假禾谷镰孢转录因子FpAPSES的鉴定与功能分析

doi:10.3864/j.issn.0578-1752.2021.16.006

摘要/Abstract

摘要：

【目的】假禾谷镰孢（Fusarium pseudograminearum）侵染小麦引起的小麦茎基腐病严重影响中国小麦的安全生产。查找假禾谷镰孢中的APSES转录因子,分析其在病原菌致病过程中的作用,为解析假禾谷镰孢的致病机制及小麦茎基腐病的防治提供理论依据。【方法】从GenBank获得物种中已知的APSES氨基酸序列,利用BLASTP方法在假禾谷镰孢中查找APSES同源蛋白,利用Pfam软件预测蛋白结构域,采用MEGA5.05构建APSES蛋白的系统进化树。利用实时荧光定量PCR（qRT-PCR）方法分析A组APSES转录因子基因FpAPSES1和FpAPSES4在假禾谷镰孢侵染过程中的表达。通过PEG介导的原生质体转化和PCR筛选FpAPSES1和FpAPSES4基因缺失的突变体菌株。在PDA培养基上测定假禾谷镰孢野生型菌株Wz2-8A、Δfpapses1和Δfpapses4突变体菌株的菌丝形态和生长速率;测定在CMC培养液中培养后的分生孢子产生情况、形态以及在无菌水中的萌发率;利用菌丝块和分生孢子接种小麦胚芽鞘和大麦叶片以及盆栽试验测定其致病性;采用ELISA方法测定小米培养基中脱氧雪腐镰刀菌烯醇（DON）的含量。【结果】假禾谷镰孢中有4个APSES同源蛋白,均含有保守的DNA结合结构域HTH。与其他物种的APSES蛋白构建系统进化树,发现FpAPSES1、FpAPSES2和FpAPSES4属于A组APSES,FpAPSES3分布在C组。FpAPSES1和FpAPSES4在侵染阶段诱导表达,推测可能参与假禾谷镰孢的致病。分别获得2个FpAPSES1和FpAPSES4基因缺失的突变体菌株Δfpapses1-T10、Δfpapses1-T27和Δfpapses4-T1、Δfpapses4-T2。表型测定结果显示,与野生型相比,FpAPSES1和FpAPSES4基因缺失突变体在PDA平板上的生长速率明显减慢、色素积累明显增多;FpAPSES1基因缺失突变体菌丝形态无差异,而FpAPSES4基因缺失突变体菌丝弯曲、分支明显增多;FpAPSES1和FpAPSES4基因缺失突变体在CMC液体中的分生孢子产生明显减少,分别比野生型下降了99.5%和97.4%,产生的分生孢子变短、隔膜减少、萌发率有所降低;与野生型相比,FpAPSES1和FpAPSES4基因缺失突变体菌丝体和分生孢子对小麦胚芽鞘的致病力明显降低,菌丝在小麦胚芽鞘表皮细胞中的扩展明显受阻;FpAPSES1和FpAPSES4基因缺失突变体对大麦叶片和小麦根部的致病力也明显降低;FpAPSES1和FpAPSES4基因缺失突变体在小米培养基中产生的DON毒素分别比野生型下降了约78%和44%。【结论】编码A组APSES同源蛋白的FpAPSES1和FpAPSES4对假禾谷镰孢的菌丝生长、分生孢子产生和致病力均具有重要作用。

关键词: 小麦茎基腐病, 假禾谷镰孢, APSES转录因子, 致病力

Abstract:

