基于T-DNA插入鉴定和解析假禾谷镰孢FpRCO1基因在调控病原菌生长及致病过程中的作用

doi:10.1016/j.jia.2024.01.001

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (9): 3055-3065.DOI: 10.1016/j.jia.2024.01.001

基于T-DNA插入鉴定和解析假禾谷镰孢FpRCO1基因在调控病原菌生长及致病过程中的作用

收稿日期:2023-07-16 接受日期:2023-11-20 出版日期:2024-09-20 发布日期:2024-08-21

Identification and characterization of FpRco1 in regulating vegetative growth and pathogenicity based on T-DNA insertion in Fusarium pseudograminearum

Haiyang Li^{1, 2*}, Yuan Zhang^{1, 2*}, Cancan Qin^{1, 2*}, Zhifang Wang^{1, 2}, Lingjun Hao^{1, 2}, Panpan Zhang^{1, 2}, Yongqiang Yuan^{1, 2}, Chaopu Ding^{1, 2}, Mengxuan Wang^{1, 2}, Feifei Zan^{1, 2}, Jiaxing Meng^{1, 2}, Xunyu Zhuang^{1, 2}, Zheran Liu^{1, 2}, Limin Wang^{1, 2}, Haifeng Zhou^{1, 2}, Linlin Chen^{1, 2}, Min Wang^{1, 2}, Xiaoping Xing^{1, 2}, Hongxia Yuan^{1, 2}, Honglian Li^{1, 2,} ^3#, Shengli Ding^{1, 2#}

¹College of Plant Protection, Henan Agricultural University, Zhengzhou 450046, China
²National Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450046, China
³Henan Plant Health Protection Technology Engineering Research Center, Zhenzhou 450046, China

Received:2023-07-16 Accepted:2023-11-20 Online:2024-09-20 Published:2024-08-21
About author:Haiyang Li, E-mail: haiyang-li@henau.edu.cn; Yuan Zhang, E-mail: 17837184209@163.com; Cancan Qin, E-mail: a18839327647@163.com; #Correspondence Honglian Li, E-mail: honglianli@sina.com; Shengli Ding, Tel: +86-371-56552870, E-mail: shengliding@henau.edu.cn * These authors contributed equally to this study.
Supported by:
This research was supported by grants from the National Natural Science Foundation of China (31901835), the Science and Technology Planning Project of Henan Province of China (212102110145), and the International (Regional) Cooperation and Exchange Program of the National Natural Science Foundation of China (31961143018). At the same time, we thank Professor Youliang Peng, China Agricultural University for providing the vector PYIP102.

摘要/Abstract

摘要：

假禾谷镰孢（Fusarium pseudograminearum）侵染小麦茎基部及根部引起严重的茎基腐病，同时还侵染小麦穗部造成赤霉病，在茎基部和穗部产生毒素，该病害对小麦的产量与质量造成严重的危害，目前已成为小麦生产中的主要病害。尽管如此，对假禾谷镰孢致病分子机制的研究还十分匮乏。为了快速鉴定参与调控假禾谷镰孢致病的基因，本研究利用T-DNA插入正向遗传学的方法建立了假禾谷镰孢的突变体库；通过病原菌接种大麦叶片筛选致病力下降的突变体，进而通过PCR扩增技术分析致病力下降突变体中被破坏的基因。通过筛选，本研究共获到9个致病力下降的假禾谷镰孢突变体，其中一个突变体中与酵母RCO1基因同源的FpRCO1基因被破坏。FpRco1是假禾谷镰孢Rpd3S组蛋白去乙酰化酶复合体的一个重要组分，为了证明编码该蛋白的FpRCO1基因的缺失与假禾谷镰孢的致病力相关，本研究采用split-marker的方法，在野生型WZ-8A菌株中，对该基因进行了敲除，获得了敲除突变体DFprco1。敲除突变体DFprco1生长减慢、产孢减少和致病力下降；回补菌株表型完全恢复到野生型的水平。在不同的外源压力测试实验中，与野生型相比，突变体DFprco1显示对NaCl、sorbitol和SDS更加的敏感，对H₂O₂不敏感的性状。为了进一步分析突变体DFprco1致病力下降的原因，本研究对假禾谷镰孢野生型菌株及突变体DFprco1在侵染阶段的基因表达水平进行了转录组分析，分析结果显示，突变体DFprco1大量基因的表达水平发生了变化，特别是参与调控DON毒素（Dyoxynivanelol, DON，脱氧镰刀雪腐烯醇）合成的TRI基因(trichothecene biosynthetic (TRI) gene, 单端孢霉烯簇生物合成) 表达水平出现显著下调。通过对假禾谷镰孢的野生型菌株及突变体DFprco1侵染的小麦粒中的DON毒素进行检测发现，突变体DFprco1侵染的小麦粒中DON含量显著减少。总之，本研究揭示了FpRCO1基因在调控假禾谷镰孢的生长发育、无性繁殖、DON的合成及致病方面扮演着重要的角色；同时本研究为深入解析假禾谷镰孢致病的分子机制提供了新的方法，为防控该重大病害提供新思路。

