肌动蛋白结合蛋白FgAbp1参与禾谷镰孢生长、发育和毒素体形成

doi:10.3864/j.issn.0578-1752.2021.13.006

摘要/Abstract

摘要：

【目的】Abp1作为肌动蛋白结合蛋白家族成员之一,在各种真核生物肌动蛋白骨架形成过程中具有核心作用。本研究旨在分析禾谷镰孢（Fusarium graminearum）中肌动蛋白结合蛋白FgAbp1在生长发育、对新型杀菌剂氰烯菌酯敏感性以及毒素体形成等生物学过程中的功能。【方法】利用融合PCR和酵母空隙修复技术分别构建FgAbp1基因敲除打靶片段和融合荧光蛋白载体,再通过聚乙二醇介导的原生质体转化的方法获取基因缺失突变体ΔFgAbp1和荧光标记菌株。观察比较野生型PH-1、突变体ΔFgAbp1和回补体ΔFgAbp1-C的菌丝生长、有性生殖以及无性繁殖,并测定基因敲除突变体ΔFgAbp1对杀菌剂氰烯菌酯的敏感性。通过融合绿色荧光蛋白,明确FgAbp1在菌丝中的分布情况;利用透射电子显微镜观察分析FgAbp1对细胞中液泡/囊泡形态的影响。在非产毒环境和诱导产毒条件下,通过双荧光共定位分析FgAbp1在禾谷镰孢毒素体形成过程中的作用。【结果】禾谷镰孢中,FgAbp1主要呈颗粒状定位于近细胞膜处。在MM培养基中,基因敲除突变体ΔFgAbp1的生长速率与野生型相比降低了15%,但在营养丰富的CM中ΔFgAbp1的生长速率却减慢了38%。突变体ΔFgAbp1在无性繁殖以及有性生殖过程中相较野生型没有明显缺陷。然而在0.5 μg·mL^-1氰烯菌酯的作用下,ΔFgAbp1的菌丝生长完全受到抑制,并且分生孢子萌发率显著下降。此外,敲除基因FgAbp1导致细胞中液泡不能正常形成且囊泡增多。在非产毒条件下,FgAbp1与DON毒素合成关键酶Tri1共定位于囊泡中;在诱导产毒条件下,FgAbp1与Tri1则共定位于毒素体中。此外,敲除基因FgAbp1会导致毒素体不能正常形成。【结论】肌动蛋白结合蛋白FgAbp1在禾谷镰孢营养生长、发育、氰烯菌酯敏感性以及毒素体形成过程中发挥着重要作用。

关键词: 禾谷镰孢, FgAbp1, 氰烯菌酯, 毒素体

Abstract:

【Objective】Abp1 is one of the actin binding proteins that plays a central role in actin cytoskeleton of diverse eukaryotic organisms. The objective of this study is to analyze functions of the actin binding protein FgAbp1 in growth and development, sensitivity to the novel fungicide phenamacril and toxisome formation inFusarium graminearum.【Method】Targeted gene deletion construct and fluorescent protein fusion vectors were generated by double-joint PCR and budding yeast gap repair system, respectively. Then, the mutant ΔFgAbp1 and fluorescently labeled strains were obtained using polyethylene glycol (PEG) mediated protoplast transformation. Mycelia growth, sexual/asexual reproduction and sensitivity to phenamacril of wild type PH-1, the mutant ΔFgAbp1 and complemented strain ΔFgAbp1-C were investigated. Localization of FgAbp1 in hyphae was examined through fusion green fluorescent protein. Transmission electron microscopy was carried out to assay the role of FgAbp1 in vacuole/vesicle morphology. Under noninducing medium and DON biosynthesis induction conditions, the role of FgAbp1 in the toxisome formation of F. graminearum was performed by dual fluorescence colocalization assay.【Result】FgAbp1 is primarily localized near the cell membrane in patches of F. graminearum. In MM medium, the growth rate of gene knockout mutant ΔFgAbp1 was reduced by 15% compared with the wild type. But in the nutrient-rich CM, the growth rate of ΔFgAbp1 was decreased by 38%. The mutant ΔFgAbp1 had no obvious defects in sexual and asexual reproduction in comparison with the wild type, while the mycelial growth of ΔFgAbp1 was completely inhibited and the conidia showed significant reduction of germination rate with 0.5 μg·mL ^-1 phenamacril treatment. Moreover,FgAbp1 deletion resulted in a high vesicle number and a block of normal vacuole formation. During growth in a toxin noninducing condition, FgAbp1 and the DON biosynthetic key enzyme Tri1 co-fluoresced in vesicles. Unexpectedly, FgAbp1 and Tri1 cellular co-localized in toxisomes under DON biosynthesis inducing conditions. Furthermore, disruption of FgAbp1resulted in abnormal toxisomes.【Conclusion】The actin binding protein FgAbp1 plays an important role in vegetative growth, development, phenamacril tolerance and toxisome formation inF. graminearum.

