陆地棉R2R3-MYB基因家族表达分析及GhMYB3D5基因在调控黄萎病抗性中的功能研究

doi:10.1016/j.jia.2024.07.040

摘要/Abstract

摘要：

黄萎病（Verticillium wilt, VW）可导致棉花产量大幅下降，提高植物对黄萎病的抗性是一个世界性难题。在作物生长过程中，需要有效地分配有限的能量资源以平衡生长和防御。目前，鲜有特异性对大丽轮枝菌作出响应的转录因子及其分子机制被报道。本研究发现，与其他类型的MYB转录因子相比，棉花中大部分R2R3型MYB转录因子在受到V. dahliae侵染后发生显著的表达变化。其中，一种新的R2R3-MYB转录因子GhMYB3D5特异性响应V. dahliae的侵染。在没有V. dahliae存在的情况下，GhMYB3D5在15种棉花组织中均不表达；在植株被V. dahliae侵染下，该基因被显著诱导表达。本研究明确了GhMYB3D5正向调控棉花黄萎病的功能及其抗性机制：在植株感染V. dahliae后，上调表达的GhMYB3D5转录因子结合GhADH1基因的启动子，并激活GhADH1基因的表达；并且GhMYB3D5转录因子与GhADH1蛋白可发生物理相互作用，从而进一步增强了GhMYB3D5对其靶基因GhADH1的转录激活强度。进一步发现转录调控模块GhMYB3D5-GhADH1通过提高植株木质素生物合成相关基因（GhPAL、GhC4H、Gh4CL和GhPOD/ GhLAC）的转录水平促进木质素积累，从而增强植株对VW的抗性。综上所述，本研究结果表明GhMYB3D5促进了防御诱导的木质素的积累，该调控方式认为是协调植物免疫和生长的有效方式。

Abstract:

Improving plant resistance to Verticillium wilt (VW), which causes massive losses in Gossypium hirsutum, is a global challenge. Crop plants need to efficiently allocate their limited energy resources to maintain a balance between growth and defense. However, few transcriptional regulators specifically respond to Verticillium dahliae and the underlying mechanism has not been identified in cotton. In this study, we found that the that expression of most R2R3-MYB members in cotton is significantly changed by V. dahliae infection relative to the other MYB types. One novel R2R3-MYB transcription factor (TF) that specifically responds to V. dahliae, GhMYB3D5, was identified. GhMYB3D5 was not expressed in 15 cotton tissues under normal conditions, but it was dramatically induced by V. dahliae stress. We functionally characterized its positive role and underlying mechanism in VW resistance. Upon V. dahliae infection, the up-regulated GhMYB3D5 bound to the GhADH1 promoter and activated GhADH1 expression. In addition, GhMYB3D5 physically interacted with GhADH1 and further enhanced the transcriptional activation of GhADH1. Consequently, the transcriptional regulatory module GhMYB3D5-GhADH1 then promoted lignin accumulation by improving the transcriptional levels of genes related to lignin biosynthesis (GhPAL, GhC4H, Gh4CL, and GhPOD/GhLAC) in cotton, thereby enhancing cotton VW resistance. Our results demonstrated that the GhMYB3D5 promotes defense-induced lignin accumulation, which can be regarded as an effective way to orchestrate plant immunity and growth.

Key words: cotton , Verticillium wilt resistance , GhMYB3D5 , GhADH1 , transcriptional regulatory module

Jie Liu, Zhicheng Wang, Bin Chen, Guoning Wang, Huifeng Ke, Jin Zhang, Mengjia Jiao, Yan Wang, Meixia Xie, Yanbin Li, Dongmei Zhang, Xingyi Wang, Qishen Gu, Zhengwen Sun, Liqiang Wu, Xingfen Wang, Zhiying Ma, Yan Zhang. 陆地棉R2R3-MYB基因家族表达分析及GhMYB3D5基因在调控黄萎病抗性中的功能研究[J]. Journal of Integrative Agriculture, 2024, 23(10): 3294-3310.