Abstract:

Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot (FCR) in wheat and poses a significant threat to wheat production in terms of grain yield and quality. However, the mechanism by which F. pseudograminearum infects wheat remains unclear. In this study, we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F. pseudograminearum. By screening this mutant library, we identified nine independent mutants that displayed impaired pathogenesis in barley leaves. Among these mutants, one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1, encoding essential component of the Rpd3S histone deacetylase complex in F. pseudograminearum. To further investigate the role of FpRCO1 in F. pseudograminearum, we employed a split-marker approach to knock out FpRCO1 in F. pseudograminearum WZ-8A. FpRCO1 deletion mutants exhibit reduced vegetative growth, conidium production, and virulence in wheat coleoptiles and barley leaves, whereas the complementary strain restores these phenotypes. Moreover, under stress conditions, the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl, sorbitol, and SDS, but possessed reduced sensitivity to H₂O₂ compared to these characteristics in the wild-type strain. RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression (particularly the downregulation of TRI gene expression), thus resulting in significantly reduced deoxynivalenol (DON) production. In summary, our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development, asexual reproduction, DON production, and pathogenicity of F. pseudograminearum. This study provides valuable insights into the molecular mechanisms underlying F. pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.

Key words: Fusarium pseudograminearum , T-DNA insertion , Rpd3S complex , FpRCO1 , pathogenicity , DON production

Haiyang Li, Yuan Zhang, Cancan Qin, Zhifang Wang, Lingjun Hao, Panpan Zhang, Yongqiang Yuan, Chaopu Ding, Mengxuan Wang, Feifei Zan, Jiaxing Meng, Xunyu Zhuang, Zheran Liu, Limin Wang, Haifeng Zhou, Linlin Chen, Min Wang, Xiaoping Xing, Hongxia Yuan, Honglian Li, Shengli Ding. 基于T-DNA插入鉴定和解析假禾谷镰孢FpRCO1基因在调控病原菌生长及致病过程中的作用[J]. Journal of Integrative Agriculture, 2024, 23(9): 3055-3065.

Haiyang Li, Yuan Zhang, Cancan Qin, Zhifang Wang, Lingjun Hao, Panpan Zhang, Yongqiang Yuan, Chaopu Ding, Mengxuan Wang, Feifei Zan, Jiaxing Meng, Xunyu Zhuang, Zheran Liu, Limin Wang, Haifeng Zhou, Linlin Chen, Min Wang, Xiaoping Xing, Hongxia Yuan, Honglian Li, Shengli Ding. Identification and characterization of FpRco1 in regulating vegetative growth and pathogenicity based on T-DNA insertion in Fusarium pseudograminearum[J]. Journal of Integrative Agriculture, 2024, 23(9): 3055-3065.

参考文献

Akinsanmi O A, Backhouse D, Simpfendorfer S, Chakraborty S. 2006. Genetic diversity of Australian Fusarium graminearum and F. pseudograminearum. Plant Pathology, 55, 494–504.

Antontseva E V, Bondar N P. 2021. Chromatin remodeling in oligodendrogenesis. Vavilovskii Zhurnal Genet Selektsii, 25, 573–579.

