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Journal of Integrative Agriculture  2024, Vol. 23 Issue (9): 3055-3065    DOI: 10.1016/j.jia.2024.01.001
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Identification and characterization of FpRco1 in regulating vegetative growth and pathogenicity based on T-DNA insertion in Fusarium pseudograminearum

Haiyang Li1, 2*, Yuan Zhang1, 2*, Cancan Qin1, 2*, Zhifang Wang1, 2, Lingjun Hao1, 2, Panpan Zhang1, 2, Yongqiang Yuan1, 2, Chaopu Ding1, 2, Mengxuan Wang1, 2, Feifei Zan1, 2, Jiaxing Meng1, 2, Xunyu Zhuang1, 2, Zheran Liu1, 2, Limin Wang1, 2, Haifeng Zhou1, 2, Linlin Chen1, 2, Min Wang1, 2, Xiaoping Xing1, 2, Hongxia Yuan1, 2, Honglian Li1, 2, 3#, Shengli Ding1, 2#

1 College of Plant Protection, Henan Agricultural University, Zhengzhou 450046, China

2 National Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450046, China

3 Henan Plant Health Protection Technology Engineering Research Center, Zhenzhou 450046, China

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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 Fpseudograminearum 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 Fpseudograminearum.  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 Fpseudograminearum, we employed a split-marker approach to knock out FpRCO1 in Fpseudograminearum 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 Fpseudograminearum.  This study provides valuable insights into the molecular mechanisms underlying Fpseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.


Keywords:  Fusarium pseudograminearum       T-DNA insertion       Rpd3S complex       FpRCO1       pathogenicity       DON production  
Received: 16 July 2023   Accepted: 20 November 2023
Fund: 
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.
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.

Cite this article: 

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. 2024. Identification and characterization of FpRco1 in regulating vegetative growth and pathogenicity based on T-DNA insertion in Fusarium pseudograminearum. Journal of Integrative Agriculture, 23(9): 3055-3065.

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