Jie Liu, Zhicheng Wang, Bin Chen, Guoning Wang, Huifeng Ke, Jin Zhang, Mengjia Jiao, Yan Wang, Meixia Xie, Yanbin Li, Dongmei Zhang, Xingyi Wang, Qishen Gu, Zhengwen Sun, Liqiang Wu, Xingfen Wang, Zhiying Ma, Yan Zhang. Expression analysis of the R2R3-MYB gene family in upland cotton and functional study of GhMYB3D5 in regulating Verticillium wilt resistance[J]. Journal of Integrative Agriculture, 2024, 23(10): 3294-3310.

参考文献

Buscaill P, Rivas S. 2014. Transcriptional control of plant defence responses. Current Opinion in Plant Biology, 20, 35–46.

Chen B, Zhang Y, Yang J, Zhang M, Ma Q M, Wang X F, Ma Z Y. 2020. The G-protein a subunit GhGPA positively regulates Gossypium hirsutum resistance to Verticillium dahliae via induction of SA and JA signaling pathways and ROS accumulation. The Crop Journal, 9, 823–833.

Chen F, Wang P, An Y, Huang J, Xu Y. 2015. Structural insight into the conformational change of alcohol dehydrogenase from Arabidopsis thaliana L. during coenzyme binding. Biochimie, 108, 33–39.

Chen J Y, Xiao H L, Gui Y J, Zhang D D, Li L, Bao Y M, Dai X F. 2016. Characterization of the Verticillium dahliae exoproteome involves in pathogenicity from cotton-containing medium. Frontiers in Microbiology, 7, 1709.

Chen Y J, Perera V, Christiansen M W, Holme I B, Gregersen P L, Grant M R, Collinge D B, Lyngkjær M F. 2013. The barley HvNAC6 transcription factor affects ABA accumulation and promotes basal resistance against powdery mildew. Plant Molecular Biology, 83, 577–590.

Cheng F, Hu T, An Y, Huang J, Xu Y. 2013. Purification and enzymatic characterization of alcohol dehydrogenase from Arabidopsis thaliana. Protein Expression and Purification, 90, 74–77.

Cheng H Q, Han L B, Yang C L, Wu X M, Zhong N Q, Wu J H, Wang F X, Wang H Y, Xia G X. 2016. The cotton MYB108 forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection. Journal of Experimental Botany, 67, 1935–1950.

Clough S J, Bent A F. 1998. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 16, 735–743.

Denancé N, Sánchez-Vallet A, Goffner D, Molina A. 2013. Disease resistance or growth: The role of plant hormones in balancing immune responses and fitness costs. Frontiers in Plant Science, 4, 155.

Deng G M, Zhang S, Yang Q S, Gao H J, Sheng O, Bi F C, Li C Y, Dong T, Yi G J, He W D, Hu C H. 2020. MaMYB4, an R2R3-MYB repressor transcription factor, negatively regulates the biosynthesis of anthocyanin in banana. Frontiers in Plant Science, 11, 600704.

Dolferus R, Jacobs M, Peacock W J, Dennis E S. 1994. Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene. Plant Physiology, 105, 1075–1087.

Dong N Q, Sun Y, Guo T, Shi C L, Zhang Y M, Kan Y, Xiang Y H, Zhang H, Yang Y B, Li Y C, Zhao H Y, Yu H X, Lu Z Q, Wang Y, Ye W W, Shan J X, Lin H X. 2020. UDP-glucosyltransferase regulates grain size and abiotic stress tolerance associated with metabolic flux redirection in rice. Nature Communications, 11, 2629.

Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. 2010. MYB transcription factors in Arabidopsis. Trends Plant Science, 15, 573–581.

Eddy S R. 2011. Accelerated profile HMM searches. PLoS Computational Biology, 7, e1002195.

Finn R D, Bateman A, Clements J, Coggill P, Eberhardt R Y, Eddy S R, Heger A, Hetherington K, Holm L, Mistry J. 2014. Pfam: the protein families database. Nucleic Acids Research, 42, D222–D230.

Fradin E F, Thomma B P. 2006. Physiology and molecular aspects of verticillium wilt diseases caused by V. dahliae and V. albo-atrum. Molecular Plant Pathology, 7, 71–86.

Fraser C M, Chapple C. 2011. The phenylpropanoid pathway in Arabidopsis. The Arabidopsis Book, 9, e0152.