Bragard C, Baptista P, Chatzivassiliou E, Di Serio F, Gonthier P, Jaques Miret J A, Justesen A F, MacLeod A, Magnusson C S, Milonas P, Navas-Cortes J A, Parnell S, Potting R, Stefani E, Thulke H H, Van der Werf W, Civera A V, Yuen J, Zappalà L, Migheli Q, et al. 2022. Pest cate gorisation of Fusarium pseudograminearum. EFSA Journal, 20, e07399.

Busby T M, Miller K Y, Miller B L. 1996. Suppression and enhancement of the Aspergillus nidulans medusa mutation by altered dosage of the bristle and stunted genes. Genetics, 143, 155–163.

Calderini D F, Castillo F M, Arenas M A, Molero G, Reynolds M P, Craze M, Bowden S, Milner M J, Wallington E J, Dowle A, Gomez L D, McQueen-Mason S J. 2021. Overcoming the trade-off between grain weight and number in wheat by the ectopic expression of expansin in developing seeds leads to increased yield potential. New Phytologist, 230, 629–640.

Chen L L, Ma Y M, Zhao J Y, Geng X J, Chen W B, Ding S L, Li H Y, Li H L. 2020. The bZIP transcription factor FpAda1 is essential for fungal growth and conidiation in Fusarium pseudograminearum. Current Genetics, 66, 507–515.

Chen X F, Kuryan B, Kitada T, Tran N, Li J Y, Kurdistani S, Grunstein M, Li B, Carey M. 2012. The Rpd3 core complex is a chromatin stabilization module. Current Biology, 22, 56–63.

Deng Y Y, Li W, Zhang P, Sun H Y, Zhang X X, Zhang A X, Chen H G. 2020. Fusarium pseudograminearum as an emerging pathogen of crown rot of wheat in eastern China. Plant Pathology, 69, 240–248

Gardiner D M, Rusu A, Benfield H A, Kazan K. 2021. Map-based cloning identifies velvet a as a critical component of virulence in Fusarium pseudograminearum during infection of wheat heads. Fungal Biology, 3, 191–200.

Hooft J M, Bureau D P. 2021. Deoxynivalenol: Mechanisms of action and its effects on various terrestrial and aquatic species. Food and Chemical Toxicology, 157, 112616.

Ji L J, Li Q S, Wang Y J, Burgess L W, Sun M W, Cao K Q, Kong L X. 2019. Monitorin g of Fusarium species and trichothecene genotypes associated with Fusarium Head Blight on wheat in Hebei Province, China. Toxins (Basel), 11, 243.

Jiang H, Xia A L, Ye M, Ren J Y, Li D A, Liu H Q, Wang Q H, Lu P, Wu C L, Xu J R, Jiang C. 2020. Opposing functions of Fng1 and the Rpd3 HDAC complex in H4 acetylation in Fusarium graminearum. PLoS Genetics, 16, e1009185.

Jin J J, Duan S N, Qi Y Z, Zhen W C, Ma J. 2021. Identification of proteins associated with Fusarium crown rot resistance in wheat using label-free quantification analysis. Journal of Integrative Agriculture, 20, 3209–3221.

Kang R J, Li G N, Zhang M J, Zhang P P, Wang L M, Zhang Y S, Chen L L, Yuan H X, Ding S L, Li H H. 2020. Expression of Fusarium pseudograminearum FpNPS9 in wheat plant and its function in pathogenicity. Current Genetics, 66, 229–243.

Kazan K, Gardiner D M. 2018. Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: Recent progress and future prospects. Molecular Plant Pathology, 19, 1547–1562.

Kettle A J, Batley J, Benfield A H, Manners J M, Kazan K, Gardiner D M. 2015a. Degradation of the benzoxazolinone class of phytoalexins is important for virulence of Fusarium pseudograminearum towards wheat. Molecular Plant Pathology, 9, 946–962.

Kettle A J, Carere J, Batley J, Benfield A H, Manners J M, Kazan K, Gardiner D M. 2015b. A γ-lactamase from cereal infecting Fusarium spp. catalyses the first step in the degradation of the benzoxazolinone class of phytoalexins. Fungal Genetics and Biology, 83, 1–9.

