Overexpression of CmWRKY8-1–VP64 Fusion Protein Reduces Resistance in Response to Fusarium oxysporum by Modulating the Salicylic Acid Signaling Pathway in Chrysanthemum morifolium
Abstract
:1. Introduction
2. Results
2.1. Isolation and Sequence Analysis of CmWRKY8-1
2.2. Characteristics of CmWRKY8-1
2.3. Expression Pattern of CmWRKY8-1 after F. oxysporum Infection in C. moriflium ‘Jinba’ and Spraying Exogenous SA to Improve the Resistance of C. moriflium ‘Jinba’ to F. oxysporum
2.4. Overexpression of CmWRKY8-1-VP64 Fusion Protein Increases the Susceptibility of Chrysanthemums to F. oxysporum
2.5. Changes in Genes Involved in the SA Signaling Pathway and Alterations in Endogenous SA
2.6. Transcriptome Sequencing Analysis and Functional Enrichment of DEGs in CmWRKY8-1 Transgenic Lines
3. Discussion
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
4.2. Isolation, Identification, Culture and Inoculation of Pathogenic Fungus
4.3. Isolation and Sequence Analysis of CmWRKY8-1
4.4. Subcellular Localization of CmWRKY8-1
4.5. Transactivation Assays of CmWRKY8-1
4.6. Quantitative Real-Time PCR (qRT-PCR)
4.7. Analysis of CmWRKY8-1 Expression under Stress Treatments
4.8. Generation of CmWRKY8-1 Transgenic Chrysanthemum
4.9. Analysis of F. oxysporum Resistance in CmWRKY8-1 Transgenic Chrysanthemums
4.10. Enzyme Activity Assay and Endogenous SA Determination
4.11. RNA-Seq Analysis
4.12. Data Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Ploetz, R.C. Fusarium wilt of banana is caused by several pathogens referred to as Fusarium oxysporum f. sp. cubense. Phytopathology 2006, 96, 653–656. [Google Scholar] [CrossRef] [PubMed]
- Minuto, A.; Gullino, M.L.; Garibaldi, A. Gerbera jamesonii, Osteospermum sp. and Argyranthemum frutescens: New hosts of Fusarium oxysporum f. sp. chrysanthemi. J. Phytopathol. 2007, 155, 373–376. [Google Scholar] [CrossRef]
- Chen, X.; Shu, Y.; Luo, M.; Xiang, M.; Huang, Y.; Zhang, W.; Dong, Z. Fusarium wilt of imperial Chrysanthemum (Chrysanthemum morifolium) caused by Fusarium oxysporum in China. Plant Dis. 2020, 104, 985. [Google Scholar] [CrossRef]
- DeFalco, T.A.; Zipfel, C. Molecular mechanisms of early plant pattern-triggered immune signaling. Mol. Cell 2021, 81, 4346. [Google Scholar] [CrossRef] [PubMed]
- Zhai, K.; Di Liang, D.; Li, H.; Jiao, F.; Yan, B.; Liu, J.; Lei, Z.; Huang, L.; Gong, X.; Wang, X.; et al. NLRs guard metabolism to coordinate pattern- and effector-triggered immunity. Nature 2022, 601, 245–251. [Google Scholar] [CrossRef]
- Rushton, P.J.; Somssich, I.E.; Ringler, P.; Shen, Q.J. WRKY transcription factors. Trends Plant Sci. 2010, 15, 247–258. [Google Scholar] [CrossRef]
- Wani, S.H.; Anand, S.; Singh, B.; Bohra, A.; Joshi, R. WRKY transcription factors and plant defense responses: Latest discoveries and future prospects. Plant Cell Rep. 2021, 40, 1071–1085. [Google Scholar] [CrossRef]
- Zhang, Y.; Wang, L. The WRKY transcription factor superfamily: Its origin in eukaryotes and expansion in plants. BMC Evol. Biol. 2005, 5, 1. [Google Scholar] [CrossRef]
- Rinerson, C.I.; Rabara, R.C.; Tripathi, P.; Shen, Q.J.; Rushton, P.J. The evolution of WRKY transcription factors. BMC Plant Biol. 2015, 15, 66. [Google Scholar] [CrossRef]
- Song, H.; Sun, W.; Yang, G.; Sun, J. WRKY transcription factors in legumes. BMC Plant Biol. 2018, 18, 243. [Google Scholar] [CrossRef] [Green Version]