Key words: Fusarium graminearum, FgAbp1, phenamacril, toxisome

张承启,王晓妍,陈莉. 肌动蛋白结合蛋白FgAbp1参与禾谷镰孢生长、发育和毒素体形成[J]. 中国农业科学, 2021, 54(13): 2759-2768.

ZHANG ChengQi,WANG XiaoYan,CHEN Li. The Actin Binding Protein FgAbp1 is Involved in Growth, Development and Toxisome Formation in Fusarium graminearum[J]. Scientia Agricultura Sinica, 2021, 54(13): 2759-2768.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.13.006

https://www.chinaagrisci.com/CN/Y2021/V54/I13/2759

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参考文献 44

[1]	DEAN R, VAN KAN J A L, PRETORIUS Z A, HAMMOND-KOSACK K E, DI PIETRO A, SPANU P D, RUDD J J, DICKMAN M, KAHMANN R, ELLIS J, FOSTER G D. The top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology, 2012, 13(4):414-430. doi: 10.1111/j.1364-3703.2011.00783.x
[2]	AUDENAERT K, VANHEULE A, HOFTE M, HAESAERT G. Deoxynivalenol: A major player in the multifaceted response of Fusarium to its environment. Toxins, 2013, 6(1):1-19. doi: 10.3390/toxins6010001
[3]	BIANCHINI A, HORSLEY R, JACK M M, KOBIELUSH B, RYU D, TITTLEMIER S, WILSON W W, ABBAS H K, ABEL S, HARRISON G, MILLER J D, SHIER W T, WEAVER G. DON occurrence in grains: A north American perspective. Cereal Foods World, 2015, 60(1):32-56. doi: 10.1094/CFW-60-1-0032
[4]	PESTKA J J. Deoxynivalenol: Mechanisms of action, human exposure, and toxicological relevance. Archives of Toxicology, 2010, 84(9):663-679. doi: 10.1007/s00204-010-0579-8
[5]	陈云, 王建强, 杨荣明, 马忠华. 小麦赤霉病发生危害形势及防控对策. 植物保护, 2017, 43(5):11-17.
	CHEN Y, WANG J Q, YANG R M, MA Z H. Current situation and management strategies of Fusarium head blight in China. Plant Protection, 2017, 43(5):11-17. (in Chinese)
[6]	史建荣, 刘馨, 仇剑波, 祭芳, 徐剑宏, 董飞, 殷宪超, 冉军舰. 小麦中镰刀菌毒素脱氧雪腐镰刀菌烯醇污染现状与防控研究进展. 中国农业科学, 2014, 47(18):3641-3654.
	SHI J R, LIU X, QIU J B, JI F, XU J H, DONG F, YIN X C, RAN J J. Deoxynivalenol contamination in wheat and its management. Scientia Agricultura Sinica, 2014, 47(18):3641-3654. (in Chinese)
[7]	TANG G F, CHEN Y, XU J R, KISTLER H C, MA Z H. The fungal myosin I is essential for Fusarium toxisome formation. PLoS Pathogens, 2018, 14(1):e1006827. doi: 10.1371/journal.ppat.1006827
[8]	ZHANG C Q, CHEN Y, YIN Y N, JI H H, SHIM W B, HOU Y P, ZHOU M G, LI X D, MA Z H. A small molecule species specifically inhibits Fusarium myosin I. Environmental Microbiology, 2015, 17(8):2735-2746. doi: 10.1111/1462-2920.12711
[9]	BEREPIKI A, LICHIUS A, READ N D. Actin organization and dynamics in filamentous fungi. Nature Reviews. Microbiology, 2011, 9(12):876-887.
[10]	GARCIA B, STOLLAR E J, DAVIDSON A R. The importance of conserved features of yeast actin-binding protein 1 (Abp1p): The conditional nature of essentiality. Genetics, 2012, 191(4):1199-1211. doi: 10.1534/genetics.112.141739
[11]	DRUBIN D G, MILLER K G, BOTSTEIN D. Yeast actin-binding proteins: Evidence for a role in morphogenesis. Journal of Cell Biology, 1988, 107(6):2551-2561.
[12]	LAPPALAINEN P, KESSELS M M, COPE M J, DRUBIN D G. The ADF homology (ADF-H) domain: A highly exploited actin-binding module. Molecular Biology of the Cell, 1998, 9(8):1951-1959. doi: 10.1091/mbc.9.8.1951
[13]	STOLLAR E J, GARCIA B, CHONG P A, RATH A, LIN H, FORMAN-KAY J D, DAVIDSON A R. Structural, functional, and bioinformatic studies demonstrate the crucial role of an extended peptide binding site for the SH3 domain of yeast Abp1p. Journal of Biological Chemistry, 2009, 284(39):26918-26927. doi: 10.1074/jbc.M109.028431
[14]	GOODE B L, RODAL A A, BARNES G, DRUBIN D G. Activation of the Arp2/3 complex by the actin filament binding protein Abp1p. Journal of Cell Biology, 2001, 153(3):627-634.