Fu Z W, Li J H, Gao X, Wang S J, Yuan T T, Lu Y T. 2024. Pathogen-induced methylglyoxal negatively regulates rice bacterial blight resistance by inhibiting OsCDR1 protease activity. Molecular Plant, 17, 325–341.

Gao X, Wheeler T, Li Z, Kenerley C M, He P, Shan L. 2011. Silencing GhNDR1 and GhMKK2 compromises cotton resistance to verticillium wilt. The Plant Journal, 66, 293–305.

Gaudinier A, Rodriguez-Medina J, Zhang L, Olson A, Liseron-Monfils C, Bågman A M, Foret J, Abbitt S, Tang M, Li B. 2018. Transcriptional regulation of nitrogen-associated metabolism and growth. Nature, 563, 259–264.

Gu K D, Zhang Q Y, Yu J Q, Wang J H, Zhang F J, Wang C K, Zhao Y W, Sun C H, You C X, Hu D G, Hao Y J. 2021. R2R3-MYB transcription factor MdMYB73 confers increased resistance to the fungal pathogen Botryosphaeria dothidea in apples via the salicylic acid pathway. Journal of Agricrotural Food Chemistry, 69, 447–458.

Guo D, Zhang J, Wang X, Han X, Wei B, Wang J, Li B, Yu H, Huang Q, Gu H, Qu L J, Qin G. 2015. The WRKY transcription factor WRKY71/EXB1 controls shoot branching by transcriptionally regulating RAX genes in Arabidopsis. The Plant Cell, 27, 3112–3127.

Hellens R P, Edwards E A, Leyland N R, Bean S, Mullineaux P M. 2000. pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Molecular Biology, 42, 819–832.

Hu D G, Li Y Y, Zhang Q Y, Li M, Sun C H, Yu J Q, Hao Y J. 2017. The R2R3-MYB transcription factor MdMYB73 is involved in malate accumulation and vacuolar acidification in apple. The Plant Journal, 91, 443–454.

Kim D, Jeon S J, Yanders S, Park S C, Kim H S, Kim S. 2022. MYB3 plays an important role in lignin and anthocyanin biosynthesis under salt stress condition in Arabidopsis. Plant Cell Reports, 41, 1549–1560.

Klosterman S J, Atallah Z K, Vallad G E, Subbarao K V. 2009. Diversity, pathogenicity, and management of Verticillium species. Annual Review of Phytopathology, 47, 39–62.

Konishi M,Yanagisawa S. 2013. Arabidopsis NIN-like transcription factors have a central role in nitrate signalling. Nature Communications, 4, 1617.

Lanot A, Hodge D, Lim E K, Vaistij F E, Bowles D J. 2008. Redirection of flux through the phenylpropanoid pathway by increased glucosylation of soluble intermediates. Planta, 228, 609–616.

Lim C W, Luan S, Lee S C. 2014. A prominent role for RCAR3-mediated ABA signaling in response to Pseudomonas syringae pv. tomato DC3000 infection in Arabidopsis. Plant and Cell Physiology, 55, 1691–1703.

Lin H, Wang M, Chen Y, Nomura K, Hui S, Gui J, Zhang X, Wu Y, Liu J, Li Q, Deng Y, Li L, Yuan M, Wang S, He S Y, He Z. 2022. An MKP-MAPK protein phosphorylation cascade controls vascular immunity in plants. Science Advances, 8, eabg8723.

Liu L, Song Y, Xu J, Li D, Li G, An L. 2017. Differential expression by chromatin modifications of alcohol dehydrogenase 1 of Chorispora bungeana in cold stress. Gene, 636, 1–16.

Liu T, Chen T, Kan J, Yao Y, Guo D, Yang Y, Ling X, Wang J, Zhang B. 2022. The GhMYB36 transcription factor confers resistance to biotic and abiotic stress by enhancing PR1 gene expression in plants. Plant Biotechnology Journal, 20, 722–735.

Liu T, Song T, Zhang X, Yuan H, Su L, Li W, Xu J, Liu S, Chen L, Chen T, Zhang M, Gu L, Zhang B, Dou D. 2014. Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis. Nature Communications, 5, 4686.

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

Lu M, Yu S, Lian J, Wang Q, He Z, Feng Y, Yang X. 2021. Physiological and metabolomics responses of two wheat (Triticum aestivum L.) genotypes differing in grain cadmium accumulation. The Science of the Total Environment, 769, 145345.

Marchive C, Roudier F, Castaings L, Bréhaut V, Blondet E, Colot V, Meyer C, Krapp A. 2013. Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants. Nature Communications, 4, 1713.

McCarthy R L, Zhong R, Fowler S, Lyskowski D, Piyasena H, Carleton K, Spicer C, Ye Z H. 2010. The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, are involved in the regulation of secondary wall biosynthesis. Plant and Cell Physiology, 51, 1084–1090.

Miller G, Schlauch K, Tam R, Cortes D, Torres M A, Shulaev V, Dangl J L, Mittler R. 2009. The plant NADPH oxidase RBOHD mediates rapid systemic signaling in response to diverse stimuli. Science Signaling, 2, ra45.

Miura K, Lee J, Jin J B, Yoo C Y, Miura T, Hasegawa P M. 2009. Sumoylation of ABI5 by the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates abscisic acid signaling. Proceedings of the National Academy of Sciences of the United States of America, 106, 5418–5423.

Papdi C, Abrahám E, Joseph M P, Popescu C, Koncz C, Szabados L. 2008. Functional identification of Arabidopsis stress regulatory genes using the controlled cDNA overexpression system. Plant Physiology, 147, 528–542.

Pathuri I P, Reitberger I E, Hückelhoven R, Proels R K. 2011. Alcohol dehydrogenase 1 of barley modulates susceptibility to the parasitic fungus Blumeria graminis f. sp. hordei. Journal of Experimental Botany, 62, 3449–3457.

Pei Y, Li X, Zhu Y, Ge X, Sun Y, Liu N, Jia Y, Li F, Hou Y. 2019. GhABP19, a novel germin-like protein from Gossypium hirsutum, plays an important role in the regulation of resistance to verticillium and fusarium wilt pathogens. Frontiers in Plant Science, 10, 583–601.

Pratyusha D S, Sarada D V L. 2022. MYB transcription factors-master regulators of phenylpropanoid biosynthesis and diverse developmental and stress responses. Plant Cell Reports, 41, 2245–2260.

Preston J, Wheeler J, Heazlewood J, Li S F, Parish R W. 2004. AtMYB32 is required for normal pollen development in Arabidopsis thaliana. The Plant Journal, 40, 979–995.

Qiu Z, Yan S, Xia B, Jiang J, Yu B, Lei J, Chen C, Chen L, Yang Y, Wang Y, Tian S, Cao B. 2019. The eggplant transcription factor MYB44 enhances resistance to bacterial wilt by activating the expression of spermidine synthase. Journal of Experimental Botany, 70, 5343–5354.

Ren Z, Fang M, Muhae-Ud-Din G, Gao H, Yang Y, Liu T, Chen W, Gao L. 2021. Metabolomics analysis of grains of wheat infected and noninfected with Tilletia controversa Kühn. Scientific Reports, 11, 18876.

Le Roy J, Huss B, Creach A, Hawkins S,Neutelings G. 2016. Glycosylation is a major regulator of phenylpropanoid availability and biological activity in plants. Frontiers in Plant Science, 7, 735.

Sánchez-Vallet A, López G, Ramos B, Delgado-Cerezo M, Riviere M P, Llorente F, Fernández P V, Miedes E, Estevez J M, Grant M, Molina A. 2012. Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina. Plant Physiology, 160, 2109–2124.

Shaban M, Miao Y, Ullah A, Khan A Q, Menghwar H, Khan A H, Ahmed M M, Tabassum M A, Zhu L. 2018. Physiological and molecular mechanism of defense in cotton against Verticillium dahliae. Plant Physiology and Biochemistry, 125, 193–204.

Shen J L, Xiao S H, Xi H, Nurimanguli A, Hu Q, Zhang X J, Yang Z G, Nie X H, Zhu L F. 2020. GhMYB43 negatively regulates lignin biosynthesis and jasmonic acid signaling. Cotton Science, 32, 522–537.

Shi H, Liu W, Yao Y, Wei Y, Chan Z. 2017. Alcohol dehydrogenase 1 (ADH1) confers both abiotic and biotic stress resistance in Arabidopsis. Plant Science, 262, 24–31.

Sinha E, Michalak A, Balaji V. 2017. Eutrophication will increase during the 21st century as a result of precipitation changes. Science, 357, 405–408.

Wang L, Ran L, Hou Y, Tian Q, Li C, Liu R, Fan D, Luo K. 2017. The transcription factor MYB115 contributes to the regulation of proanthocyanidin biosynthesis and enhances fungal resistance in poplar. The New Phytologist, 215, 351–367.

Wang P, Zhou L, Jamieson P, Zhang L, Zhao Z, Babilonia K, Shao W, Wu L, Mustafa R, Amin I, Diomaiuti A, Pontiggia D, Ferrari S, Hou Y, He P, Shan L. 2020. The cotton wall-associated kinase GhWAK7A mediates responses to fungal wilt pathogens by complexing with the chitin sensory receptors. The Plant Cell, 32, 3978–4001.

Wang X C, Wu J, Guan M L, Zhao C H, Geng P, Zhao Q. 2020. Arabidopsis plays dual roles in flavonoid biosynthesis. The Plant Journal, 101, 637–652.

Wang Y, Liang C, Wu S, Zhang X, Tang J, Jian G, Jiao G, Li F, Chu C. 2016. Significant improvement of cotton Verticillium wilt resistance by manipulating the expression of gastrodia antifungal proteins. Molecular Plant, 9, 1436–1439.

Wei T, Tang Y, Jia P, Zeng Y, Wang B, Wu P, Quan Y, Chen A, Li Y, Wu J. 2021. A cotton lignin biosynthesis gene, GhLAC4, fine-tuned by ghr-miR397 modulates plant resistance against Verticillium dahliae. Frontiers in Plant Science, 12, 743795.

Wildermuth M C. 2010. Modulation of host nuclear ploidy: A common plant biotroph mechanism. Current Opinion in Plant Biology, 13, 449–458.

Xiao S, Hu Q, Shen J, Liu S, Yang Z, Chen K, Klosterman S J, Javornik B, Zhang X, Zhu L. 2021. GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae. Plant Cell Reports, 40, 735–751.

Xie M, Zhang J, Tschaplinski T J, Tuskan G A, Chen J G, Muchero W. 2018. Regulation of lignin biosynthesis and its role in growth-defense tradeoffs. Frontiers in Plant Science, 9, 1427.

Yang C Y. 2014. Hydrogen peroxide controls transcriptional responses of ERF73/HRE1 and ADH1 via modulation of ethylene signaling during hypoxic stress. Planta, 239, 877–885.

Yang L, Wang Z, Zhang A, Bhawal R, Li C, Zhang S, Cheng L, Hua J. 2022. Reduction of the canonical function of a glycolytic enzyme enolase triggers immune responses that further affect metabolism and growth in Arabidopsis. The Plant Cell, 34, 1745–1767.

Yang Z, Ge X, Li W, Jin Y, Liu L, Hu W, Liu F, Chen Y, Peng S, Li F. 2021. Cotton D genome assemblies built with long-read data unveil mechanisms of centromere evolution and stress tolerance divergence. BMC Biology, 19, 115.

Zhang M, Wang J, Luo Q, Yang C, Yang H, Cheng Y J. 2021. CsMYB96 enhances citrus fruit resistance against fungal pathogen by activating salicylic acid biosynthesis and facilitating defense metabolite accumulation. Journal of Plant Physiology, 264, 153472.

Zhang Y, Yin X, Xiao Y, Zhang Z, Li S, Liu X, Zhang B, Yang X, Grierson D, Jiang G, Klee H J, Chen K. 2018. An ethylene response factor-MYB transcription complex regulates furaneol biosynthesis by activating quinone oxidoreductase expression in strawberry. Plant Physiology, 178, 189–201.

Zhong R,Ye Z H. 2007. Regulation of cell wall biosynthesis. Current Opinion in Plant Biology, 10, 564–572.

Zhu Y, Hu X, Wang P, Wang H, Ge X, Li F, Hou Y. 2022. GhODO1, an R2R3-type MYB transcription factor, positively regulates cotton resistance to Verticillium dahliae via the lignin biosynthesis and jasmonic acid signaling pathway. International Journal of Biological Macromolecules, 201, 580–591.