ATP硫酸化酶基因MoMET3在稻瘟病菌生长发育和致病过程中的功能分析

doi:10.16819/j.1001-7216.2017.7018

摘要/Abstract

摘要：

目的真菌对硫元素的利用始于ATP硫酸化酶对硫酸根离子的活化。对稻瘟病菌ATP硫酸化酶在病菌生长发育和致病过程中的功能进行分析,可为稻瘟病的防治提供药物靶标。方法采用同源重组方法构建基因敲除载体,并对突变体的表型进行分析。结果 MoMET3基因是病菌营养生长和分生孢子形成所必需的,但不是毒力因子。稻瘟病菌ATP硫酸化酶编码基因MoMET3缺失后,突变体营养生长速率减慢,产孢量降低,但是突变体对寄主的毒力不受影响;虽然基因缺失不影响突变体分生孢子萌发和附着胞成熟,但突变体在硫营养限制时孢子萌发后次生菌丝的生长明显受抑。MoMET3基因互补后,突变体恢复正常生长,能利用硫酸盐。结论无机硫的还原对分生孢子的萌发和附着胞的成熟并不是必需的,分生孢子内储存的还原态硫化合物可以满足这一过程中硫营养的需求,但病菌侵入寄主组织后需要吸收利用寄主源还原态硫以利于侵染菌丝的扩展。

关键词: 稻瘟病菌, ATP硫酸化酶, MoMET3, 功能分析

Abstract:

【Objective】 Sulfur is generally made biologically available by activation with ATP in a reaction catalyzed by ATP sulfurylase in fungi. The function of ATP sulfurylase in the growth and pathogenecity of Magnaporthe oryzae was analyzed to lay a foundation for target determination for rice blast prevention.【Method】The knockout vector was constructed by homologous recombination method, and the phenotype of the mutant was analyzed.【Result】MoMET3 was required for vegetative growth and conidiation, but not a virulence factor. The growth rate and conidiation of MoMET3- deleted mutants were significantly decreased in complete medium. Loss of MoMET3 did not affect conidial germination and appressiorial formation, but the growth of secondary hyphae was inhibited in the minimal medium. After complementation of MoMET3 gene, the mutant returned to normal growth and can utilize sulfate. 【Conclusion】These evidences revealed that the reduction of inorganic sulfur is not necessary for the process of conidial germination and appressiorial formation. The endogenous reduced sulfur in conidia was sufficient to meet the sulfur needs in the process of conidial germination and appressiorial formation, and utilization of host-derived reduced sulfur played an important role in the hypha expansion of the blast fungus in hosts.

Key words: Magnaporthe oryzae, ATP sulfurylase, MoMET3, functional analysis

中图分类号:

冯向阳, 张震, 柴荣耀, 邱海萍, 王教瑜, 毛雪琴, 王艳丽, 孙国昌. ATP硫酸化酶基因MoMET3在稻瘟病菌生长发育和致病过程中的功能分析[J]. 中国水稻科学, 2017, 31(5): 542-550.

Xiangyang FENG, Zhen ZHANG, Rongyao CHAI, Haiping QIU, Jiaoyu WANG, Xueqin MAO, Yanli WANG, Guochang SUN. Functional Analysis of MoMET3 in Growth, Development and Pathogenicity of Magnaporthe oryzae[J]. Chinese Journal OF Rice Science, 2017, 31(5): 542-550.

图/表 8

表1 本研究所用引物

Table 1 Primers used in this study.

引物Primer	序列 Sequence(5'-3')	功能 Function	目的片段大小 Size of the target fragment / kb
APSP11	ATTATTATGGAGAAACTCGAGGATGCTCCTGAGTCGCCGTCATCGA	扩增MoMET3上游片段	1.5
APSP12	TAGGGTACCGAGCTCGAATTCGAGGGTTGGCCATTATTGGCGTGCGTG	扩增MoMET3上游片段	1.5
APSP17	TCAATATCATCTTCTGGATCCCGTTTGTAAAACAGAAGCCTTGCACT	扩增MoMET3下游片段	1.5
APSP18	GCCAAGCTTGCATGCCTGCAGTGGGACGAAGACTGCAGATCGCTATCG	扩增MoMET3下游片段	1.5
BAR-F	GAATTCGAGCTCGGTACCCTAAATCTCGGTGACGGGCAGGACCGG	扩增BAR表达盒	0.9
BAR-R	GGATCCAGAAGATGATATTGAAGGAGCACTTTTTGGGCTTGGCTG	扩增BAR表达盒	0.9
BAR-CF	CTCGGTGACGGGCAGGACCGG	验证T-DNA是否整合到稻瘟病菌基因组中	0.9
BAR-CR	GGAGCACTTTTTGGGCTTGGCTG	验证T-DNA是否整合到稻瘟病菌基因组中	0.9
APSP41	TACAATGGCGTCGTCAAGGA	扩增MoMET3基因片段	0.8
APSP42	AGGTAGGTCATCATCTGGAAGG	扩增MoMET3基因片段	0.8
APSP45	CTATCGGCGGGTAATAAGGGAACTG	验证同源重组事件的发生情况	2.0/2.7
APSP46	GGTTCTCACCGTCTATTCCTTATCAC	验证同源重组事件的发生情况	2.0/2.7
APSP49	ATTATTATGGAGAAACTCGAGGATGCTCCTGAGTCGCCGTCATCGA	扩增用于互补的MoMET3片段	3.6
APSP50	CCCGGGTACCGAGCTCGAATTCGTTGGTGATGATGTATGTGCGACCG	扩增用于互补的MoMET3片段	3.6

图1 MoMET3基因缺失突变体的获得

Fig. 1. Generation of the MoMET3 deletion mutants.

图2 供试菌株在CM平板上的菌落形态与产孢情况(Mean±SD, n=6)

Fig. 2. Morphology and conidiation of the tested M. oryzae strains on CM medium(Mean±SD, n=6).

图3 供试菌株对硫营养的利用情况

Fig. 3. Utilization of sulfur compounds of the tested M. oryzae strains on minimal medium.

图4 供试菌株在基本培养基上的菌丝形态

Fig. 4. Morphology of M. oryzae on minimal medium.

图5 供试菌株分生孢子的萌发和附着胞的分化(Mean±SD, n=8)

Fig. 5. Conidial germination and appressorial formation of MoMET3 deletion mutants(Mean±SD, n=8)

图6 MoMET3基因缺失突变体的致病性

Fig. 6. Pathogenicity test of MoMET3 deletion mutants.

图7 供试菌株对硫营养的利用情况和在CM平板上的菌落形态

Fig. 7. Utilization of sulfur compounds of the tested M. oryzae strains on minimal medium and the morphology of the tested M. oryzae strains on CM medium.

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