[15]	FAZI B, COPE M, DOUANGAMATH A, FERRACUTI S, SCHIRWITZ K, ZUCCONI A, DRUBIN D G, WILMANNS M, CESARENI G, CASTAGNOLI L. Unusual binding properties of the SH3 domain of the yeast actin-binding protein Abp1: Structural and functional analysis. Journal of Biological Chemistry, 2002, 277(7):5290-5298. doi: 10.1074/jbc.M109848200
[16]	STEFAN C J, PADILLA S M, AUDHYA A, EMR S D. The phosphoinositide phosphatase Sjl2 is recruited to cortical actin patches in the control of vesicle formation and fission during endocytosis. Molecular and Cellular Biology, 2005, 25(8):2910-2923. doi: 10.1128/MCB.25.8.2910-2923.2005
[17]	HAYNES J, GARCIA B, STOLLAR E J, RATH A, ANDREWS B J, DAVIDSON A R. The biologically relevant targets and binding affinity requirements for the function of the yeast actin-binding protein 1 Src-homology 3 domain vary with genetic context. Genetics, 2007, 176(1):193-208. doi: 10.1534/genetics.106.070300
[18]	KESSELS M M, ENGQVIST-GOLDSTEIN A E Y, DRUBIN D G, QUALMANN B. Mammalian Abp1, a signal-responsive F-actin- binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin. Journal of Cell Biology, 2001, 153(2):351-366.
[19]	MISE-OMATA S, MONTAGNE B, DECKERT M, WIENANDS J, ACUTO O. Mammalian actin binding protein 1 is essential for endocytosis but not lamellipodia formation: Functional analysis by RNA interference. Biochemical and Biophysical Research Communications, 2003, 301(3):704-710. doi: 10.1016/S0006-291X(02)02972-8
[20]	KESSELS M M, ENGQVIST-GOLDSTEIN A E Y, DRUBIN D G. Association of mouse actin-binding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1 activation. Molecular Biology of the Cell, 2000, 11(1):393-412. doi: 10.1091/mbc.11.1.393
[21]	CORTESIO C L, PERRIN B J, BENNIN D A, HUTTENLOCHER A. Actin-binding protein-1 interacts with WASp-interacting protein to regulate growth factor-induced dorsal ruffle formation. Molecular Biology of the Cell, 2010, 21(1):186-197. doi: 10.1091/mbc.e09-02-0106
[22]	FENSTER S D, KESSELS M M, QUALMANN B, CHUNG W J, NASH J, GUNDELFINGER E D, GARNER C C. Interactions between Piccolo and the actin/dynamin-binding protein Abp1 link vesicle endocytosis to presynaptic active zones. Journal of Biological Chemistry, 2003, 278(22):20268-20277. doi: 10.1074/jbc.M210792200
[23]	HAN J, KORI R, SHUI J W, CHEN Y R, YAO Z B, TAN T H. The SH3 domain-containing adaptor HIP-55 mediates c-Jun N-terminal kinase activation in T cell receptor signaling. Journal of Biological Chemistry, 2003, 278(52):52195-52202. doi: 10.1074/jbc.M305026200
[24]	HOLTZMAN D A, YANG S, DRUBIN D G. Synthetic-lethal interactions identify two novel genes, SLA1and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae. Journal of Cell Biology, 1993, 122(3):635-644.
[25]	TUO S, NAKASHIMA K, PRINGLE J R. Role of endocytosis in localization and maintenance of the spatial markers for bud-site selection in yeast. PLoS ONE, 2013, 8(9):e72123. doi: 10.1371/journal.pone.0072123
[26]	COPE M J, YANG S, SHANG C, DRUBIN D G. Novel protein kinases Ark1p and Prk1p associate with and regulate the cortical actin cytoskeleton in budding yeast. Journal of Cell Biology, 1999, 144(6):1203-1218.
[27]	WESP A, HICKE L, PALECEK J, LOMBARDI R, AUST T, MUNN A L, RIEZMAN H. End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae. Molecular Biology of the Cell, 1997, 8(11):2291-2306. doi: 10.1091/mbc.8.11.2291
[28]	AGHAMOHAMMADZADEH S, SMACZYNSKA-DE ROOIJ I I, AYSCOUGH K R. An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited. PLoS ONE, 2014, 9(7):e103311. doi: 10.1371/journal.pone.0103311