Kettle A J, Carere J, Batley J, Manners J M, Kazan K, Gardiner D M. 2016. The Fdb3 t ranscription factor of the Fusarium Detoxification of Benzoxazolinone gene cluster is required for MBOA but not BOA degradation in Fusarium pseudograminearum. Fungal Genetics and Biology, 88, 44–53.

Lee M K, Kwon N J, Lee I S, Jung S, Kim S C, Yu J H. 2016. Negative regulation and developmental competence in Aspergillus. Scientific Reports, 6, 28874.

Li H L, Yuan H X, Fu B, Xing X P, Sun B J, Tang W H. 2012. First report of Fusarium pseudograminearum causing Crown Rot of wheat in Henan, China. Plant Disease, 96, 1065.

Li K, Liu D M, Pan X, Yan S W, Song J Q, Liu D W, Wang Z F, Xie Y, Dai J L, Liu J H, Li H L, Zhang X T, Gao F. 2022. Deoxynivalenol biosynthesis in Fusarium pseudograminearum significantly repressed by a megabirnavirus. Toxins (Basel), 14, 503.

Lv B, Zheng L, Liu H, Tang J T, Hsiang T, Huang J B. 2016. Use of random T-DNA mutagenesis in identification of gene UvPRO1, a regulator of conidiation, stress response, and virulence in Ustilaginoidea virens. Frontiers in Microbiology, 7, 2086.

Ma H J, Li L, Gai Y P, Zhang X Y, Chen Y N, Zhuo X K, Cao Y Z, Jiao C, Gmitter Jr F G, Li H Y. 2021. Histone acetyltransferases and deacetylases are required for virulence, conidiation, DNA damage repair, and multiple stresses resistance of Alternaria alternata. Frontiers in Microbiology, 12, 783633.

Meng S, Liu Z Q, Shi H B, Wu Z L, Qiu J H, Wen H, Lin F C, Tao Z, Luo C X, Kou Y J. 2021. UvKmt6-mediated H3K27 trimethylation is required for development, pathogenicity, and stress response in Ustilaginoidea virens. Virulence, 12, 2972–2988.

Meng Y, Patel G, Heist M, Betts M F, Tucker S L, Galadima N, Donofrio N M, Brown D, Mitchell T K, Li L, Xu J R, Orbach M, Thon M, Dean R A, Farman M L. 2007. A systematic analysis of T-DNA insertion events in Magnaporthe oryzae. Fungal Genetics and Biology, 44, 1050–1064.

Monds R D, Cromey M G, Lauren D R, di Menna M, Marshall J. 2005. Fusarium graminearum, F. cortaderiae and F. pseudograminearum in New Zealand: Molecular phylogenetic analysis, mycotoxin chemotypes and co-existence of species. Mycological Research, 109, 410–420.

Obanor F, Chakraborty S. 2014. Aetiology and toxigenicity of Fusarium graminearum and F. pseudograminearum causing crown rot and head blight in Australia under natural and artificial infection. Plant Pathology, 63, 1218–1229.

Obanor F, Neate S, Simpfendorfer S, Sabburg R, Wilson P, Chakraborty S. 2013. Fusarium graminearum and Fusarium pseudograminearum caused the 2010 head blight epidemics in Australia. Plant Pathology, 62, 79–91.

Powell J J, Carere J, Fitzgerald T L, Stiller J, Covarelli L, Xu Q, Gubler F, Colgrave M L, Gardiner D M, Manners J M, Henry R J, Kazan K. 2017. The Fusari um crown rot pathogen Fusarium pseudograminearum triggers a suite of transcriptional and metabolic changes in bread wheat (Triticum aestivum L.). Annals Botany, 119, 853–867.

Ruan C, Cui H C, Lee C H, Li S, Li B. 2016. Homodimeric PHD domain-containing Rco1 subunit constitutes a critical interaction Hub within the Rpd3S histone deacetylase complex. Journal of Biological Chemistry, 291, 5428–5438.

Tunali B, Obanor F, Erginbas G, Westecott R A, Nicol J, Chakraborty S. 2012. Fitness of three Fusarium pathogens of wheat. FEMS Microbiology Ecology, 81, 596–609.

