Please wait a minute...
Journal of Integrative Agriculture  2024, Vol. 23 Issue (12): 4093-4106    DOI: 10.1016/j.jia.2023.09.005
Plant Protection Advanced Online Publication | Current Issue | Archive | Adv Search |
A potential hyphal fusion protein complex with an important role in development and virulence interacts with autophagy-related proteins in Fusarium pseudograminearum
Linlin Chen1, 2, Yixuan Shan1, Zaifang Dong1, Yake Zhang1, Mengya Peng1, Hongxia Yuan1, Yan Shi1, Honglian Li1, 2, Xiaoping Xing1#

1 College of Plant Protection, Henan Agricultural University, Zhengzhou 450000, China

2 National Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450000, China

Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  

菌丝融合在子囊真菌中普遍存在,调控多种发育过程。但是菌丝融合在植物病原真菌中的生物功能及分子机制尚不明了。本研究基于假禾谷镰孢T-DNA插入突变体库的筛选,鉴定到一个菌丝融合蛋白FpHam-2。利用酵母双杂交方法发现FpHam-2与另外两个菌丝融合蛋白FpHam-3和FpHam-4直接互作,且其作为STRIPAK复合体的核心元件在真核生物中非常保守。利用假禾谷镰孢遗传转化分别获得FpHam-2FpHam-3FpHam-4缺失的突变体菌株。表型分析发现,三种突变体具有相似的生物学缺陷表型,与野生型和回补菌株相比,基因缺失突变体的菌丝生长速度减慢、不能发生菌丝融合、对细胞膜、细胞壁和氧化胁迫的耐受性降低,不能产生分生孢子,且致病性降低。推测FpHam-2、FpHam-3和FpHam-4可能作为一种蛋白复合体起作用。通过酵母双杂交方法进一步验证FpHam-2与细胞自噬相关蛋白FpAtg3、FpAtg28和FpAtg33直接互作。假禾谷镰孢饥饿诱导后,野生型和回补菌株有大量自噬体被MDC染料标记,而FpHam-2缺失突变体的细胞自噬明显减弱。综合上述结果,本研究阐明了菌丝融合蛋白FpHam-2、FpHam-3和FpHam-4在假禾谷镰孢生长、产孢和致病中的重要作用,并初步揭示了其作用的潜在分子机制。



Abstract  

Hyphal fusion (anastomosis) is a common process serving many important functions at various developmental stages in the life cycle of ascomycetous fungi.  However, the biological roles and molecular mechanisms in plant pathogenic fungi were widely unknown.  In this study, a hyphal fusion protein FpHam-2 was screened from a T-DNA insertion mutant library of Fusarium pseudograminearum, and FpHam-2 interacts with another 2 hyphal fusion protein homologues FpHam-3 and FpHam-4.  Each of these 3 genes deletion mutant revealed in similar defective phenotypes compared with the WT and complemented strains, including reduction in growth rate, defects in hyphal fusion and conidiation, more sensitive for cell membrane, cell wall and oxidative stress responses, and decreased in virulence.  The yeast two-hybrid assay was used to identify that FpHam-2 interacts with 3 autophagy-related proteins, including FpAtg3, FpAtg28 and FpAtg33.  Furthermore, FpHam-2-deletion mutant showed decreased accumulation of autophagic bodies in hypha.  In conclusion, FpHam-2, FpHam-3 and FpHam-4 have an essential role for hyphal fusion and regulating the growth, conidiation and virulence in Fpseudograminearum.


Keywords:  Fusarium pseudograminearum        hyphal fusion        FpHam-2        virulence        autophagy-related proteins  
Received: 25 March 2023   Accepted: 28 June 2023
Fund: This project is supported by the grants from the National Natural Science Foundation of China (U2004140) and the Henan Provincial Science and Technology Major Project, China (221100110100).
About author:  Linlin Chen, E-mail: llchensky@163.com; #Correspondence Xiaoping Xing, Tel: +86-371-56552870, E-mail: xxp168@163.com

Cite this article: 

Linlin Chen, Yixuan Shan, Zaifang Dong, Yake Zhang, Mengya Peng, Hongxia Yuan, Yan Shi, Honglian Li, Xiaoping Xing. 2024. A potential hyphal fusion protein complex with an important role in development and virulence interacts with autophagy-related proteins in Fusarium pseudograminearum. Journal of Integrative Agriculture, 23(12): 4093-4106.

