PdCYP51B 195 bp增强子在获得指状青霉DMI抗性和增加真菌毒力中的意义,Pesticide Biochemistry and Physiology

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PdCYP51B 195 bp增强子在获得指状青霉DMI抗性和增加真菌毒力中的意义
Pesticide Biochemistry and Physiology ( IF 4.2 ) Pub Date : 2020-05-01 , DOI: 10.1016/j.pestbp.2020.01.003
Marta de Ramón-Carbonell ₁ , Paloma Sánchez-Torres ₂

Affiliation

对来自指状青霉的两个甾醇 14α-脱甲基酶基因 PdCYP51A 和 PdCYP51B 进行了评估，发现 95% 的抑霉唑 (IMZ) 抗性分离株在 PdCYP51B 启动子中携带 195 bp 插入。我们通过过表达对两种甾醇 14α-去甲基酶进行了功能表征。过表达突变体的分子分析表明，PdCYP51B 插入的引入比先前描述的赋予 DMI 杀菌剂抗性的五串联重复 PdCYP51A 序列更稳定。这两种增强子可以共存于最初包含 195 bp PdCYP51B 插入的 P. digitatum 分离株中，但 195 bp PdCYP51B 增强子的引入促进了 PdCYP51A 的五串联重复序列的丢失。195-bp PdCYP51B 的掺入导致来自已经具有抗性的分离株的突变体的 DMI 杀真菌剂抗性增加，并赋予来自敏感分离株的突变体对 DMI 的抗性。除了来自五串联重复 PdCYP51A 序列的那些外，这两个基因的转录评估显示在所有过表达突变体中都有明显的诱导，而 PdCYP51A 表达急剧下降。与无菌生长相比，在柑橘感染期间只有 PdCYP51B 表现出上调，并且 PdCYP51B 在真菌毒力中的作用得到进一步加强，因为低毒力的菌株在过表达突变体中表现出增加的感染性。该研究通过替换具有相同推定功能的同源基因，表明 PdCYP51B 增强子在 DMI 抗性和真菌毒力的获得中起主要作用。

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Significance of 195 bp-enhancer of PdCYP51B in the acquisition of Penicillium digitatum DMI resistance and increase of fungal virulence

Two sterol 14α-demethylase genes from Penicillium digitatum, PdCYP51A and PdCYP51B, were evaluated and revealed that 95% of Imazalil (IMZ)-resistant isolates carried a 195-bp insertion in the PdCYP51B promoter. We functionally characterized both sterol 14α-demethylases by overexpression. Molecular analysis of overexpression mutants showed that the introduction of PdCYP51B insertion is more stable than the five-tandem repeat PdCYP51A sequence previously described that confers DMI fungicide resistance. The both enhancers can coexist in P. digitatum isolates that initially contained the 195-bp PdCYP51B insertion but the introduction of 195-bp PdCYP51B enhancer promoted the loss of the five-tandem repeat of PdCYP51A. The incorporation of 195-bp PdCYP51B resulted in an increase of DMI fungicide resistance in mutants from already resistant isolates and confers resistance to DMIs in mutants from sensitive isolates. Transcription evaluation of the both genes showed noticeable induction in all overexpression mutants, except for those coming from the five-tandem repeat PdCYP51A sequence, whereas PdCYP51A expression dropped dramatically. Only PdCYP51B exhibited up-regulation during citrus infection compared to axenic growth, and the role of PdCYP51B in fungal virulence was further reinforced since strains with low virulence showed increased infectivity in overexpression mutants. This study suggested the predominant role of the PdCYP51B enhancer in the acquisition of DMI resistance and fungal virulence, by replacing homologues genes with same putative function.

更新日期：2020-05-01

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