- Ülker, B.; Somssich, I.E. WRKY transcription factors: From DNA binding towards biological function. Curr. Opin. Plant Biol. 2004, 7, 491–498. [Google Scholar] [CrossRef]
- Jiang, J.; Ma, S.; Ye, N.; Jiang, M.; Cao, J.; Zhang, J. WRKY transcription factors in plant responses to stresses. J. Integr. Plant Biol. 2017, 59, 86–101. [Google Scholar] [CrossRef]
- Chen, L.; Zhang, L.; Yu, D. Wounding-induced wrky8 is involved in basal defense in Arabidopsis. Mol. Plant-Microbe Interact. 2010, 23, 558–565. [Google Scholar] [CrossRef]
- Zheng, Z.; Abu Qamar, S.; Chen, Z.; Mengiste, T. Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J. 2006, 48, 592–605. [Google Scholar] [CrossRef]
- Jiang, Y.; Yu, D. The WRKY57 transcription factor affects the expression of Jasmonate ZIM-domain genes transcriptionally to compromise Botrytis cinerea resistance. Plant Physiol. 2016, 171, 2771–2782. [Google Scholar] [CrossRef]
- Wang, W.J.; Li, T.; Chen, Q.; Yao, S.X.; Deng, L.L.; Zeng, K.F. CsWRKY25 improves resistance of citrus fruit to Penicillium digitatum via modulating reactive oxygen species production. Front Plant Sci. 2022, 12, 818198. [Google Scholar] [CrossRef]
- Chakraborty, J.; Ghosh, P.; Sen, S.; Nandi, A.K.; Das, S. 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, 411–431. [Google Scholar] [CrossRef]
- Zhang, W.; Gao, T.; Li, P.; Tian, C.; Song, A.; Jiang, J.; Guan, Z.; Fang, W.; Chen, F.; Chen, S. Chrysanthemum CmWRKY53 negatively regulates the resistance of chrysanthemum to the aphid Macrosiphoniella sanborni. Hortic. Res. 2020, 7, 1–9. [Google Scholar] [CrossRef]
- Vlot, A.C.; Dempsey, D.M.A.; Klessig, D.F. Salicylic acid, a multifaceted hormone to combat disease. Annu. Rev. Phytopathol. 2009, 47, 177–206. [Google Scholar] [CrossRef]
- Bi, M.; Li, X.; Yan, X.; Liu, D.; Gao, G.; Zhu, P.; Mao, H. Chrysanthemum WRKY15-1 promotes resistance to Puccinia horiana Henn. via the salicylic acid signaling pathway. Hortic. Res. 2021, 8, 1–11. [Google Scholar] [CrossRef]
- Zhao, X.; Qi, C.; Jiang, H.; Zhong, M.; You, C.; Li, Y.; Hao, Y. MdWRKY15 improves resistance of apple to Botryosphaeria dothidea via the salicylic acid-mediated pathway by directly binding the MdICS1 promoter. J. Integr. Plant Biol. 2020, 62, 527–543. [Google Scholar] [CrossRef] [PubMed]
- Yaghmai, R.; Cutting, G.R. Optimized regulation of gene expression using artificial transcription factors. Mol. Ther. 2002, 5, 685–694. [Google Scholar] [CrossRef] [PubMed]
- Lowder, L.G.; Zhang, D.; Baltes, N.J.; Paul, J.W.; Tang, X.; Zheng, X.; Voytas, D.F.; Hsieh, T.-F.; Zhang, Y.; Qi, Y. A CRISPR/Cas9 Toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 2015, 169, 971–985. [Google Scholar] [CrossRef] [PubMed]
- Piatek, A.; Ali, Z.; Baazim, H.; Li, L.; Abulfaraj, A.; Al-Shareef, S.; Aouida, M.; Mahfouz, M.M. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol. J. 2015, 13, 578–589. [Google Scholar] [CrossRef]
- Ren, C.; Li, H.; Liu, Y.; Li, S.; Liang, Z. Highly efficient activation of endogenous gene in grape using CRISPR/dCas9-based transcriptional activators. Hortic. Res. 2022, 9, uhab037. [Google Scholar] [CrossRef]
- Wang, J.; Wu, F.; Zhu, S.; Xu, Y.; Cheng, Z.; Wang, J.; Li, C.; Sheng, P.; Zhang, H.; Cai, M.; et al. Overexpression of OsMYB1R1-VP64 fusion protein increases grain yield in rice by delaying flowering time. FEBS Lett. 2016, 590, 3385–3396. [Google Scholar] [CrossRef]
- Hadizadeh, H.; Samiei, L.; Shakeri, A. Chrysanthemum, an ornamental genus with considerable medicinal value: A comprehensive review. S. Afr. J. Bot. 2022, 144, 23–43. [Google Scholar] [CrossRef]
