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| Mutations in FgPrp6 suppressive to the Fgprp4 mutant in Fusarium graminearum |
| LI Chao-hui1, 2, FAN Zhi-li1, HUANG Xin-yi1, WANG Qin-hu1, JIANG Cong1, XU Jin-rong1, 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
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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FgPrp6E308K-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/FgPRP6Δ199-221-GFP或Fgprp6/FgPRP6Δ250-262-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/FgPRP6Δ199-221-GFP和Fgprp6/FgPRP6Δ250-262-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/FgPRP6Δ199–221-GFP or Fgprp6/FgPRP6Δ250–262-GFP showed that 28 and 35% introns had splicing defects, respectively, which may be responsible for their defects in plant infection.
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Received: 19 January 2021
Accepted: 12 May 2021
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| Fund: 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). |
| About author:
Correspondence JIN Qiao-jun, Tel: +86-29-87082710, E-mail: jqiaojun@nwafu.edu.cn |
Cite this article:
LI Chao-hui, FAN Zhi-li, HUANG Xin-yi, WANG Qin-hu, JIANG Cong, XU Jin-rong, JIN Qiao-jun.
2022.
Mutations in FgPrp6 suppressive to the Fgprp4 mutant in Fusarium graminearum. Journal of Integrative Agriculture, 21(5): 1375-1388.
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