Vm-milRN7调控苹果树腐烂病菌致病的功能及机理

doi:10.3864/j.issn.0578-1752.2024.10.007

摘要/Abstract

摘要：

【背景】microRNA-like RNA（milRNA）是真菌中普遍存在的一类与动植物microRNA具有相似生成和作用机制的调控因子，广泛参与其生长、发育，以及植物病原真菌的侵染致病等生命活动。苹果树腐烂病菌（苹果黑腐皮壳Valsa mali）引起的腐烂病是影响苹果生产的最具毁灭性的病害。【目的】探究Vm-milRN7调控苹果树腐烂病菌致病的功能及机理，为苹果树腐烂病的靶向性抗病育种提供理论依据。【方法】以03-8菌株基因组DNA为模板扩增Vm-milRN7前体序列并构建Vm-milRN7前体过表达载体；扩增Vm-milRN7前体上下游序列并利用Double-joint PCR技术构建Vm-milRN7前体敲除盒。利用PEG介导的原生质体转化法构建Vm-milRN7前体过表达及敲除突变体。通过培养皿内生长试验及离体枝条、叶片接种试验分析Vm-milRN7前体过表达及敲除突变体对病菌营养生长及致病的功能。利用qRT-PCR以及烟草共转化试验验证Vm-milRN7对其靶标基因Vm-09496的调控作用；使用生物信息学软件对Vm-09496进行蛋白序列特征和系统发育分析。为解析其功能，创制Vm-09496的敲除突变体以及回补菌株，并鉴定其表型。【结果】利用PEG介导的遗传转化技术获得Vm-milRN7过表达转化株以及敲除突变体。营养生长观察发现，与野生型菌株相比，Vm-milRN7过表达转化株生长表型无明显差异，而敲除突变体营养生长速率显著降低；致病力检测发现，Vm-milRN7过表达转化株对苹果叶片的致病力显著增强，敲除突变体对苹果叶片和枝条的致病力均显著降低。qRT-PCR和烟草共转化试验分析发现，Vm-milRN7能够抑制其候选靶标基因Vm-09496的表达。生物信息学分析发现该基因编码一个假想蛋白，且与梨黑腐皮壳（V. pyri）中的VP1G_09956进化关系最近。创制Vm-09496的敲除突变体和回补菌株，发现与野生型菌株相比，敲除突变体对枝条和叶片的致病力均显著提高，而回补菌株营养生长速率及致病力恢复至野生型水平。【结论】Vm-milRN7通过调控靶标基因Vm-09496的降解参与苹果树腐烂病菌致病过程。靶标基因Vm-09496是影响腐烂病菌侵染的重要内源基因，在苹果树腐烂病菌内负向调控致病力。

关键词: 苹果黑腐皮壳, milRNA, 敲除突变体, 致病力, 转录后调控

Abstract:

【Background】MicroRNA-like RNAs (milRNAs) are a class of regulatory factors commonly found in fungi with similar generation and action mechanism as plant and animal microRNAs, which are widely involved in their growth and development, as well as in life activities such as infection and pathogenesis of plant pathogenic fungi. The apple Valsa canker caused by Valsa mali is the most destructive disease affecting apple production.【Objective】 The research aims to explore the function and mechanism of Vm-milRN7 in regulating the pathogenicity of V. mali, and to provide a theoretical basis for targeted disease resistance breeding of apple Valsa canker.【Method】 Vm-milRN7 precursor overexpression vector was constructed by amplifying Vm-milRN7 precursor sequence using genomic DNA of strain 03-8 as a template; upstream and downstream sequences of Vm-milRN7 precursor were amplified and Vm-milRN7 precursor knockout mutants were constructed by using Double-joint PCR technique. The Vm-milRN7 precursor overexpression strains and knockout mutants were constructed by PEG-mediated protoplast transformation. The vegetative growth rate of Vm-milRN7 precursor overexpression strains and knockout mutants was determined by cultivation on PDA medium, and the pathogenicity of these strains was verified by inoculation on apple twigs and leaves. The regulatory relationship between Vm-milRN7 and its potential target gene Vm-09496 was identified by qRT-PCR and co-infiltration experiment in Nicotiana benthamiana leaves; protein sequence characterization and phylogenetic analysis of Vm-09496 were performed using bioinformatics software. In order to analyze its function, Vm-09496 knockout mutants and complement strains were created and their phenotypes were characterized.【Result】 PEG-mediated genetic transformation was used to create Vm-milRN7 overexpression strains and knockout mutants. Vm-milRN7 overexpression strains showed no apparent alteration in vegetative growth rate compared with that of the wild-type strains, whereas the knockout mutants had a significantly lower vegetative growth rate. Compared with the wild-type strains, the overexpression strains showed a significant increase in the pathogenicity of V. mali on apple leaves, while the knockout mutants showed a significant decrease in the pathogenicity of V. mali on apple leaves and twigs. Further, qRT-PCR and co-infiltration in N. benthamiana leaves assay showed that Vm-milRN7 inhibited the expression of its potential target gene Vm-09496. Bioinformatics analysis revealed that the gene encodes an imaginary protein and is evolutionarily most closely related to VP1G_09956 in V. pyri. In order to analyze its function, the Vm-09496 knockout mutants and complement strains were created, and the pathogenicity of the knockout mutants on both twigs and leaves was significantly increased compared with that of the wild-type strain, while the vegetative growth rate and pathogenicity of the complement strains recovered to the wild-type level.【Conclusion】 Vm-milRN7 may be involved in the pathogenity of V. mali by regulating the expression of the target gene Vm-09496. The target gene Vm-09496 is an important endogenous gene affecting V. mali infection and negatively regulates V. mali pathogenicity.