- Song, A.; Li, P.; Jiang, J.; Chen, S.; Li, H.; Zeng, J.; Shao, Y.; Zhu, L.; Zhang, Z.; Chen, F. Phylogenetic and transcription analysis of chrysanthemum WRKY transcription Factors. Int. J. Mol. Sci. 2014, 15, 14442–14455. [Google Scholar] [CrossRef]
- Guan, Y.; He, X.; Wen, D.; Chen, S.; Chen, F.; Chen, F.; Jiang, Y. Fusarium oxysporum infection on root elicit aboveground terpene production and salicylic acid accumulation in Chrysanthemum morifolium. Plant Physiol. Biochem. 2022, 190, 11–23. [Google Scholar] [CrossRef]
- Janská, A.; Maršík, P.; Zelenková, S.; Ovesná, J. Cold stress and acclimation—What is important for metabolic adjustment? Plant Biol. 2010, 12, 395–405. [Google Scholar] [CrossRef]
- Saruhan, N.; Saglam, N.S.; 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. [Google Scholar] [CrossRef]
- Janda, T.; Szalai, G.; Pál, M. Salicylic acid signalling in plants. Int. J. Mol. Sci. 2020, 21, 2655. [Google Scholar] [CrossRef]
- Wang, Z.; Deng, J.; Liang, T.; Su, L.; Zheng, L.; Chen, H.; Liu, D. Lilium regale Wilson WRKY3 modulates an antimicrobial peptide gene, LrDef1, during response to Fusarium oxysporum. BMC Plant Biol. 2022, 22, 257. [Google Scholar] [CrossRef]
- Wang, L.; Guo, D.; Zhao, G.; Wang, J.; Zhang, S.; Wang, C.; Guo, X. Group IIc WRKY transcription factors regulate cotton resistance to Fusarium oxysporum by promoting GhMKK2 -mediated flavonoid biosynthesis. New Phytol. 2022, 236, 249–265. [Google Scholar] [CrossRef]
- Chakraborty, J.; Sen, S.; Ghosh, P.; Jain, A.; Das, S. 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. [Google Scholar] [CrossRef]
- Konermann, S.; Brigham, M.D.; Trevino, A.E.; Joung, J.; Abudayyeh, O.O.; Barcena, C.; Hsu, P.D.; Habib, N.; Gootenberg, J.S.; Nishimasu, H.; et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 2015, 517, 583–588. [Google Scholar] [CrossRef]
- Cao, H.; Bowling, S.A.; Gordon, A.S.; Dong, X. Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell 1994, 6, 1583–1592. [Google Scholar] [CrossRef]
- Cao, H.; Glazebrook, J.; Clarke, J.D.; Volko, S.; Dong, X. The arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 1997, 88, 57–63. [Google Scholar] [CrossRef]
- Wildermuth, M.C.; Dewdney, J.; Wu, G.; Ausubel, F.M. Erratum: Corrigendum: Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 2001, 417, 571. [Google Scholar] [CrossRef]
- Gawroński, P.; Górecka, M.; Bederska, M.; Rusaczonek, A.; Ślesak, I.; Kruk, J.; Karpiński, S. Isochorismate synthase 1 is required for thylakoid organization, optimal plastoquinone redox status, and state transitions in Arabidopsis thaliana. J. Exp. Bot. 2013, 64, 3669–3679. [Google Scholar] [CrossRef]
- Dong, H.; Tan, J.; Li, M.; Yu, Y.; Jia, S.; Zhang, C.; Wu, Y.; Liu, Y. Transcriptome analysis of soybean WRKY TFs in response to Peronospora manshurica infection. Genomics 2019, 111, 1412–1422. [Google Scholar] [CrossRef] [PubMed]
- Wang, C.; He, X.; Li, Y.; Wang, L.; Guo, X.; Guo, X. The cotton MAPK kinase GhMPK20 negatively regulates resistance to Fusarium oxysporum by mediating the MKK4-MPK20-WRKY40 cascade. Mol. Plant Pathol. 2018, 19, 1624–1638. [Google Scholar] [CrossRef] [PubMed]
- Xiong, X.; Sun, S.; Li, Y.; Zhang, X.; Sun, J.; Xue, F. The cotton WRKY transcription factor GhWRKY70 negatively regulates the defense response against Verticillium dahliae. Crop. J. 2019, 7, 393–402. [Google Scholar] [CrossRef]
