AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata

Aims Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. Methods and results A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6 h) and a 1.46-fold upregulation during the infection hyphae formation period (8 h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. Conclusions AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. Significance and impact of study Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.