苹果树腐烂病菌含cupin结构域蛋白Vmcupin1的鉴定及功能分析

Identification and functional analysis of cupin domain-containing protein Vmcupin1 in Valsa mali

由苹果黑腐皮壳(Valsa mali)侵染引起的苹果树腐烂病是严重威胁我国苹果产业健康发展的重大枝干病害。揭示病菌致病机理有助于制定病害防控新策略。含cupin结构域蛋白是一个大的蛋白家族,广泛参与植物发育和逆境胁迫响应等多种生命活动。有关植物病原真菌中该类蛋白的研究报道很少,其参与菌丝生长和侵染致病的生物学功能尚不明确。基于V.mali与苹果树皮互作的转录组分析,发现一个在病菌侵染过程中显著上调表达的基因。蛋白序列特征分析发现,该蛋白含有1个典型的cupin结构域,且与其他物种含cupin结构域蛋白高度同源,将之命名为Vmcupin1。实时荧光定量分析发现Vmcupin1在病菌侵染过程中显著上调表达。创制了Vmcupin1缺失突变体和回补菌株,发现该基因缺失后,病菌生长速率在一定程度上降低,其致病力和适应H2O2和NaCl胁迫的能力显著降低,表明Vmcupin1在病菌营养生长、侵染致病,以及逆境胁迫中发挥重要功能。研究结果丰富了真菌含cupin结构域蛋白功能的认知,有助于全面解析腐烂病菌致病机理。

Apple tree valsa canker,caused by Valsa mali,is a serious branch disease threatening the healthy development of the apple industry in China.Revealing its pathogenic mechanism has important theoretical significance for formulating new strategies for disease control.Cupin domain-containing proteins belong to a large protein family,and are involved in many biological processes in plants,such as development and stress-related responses.There are few studies on this kind of protein in plant pathogenic fungi,and its biological function in mycelial growth and infection is still unclear.Based on the transcriptome analysis between V.mali and apple twig bark tissues,a candidate gene was significantly up-regulated during pathogen infection.The gene encoded a cupin domain-containing protein,which was highly homologous with cupin domain-containing proteins from other organisms,and it was named Vmcupin1.Gene expression analysis by qRT-PCR revealed that Vmcupin1 was significantly up-regulated during pathogen infection.Furthermore,Vmcupin1 deletion mutants and complement transformants were created,respectively.It was found that the growth rate of mutants was slightly reduced while the pathogenicity and ability to adapt to H2O2 and NaCl stress were significantly reduced,indicating that Vmcupin1 plays an important role in the vegetative growth,pathogenicity and stress response of V.mali.These results lay an important foundation for the comprehensive analysis of pathogenic mechanism of V.mali and the enrichment of functional cognition of fungal cupin domain-containing proteins.