- Duan, Y.; Jiang, Y.; Ye, S.; Karim, A.; Ling, Z.; He, Y.; Yang, S.; Luo, K. PtrWRKY73, a salicylic acid-inducible poplar WRKY transcription factor, is involved in disease resistance in Arabidopsis thaliana. Plant Cell Rep. 2015, 34, 831–841. [Google Scholar] [CrossRef]
- Gao, G.; Jin, R.; Liu, D.; Zhang, X.; Sun, X.; Zhu, P.; Mao, H. CmWRKY15-1 promotes resistance to Chrysanthemum white rust by regulating CmNPR1 expression. Front. Plant Sci. 2022, 13, 865607. [Google Scholar] [CrossRef]
- Cheng, P.L.; Liu, Y.N.; Yang, Y.M.; Chen, H.; Cheng, H.; Hu, Q.; Zhang, Z.X.; Gao, J.J.; Zhang, J.X.; Ding, L.; et al. CmBES1 is a regulator of boundary formation in chrysanthemum ray florets. Hortic. Res. 2020, 7, 129. [Google Scholar] [CrossRef]
- Gao, H.; Song, A.; Zhu, X.; Chen, F.; Jiang, J.; Chen, Y.; Sun, Y.; Shan, H.; Gu, C.; Li, P.; et al. The heterologous expression in Arabidopsis of a chrysanthemum Cys2/His2 zinc finger protein gene confers salinity and drought tolerance. Planta 2012, 235, 979–993. [Google Scholar] [CrossRef]
- Yoo, S.D.; Cho, Y.H.; Sheen, J. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protoc. 2007, 2, 1565–1572. [Google Scholar] [CrossRef]
- Gu, C.; Chen, S.; Liu, Z.; Shan, H.; Luo, H.; Guan, Z.; Chen, F. Reference gene selection for quantitative real-time PCR in Chrysanthemum subjected to biotic and abiotic stress. Mol. Biotechnol. 2011, 49, 192–197. [Google Scholar] [CrossRef]
- Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29, e45. [Google Scholar] [CrossRef]
- Zhu, L.; Guan, Y.; Zhang, Z.; Song, A.; Chen, S.; Jiang, J.; Chen, F. CmMYB8 encodes an R2R3 MYB transcription factor which represses lignin and flavonoid synthesis in chrysanthemum. Plant Physiol. Biochem. 2020, 149, 217–224. [Google Scholar] [CrossRef]
- Langmead, B.; Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 2012, 9, 357–359. [Google Scholar] [CrossRef]
- Kim, D.; Langmead, B.; Salzberg, S.L. HISAT: A fast spliced aligner with low memory requirements. Nat. Methods 2015, 12, 357–360. [Google Scholar] [CrossRef]
- Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef]
- Altschul, S.F.; Madden, T.L.; Schäffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389–3402. [Google Scholar] [CrossRef]
- Gene Ontology Consortium. The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res. 2004, 32, D258–D261. [Google Scholar] [CrossRef]
- Li, B.; Dewey, C.N. RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform. 2011, 12, 323. [Google Scholar] [CrossRef] [Green Version]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Miao, W.; Ge, L.; Wang, Y.; Li, S.; Sun, D.; Liu, Y.; Guan, Z.; Chen, S.; Fang, W.; Chen, F.; 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. https://doi.org/10.3390/ijms24043499
Miao W, Ge L, Wang Y, Li S, Sun D, Liu Y, Guan Z, Chen S, Fang W, Chen F, 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. International Journal of Molecular Sciences. 2023; 24(4):3499. https://doi.org/10.3390/ijms24043499
Chicago/Turabian StyleMiao, Weihao, Lijiao Ge, Yuean Wang, Song Li, Daojin Sun, Ye Liu, Zhiyong Guan, Sumei Chen, Weimin Fang, Fadi Chen, and et al. 2023. "Overexpression of CmWRKY8-1–VP64 Fusion Protein Reduces Resistance in Response to Fusarium oxysporum by Modulating the Salicylic Acid Signaling Pathway in Chrysanthemum morifolium" International Journal of Molecular Sciences 24, no. 4: 3499. https://doi.org/10.3390/ijms24043499