Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (5): 1375-1388.DOI: 10.1016/S2095-3119(21)63731-0

• • 上一篇下一篇

收稿日期:2021-01-19 接受日期:2021-05-12 出版日期:2022-05-01 发布日期:2021-05-12

Mutations in FgPrp6 suppressive to the Fgprp4 mutant in Fusarium graminearum

LI Chao-hui^{1, 2}, FAN Zhi-li¹, HUANG Xin-yi¹, WANG Qin-hu¹, JIANG Cong¹, XU Jin-rong^{1, 3}, JIN Qiao-jun¹

¹ NWAFU-PU Joint Research Center/State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling 712100, P.R.China
² Institute of Plant Protection/Jiangsu Key Laboratory for Food Quality and Safety–State Key Laboratory Cultivation Base of Ministry of Science and Technology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China
³ Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN47907, USA

Received:2021-01-19 Accepted:2021-05-12 Online:2022-05-01 Published:2021-05-12
About author: Correspondence JIN Qiao-jun, Tel: +86-29-87082710, E-mail: jqiaojun@nwafu.edu.cn
Supported by:
This work was supported by the grants from the National Natural Science Foundation of China (31600117) and the Natural Science Basic Research Program of Shaanxi, China (2020JM-165).

摘要/Abstract

摘要：

剪接因子Prp6是剪接三聚体U4/U6.U5中的关键蛋白，在人类细胞和裂殖酵母中，它也是调控前体mRNA剪接的激酶Prp4的底物。前期研究发现引起小麦赤霉病的禾谷镰孢菌FgPrp6蛋白序列的自发突变（角突变）可以部分恢复Fgprp4突变体的表型。禾谷镰孢菌FgPrp4激酶调控剪接效率，其敲除突变体生长缓慢且丧失产孢、有性生殖和致病能力。为了进一步探索FgPrp6与激酶FgPrp4的关系，本研究通过对随机收集的240株Fgprp4角突变子的FgPRP6基因进行PCR产物测序，鉴定了20个角变子中的12个突变。其中3个突变位点在FgPrp6蛋白的N端结构域和HAT重复结构域的连接处，7个突变位点位于前两个HAT重复区域。对角变子的转录组数据分析结果表明FgPrp6上不同位置的角突变对Fgprp4突变体前体mRNA剪接缺陷的恢复程度不同。通过在野生型菌株中转入FgPrp6^E308K-GFP载体或在内源FgPrp6上原位引入R230H突变，同时敲除FgPRP4的方法证实FgPrp6上的E308K或R230H都可以抑制Fgprp4。本研究利用co-IP和BiFc的方法证明禾谷镰孢菌的FgPrp6和激酶FgPrp4可以在体内互作，并进一步利用磷酸化抗体检测体内FgPrp6磷酸化水平的方法实验证明FgPrp6为FgPrp4的底物。通过将FgPrp6上保守的Prp4磷酸化位点和预测的磷酸化位点突变为A的方法验证这些位点的功能，结果表明T261，T219&T221和T199&T200对FgPrp6在菌落生长和有性生殖中的功能无关紧要，但是对其在侵染植物阶段的功能至关重要。扫描电镜和共聚焦显微镜观察发现它们主要在禾谷镰孢菌侵染生长，如侵染垫的形成和在植物细胞间的扩展中发挥作用。通过对野生型禾谷镰孢菌、Fgprp6/FgPRP6^Δ199-221-GFP或Fgprp6/FgPRP6^Δ²⁵⁰^-262-GFP侵染三天的小麦麸片的转录组数据分析禾谷镰孢菌的前体mRNA剪接缺陷发现Fgprp6/FgPRP6^Δ199-221-GFP和Fgprp6/FgPRP6^Δ250-262-GFP菌株中各有28%和35%的内含子剪接具有缺陷，推测这种剪接缺陷是突变体侵染生长缺陷的原因。该研究为将来进一步解析禾谷镰孢菌前体mRNA剪接调控及剪接与致病性的关系奠定了基础。

Abstract: The pre-mRNA processing factor Prp6 is an essential component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP). In a previous study, mutations were identified in the PRP6 ortholog in four suppressors of Fgprp4 that was deleted of the only kinase FgPrp4 among the spliceosome components in the plant pathogenic fungus Fusarium graminearum. In this study, we identified additional suppressor mutations in FgPrp6 and determined the suppressive effects of selected mutations. In total, 12 mutations of FgPRP6 were identified in 20 suppressors of Fgprp4 by sequencing analysis. Whereas three mutation sites are in the linker region of FgPrp6, seven are in the first two HAT repeats. RNA-seq analysis showed that suppressor mutations on different sites caused different splicing efficiency recovery. The suppressive effects of E308K and R230H were verified. Similar to human and fission yeast, the FgPrp6 was phosphorylated by the FgPrp4 kinase. Interestingly, the conserved Prp4-phosphorylation sites T261, T219&T221, and predicted phosphorylation sites T199&T200 on FgPrp6 were dispensable for the function of FgPrp6 in hyphal growth and sexual reproduction but important in plant infection. They are required for the infectious growth of F. graminearum in wheat lemma. RNA-seq analysis of the wheat lemma infected with Fgprp6/FgPRP6^Δ199–221-GFP or Fgprp6/FgPRP6^Δ250–262-GFP showed that 28 and 35% introns had splicing defects, respectively, which may be responsible for their defects in plant infection.