【Objective】Fusarium crown rot (FCR) caused by Fusarium pseudograminearum seriously affects wheat production in China. The objective of this study is to find and analyze the functions of APSES transcription factors in F. pseudograminearum, and to provide theoretical basis for revealing the pathogenic mechanism of F. pseudograminearum and prevention and treatment of FCR.【Method】The known APSES proteins were obtained from GenBank, and APSES candidates in F. pseudograminearum were found by BLASTP. Domains of FpAPSES proteins were determined using Pfam. The phylogenetic tree of APSES proteins was constructed using MEGA 5.05. The relative expression levels of FpAPSES1 and FpAPSES4 during infection were examined by quantitative RT-PCR (qRT-PCR). FpAPSES1 and FpAPSES4 deletion mutants were generated by polyethylene glycol (PEG)-mediated protoplast fungal transformation, and screened by PCR. The wild type, Δfpapses1 and Δfpapses4 strains were activated on PDA medium, and cultured on PDA for mycelial growth and morphology assays; mycelial blocks were introduced into liquid CMC medium to assess conidiation and conidia morphology; conidia were cultured in sterile distilled water to explore conidia germination; mycelial blocks or conidia suspension were prepared and inoculated wheat coleoptiles and barely leaves to explore pathogenicity. The pot-culture experiment was also used for virulence assay. ELISA was used to detect deoxynivalenol (DON) in inoculation millet. 【Result】 Four APSES candidates were found in F. pseudograminearum, which all contained the conserved DNA binding domain HTH. The phylogenetic tree analysis showed that FpAPSES1, FpAPSES2 and FpAPSES4 were divided into A group, while FpAPSES3 belonged to the C group. The qRT-PCR analysis revealed that FpAPSES1 and FpAPSES4 were up-regulated at infection stages, which suggested that FpAPSES1 and FpAPSES4 might play important roles in infection. Two FpAPSES1 deletion mutants (Δfpapses1-T10 and Δfpapses1-T27) and two FpAPSES4 deletion mutants (Δfpapses4-T1 and Δfpapses4-T2) were obtained. Compared with the wild type strain, both FpAPSES1- and FpAPSES4-deleted mutants exhibited significantly reduced colony growth rates. FpAPSES1-deleted mutants had normal hyphal branches, while FpAPSES4-deleted mutants exhibited curved and more branched hypha. Both FpAPSES1- and FpAPSES4-deleted mutants exhibited significantly reduced conidiation in CMC, and the conidia numbers were respectively reduced by 99.5% and 97.4% comparing with that of the wild type. Furthermore, conidia of FpAPSES1- and FpAPSES4-deleted mutants were shorter, less septa and lower germination rates. The wheat coleoptiles were point-inoculated with mycelial blocks or conidia, and the virulence of FpAPSES1- and FpAPSES4-deleted mutants was significantly reduced comparing to the wild type. Reduced pathogenicity was further observed by barely leaves inoculation and pot culture experiment. The DON levels in millet upon infection with FpAPSES1- and FpAPSES4-deleted mutants were reduced by 78% and 44% comparing to the wild type, respectively.【Conclusion】Both A group APSES homologs FpAPSES1 and FpAPSES4 play important roles in growth, conidiation and pathogenicity of F. pseudograminearum.

Key words: Fusarium crown rot, Fusarium pseudograminearum, APSES transcription factor, pathogenicity

赵静雅,夏荟清,彭梦雅,凡卓,殷悦,徐赛博,张楠,陈文波,陈琳琳. 假禾谷镰孢转录因子FpAPSES的鉴定与功能分析[J]. 中国农业科学, 2021, 54(16): 3428-3439.

ZHAO JingYa,XIA HuiQing,PENG MengYa,FAN Zhuo,YIN Yue,XU SaiBo,ZHANG Nan,CHEN WenBo,CHEN LinLin. Identification and Functional Analysis of Transcription Factors FpAPSES in Fusarium pseudograminearum[J]. Scientia Agricultura Sinica, 2021, 54(16): 3428-3439.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.16.006

