CHD in Alternaria alternata: gene cloning and degradation performance against phenolic acids in pear fruit peel

Abstract:

[Background] To successfully infect and colonize the host tissue, phytopathogenic fungi need to detoxify the antifungal secondary metabolites such as phenolic compounds in the host. Studies have shown some pathogenic fungi can degrade phenolic compounds in the host by synthesizing catechol 1,2-dioxygenase (CHD). However, whether the Alternaria alternata can degrade phenols in pear fruit peel and the mechanism remain unclear. [Objective] To clone CHD from A. alternata, conduct bioinformatics analysis of CHD, and study the degradation performance of phenolic acids in pear fruit peel by CHD, thus providing support for further clarifying the molecular mechanism of this enzyme. [Methods] CHD was cloned from A. alternata by homologous cloning. Smart and SOPMA were used for bioinformatics analysis. The changes in phenolic acid content after metabolism of A. alternata were determined by quaternary ultra-fast liquid chromatography, and the expression level of CHD during the metabolism of phenolic acids was analyzed by RT-qPCR. [Results] Two CHD genes were cloned from A. alternata and designated as AaCHD1 and AaCHD2. The proteins encoded by the two genes both contained C-terminal and N-terminal conserved domains of dioxygenases. The phylogenetic analysis showed that AaCHD1 and AaCHD2 were highly homologous to the CHD amino acid sequences in Stemphylium lycopersici and Cochliobolus heterostrophus, respectively. Furthermore, A. alternata can grow with phenolic acids as the sole carbon source and differentially metabolize phenolic acids in pear fruit peel. Specifically, it showed stronger degradation effects on ferulic acid, chlorogenic acid, and caffeic acid than on p-coumaric acid. At the same time, exogenous phenolic acids up-regulated the expression of AaCHD1 and AaCHD2, and exogenous caffeic acid had the strongest up-regulatory effect, followed by ferulic acid and p-coumaric acid. [Conclusion] AaCHD in A. alternata played a role in degrading phenolic acids in pear fruit peel, and the degradation effects vary with different phenolic acids, which provides a theoretical basis for comprehensively revealing the molecular mechanism of AaCHD in overcoming the defense effect of phenolic acids in pear peel.