Key words: Valsa mali, milRNA, knockout mutant, pathogenicity, post-transcriptional regulation

张健, 赵彬森, 冯浩, 黄丽丽. Vm-milRN7调控苹果树腐烂病菌致病的功能及机理[J]. 中国农业科学, 2024, 57(10): 1930-1942.

ZHANG Jian, ZHAO BinSen, FENG Hao, HUANG LiLi. Function and Mechanism Analysis of Vm-milRN7 Regulating the Pathogenicity of Valsa mali[J]. Scientia Agricultura Sinica, 2024, 57(10): 1930-1942.

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引物名称Primer name	引物序列Primer sequence (5′-3′)
Vm-milRN7OE-F	GTAGGAACCCAATCTTCAAATTCGTCTCCAAGCA
Vm-milRN7OE-R	TGAATGTTGAGTGGAATGATAGCAGTTCCAATTC
Vm-milRN7-1F	GACCGTATCGGTATCACCTAACG
Vm-milRN7-2R	CAGATACGGCAGAGAAATCGCAACCTCACAGCAAATCTCACGACCACTTC
Vm-milRN7-3F	GTTTAGATTCCAAGTGTCTACTGCTGGCCAGGATCGACAGACTGAACAGAA
Vm-milRN7-4R	CTAGAGGAATAACGAGGAACGGA
Vm-milRN7-5F	CCGCCCAAGTATTTCCGACC
Vm-milRN7-6R	AAACCTCTCCGCCGCCTAGA
Vm-milRN7-7F	AGTGGAGGCATAGCAATCAATT
Vm-milRN7-8R	TTCAACAGGTCCAAGGTTTAGG
Vm-09496-1F	CTGCTTGGCATGTTCCCTTAC
Vm-09496-2R	CAGATACGGCAGAGAAATCGCAACCTCCCCGCTTTGCTTACTTTGTGT
Vm-09496-3F	GTTTAGATTCCAAGTGTCTACTGCTGGCGGACCACATCACTACCTACTCG
Vm-09496-4R	TATTGCTCTATCCTGGCTTTCT
Vm-09496-5F	CCGATACCGACAGCCTTTACTT
Vm-09496-6R	GCTCATGGTCTGAATCTCCACC
Vm-09496-7F	TTTGGTGCCACTGTCGTCTATC
Vm-09496-8R	AACGGGCTTCTCAATCTCATGT
HB-Vm-09496-F	TCTCATCACCATCACCATCACCAGGATTGAAGGTCCCGGTGCG
HB-Vm-09496-R	TCGCCCTTGCTCACCCTCGAGGGAAGGGCTTTTTTCATTAGCAGCACA
Pri-N7-F	CATTTCATTTGGAGAGGACCATGACACAGTGAAGTGGTCGTGAGATT
Pri-N7-R	TCTAGTTCATCTAGAGGATCAAGCAGTTCCAATTCGATGTATTC
Tar-Vm-09496-F	CATTTCATTTGGAGAGGACCATGTTCCGCCTCGACTCCCGCCACGTCG
Tar-Vm-09496-R	GTTGTGTTGAGAATTCTCGAGGCGGTGCCGAACTCCTGGGCCCCT
RT-Vm-milRN7	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTAAATC
qRT-Vm09496-F	TTGTGTTTGGTCTGGTGTTGC
qRT-Vm09496-R	CGAGTCTGATTCCGTTTTCGT
G850-F	TCGGCTATGACTGGGCACAACA
G852-R	GAGCGGCGATACCGTAAAGCAC
G855-R	TGTTGGGTTTGAGCTAGGTGGGGTGA
G856-F	GAATGGTCAAATCAAACTGCTAGATAT
RT-VmU6	GCTTTCTTTGGTATAGCGTGTCA
VmU6-F	GGTCAATTTGAAACAATACAGAGAAGAT
VmU6-R	TCTTTGGTATAGCGTGTCATCCTTAG