanli Li, Mingwei Gao, Yongcui Wang, Xiuli Wang, Chao Zhang, Hui Jiang, Ying Chen, Jiabao Wang, Junsheng Zhao. GhWRKY75 positively regulates GhPR6-5b via binding to a W-box TTGAC (C/T) to orchestrate cotton resistance to Verticillium dahliae [J]. Journal of Integrative Agriculture, 2024, 23(10): 3343-3357.

参考文献

Bailey T L, Williams N, Misleh C, Li W W. 2006. MEME: Discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Research, 34, W369–W373.

Beliën T, Van Campenhout S, Van Acker M, Volckaert G. 2005. Cloning and characterization of two endoxylanases from the cereal phytopathogen Fusarium graminearum and their inhibition profile against endoxylanase inhibitors from wheat. Biochemical and Biophysical Research Communications, 327, 407–414.

Brutus A, Reca I B, Herga S, Mattei B, Puigserver A, Chaix J C, Juge N, Bellincampi D, Giardina T. 2005. A family 11 xylanase from the pathogen Botrytis cinerea is inhibited by plant endoxylanase inhibitors XIP-I and TAXI-I. Biochemical and Biophysical Research Communications, 337, 160–166.

Chai Q C, Shang X G, Wu S, Zhu G Z, Cheng C Z, Cai C P, Wang X Y, Guo W Z. 2017. 5-Aminolevulinic acid dehydratase gene dosage affects programmed cell death and immunity. Plant Physiology, 175, 511–528.

Chai Q C, Wang X L, Gao M W, Zhao X C, Chen Y, Zhang C, Jiang H, Wang J B, Wang Y C, Zheng M, Baltaevich A M, Zhao J, Zhao J S. 2023. A glutathione S-transferase GhTT19 determines flower petal pigmentation via regulating anthocyanin accumulation in cotton. Plant Biotechnology Journal, 21, 433–448.

Chassot C, Nawrath C, Metraux J P. 2007. Cuticular defects lead to full immunity to a major plant pathogen. The Plant Journal, 49, 972–980.

Chen C, Chen H, Zhang Y, Thomas H R, Frank M H, He Y, Xia R. 2020. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 13, 1194–1202.

Chen L G, Zhang L P, Xiang S Y, Chen Y L, Zhang H Y, Yu D Q. 2021. The transcription factor WRKY75 positively regulates jasmonate-mediated plant defense to necrotrophic fungal pathogens. Journal of Experimental Botany, 72, 1473–1489.

Chen Z Y, Brown R L, Lax A R, Cleveland T E, Russin J S. 1999. Inhibition of plant–pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli. Applied and Environmental Microbiology, 65, 1320–1324.

Devaiah B N, Karthikeyan A S, Raghothama K G. 2007. WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiology, 143, 1789–1801.

Durrant W E, Dong X N. 2004. Systemic acquired resistance. Annual Review of Phytopathology, 42, 185–209.

Federici L, Di Matteo A, Fernandez-Recio J, Tsernoglou D, Cervone F. 2006. Polygalacturonase inhibiting proteins: Players in plant innate immunity? Trends in Plant Science, 11, 65–70.

Gao Q M, Venugopal S, Navarre D, Kachroo A. 2011. Low oleic acid-derived repression of jasmonic acid-inducible defense responses requires the WRKY50 and WRKY51 proteins. Plant Physiology, 155, 464–476.

Guo P R, Li Z H, Huang P X, Li B S, Fang S, Chu J F, Guo H W. 2017. A tripartite amplification loop involving the transcription factor WRKY75, salicylic acid, and reactive oxygen species accelerates leaf senescence. The Plant Cell, 29, 2854–2870.

Haq S K, Atif S M, Khan R H. 2004. Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: Natural and engineered phytoprotection. Archives of Biochemistry and Biophysics, 431, 145–159.

Hou S G, Jamieson P, He P. 2018. The cloak, dagger, and shield: Proteases in plant-pathogen interactions. The Biochemical Journal, 475, 2491–2509.

Hu B, Jin J, Guo A Y, Zhang H, Luo J, Gao G. 2015. GSDS 2.0: An upgraded gene feature visualization server. Bioinformatics, 31, 1296–1297.

Huang H, Qi S D, Qi F, Wu C A, Yang G D, Zheng C C. 2010. NtKTI1, a Kunitz trypsin inhibitor with antifungal activity from Nicotiana tabacum, plays an important role in tobacco’s defense response. The FEBS Journal, 277, 4076–4088.

Jiang H, Gao M W, Chen Y, Zhang C, Wang J B, Chai Q C, Wang Y C, Zheng J X, Wang X L, Zhao J S. 2023. Effect of the L-D1 alleles on leaf morphology, canopy structure and photosynthetic productivity in upland cotton (Gossypium hirsutum L.) Journal of Integrative Agriculture, 22, 108–119.

Jones J D, Dangl J L. 2006. The plant immune system. Nature, 444, 323–329.

Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology Evolution, 33, 1870–1874.

Li J, Brader G, Palva E T. 2004. The WRKY70 transcription factor: A node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. The Plant Cell, 16, 319–331.

Li J, Brader G, Palva E T. 2008. Kunitz trypsin inhibitor: An antagonist of cell death triggered by phytopathogens and fumonisin b1 in Arabidopsis. Molecular Plant, 1, 482–495.

Lu K K, Song R F, Guo J X, Zhang Y, Zuo J X, Chen H H, Liao C Y, Hu X Y, Ren F, Lu Y T, Liu W C. 2023. CycC1;1-WRKY75 complex-mediated transcriptional regulation of SOS1 controls salt stress tolerance in Arabidopsis. The Plant Cell, 35, 2570–2591.