[29]	LI L W, ZHANG S P, LIU X Y, YU R, LI X R, LIU M X, ZHANG H F, ZHENG X B, WANG P, ZHANG Z G. Magnaporthe oryzae Abp1, a MoArk1 kinase-interacting actin binding protein, links actin cytoskeleton regulation to growth, endocytosis, and pathogenesis. Molecular Plant-Microbe Interactions, 2019, 32(4):437-451. doi: 10.1094/MPMI-10-18-0281-R
[30]	NICHOLSON P. The Fusarium laboratory manual. Plant Pathology, 2007, 56(6):1037. doi: 10.1111/ppa.2007.56.issue-6
[31]	YU J H, HAMARI Z, HAN K H, SEO J A, REYES-DOMINGUEZ Y, SCAZZOCCHIO C. Double-joint PCR: A PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genetics and Biology, 2004, 41(11):973-981. doi: 10.1016/j.fgb.2004.08.001
[32]	PROCTOR R H, HOHN T M, MCCORMICK S P. Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Molecular Plant-Microbe Interactions, 1995, 8(4):593-601. doi: 10.1094/MPMI-8-0593
[33]	BRUNO K S, TENJO F, LI L, HAMER J E, XU J R. Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea. Eukaryotic Cell, 2004, 3(6):1525-1532. doi: 10.1128/EC.3.6.1525-1532.2004
[34]	CHEN Y, ZHENG S Y, JU Z Z, ZHANG C Q, TANG G F, WANG J, WEN Z Y, CHEN W, MA Z H. Contribution of peroxisomal docking machinery to mycotoxin biosynthesis, pathogenicity and pexophagy in the plant pathogenic fungus Fusarium graminearum. Environmental Microbiology, 2018, 20(9):3224-3245. doi: 10.1111/emi.2018.20.issue-9
[35]	TANG G F, ZHANG C Q, JU Z Z, ZHENG S Y, WEN Z Y, XU S, CHEN Y, MA Z H. The mitochondrial membrane protein FgLetm1 regulates mitochondrial integrity, production of endogenous reactive oxygen species and mycotoxin biosynthesis in Fusarium graminearum. Molecular Plant Pathology, 2018, 19(7):1595-1611. doi: 10.1111/mpp.2018.19.issue-7
[36]	TRAIL F. For blighted waves of grain: Fusarium graminearum in the postgenomics era. Plant Physiology, 2009, 149(1):103-110. doi: 10.1104/pp.108.129684
[37]	MENKE J, DONG Y H, KISTLER H C. Fusarium graminearum Tri12p influences virulence to wheat and trichothecene accumulation. Molecular Plant-Microbe Interactions, 2012, 25(11):1408-1418. doi: 10.1094/MPMI-04-12-0081-R
[38]	WINDER S J, AYSCOUGH K R. Actin-binding proteins. Journal of Cell Science, 2005, 118(4):651-654. doi: 10.1242/jcs.01670
[39]	DOS REMEDIOS C G, CHHABRA D, KEKIC M, DEDOVA I V, TSUBAKIHARA M, BERRY D A, NOSWORTHY N J. Actin binding proteins: Regulation of cytoskeletal microfilaments. Physiological Reviews, 2003, 83(2):433-473. doi: 10.1152/physrev.00026.2002
[40]	ARAUJO-BAZAN L, PENALVA M A, ESPESO E A. Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans. Molecular Microbiology, 2008, 67(4):891-905. doi: 10.1111/mmi.2008.67.issue-4
[41]	QUALMANN B, KESSELS M M, KELLY R B. Molecular links between endocytosis and the actin cytoskeleton. Journal of Cell Biology, 2000, 150(5):F111-F116.
[42]	MATSUO K, HIGUCHI Y, KIKUMA T, ARIOKA M, KITAMOTO K. Functional analysis of Abp1p-interacting proteins involved in endocytosis of the MCC component in Aspergillus oryzae. Fungal Genetics and Biology, 2013, 56:125-134. doi: 10.1016/j.fgb.2013.03.007
[43]	ZHENG Z T, HOU Y P, CAI Y Q, ZHANG Y, LI Y J, ZHOU M G. Whole-genome sequencing reveals that mutations in myosin-5 confer resistance to the fungicide phenamacril in Fusarium graminearum. Scientific Reports, 2015, 5:8248. doi: 10.1038/srep08248
[44]	CHEN Y, KISTLER H C, MA Z H. Fusarium graminearum trichothecene mycotoxins: Biosynthesis, regulation, and management. Annual Review of Phytopathology, 2019, 57:15-39. doi: 10.1146/annurev-phyto-082718-100318