Aoki T, O’Donnell K. 1999. Morphological and molecular characterization of Fusarium pseudograminearum sp. nov., formerly recognized as the group 1 population of FgraminearumMycologia91, 597.

Bentley A R, Summerell B A, Burgess L W. 2008. Sexual compatibility in Fusarium pseudograminearum (Gibberella coronicola). Mycological Research112, 1101–1106.

Catlett N L, Lee B N, Yoder O C, Turgeon B G. 2003. Split-marker recombination for efficient targeted deletion of fungal genes. Fungal Genetics Reports50, 9–11.

Clark-Cotton M R, Jacobs K C, Lew D J. 2022. Chemotropism and cell-cell fusion in fungi. Microbiology and Molecular Biology Reviews86, e0016521.

Contento A L, Xiong Y, Bassham D C. 2005. Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein. Plant Journal42, 598–608.

Corral-Ramos C, Roca M G, Di Pietro A, Roncero M I G, Ruiz-Roldan C. 2015. Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporumAutophagy11, 131–144.

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 Pathology69, 240–248.

Dettmann A, Heilig Y, Ludwig S, Schmitt K, Illgen J, Fleißner A, Valerius O, Seiler S. 2013. HAM-2 and HAM-3 are central for the assembly of the Neurospora STRIPAK complex at the nuclear envelope and regulate nuclear accumulation of the MAP kinase MAK-1 in a MAK-2-dependent manner. Molecular Microbiology90, 796–812.

EFSAPLH (European Food Safety Authority Panel on Plant Health), 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 categorisation of Fusarium pseudograminearumEFSA Journal20, e07399.

Fleissner A, Herzog S. 2016. Signal exchange and integration during self-fusion in filamentous fungi. Seminars in Cell & Developmental Biology57, 76–83.

Fleissner A, Sarkar S, Jacobson D J, Roca M G, Read N D, Glass N L. 2005. The so locus is required for vegetative cell fusion and postfertilization events in Neurospora crassaEukaryot Cell4, 920–930.

Fleissner A, Simonin A R, Glass N L. 2008. Cell fusion in the filamentous fungus, Neurospora crassaMethods in Molecular Biology475, 21–38.

Francisco C S, Zwyssig M M, Palma-Guerrero J. 2020. The role of vegetative cell fusions in the development and asexual reproduction of the wheat fungal pathogen Zymoseptoria triticiBMC Biology18, 99.

Fu C, Iyer P, Herkal A, Abdullah J, Stout A, Free S J. 2011. Identification and characterization of genes required for cell-to-cell fusion in Neurospora crassaEukaryot Cell10, 1100–1109.

Furukawa K, Fukuda T, Yamashita S, Saigusa T, Kurihara Y, Yoshida Y, Kirisako H, Nakatogawa H, Kanki T. 2018. The PP2A-like protein phosphatase Ppg1 and the Far complex cooperatively counteract CK2-mediated phosphorylation of Atg32 to inhibit mitophagy. Cell Reports23, 3579–3590.

Furukawa K, Innokentev A, Kanki T. 2021. Mitophagy regulation mediated by the Far complex in yeast. Autophagy17, 1042–1043.

Gardiner D M, Benfield A H, Stiller J, Stephen S, Aitken K, Liu C J, Kazan K. 2018. A high-resolution genetic map of the cereal crown rot pathogen Fusarium pseudograminearum provides a near-complete genome assembly. Molecular Plant Pathology19, 217–226.

Glass N L, Jacobson D J, Shiu P K. 2000. The genetics of hyphal fusion and vegetative incompatibility in filamentous ascomycete fungi. Annual Review of Genetics34, 165–186.