https://www.chinaagrisci.com/CN/Y2021/V54/I16/3428

图/表 10

表1

表2

图1

图2

图3

图4

图5

图6

图7

图 8

参考文献 32

[1]	LI H L, YUAN H X, FU B, XING X P, SUN B J, TANG W H. First report of Fusarium pseudograminearum causing crown rot of wheat in Henan, China. Plant Disease, 2012, 96(7):1065.
[2]	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. Diversity of the Fusarium pathogens associated with crown rot in the Huanghuai wheat-growing region of China. Environmental Microbiology, 2019, 21(8):2740-2754. doi: 10.1111/emi.2019.21.issue-8
[3]	KAZAN K, GARDINER D M. Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: Recent progress and future prospects. Molecular Plant Pathology, 2018, 19(7):1547-1562. doi: 10.1111/mpp.2018.19.issue-7
[4]	OBANOR F, NEATE S, SIMPFENDORFER S, SABBURG R, WILSON P, CHAKRABORTY S. Fusarium graminearum and Fusarium pseudograminearum caused the 2010 head blight epidemics in Australia. Plant Pathology, 2013, 62(1):79-91. doi: 10.1111/ppa.2012.62.issue-1
[5]	ZHAO Y, SU H, ZHOU J, FENG H H, ZHANG K Q, YANG J K. The APSES family proteins in fungi: Characterizations, evolution and functions. Fungal Genetics and Biology, 2015, 81:271-280. doi: 10.1016/j.fgb.2014.12.003
[6]	LIU J F, HUANG J G, ZHAO Y X, LIU H A, WANG D W, YANG J, ZHAO W S, TAYLOR I A, PENG Y L. Structural basis of DNA recognition by PCG2 reveals a novel DNA binding mode for winged helix-turn-helix domains. Nucleic Acids Research, 2015, 43(2):1231-1240. doi: 10.1093/nar/gku1351
[7]	QI Z Q, WANG Q, DOU X Y, WANG W, ZHAO Q, LV R L, ZHANG H F, ZHENG X B, WANG P, ZHANG Z G. MoSwi6, an APSES family transcription factor, interacts with MoMps1 and is required for hyphal and conidial morphogenesis, appressorial function and pathogenicity of Magnaporthe oryzae. Molecular Plant Pathology, 2012, 13(7):677-689. doi: 10.1111/mpp.2012.13.issue-7
[8]	MILES S, LI L, DAVISON J, BREEDEN L L. Xbp1 directs global repression of budding yeast transcription during the transition to quiescence and is important for the longevity and reversibility of the quiescent state. PLoS Genetics, 2013, 9(10):e1003854. doi: 10.1371/journal.pgen.1003854
[9]	GIMENO C J, FINK G R. Induction of pseudohyphal growth by overexpression of PHD1, a Saccharomyces cerevisiae gene related to transcriptional regulators of fungal development. Molecular and Cellular Biology, 1994, 14(3):2100-2112.
[10]	PARK H, MYERS C L, SHEPPARD D C, PHAN Q T, SANCHEZ A A, EDWARDS J E, FILLER S G. Role of the fungal Ras-protein kinase A pathway in governing epithelial cell interactions during oropharyngeal candidiasis. Cellular Microbiology, 2005, 7(4):499-510. doi: 10.1111/cmi.2005.7.issue-4
[11]	STOLDT V R, SONNEBORN A, LEUKER C E, ERNST J F. Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi. The EMBO Journal, 1997, 16(8):1982-1991. doi: 10.1093/emboj/16.8.1982
[12]	LYSOE E, PASQUALI M, BREAKSPEAR A, KISTLER H C. The transcription factor FgStuAp influences spore development, pathogenicity, and secondary metabolism in Fusarium graminearum. Molecular Plant-Microbe Interactions, 2011, 24(1):54-67. doi: 10.1094/MPMI-03-10-0075
[13]	NISHIMURA M, FUKADA J, MORIWAKI A, FUJIKAWA T, OHASHI M, HIBI T, HAYASHI N. Mstu1, an APSES transcription factor, is required for appressorium-mediated infection in Magnaporthe grisea. Bioscience, Biotechnology and Biochemistry, 2009, 73(8):1779-1786. doi: 10.1271/bbb.90146
[14]	RATH M, CRENSHAW N J, LOFTON L W, GLENN A E, GOLD S E. FvSTUA is a key regulator of sporulation, toxin synthesis, and virulence in Fusarium verticillioides. Molecular Plant-Microbe Interactions, 2020, 33(7):958-971. doi: 10.1094/MPMI-09-19-0271-R
[15]	周欣, 云英子, 郭谱胜, 吴凯莉, 陈伟钟, 李江江, 周晓莉. 尖孢镰刀菌番茄专化型中APSES转录因子FolStuA的功能分析. 分子植物育种, 2018, 16(3):772-780.
	ZHOU X, YUN Y Z, GUO P S, WU K L, CHEN W Z, LI J J, ZHOU X L. Functional analysis of APSES transcription factor FolStuA in Fusarium oxysporum f. sp. lycopersici. Molecular Plant Breeding, 2018, 16(3):772-780. (in Chinese)
[16]	GARDINER D M, BENFIELD A H, STILLER J, STEPHEN S, AITKEN K, LIU C, KAZAN K. A high-resolution genetic map of the cereal crown rot pathogen Fusarium pseudograminearum provides a near-complete genome assembly. Molecular Plant Pathology, 2018, 19(1):217-226. doi: 10.1111/mpp.2018.19.issue-1
[17]	FINN R D, BATEMAN A, CLEMENTS J, COGGILL P, EBERHARDT R Y, EDDY S R, HEGER A, HETHERINGTON K, HOLM L, MISTRY J, SONNHAMMER E L L, TATE J, PUNTA M. Pfam: The protein families database. Nucleic Acids Research, 2014, 42:D222-D230. doi: 10.1093/nar/gkt1223
[18]	HALL B G. Building phylogenetic trees from molecular data with MEGA. Molecular Biology and Evolution, 2013, 30(5):1229-1235. doi: 10.1093/molbev/mst012
[19]	CATLETT N L, LEE B, YODER O C, TURGEON B G. Split-marker recombination for efficient targeted deletion of fungal genes. Fungal Genetics Reports, 2003, 50:9-11. doi: 10.4148/1941-4765.1150
[20]	张承启, 廖露露, 齐永霞, 丁克坚, 陈莉. 禾谷镰孢核孔蛋白基因FgNup42的功能分析. 中国农业科学, 2021, 54(9):1894-1903.
	ZHANG C Q, LIAO L L, QI Y X, DING K J, CHEN L. Functional analysis of the nucleoporin gene FgNup42 in Fusarium graminearium. Scientia Agriculture Sinica, 2021, 54(9):1894-1903. (in Chinese)
[21]	LIU Z H, FRIESEN T L. Polyethylene glycol (PEG)-mediated transformation in filamentous fungal pathogens. Methods in Molecular Biology, 2012, 835:365-375.
[22]	CHEN L L, GENG X J, MA Y M, ZHAO J Y, LI T L, DING S L, SHI Y, LI H L. Identification of basic helix-loop-helix transcription factors reveals candidate genes involved in pathogenicity of Fusarium pseudograminearum. Canadian Journal of Plant Pathology, 2019, 41(2):200-208. doi: 10.1080/07060661.2018.1564941
[23]	TUNALI B, OBANOR F, ERGINBAS G, WESTECOTT R A, NICOL J, CHAKRABORTY S. Fitness of three Fusarium pathogens of wheat. FEMS Microbiology Ecology, 2012, 81(3):596-609. doi: 10.1111/j.1574-6941.2012.01388.x
[24]	CHUNG D, UPADHYAY S, BOMER B, WILKINSON H H, EBBOLE D J, SHAW B D. Neurospora crassa ASM-1 complements the conidiation defect in a stuA mutant of Aspergillus nidulans. Mycologia, 2015, 107(2):298-306. doi: 10.3852/14-079
[25]	MILLER K Y, TOENNIS T M, ADAMS T H, MILLER B L. Isolation and transcriptional characterization of a morphological modifier: The Aspergillus nidulans stunted (stuA) gene. Molecular and General Genetics, 1991, 227(2):285-292. doi: 10.1007/BF00259682
[26]	FOORD R, TAYLOR I A, SEDGWICK S G, SMERDON S J. X-ray structural analysis of the yeast cell cycle regulator Swi6 reveals variations of the ankyrin fold and has implications for Swi6 function. Nature Structural Biology, 1999, 6(2):157-165. doi: 10.1038/5845
[27]	KOCH C, MOLL T, NEUBERG M, AHORN H, NASMYTH K. A role for the transcription factors Mbp1 and Swi4 in progression from G1 to S phase. Science, 1993, 261(5128):1551-1557. doi: 10.1126/science.8372350
[28]	AMIGONI L, COLOMBO S, BELOTTI F, ALBERGHINA L, MARTEGANI E. The transcription factor Swi4 is target for PKA regulation of cell size at the G1to S transition in Saccharomyces cerevisiae. Cell Cycle, 2015, 14(15):2429-2438. doi: 10.1080/15384101.2015.1055997
[29]	JIANG L, WANG J, ASGHAR F, SNYDER N, CUNNINGHAM K W. CaGdt1 plays a compensatory role for the calcium pump CaPmr1 in the regulation of calcium signaling and cell wall integrity signaling in Candida albicans. Cell Communication and Signaling, 2018, 16(1):33. doi: 10.1186/s12964-018-0246-x
[30]	MEDINA E M, WALSH E, BUCHLER N E. Evolutionary innovation, fungal cell biology, and the lateral gene transfer of a viral KilA-N domain . Current Opinion in Genetics and Development, 2019, 58/59:103-110.
[31]	王大伟. 稻瘟病菌APSES转录因子Pcg2的作用及其机理的研究[D]. 北京: 中国农业大学, 2014.
	WANG D W. Functional characterization of the APSES transcription factor Pcg2 in the rice blast fungus[D]. Beijing: China Agricultural University, 2014. (in Chinese)
[32]	SON H, SEO Y S, MIN K, PARK A R, LEE J, JIN J M, LIN Y, CAO P, HONG S Y, KIM E K, et al. A phenome-based functional analysis of transcription factors in the cereal head blight fungus, Fusarium graminearum. PLoS Pathogens, 2011, 7(10):e1002310. doi: 10.1371/journal.ppat.1002310