引物 Primer	序列 Sequence (5′-3′)
Abp1-up-F	CAGTGTCGGGGTATTCGGAAGCG
Abp1-up-R	CAAAATAGGCATTGATGTGTTGACCTCCGATTGAGGGACGCCATCTTGAGG
Abp1-down-F	CTCGTCCGAGGGCAAAGGAATAGAGTAGGGAGTTCCCTGATGAGGATTGG
Abp1-down-R	CAACAACCTCAACCTGTAGGCCA
HPH-F	GGAGGTCAACACATCAATGCCTATT
HPH-R	CTACTCTATTCCTTTGCCCT
Abp1-nest-F	GCGTGGAACGATACGATGACGAG
Abp1-nest-R	GCTAACCGTTCTATTTAACAGTCC
Abp1-ID-F	GACCTTCGTCAGCTTCGTCTCTC
Abp1-ID-R	GCCGGGAACAGACCAGTATGGC
Abp1-GFP-F	ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTTTGGAAGCGCCAAATTCTACT
Abp1-GFP-R	CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCTGGTTAAGCTGAACGTAGTT
ID-Abp1-GFP-F	GCCGCCGCTCAGGCAGATGCC
ID-GFP-R	GTCAGCTTGCCGTAGGTGGCA
Abp1-RFP-F	ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTTTGGAAGCGCCAAATTCTACT
Abp1-RFP-R	CATGAACTCCTTGATGACGTCCTCGGAGGAGGCCATCTGGTTAAGCTGAACGTAGTT
ID-RFP-R	TTGGAGCCGTACTGGAACTG
Tri1-GFP-F	ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTGTGAGTAGGCCTCATAGCGAC
Tri1-GFP-R	CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACGTCATCCTGTACCAATTCCAAT