AUTHOR=Chen Fengping , Lin Dong , Wang Jingyuan , Li Botao , Duan Hongxia , Liu Junli , Liu Xili 

TITLE=Heterologous expression of the Monilinia fructicola CYP51 (MfCYP51) gene in Pichia pastoris confirms the mode of action of the novel fungicide, SYP-Z048

JOURNAL=Frontiers in Microbiology

VOLUME=6

YEAR=2015

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2015.00457

DOI=10.3389/fmicb.2015.00457

ISSN=1664-302X

ABSTRACT=<p>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 <italic>CYP51</italic>, <italic>ERG2</italic> and/or <italic>ERG24</italic>, and <italic>ERG27</italic>, which are involved in the ergosterol biosynthesis pathway. In the current study the <italic>ERG2</italic>, <italic>ERG24</italic>, and <italic>ERG27</italic> genes were cloned from wild type and resistant mutants of <italic>Monilinia fructicola</italic> in an attempt to clarify the target site of SYP-Z048. Comparative analysis of the deduced aa sequence of these genes, as well as <italic>CYP51</italic>, 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 <italic>CYP51</italic>, was correlated with reduced sensitivity to SYP-Z048. Heterologous expression of <italic>MfCYP51</italic>-<italic>136Y</italic> (<italic>MfCYP136Y</italic>) and <italic>MfCYP51</italic>-<italic>136F</italic> (<italic>MfCYP136F)</italic> in <italic>Pichia pastoris</italic> revealed that <italic>MfCYP136F</italic> significantly reduced sensitivity to SYP-Z048, increasing the average EC<sub>50</sub> of the transformants 11-fold relative to those carrying <italic>MfCYP136Y</italic>. However, neither the additional copy of <italic>MfCYP136Y</italic> nor multiple copies of <italic>MfCYP136F</italic> were found to reduce sensitivity relative to the empty vector control or single copy transformants, respectively. Molecular docking experiments using SYP-Z048 with <italic>HsCYP145Y</italic> and the mutated version <italic>HsCYP145F</italic> as substitutes for <italic>MfCYP136Y</italic> and <italic>MfCYP136F</italic>, respectively, indicated that the reduced affinity of <italic>HsCYP145F</italic> for SYP-Z048 resulted from the loss of a hydrogen bond between the fungicide and the active site. Taken together these results indicate that <italic>MfCYP51</italic> is the major target site of SYP-Z048 in <italic>M. fructicola</italic>, which has important implications for the resistance management of this fungicide in the field.</p>