Key words: RNA splicing , FgPrp6 , suppressor , plant infection , phosphorylation

. [J]. Journal of Integrative Agriculture, 2022, 21(5): 1375-1388.

LI Chao-hui, FAN Zhi-li, HUANG Xin-yi, WANG Qin-hu, JIANG Cong, XU Jin-rong, JIN Qiao-jun. Mutations in FgPrp6 suppressive to the Fgprp4 mutant in Fusarium graminearum[J]. Journal of Integrative Agriculture, 2022, 21(5): 1375-1388.

参考文献

Agafonov D E, Kastner B, Dybkov O, Hofele R V, Liu W T, Urlaub H, Luhrmann R, Stark H. 2016. Molecular architecture of the human U4/U6.U5 tri-snRNP. Science, 351, 1416–1420.
Bai G H, Shaner G. 2004. Management and resistance in wheat and barley to Fusarium head blight. Annual Review of Phytopathology, 42, 135–161.
Bertram K, Agafonov D E, Dybkov O, Haselbach D, Leelaram M N, Will C L, Urlaub H, Kastner B, Luhrmann R, Stark H. 2017. Cryo-EM structure of a pre-catalytic human spliceosome primed for activation. Cell, 170, 701–713.
Boenisch M J, Schäfer W. 2011. Fusarium graminearum forms mycotoxin producing infection structures on wheat. Bmc Plant Biology, 11, 110.
Boesler C, Rigo N, Anokhina M M, Tauchert M J, Agafonov D E, Kastner B, Urlaub H, Ficner R, Will C L, Luhrmann R. 2016. A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity. Nature Communications, 7, 11997.
Bottner C A, Schmidt H, Vogel S, Michele M, Kaufer N F. 2005. Multiple genetic and biochemical interactions of Brr2, Prp8, Prp31, Prp1 and Prp4 kinase suggest a function in the control of the activation of spliceosomes in Schizosaccharomyces pombe. Current Genetics, 48, 151–161.
Brown N A, Urban M, van de Meene A M, Hammond-Kosack K E. 2010. The infection biology of Fusarium graminearum: defining the pathways of spikelet to spikelet colonisation in wheat ears. Fungal Biology, 114, 555–571.
Bruno K S, Tenjo F, Li L, Hamer J E, Xu J R. 2004. Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea. Eukaryotic Cell, 3, 1525–1532.
Cuomo C A, Gueldener U, Xu J R, Trail F, Turgeon B G, Di Pietro A, Walton J D, Ma L J, Baker S E, Rep M, Adam G, Antoniw J, Baldwin T, Calvo S, Chang Y L, DeCaprio D, Gale L R, Gnerre S, Goswami R S, Hammond-Kosack K, et al. 2007. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science, 317, 1400–1402.
Dou K, Huang C F, Ma Z Y, Zhang C J, Zhou J X, Huang H W, Cai T, Tang K, Zhu J K, He X J. 2013. The PRP6-like splicing factor STA1 is involved in RNA-directed DNA methylation by facilitating the production of Pol V-dependent scaffold RNAs. Nucleic Acids Research, 41, 8489–8502.
Galisson F, Legrain P. 1993. The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP. Nucleic Acids Research, 21, 1555–1562.
Gao X, Jin Q, Jiang C, Li Y, Li C, Liu H, Kang Z, Xu J R. 2016. FgPrp4 kinase is important for spliceosome B-complex activation and splicing efficiency in Fusarium graminearum. PLoS Genetics, 12, e1005973.
Gao X, Zhang J, Song C, Yuan K, Wang J, Jin Q, Xu J R. 2018. Phosphorylation by Prp4 kinase releases the self-inhibition of FgPrp31 in Fusarium graminearum. Current Genetics, 64, 1261–1274.
Hou Z M, Xue C Y, Peng Y L, Katan T, Kistler H C, Xu J R. 2002. A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility, heterokaryon formation, and plant infection. Molecular Plant–Microbe Interactions, 15, 1119–1127.
Hu S, Zhou X, Gu X, Cao S, Wang C, Xu J R. 2014. The cAMP-PKA pathway regulates growth, sexual and asexual differentiation, and pathogenesis in Fusarium graminearum. Molecular Plant–Microbe Interactions, 27, 557–566.
Jenczmionka N J, Maier F J, Losch A P, Schafer W. 2003. Mating, conidiation and pathogenicity of Fusarium graminearum, the main causal agent of the head-blight disease of wheat, are regulated by the MAP kinase gpmk1. Current Genetics, 43, 87–95.
Li X, Fan Z, Yan M, Qu J, Xu J R, Jin Q. 2019. Spontaneous mutations in FgSAD1 suppress the growth defect of the Fgprp4 mutant by affecting tri-snRNP stability and its docking in Fusarium graminearum. Environmental Microbiology, 21, 4488–4503.
