title: Heterologous expression of the Monilinia fructicola CYP51 (MfCYP51) gene in Pichia pastoris confirms the mode of action of the novel fungicide, SYP-Z048.
abstract: The novel agricultural fungicide 3-[5-(4-chlorophenyl)-2,3-dimethyl-3-isoxazolidinyl] pyridine (SYP-Z048) developed by China Shenyang Research Institute of Chemical Industry has been confirmed to be an ergosterol biosynthesis inhibitor (EBI). Previous studies have shown that EBIs target the proteins from a range of genes, including CYP51, ERG2 and/or ERG24, and ERG27, which are involved in the ergosterol biosynthesis pathway. In the current study the ERG2, ERG24, and ERG27 genes were cloned from wild type and resistant mutants of Monilinia fructicola in an attempt to clarify the target site of SYP-Z048. Comparative analysis of the deduced aa sequence of these genes, as well as CYP51, revealed several point mutations that resulted in amino acid variation among the sensitive and resistant isolates. However, sensitivity assays indicated that only one, the substitution of phenylalanine (F) for the tyrosine (Y) at 136 in CYP51, was correlated with reduced sensitivity to SYP-Z048. Heterologous expression of MfCYP51-136Y (MfCYP136Y) and MfCYP51-136F (MfCYP136F) in Pichia pastoris revealed that MfCYP136F significantly reduced sensitivity to SYP-Z048, increasing the average EC50 of the transformants 11-fold relative to those carrying MfCYP136Y. However, neither the additional copy of MfCYP136Y nor multiple copies of MfCYP136F were found to reduce sensitivity relative to the empty vector control or single copy transformants, respectively. Molecular docking experiments using SYP-Z048 with HsCYP145Y and the mutated version HsCYP145F as substitutes for MfCYP136Y and MfCYP136F, respectively, indicated that the reduced affinity of HsCYP145F for SYP-Z048 resulted from the loss of a hydrogen bond between the fungicide and the active site. Taken together these results indicate that MfCYP51 is the major target site of SYP-Z048 in M. fructicola, which has important implications for the resistance management of this fungicide in the field.
authors.affiliation: Key Laboratory of Plant Virology of Fujian Province, Institute of Plant Virology, Fujian Agriculture and Forestry University Fuzhou, China ; Department of Plant Pathology, China Agricultural University Beijing, China.
authors.forename: Fengping
authors.initials: F
authors.lastname: Chen
authors.affiliation: Department of Plant Pathology, China Agricultural University Beijing, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Lin
authors.affiliation: Department of Plant Pathology, China Agricultural University Beijing, China.
authors.forename: Jingyuan
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Plant Pathology, China Agricultural University Beijing, China ; Taizhou Entry-Exit Inspection and Quarantine Bureau Zhejiang, China.
authors.forename: Botao
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Chemistry, College of Science, China Agricultural University Beijing, China.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Duan
authors.affiliation: State Key Laboratory of the Discovery and Development of Novel Pesticide, China Shenyang Research Institute of Chemical Industry Shenyang, China.
authors.forename: Junli
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Plant Pathology, China Agricultural University Beijing, China.
authors.forename: Xili
authors.initials: X
authors.lastname: Liu
keywords: Monilinia fructicola; SYP-Z048; Y136F; heterologous expression; mode of action; point mutation