Goudreault M, D’Ambrosio L M, Kean M J, Mullin M J, Larsen B G, Sanchez A, Chaudhry S, Chen G I, Sicheri F, Nesvizhskii A I, Aebersold R, Raught B, Gingras A C. 2009. A PP2A phosphatase high density interaction network identifies a novel Striatin-interacting phosphatase and kinase complex linked to the Cerebral Cavernous Malformation 3 (CCM3) Protein. Molecular & Cellular Proteomics8, 157–171.

Guo L, Wenner N, Kuldau G A. 2015. FvSO regulates vegetative hyphal fusion, asexual growth, fumonisin B1 production, and virulence in Fusarium verticillioidesFungal Biology119, 1158–1169.

Hassan Y I, Bullerman L B. 2009. Wheat bran as an alternative substrate for macroconidia formation by some Fusarium species. Journal of Microbiological Methods77, 134–136.

Hu Z H, Sankar D S, Vu B, Leytens A, Vionnet C, Wu W, Stumpe M, Martinez-Martinez E, Stork B, Dengjel J. 2021. ULK1 phosphorylation of striatin activates protein phosphatase 2A and autophagy. Cell Reports36, 109762.

Innokentev A, Furukawa K, Fukuda T, Saigusa T, Inoue K, Yamashita S I, Kanki T. 2020. Association and dissociation between the mitochondrial Far complex and Atg32 regulate mitophagy. eLife9, e63694.

Ishikawa F H, Souza E A, Read N D, Roca M G. 2010. Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianumFungal Biology114, 2–9.

Kazan K, Gardiner D M. 2018. Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: Recent progress and future prospects. Molecular Plant Pathology19, 1547–1562.

Kemp H A, Sprague G F. 2003. Far3 and five interacting proteins prevent premature recovery from pheromone arrest in the budding yeast Saccharomyces cerevisiaeMolecular and Cellular Biology23, 1750–1763.

Kuck U, Beier A M, Teichert I. 2016. The composition and function of the striatin-interacting phosphatases and kinases (STRIPAK) complex in fungi. Fungal Genetics and Biology90, 31–38.

Kuck U, Radchenko D, Teichert I. 2019. STRIPAK, a highly conserved signaling complex, controls multiple eukaryotic cellular and developmental processes and is linked with human diseases. Biological Chemistry400, 1005–1022.

Kuck U, Stein V. 2021. STRIPAK, a key regulator of fungal development, operates as a multifunctional signaling Hub. Journal of Fungi7, 443.

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 Disease96, 1065.

Neisch A, Neufeld T, Hays T. 2016. A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation. Molecular Biology of the Cell27, 441–461.

Nordzieke D E, Fernandes T R, El Ghalid M, Turra D, Di Pietro A. 2019. NADPH oxidase regulates chemotropic growth of the fungal pathogen Fusarium oxysporum towards the host plant. New Phytologist224, 1600–1612.

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 Pathology62, 79–91.

Pinan-Lucarre B, Clave C. 2008. Monitoring autophagy in the filamentous fungus Podospora anserinaMethods in Enzymology451, 251–270.

Pracheil T, Liu Z. 2013. Tiered assembly of the yeast Far3-7-8-9-10-11 complex at the endoplasmic reticulum. Journal of Biological Chemistry288, 16986–16997.

Read N D, Lichius A, Shoji J Y, Goryachev A B. 2009. Self-signalling and self-fusion in filamentous fungi. Current Opinion in Microbiology12, 608–615.

Reschka E J, Nordzieke S, Valerius O, Braus G H, Poggeler S. 2018. A novel STRIPAK complex component mediates hyphal fusion and fruiting-body development in filamentous fungi. Molecular Microbiology110, 513–532.

Sankar D S, Hu Z H, Dengjel J. 2022. The complex interplay between ULK1 and protein phosphatases in autophagy regulation. Autophagy18, 455–456.

Schiestl R H, Gietz R D. 1989. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Current Genetics16, 339–346.

Seo G Y, Han H, Vargas R E, Yang B, Li X, Wang W Q. 2020. MAP4K interactome reveals STRN4 as a key STRIPAK complex component in Hippo pathway regulation. Cell Reports32, 107860.

Silar P. 2005. Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant. Mycological Research109, 137–149.

Simonin A R, Rasmussen C G, Yang M, Glass N L. 2010. Genes encoding a striatin-like protein (ham-3) and a forkhead associated protein (ham-4) are required for hyphal fusion in Neurospora crassaFungal Genetics and Biology47, 855–868.

Tunali B, Obanor F, Erginbas G, Westecott R A, Nicol J, Chakraborty S. 2012. Fitness of three Fusarium pathogens of wheat. FEMS Microbiology Ecology81, 596–609.

Vangalis V, Knop M, Typas M A, Papaioannou I A. 2021a. Establishment of conidial fusion in the asexual fungus Verticillium dahliae as a useful system for the study of non-sexual genetic interactions. Current Genetics67, 471–485.

Vangalis V, Likhotkin I, Knop M, Typas M A, Papaioannou I A. 2021b. Starvation-induced cell fusion and heterokaryosis frequently escape imperfect allorecognition systems in an asexual fungal pathogen. BMC Biology19, 169.

Vangalis V, Papaioannou I A, Markakis E A, Knop M, Typas M A. 2021c. The NADPH oxidase A of Verticillium dahliae is essential for pathogenicity, normal development, and stress tolerance, and it interacts with Yap1 to regulate redox homeostasis. Journal of Fungi (Basel), 7, 740.

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 Fusarium pseudograminearum on pathogenesis in wheat. Journal of Integrative Agriculture16, 2504–2512.

Wang Y J, Liu X, Xu Y J, Gu Y Y, Zhang X Y, Zhang M X, Wen W, Lee Y W, Shi J R, Mohamed S R, Goda A A, Wu H J, Gao X W, Gu Q. 2022. The autophagy-related proteins FvAtg4 and FvAtg8 are involved in virulence and fumonisin biosynthesis in Fusarium verticillioidesVirulence13, 764–780.

Xiang Q, Rasmussen C, Glass N L. 2002. The ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in Neurospora crassaGenetics160, 169–180.

Xing X P, Zhang P P, Ding S L, Yuan H X, Chen L L, Li H L. 2017. Optimizing of agrobacterium tumefaciens-mediated genetic transformation sytem in Fusarium pseudograminearumJournal of Agricultural Biotechnology25, 1887–1894. (in Chinese)

Xu F, Song Y L, Yang G Q, Wang J M, Liu L L, Li Y H. 2015. First report of Fusarium pseudograminearum from wheat heads with Fusarium head blight in north China plain. Plant Disease99, 156.

Zhang J, Xia M C, Xue B G, Goodwin P H, Sun R H, Quan X, Lu W G, Yang L R. 2018. First report of Fusarium pseudograminearum causing root rot on soybean (Glycine max) in Henan, China. Plant Disease102, 1454.

Zheng H W, Miao P F, Lin X L, Li L P, Wu C X, Chen X M, Abubakar Y, Norvienyeku J, Li G, Zhou J, Wang Z H, Zheng W H. 2018. Small GTPase Rab7-mediated FgAtg9 trafficking is essential for autophagy- dependent development and pathogenicity in Fusarium graminearumPLoS Genetics14, e1007546.

Zhou H F, He X L, Wang S, Ma Q Z, Sun B J, Ding S L, Chen L L, Zhang M, Li H L. 2019. Diversity of the Fusarium pathogens associated with crown rot in the Huanghuai wheat-growing region of China. Environmental Microbiology21, 2740–2754.

Zhu X M, Li L, Cai Y Y, Wu X Y, Shi H B, Liang S, Qu Y M, Naqvi N I, Del Poeta M, Dong B, Lin F C, Liu X H. 2021. A VASt-domain protein regulates autophagy, membrane tension, and sterol homeostasis in rice blast fungus. Autophagy17, 2939–2961.

[1] CAO Peng, CHEN Jia-lan, LI Ning-ning, ZHANG Shuang-xi, WANG Rong-bo, LI Ben-jin, LIU Pei-qing, AN Yu-yan, ZHANG Mei-xiang. Seedling Petri-dish inoculation method: A robust, easy-to-use and reliable assay for studying plant–Ralstonia solanacearum interactions[J]. >Journal of Integrative Agriculture, 2023, 22(12): 3709-3719.
No Suggested Reading articles found!