Liu H, Wang Q, He Y, Chen L, Hao C, Jiang C, Li Y, Dai Y, Kang Z, Xu J R. 2016. Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes. Genome Research, 26, 499–509.
Liu S, Li P, Dybkov O, Nottrott S, Hartmuth K, Luhrmann R, Carlomagno T, Wahl M C. 2007. Binding of the human Prp31 Nop domain to a composite RNA-protein platform in U4 snRNP. Science, 316, 115–120.
Liu S B, Rauhut R, Vornlocher H P, Luhrmann R. 2006. The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP. Rna - A Publication of the Rna Society, 12, 1418–1430.
Lutzelberger M, Bottner C A, Schwelnus W, Zock-Emmenthal S, Razanau A, Kaufer N F. 2010. The N-terminus of Prp1 (Prp6/U5-102 K) is essential for spliceosome activation in vivo. Nucleic Acids Research, 38, 1610–1622.
Makarov E M, Makarova O V, Achsel T, Luhrmann R. 2000. The human homologue of the yeast splicing factor Prp6p contains multiple TPR elements and is stably associated with the U5 snRNP via protein–protein interactions. Journal of Molecular Biology, 298, 567–575.
Nguyen T H, Galej W P, Bai X C, Oubridge C, Newman A J, Scheres S H, Nagai K. 2016. Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 A resolution. Nature, 530, 298–302.
Ohi M D, Ren L, Wall J S, Gould K L, Walz T. 2007. Structural characterization of the fission yeast U5.U2/U6 spliceosome complex. Proceedings of the National Academy of Sciences of the United States of America, 104, 3195–3200.
Proctor R H, Hohn T M, Mccormick S P. 1995. Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Molecular Plant–Microbe Interactions, 8, 593–601.
Schneider M, Hsiao H H, Will C L, Giet R, Urlaub H, Luhrmann R. 2010. Human PRP4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation. Nature Structural & Molecular Biology, 17, 216–221.
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, Lee S H, Cho A, Lee S, Kim M G, Kim Y, Kim J E, Kim J C, Choi G J, Yun S H, Lim J Y, Kim M, et al. 2011. A phenome-based functional analysis of transcription factors in the cereal head blight fungus, Fusarium graminearum. PLoS Pathogens, 7, e1002310.
Sun M, Zhang Y, Wang Q, Wu C, Jiang C, Xu J R. 2018. The tri-snRNP specific protein FgSnu66 is functionally related to FgPrp4 kinase in Fusarium graminearum. Molecular Microbiology, 109, 494–508.
Wang C, Zhang S, Hou R, Zhao Z, Zheng Q, Xu Q, Zheng D, Wang G, Liu H Q, Gao X, Ma J W, Kistler H C, Kang Z S, Xu J R. 2011. Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum. PLoS Pathogens, 7, e1002460.
Will C L, Luhrmann R. 2011. Spliceosome structure and function. Cold Spring Harbor Perspectives in Biology, 3, a003707.
Xu Y B, Li H P, Zhang J B, Song B, Chen F F, Duan X J, Xu H Q, Liao Y C. 2010. Disruption of the chitin synthase gene CHS1 from Fusarium asiaticum results in an altered structure of cell walls and reduced virulence. Fungal Genetics and Biology, 47, 205–215.
Yun Y, Liu Z, Yin Y, Jiang J, Chen Y, Xu J R, Ma Z. 2015. Functional analysis of the Fusarium graminearum phosphatome. New Phytologist, 207, 119–134.
Zhang Y, Gao X, Sun M, Liu H, Xu J R. 2017. The FgSRP1 SR-protein gene is important for plant infection and pre-mRNA processing in Fusarium graminearum. Environmental Microbiology, 19, 4065–4079.
Zhao X H, Xu J R. 2007. A highly conserved MAPK-docking site in Mst7 is essential for Pmk1 activation in Magnaporthe grisea. Molecular Microbiology, 63, 881–894.
Zheng D, Zhang S, Zhou X, Wang C, Xiang P, Zheng Q, Xu J R. 2012. The FgHOG1 pathway regulates hyphal growth, stress responses, and plant infection in Fusarium graminearum. PLoS ONE, 7, e49495.
Zhou X, Li G, Xu J R. 2011. Efficient approaches for generating GFP fusion and epitope-tagging constructs in filamentous fungi. Methods in Molecular Biology, 722, 199–212.
Zhou X, Zhang H, Li G, Shaw B, Xu J R. 2012. The cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae. PLoS Pathogens, 8, e1002911.
Zhou X Y, Liu W D, Wang C F, Xu Q J, Wang Y, Ding S L, Xu J R. 2011. A MADS-box transcription factor MoMcm1 is required for male fertility, microconidium production and virulence in Magnaporthe oryzae. Molecular Microbiology, 80, 33–53.

相关文章 0

编辑推荐

Metrics