Functional analysis of the putative ribosome biogenesis factor MoRei1in <i>Magnaporthe oryzae</i>

doi:10.3785/j.issn.1008-9209.2021.06.153

Journal of Zhejiang University (Agriculture and Life Sciences)

2022, Vol. 48

Issue (4): 443-452 DOI: 10.3785/j.issn.1008-9209.2021.06.153

Plant protection

Functional analysis of the putative ribosome biogenesis factor MoRei1in Magnaporthe oryzae

Shuai TANG(

),Zhe XU,Wuyun Lü,Qi TONG,Yu XIAO,Zhengyi WANG(

)

State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China

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Abstract

To further explore the biological roles of MoRei1in the rice blast fungus Magnaporthe oryzae, we carried out MoREI1 gene deletion, phenotypic analysis of ΔMorei1 mutants, gene complementation and identification of its interaction proteins. The results showed that the ΔMorei1 mutants were defective in sexual reproduction, appressorium formation and pathogenicity. Deletion of MoREI1 led to significant increase of sensitivity to cell wall damaging and oxidative stress agents, and delay of glycogen mobilization and degradation during appressorium development. Moreover, the MoREI1 gene could partially complement the defects of Saccharomyces cerevisiae Δrei1 mutants, indicating that MoRei1 is a functional homolog of yeast Rei1, and may share a similar role in ribosome biogenesis. MoRei1 physically interacts with MoAlb1, a predicted nuclear export factor. Interestingly, deletion of MoREI1 resulted in the impairment of nuclear localization of MoAlb1. These results lay a foundation to clarify the roles of MoRei1 in fungal morphogenesis and pathogenicity in M. oryzae.

Key words： Magnaporthe oryzae ribosome biogenesis factor MoRei1 functional analysis MoAlb1 protein

Received: 15 June 2021 Published: 03 September 2022

CLC:

S 432.1

Corresponding Authors: Zhengyi WANG E-mail: 21816071@zju.edu.cn;zhywang@zju.edu.cn

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Cite this article:

Shuai TANG,Zhe XU,Wuyun Lü,Qi TONG,Yu XIAO,Zhengyi WANG. Functional analysis of the putative ribosome biogenesis factor MoRei1in Magnaporthe oryzae. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(4): 443-452.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2021.06.153 OR https://www.zjujournals.com/agr/Y2022/V48/I4/443

稻瘟病菌假定核糖体生成因子MoRei1功能研究

为深入探究稻瘟病菌假定核糖体生成因子MoRei1的生物学功能，通过敲除MoREI1基因、分析ΔMorei1突变体表型、鉴定基因互补及其互作蛋白功能等对其进行了研究。结果表明：敲除MoREI1基因后，稻瘟病菌ΔMorei1突变体的有性生殖能力丧失，附着胞形成率显著下降，致病力减弱；ΔMorei1突变体对细胞壁和氧化胁迫因子的敏感性增强，附着胞发育过程中糖原的移动和降解速率下降。稻瘟病菌MoREI1基因可恢复酿酒酵母REI1基因缺失突变体的部分表型，说明MoRei1与酵母核糖体生成因子Rei1在功能上具有同源性，并可能在核糖体生成过程中起类似的作用。稻瘟病菌MoRei1蛋白与预测的核输出因子MoAlb1间存在物理互作，且MoREI1基因缺失可影响稻瘟病菌MoAlb1蛋白的亚细胞定位。上述研究结果对阐明MoRei1在稻瘟病菌形态分化和致病性中的作用及其机制有重要意义。

关键词： 稻瘟病菌, 核糖体生成因子MoRei1, 功能分析, MoAlb1蛋白

Table 1 Primers used in this study

Fig. 1 Phylogenetic analysis of Rei1 homologs from different fungal species

Fig. 2 Growth status of the wild type BY4741, mutant Δrei1 and complementation strains Δreil/MoREI1 of S. cerevisiae at different temperaturesNegative control: Mutant strains of S. cerevisiae YBR267W inserted by the empty plasmid pYES2.

Fig. 3 Sexual reproduction tests of the wild type strainGuy11 and ΔMorei1-22 mutant of M. oryzae

Fig. 4 Influence of MoREI1 deletion on pathogenicity, appressorium formation and penetration of M. oryzaeA. Lesions on rice leaf at 5 d after spraying conidia; B. Morphology of appressorium (24 h); C. Formation rate of appressorium (24 h); D. Types of the invasion hyphae (AP: Appressorium; IH: Invasion hypha); E. Penetration assays of the barley epidermis and the proportions of different invasion hyphae (48 h). Double asterisks (**) indicate significant differences at the 0.01 probability level.

Fig. 5 Growth status of the wild type strain Guy11 and ΔMorei1-22 of M. oryzae on the stress media that contain the cell wall or oxidative stress factorsA. Growth status of various tested strains on the stress media; B. Growth inhibition rate of the tested strains. CM: Complete medium; CR: Congo red. Single asterisk (*) indicates significant differences at the 0.05 probability level, and the same as below.

Fig. 6 Influence of MoREI1 deletion on mobilization and degradation of glycogens during appressorium formation of M. oryzaeA. Staining of the conidia with KI/I₂ solution; B. Statistic of glycogen mobilization in conidia of different tested strains; C. Statistic of glycogen degradation in appressoria of different tested strains.

Fig. 7 Identification of the interaction protein of MoRei1Positive control: Wild type strains of S. cerevisiae BY4741 inserted by the plasmid pGADT7+pGBKT7-53; Negative control: Wild type strains of S. cerevisiae BY4741 inserted by the plasmid pGADT7+pGBKT7-lam. AD: Activation domain; BD: DNA binding domain.

Fig. 8 Influence of MoREI1 deletion on thenuclear localization of MoAlb1


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