Wang L M, Xie S P, Zhang Y S, Kang R J, Zhang M J, Wang M, Li H Y, Chen L L, Yuan H X, Ding S L, Liang S, Li H L. 2020. The FpPPR1 gene encodes a pentatricopeptide repeat protein that is essential for asexual development, sporulation, and pathogenesis in Fusarium pseudograminearum. Frontiers in Genetics, 11, 535622.

Wang L M, Zhang Y F, Du Z L, Kang R J, Chen L L, Xing X P, Yuan H X, Ding S L, Li H L. 2017. FpPDE1 function of Fsarium pseudograminearum on pathogenesis in wheat. Journal of Integrative Agriculture, 16, 2504–2512.

Wang Q N, Huang P P, Zhou S Y. 2020. Functional characterization of the catalytic and bromodomain of FgGCN5 in development, DON production and virulence of Fusarium graminearum. Journal of Integrative Agriculture, 19, 2477–2487.

Wang S, Wu X M, Liu C H, Shang J Y, Gao F, Guo H S. 2020. Verticillium dahliae chromatin remodeling facilitates the DNA damage repair in response to plant ROS stress. PLoS Pathogens, 16, e1008481.

Wollenberg R D, Sondergaard T E, Nielsen M R, Knutsson S, Pedersen T B, Westphal K R, Wimmer R, Gardiner D M, Sorensen J L. 2019. There it is! Fusarium pseudograminearum did not lose the fusaristatin gene cluster after all. Fungal Biology, 123, 10–17.

Wu Y X, Wang Y J, Han S, Li Q S, Kong L X. 2023. The global regulator FpLaeB is required for the regulation of growth, development, and virulence in Fusarium pseudograminearum. Frontiers in Plant Science, 14, 1132507.

Xia H Q, Chen L L, Fan Z, Peng M Y, Zhao J Y, Chen W B, Li H Y, Shi Y, Ding S L, Li H L. 2021. Heat stress tolerance gene FpHsp104 affects conidiation and pathogenicity of Fusarium pseudograminearum. Frontiers in Microbiology, 12, 695535.

Xing X P, Zhang P P, Ding S L, Yuan H X, Chen L L, Li H L. 2017. Optimize the Agrobacterium tumefaciens-mediated transformation technology system of F. pseudograminearum. Journal of Agricultural Biotechnology, 25, 8.

Yang X J, Seto E. 2008. The Rpd3/Hda1 family of lysine deacetylases: From bacteria and yeast to mice and men. Nature Reviews Molecular Cell Biology, 9, 206–218.

Yeheskely-Hayon D, Kotler A, Stark M, Hashimshony T, Sagee S, Kassir Y. 2013. The roles of the catalytic and noncatalytic activities of Rpd3L and Rpd3S in the regulation of gene transcription in yeast. PLoS ONE, 8, e85088.

Zhang N, Yang Z Z, Zhang Z H, Liang W X. 2020. BcRPD3-mediated histone deacetylation is involved in growth and pathogenicity of Botrytis cinerea. Frontiers in Microbiology, 11, 1832.

Zhang Y S, Wang L M , Liang S, Zhang P P, Kang R J, Zhang M J, Wang M, Chen L L, Yuan H X, Ding S L, Li H L. 2020. FpDep1, a component of Rpd3L histone deacetylase complex, is important for vegetative development, ROS accumulation, and pathogenesis in Fusarium pseudograminearum. Fungal Genetics and Biology, 135, 103299.

Zhao J Y, Peng M Y, Chen W B, Xing X P, Shan Y X, Fan Z, Shi Y, Li H Y, Yang X, Li H L, Chen L L. 2022. Transcriptome analysis and functional validation identify a putative bZIP transcription factor, Fpkapc, that regulates development, stress responses, and virulence in Fusarium pseudograminearum. Phytopathology, 112, 1299–1309.

基于T-DNA插入鉴定和解析假禾谷镰孢FpRCO1基因在调控病原菌生长及致病过程中的作用

Identification and characterization of FpRco1 in regulating vegetative growth and pathogenicity based on T-DNA insertion in Fusarium pseudograminearum

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics