INRA UR1290, BIOGER CPP, Thiverval-Grignon, France.
PLoS One. 2012;7(8):e42520. doi: 10.1371/journal.pone.0042520. Epub 2012 Aug 13.
Dicarboximides and phenylpyrroles are commonly used fungicides against plant pathogenic ascomycetes. Although their effect on fungal osmosensing systems has been shown in many studies, their modes-of-action still remain unclear. Laboratory- or field-mutants of fungi resistant to either or both fungicide categories generally harbour point mutations in the sensor histidine kinase of the osmotic signal transduction cascade.In the present study we compared the mechanisms of resistance to the dicarboximide iprodione and to pyrrolnitrin, a structural analogue of phenylpyrrole fungicides, in Botrytis cinerea. Pyrrolnitrin-induced mutants and iprodione-induced mutants of B. cinerea were produced in vitro. For the pyrrolnitrin-induced mutants, a high level of resistance to pyrrolnitrin was associated with a high level of resistance to iprodione. For the iprodione-induced mutants, the high level of resistance to iprodione generated variable levels of resistance to pyrrolnitrin and phenylpyrroles. All selected mutants showed hypersensitivity to high osmolarity and regardless of their resistance levels to phenylpyrroles, they showed strongly reduced fitness parameters (sporulation, mycelial growth, aggressiveness on plants) compared to the parental phenotypes. Most of the mutants presented modifications in the osmosensing class III histidine kinase affecting the HAMP domains. Site directed mutagenesis of the bos1 gene was applied to validate eight of the identified mutations. Structure modelling of the HAMP domains revealed that the replacements of hydrophobic residues within the HAMP domains generally affected their helical structure, probably abolishing signal transduction. Comparing mutant phenotypes to the HAMP structures, our study suggests that mutations perturbing helical structures of HAMP2-4 abolish signal-transduction leading to loss-of-function phenotype. The mutation of residues E529, M427, and T581, without consequences on HAMP structure, highlighted their involvement in signal transduction. E529 and M427 seem to be principally involved in osmotic signal transduction.
二羧酸酰亚胺和苯并吡咯类是常用的防治植物病原子囊菌的杀菌剂。尽管许多研究已经表明它们对真菌渗透感应系统的作用，但它们的作用机制仍然不清楚。实验室或田间对这两类杀菌剂都具有抗性的真菌突变体通常在渗透信号转导级联的传感器组氨酸激酶中存在点突变。在本研究中，我们比较了对二羧酸酰亚胺异菌脲和苯并吡咯类杀菌剂吡咯霉素的抗性机制，供试菌株为葡萄孢菌。在体外产生了吡咯霉素诱导的突变体和异菌脲诱导的突变体。对于吡咯霉素诱导的突变体，对吡咯霉素的高水平抗性与对异菌脲的高水平抗性相关。对于异菌脲诱导的突变体，对异菌脲的高水平抗性产生了对吡咯霉素和苯并吡咯类的可变水平的抗性。所有选定的突变体对高渗透压表现出超敏性，并且无论它们对苯并吡咯类的抗性水平如何，与亲本表型相比，它们的适应性参数（孢子形成、菌丝生长、对植物的侵袭性）都明显降低。大多数突变体在第三类组氨酸激酶的渗透压感应域中发生了修饰，影响了 HAMP 结构域。应用定点突变技术对 bos1 基因进行了验证，其中有 8 个突变得到了验证。HAMP 结构域的结构建模表明，HAMP 结构域内疏水性残基的取代通常会影响它们的螺旋结构，可能会破坏信号转导。将突变体表型与 HAMP 结构进行比较，我们的研究表明，突变体扰乱 HAMP2-4 的螺旋结构会导致信号转导中断，从而导致功能丧失表型。对 HAMP 结构没有影响的 E529、M427 和 T581 残基的突变，突显了它们在信号转导中的参与。E529 和 M427 似乎主要参与渗透信号转导。