引物Primer	引物序列Primer sequence (5′-3′)
FpAPSES1-F1	GTGGGTCTCATGATGATTC
FpAPSES4-F1	GAGTCTTCAACCCCTCTC
FpAPSES1-R1	CAATATCATCTTCTGTCGACGTTGAAAGATTGTTGAGATG
FpAPSES4-R1	CAATATCATCTTCTGTCGACGTTGGCGACTAAGATGAAGC
FpAPSES1-F2	ATAGAGTAGATGCCGACCGCGGGTTCTACCGGCACTCACCTCTC
FpAPSES4-F2	ATAGAGTAGATGCCGACCGCGGGTTCTTAACATCATTGGCCTATC
FpAPSES1-R2	CATACAAACATGCTGCCTCTC
FpAPSES4-R2	CAATTGTCCGGTGCTGTC
YG-F	GATGTAGGAGGGCGTGGATATGTCCT
HY-R	GTATTGACCGATTCCTTGCGGTCCGAA
HYG-F	GTCGACAGAAGATGATATTG
HYG-R	GAACCCGCGGTCGGCATCTACTCTAT
FpAPSES1-F3	CATTAGGCTCTTCGGGTGTG
FpAPSES4-F3	CACACCAACCCAATCCCTC
FpAPSES1-R3	CATACGAGACTCTACAACAG
FpAPSES4-R3	GTTCTGGTTGCTGGTCCTG
FpAPSES1-G1	CAACGACTCATGGCTCAATG
FpAPSES4-G1	CATGCATATTCAGAACCCAG
FpAPSES1-G2	GTAAGGGAGAGGTTCAAGAG
FpAPSES4-G2	GTGCTGTGTGATCCTCTG
H1R	GCTGATCTGACCAGTTGC
H1F	GTCGATGCGACGCAATCGT
H2F	TTCCTCCCTTTATTTCAGATTCAA
H2R	ATGTTGGCGACCTCGTATTGG
TEF1a-RTF	TCACCACTGAAGTCAAGTCC
TEF1a-RTR	ACCAGCGACGTTACCACGTC
FpAPSES1-RTF	AACGTGGATATGTATGCGGC
FpAPSES4-RTF	GCTATGGGTGATTTGGATGTCG
FpAPSES1-RTR	TCGACATCTG GAGACATCTC
FpAPSES4-RTR	GTTCTTTCATGACCATAGG

蛋白名称 Protein name	基因ID Gene ID	转录号 Transcription number	蛋白大小 Size (aa)	蛋白分子量Molecular weight (kD)	蛋白等电点 Isoelectric point (PI)
FpAPSES1	XP_009261816.1	FPSE_10424	794	86.28	6.02
FpAPSES2	XP_009258602.1	FPSE_07209	420	46.08	5.78
FpAPSES3	XP_009254016.1	FPSE_02622	702	77.12	6.34
FpAPSES4	XP_009258789.1	FPSE_07396	675	75.55	5.30