CmWRKY6–1–CmWRKY15-like transcriptional cascade negatively regulates the resistance to Fusarium oxysporum infection in Chrysanthemum morifolium

Abstract

Chrysanthemum Fusarium wilt is a soil-borne disease that causes serious economic losses to the chrysanthemum industry. However, the molecular mechanism underlying the response of chrysanthemum WRKY to Fusarium oxysporum infection remains largely unknown. In this study, we isolated CmWRKY6–1 from chrysanthemum ‘Jinba’ and identified it as a transcriptional repressor localized in the nucleus via subcellular localization and transcriptional activation assays. We found that CmWRKY6–1 negatively regulated resistance to F. oxysporum and affected reactive oxygen species (ROS) and salicylic acid (SA) pathways using transgenic experiments and transcriptomic analysis. Moreover, CmWRKY6–1 bound to the W-box element on the CmWRKY15-like promoter and inhibited its expression. Additionally, we observed that CmWRKY15-like silencing in chrysanthemum reduced its resistance to F. oxysporum via transgenic experiments. In conclusion, we revealed the mechanism underlying the CmWRKY6–1–CmWRKY15-like cascade response to F. oxysporum infection in chrysanthemum and demonstrated that CmWRKY6–1 and CmWRKY15-like regulates the immune system.

Acknowledgements

This research was supported by National Natural Science Foundation of China (32072603), China Agriculture Research System (CARS-23-A18), The JBGS Project of Seed Industry Revitalization in Jiangsu Province [JBGS(2021)094] and Jiangsu Agriculture Science and Technology Innovation Fund [CX(22)2033].

Author contributions

S.Z., Y.Liu., Z.G., Y.Liao., S.C., W.F., F.C., and W.M. designed the research. W.M., X.X., Y.W., and Y.Y. performed experiments. W.M. and L.G. analysed data. W.M. wrote the manuscript. All authors read and approved the final manuscript.

Data availability

All transcriptomic sequencing data associated with this study have been submitted to the NCBI SRA under the accession number PRJNA946259.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

References

[1] Steinkellner S, Mammerler R, Vierheilig H . Germination of Fusarium oxysporum in root exudates from tomato plants chanllenged with different Fusarium oxysporum strains . Euro J Plant Pathol. 2008; 122: 395-401.

[2] Li CY, Chen S, Zuo CW et al. The use of GFP—transformed isolates to study infection of banana with Fusarium oxysporum f. sp. cubense race 4 . Eur J Plant Pathol. 2011; 131: 327-40.

[3] Zhang Y, Wang X, Jin C et al. Comparative transcriptome and proteome analysis of lily clones inoculated with Fusarium oxysporum f. sp. lilii . Ornam Plant Res. 2022; 2: 23.

[4] DeFalco TA, Zipfel C . Molecular mechanisms of early plant pattern—triggered immune signaling. Mol Cell. 2021; 81: 4346.

[5] Monteiro F, Nishimura MT . Structural, functional, and genomic diversity of plant NLR proteins: an evolved resource for rational engineering of plant immunity. Annu Rev Phytopathol. 2018; 56: 243-67.

[6] Qi YP, Tsuda K, Glazebrook J et al. Physical association of pattern—triggered immunity (PTI) and effector—triggered immunity (ETI) immune receptors in Arabidopsis. Mol Plant Pathol. 2011; 12: 702-8.

[7] Rushton PJ, Somssich IE, Ringler P et al. WRKY transcription factors. Trends Plant Sci. 2010; 15: 247-58.

[8] Chen XJ, Li C, Wang H et al. WRKY transcription factors: evolution, binding, and action. Phytopathol Res. 2019; 1: 13.

[9] Li WX, Pang SY, Lu ZG et al. Function and mechanism of WRKY transcription factors in abiotic stress responses of plants. Plants. 2020; 9: 1515.

[10] Bakshi M, Oelmüller R . WRKY transcription factors Jack of many trades in plants. Plant Signal Behav. 2014; 9: e27700.

[11] Zhang YJ, Wang LJ . The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evol Biol. 2005; 5: 1.

[12] Phukan UJ, Jeena GS, Shukla RK . WRKY transcription factors: molecular regulation and stress responses in plants. Front Plant Sci. 2016; 7: 760.

[13] Birkenbihl RP, Diezel C, Somssich IE . Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea infection . Plant Physiol. 2012; 159: 266-85.

[14] Chen XT, Liu J, Lin GF et al. Overexpression of AtWRKY28 and AtWRKY75 in Arabidopsis enhances resistance to oxalic acid and Sclerotinia sclerotiorum . Plant Cell Rep. 2013; 32: 1589—99.

[15] Chujo T, Takai R, Akimoto—Tomiyama C et al. Involvement of the elicitor—induced gene OsWRKY53 in the expression of defense—related genes in rice . Biochim Biophys Acta. 2007; 1769: 497-505.

[16] Shimono M, Sugano S, Nakayama A et al. Rice WRKY45 plays a crucial role in benzothiadiazole—inducible blast resistance. Plant Cell. 2007; 19: 2064-76.

[17] Jiang JJ, Ma SH, Ye NH et al. WRKY transcription factors in plant responses to stresses. J Integr Plant Biol. 2017; 59: 86-101.

[18] Joshi A, Jeena GS, Shikha et al. Ocimum sanctum, OscWRKY1, regulates phenylpropanoid pathway genes and promotes resistance to pathogen infection in Arabidopsis . Plant Mol Biol. 2022; 110: 235-51.

[19] Chakraborty J, Priya P, Dastidar SG et al. Physical interaction between nuclear accumulated CC—NB—ARC—LRR protein and WRKY64 promotes EDS1 dependent fusarium wilt resistance in chickpea. Plant Sci. 2018; 276: 111-33.

[20] Dong H, Tan J, Li M et al. Transcriptome analysis of soybean WRKY TFs in response to Peronospora manshurica infection . Genomics. 2019; 111: 1412—22.

[21] Tarja K, Günter B, Jing L et al. Chlorophyllase1 a damage control enzyme affects the balance between defense pathways in plants. Plant Cell. 2005; 17: 282-94.

[22] Hou X, Xie KB, Yao JL et al. A homolog of human ski—interacting protein in rice positively regulates cell viability and stress tolerance. Proc Natl Acad Sci U S A. 2009; 106: 6410-5.

[23] Sahu PK, Jayalakshmi K, Tilgam J et al. ROS generated from biotic stress: effects on plants and alleviation by endophytic microbes. Front Plant Sci. 2022; 13: 1042936.

[24] Janská A, Maršík P, Zelenková S et al. Cold stress and acclimation—what is important for metabolic adjustment? Plant Biol. 2010; 12: 395-405.

[25] Saruhan N, Saglam NS, Kadioglu A . Salicylic acid pretreatment induces drought tolerance and delays leaf rolling by inducing antioxidant systems in maize genotypes. Acta Physiol Plant. 2012; 34: 97-106.

[26] Adachi H, Nakano T, Miyagawa N et al. WRKY transcription factors phosphorylated by MAPK regulate a plant immune NADPH oxidase in Nicotiana benthamiana . Plant Cell. 2015; 27: 2645—63.

[27] Cui J, Jiang N, Meng J et al. LncRNA33732—respiratory burst oxidase module associated with WRKY1 in tomato— Phytophthora infestans interactions . Plant J. 2019; 97: 933-46.

[28] Saleem M, Fariduddin Q, Castroverde CDM . Salicylic acid: a key regulator of redox signalling and plant immunity. Plant Physiol Biochem. 2021; 168: 381-97.

[29] Vlot AC, Dempsey DMA, Klessig DF . Salicylic acid, a multifaceted hormone to combat disease. Annu Rev Phytopathol. 2009; 47: 177-206.

[30] Herrera—Vásquez A, Salinas P, Holuigue L . Salicylic acid and reactive oxygen species interplay in the transcriptional control of defense genes expression. Front Plant Sci. 2015; 6: 171.

[31] Mateo A, Funck D, Mühlenbock P et al. Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis. J Exp Bot. 2006; 57: 1795-807.

[32] Chakraborty J, Sen S, Ghosh P et al. Inhibition of multiple defense responsive pathways by CaWRKY70 transcription factor promotes susceptibility in chickpea under Fusarium oxysporum stress condition . BMC Plant Biol. 2020; 20: 319.

[33] Miao WH, Ge LJ, Wang YA et al. Overexpression of CmWRKY8—1—VP64 fusion protein reduces resistance in response to Fusarium oxysporum by modulating the salicylic acid signaling pathway in Chrysanthemum morifolium . Int J Mol Sci. 2023; 24: 3499.

[34] Xin JJ, Liu Y, Li HY et al. CmMLO17 and its partner CmKIC potentially support Alternaria alternata growth in Chrysanthemum morifolium . Hortic Res. 2021; 8: 101.

[35] Sumitomo K, Shirasawa K, Isobe S et al. A genome—wide association and fine—mapping study of white rust resistance in hexaploid chrysanthemum cultivars with a wild diploid reference genome. Hortic Res. 2022; 9: uhac170.

[36] Song AP, Li PL, Jiang JF et al. Phylogenetic and transcription analysis of chrysanthemum WRKY transcription factors. Int J Mol Sci. 2014; 15: 14442—55.

[37] Bi MM, Li XY, Yan X et al. Chrysanthemum WRKY15—1 promotes resistance to Puccinia horiana Henn. Via the salicylic acid signaling pathway . Hortic Res. 2021; 8: 6.

[38] Gao G, Jin RB, Liu D et al. CmWRKY15—1 promotes resistance to chrysanthemum white rust by regulating CmNPR1 expression . Front Plant Sci. 2022; 13: 865607.

[39] Peng Y, Bartley LE, Canlas P et al. OsWRKY IIa transcription factors modulate Rice innate immunity. Rice. 2010; 3: 36-42.

[40] Wang M, Tang WX, Xiang L et al. Involvement of MdWRKY40 in the defense of mycorrhizal apple against Fusarium solani . BMC Plant Biol. 2022; 22: 385.

[41] Wang Y, Wang XH, Fang J et al. VqWRKY56 interacts with VqbZIPC22 in grapevine to promote proanthocyanidin biosynthesis and increase resistance to powdery mildew. New Phytol. 2023; 237: 1856—75.

[42] Katagiri F, Tsuda K . Understanding the plant immune system. Mol Plant—Microbe Interact. 2010; 23: 1531-6.

[43] Barna B, Fodor J, Harrach BD et al. The Janus face of reactive oxygen species in resistance and susceptibility of plants to necrotrophic and biotrophic pathogens. Plant Physiol Biochem. 2012; 59: 37-43.

[44] Nath M, Yadav S, Sahoo RK et al. PDH45 transgenic rice maintain cell viability through lower accumulation of Na ⁺, ROS and calcium homeostasis in roots under salinity stress . J Plant Physiol. 2016; 191: 1-11.

[45] Lefevere H, Bauters L, Gheysen G . Salicylic acid biosynthesis in plants. Front Plant Sci. 2020; 11: 338.

[46] Guan YQ, He X, Wen D et al. Fusarium oxysporum infection on root elicit aboveground terpene production and salicylic acid accumulation in Chrysanthemum morifolium . Plant Physiol Biochem. 2022; 190: 11-23.

[47] Hsin KT, Hsieh MC, Lee YH et al. Insight into the phylogeny and binding ability of WRKY transcription factors. Int J Mol Sci. 2022; 23: 2895.

[48] Chakraborty J, Ghosh P, Sen S et al. CaMPK9 increases the stability of CaWRKY40 transcription factor which triggers defense response in chickpea upon Fusarium oxysporum f. sp. ciceri Race1 infection . Plant Mol Biol. 2019; 100: 411—31.

[49] Liu ZQ, Shi LP, Yang S et al. A conserved double—W box in the promoter of CaWRKY40 mediates autoregulation during response to pathogen attack and heat stress in pepper . Mol Plant Pathol. 2021; 22: 3-18.

[50] Li CW, Su JS, Zhao N et al. CmERF5—CmRAP2.3 transcriptional cascade positively regulates waterlogging tolerance in Chrysanthemum morifolium . Plant Biotechnol J. 2023; 21: 270-82.

[51] Sun Y, Xia XL, Jiang JF et al. Salicylic acid—induced changes in physiological parameters and genes of the flavonoid biosynthesis pathway in Artemisia vulgaris and Dendranthema nankingense during aphid feeding . Genet Mol Res. 2016; 15: 1.

[52] Kwak JM, Nguyen V, Schroeder JI . The role of reactive oxygen species in hormonal responses. Plant Physiol. 2006; 141: 323-9.

[53] Yoo SD, Cho YH, Sheen J . Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2007; 2: 1565-72.

[54] Gu CS, Chen SM, Liu ZL et al. Reference gene selection for quantitative real—time PCR in chrysanthemum subjected to biotic and abiotic stress. Mol Biotechnol. 2011; 49: 192-7.

[55] Zhai LS, Zhu XC, Yang SJ et al. Constitutive expression of a chrysanthemum phospholipase Dα gene in Chrysanthemum morifolium enhances drought tolerance . Ornam Plant Res. 2021; 1: 8.

[56] Pfaffl MW . A new mathematical model for relative quantification in real—time RT—PCR. Nucleic Acids Res. 2001; 29: e45.

[57] Zhu L, Guan YX, Zhang ZH et al. CmMYB8 encodes an R2R3 MYB transcription factor which represses lignin and flavonoid synthesis in chrysanthemum . Plant Physiol Biochem. 2020; 149: 217-24.

[58] Xu SJ, Wu Z, Zhao JY et al. High—efficiency Agrobacterium—mediated transformation of chrysanthemum via vacuum infiltration of internode . Ornam Plant Res. 2022; 2: 1-7.

[59] Li B, Dewey CN . RSEM: accurate transcript quantification from RNA—Seq data with or without a reference genome. Bioinformatics. 2011; 12: 323.