Ma Z C, Zhu L, Song T Q, Wang Y, Zhang Q, Xia Y Q, Qiu M, Lin Y C, Li H Y, Kong L, Fang Y F, Ye W W, Wang Y, Dong S M, Zheng X B, Tyler B M, Wang Y C. 2017. A paralogous decoy protects Phytophthora sojae apoplastic effector PsXEG1 from a host inhibitor. The Plant Cell, 355, 710–714.

Mao P, Duan M R, Wei C H, Li Y. 2007. WRKY62 transcription factor acts downstream of cytosolic NPR1 and negatively regulates jasmonate-responsive gene expression. Plant and Cell Physiology, 48, 833–842.

Misas-Villamil J C, Van Der Hoorn R A. 2008. Enzyme-inhibitor interactions at the plant–pathogen interface. Current Opinion in Plant Biology, 11, 380–388.

Pandey S P, Somssich I E. 2009. The role of WRKY transcription factors in plant immunity. Plant Physiology, 150, 1648–1655.

Pieterse C M, Van Der Does D, Zamioudis C, Leon-Reyes A, Van Wees S C. 2012. Hormonal modulation of plant immunity. Annual Review of Cell and Developmental Biology, 28, 489–521.

Rishmawi L, Pesch M, Juengst C, Schauss A C, Schrader A, Hülskamp M. 2014. Non-cell-autonomous regulation of root hair patterning genes by WRKY75 in Arabidopsis. Plant Physiology, 165, 186–195.

Rodriguez-Sifuentes L, Marszalek J E, Chuck-Hernandez C, Serna-Saldivar S O. 2020. Legumes protease inhibitors as biopesticides and their defense mechanisms against biotic factors. International Journal of Molecular Sciences, 21, 3322

Ryan C A. 1990. Protease inhibitors in plants: Genes for improving defenses against insects and pathogens. Annual Review of Phytopathology, 28, 425–449.

Sels J, Mathys J, De Coninck B M, Cammue B P, De Bolle M F. 2008. Plant pathogenesis-related (PR) proteins: A focus on PR peptides. Plant Physiology and Biochemistry, 46, 941–950.

Shang X G, Yu Y J, Zhu L J, Liu H Q, Chai Q C, Guo W Z. 2020. A cotton NAC transcription factor GhirNAC2 plays positive roles in drought tolerance via regulating ABA biosynthesis. Plant Science, 296, 110498.

Shang X G, Zhu L J, Duan Y J, He Q F, Zhao M Y, Yu Y J, Guo W Z. 2022. An easy and rapid transformation protocol for transient expression in cotton fiber. Frontiers in Plant Science, 13, 837994.

Stael S, Kmiecik P, Willems P, Van Der Kelen K, Coll N S, Teige M, Van Breusegem F. 2015. Plant innate immunity–sunny side up? Trends in Plant Science, 20, 3–11.

Tian X M, Han P, Wang J, Shao P X, An Q S, Nurimanguli A, Yang Q Y, You C Y, Lin H R, Zhu L F, Pan Z Y, Nie X H. 2023. Association mapping of lignin response to Verticillium wilt through an eight-way MAGIC population in Upland cotton. Journal of Integrative Agriculture, 22, 1324–1337.

Wang G L, Xu J, Li L C, Guo Z, Si Q X, Zhu G Z, Wang X Y, Guo W Z. 2019. GbCYP86A1-1 from Gossypium barbadense positively regulates defence against Verticillium dahliae by cell wall modification and activation of immune pathways. Plant Biotechnology Journal, 18, 222–238.

Wang H P, Chen W Q, Xu Z Y, Chen M F, Yu D Q. 2023. Functions of WRKYs in plant growth and development. Trends in Plant Science, 28, 630–645.

Wang Y Q, Liang C Z, Wu S J, Jian G L, Zhang X Y, Zhang H Y, Tang J Y, Li J, Jiao G L, Li F G, Chu C C. 2020. Vascular-specific expression of Gastrodia antifungal protein gene significantly enhanced cotton Verticillium wilt resistance. Plant Biotechnology Journal, 18, 1498–1500.

Yang M M, Wang Y X, Chen C, Xin X, Dai S S, Meng C, Ma N N. 2024. Transcription factor WRKY75 maintains auxin homeostasis to promote tomato defense against Pseudomonas syringae. Plant Physiology, 00, 1–16.

Ye X Y, Ng T B, Rao P F. 2001. A Bowman-Birk-type trypsin-chymotrypsin inhibitor from broad beans. Biochemical and Biophysical Research Communications, 289, 91–96.

Zhang C Y, Fang H, Shi X T, He F, Wang R Y, Fan J B, Bai P F, Wang J Y, Park C H, Bellizzi M, Zhou X P. 2020. A fungal effector and a rice NLR protein have antagonistic effects on a Bowman-Birk trypsin inhibitor. Plant Biotechnology Journal, 18, 2354–2363.

Zhang G L, Zhao Z Q, Ma P P, Qu Y Y, Sun G Q, Chen Q J. 2021. Integrative transcriptomic and gene co-expression network analysis of host responses upon Verticillium dahliae infection in Gossypium hirsutum. Scientific Reports, 11, 20586.

Zhang L P, Chen L G, Yu D Q. 2018. Transcription factor WRKY75 interacts with DELLA proteins to affect flowering. Plant Physiology, 176, 790–803.