The CYP51F1 Gene of Leptographium qinlingensis: Sequence Characteristic, Phylogeny and Transcript Levels

Abstract

Leptographium qinlingensis is a fungal associate of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandi) that must overcome the terpenoid oleoresin defenses of host trees. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include export and the use of these compounds as a carbon source. As one of the fungal cytochrome P450 proteins (CYPs), which play important roles in general metabolism, CYP51 (lanosterol 14-α demethylase) can catalyze the biosynthesis of ergosterol and is a target for antifungal drug. We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae). The transcription level of CYP51F1 following treatment with terpenes and pine phloem extracts was upregulated, while using monoterpenes as the only carbon source led to the downregulation of CYP5F1 expression. The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1). The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

Protective effects of selenium against zearalenone-induced apoptosis in chicken spleen lymphocyte via an endoplasmic reticulum stress signaling pathway

“…The 260/280 ratio of all samples was between 1.8 and 2.0. Purified RNA was subjected to reverse transcription to cDNA by FastKing RT Kit (With gDNase) (Tiangen Biotech, Beijing, China) following the manufacturer's instructions (Dai et al 2015).…”

Phylogeny of Leptographium Qinlingensis Cytochrome P450 Genes and Their Expression When Grown on Different Media or Treated With Terpenoids

“…The host chemical compound tolerance of L. qinlingensis was determined with the MIC test, and the induction of CYP genes by monoterpenes and pine extract was identi ed. This fungus is symbiotic with D. armandi and has considerable similarity with G. clavigera, which can retrieve nutrition from pine wood and utilize monoterpenes as a carbon source (DiGuistini et al 2011;Lah et al 2013;Dai et al 2015b). Some CYP genes might be involved in fatty acid metabolism and detoxify terpenes and phenolics, similar to other blue-stained fungi, which also indicates the pathogenic properties of L. qinlingensis in Chinese white pine.…”

“…For six P450 genes and the reference gene EF (Dai et al 2015b), speci c primers were designed using Primer Premier 5.0 (Table S2). To estimate the qPCR e ciency and validate the primers for each gene, a linear regression analysis was performed between the mean values of the quanti cation cycles (Cq) of different dilutions (1.0, 10 − 1 , 10 − 2 , 10 − 3 , and 10 − 4 ) of cDNAs and the initial concentration.…”

“…The constitutive and induced defensive system of P. armandi consists of a multitude of monoterpenes, sesquiterpenes and diterpenes (Chen et al 2006) and can stop or delay beetles from entering trees (Erbilgin et al 2003). Moreover, monoterpenes present in the resin, including pinene, limonene and carene, can injure or kill beetles and inhibit fungal growth (Reid and Purcell 2011;Dai et al 2015aDai et al , 2015b. Previous studies have shown that symbiotic fungi destroy bleeding cells, block resin canals in host trees and kill epithelial cells, thereby resulting in disorders of the nutrient and water metabolism of the host (Chen and Tang 2002).…”

“…Treatment with a terpenoid blend or pine phloem extract for associated fungi of bark beetles (D. ponderosae and D. armandi) always induces speci c CYPs (Lah et al 2013;Dai et al 2015b). Thus, cytochrome P450 enzymes that are highly induced by terpenes and metabolize or utilize monoterpenes are considered the major mechanisms that enable fungal resistance to monoterpenes (Lah et al 2013;Wang et al 2014).…”