Abstract: 【Objective】The objective of this study is to clarify the function of cAMP phosphodiesterase (PDE) and cAMP signal pathway in regulating the pathogenicity of Setosphaeria turcica, the genes encoding cAMP phosphodiesterase were cloned and the expression pattern of these genes were analyzed during the development of infective structures and the early stage of infecting the host. 【Method】Based on conserved sequence of PDE genes from Saccharomyces cerevisiae, Candida albicans, Botrytis cinerea, Magnaporthe oryzae, Metarhizium anisopliae and Aspergillus niger, the homologous fragments were amplified by degenerate primers PCR, and the full length PDE genes and flanking sequences were obtained by combining RACE and Genome Walking. Multiple sequence alignment for prediction coding product of the protein was constructed by MEGA 5.0 software and phylogenetic tree was constructed by adjacent method. Gene structure was analyzed by GSDS. Physical and chemical properties were analyzed by ProtParam. Secondary structure was predicted by SOMPA. Conserved domains were analyzed by SMART on line. The material of different development periods from S. turcica was collected under the induction on artificial hydrophobic medium and on the susceptible host surface. Quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression patterns.【Result】There were one high-affinity phosphodiesterase gene (StH-PDE) and one low-affinity phosphodiesterase gene (StL-PDE) in the genome of S. turcica. Full-length of StH-PDE was 3 208 bp, which consisted of 5 introns and 6 exons, the coding region was 2 898 bp. The full length of StL-PDE was 5 054 bp, which contained 4 introns and 5 exons, with the coding region of 3 090 bp. StH-PDE protein contained conservative type I cAMP phosphodiesterase, and StL-PDE contained L-PDE specific type II cAMP phosphodiesterase catalytic domain. PDE homologous genes in different plant pathogenic fungi presented a high degree of similarity. StH-PDE and the high affinity phosphodiesterase gene from Magnaporthe grisea, Cordyceps militaris, Metarhizium acridum were clustered in the same evolution. StL-PDE and the low affinity phosphodiesterase gene from Ascochyta rabiri, Scedosporium apiospermum, Fusarium oxysporum, Metarhizium album were clustered in the same evolution. Compared with the mycelia, the expression levels of StL-PDE and StH-PDE in conidia were significantly up-regulated, and StL-PDE and StH-PDE were up-regulated by about 52-fold and 2-fold under the induction on artificial hydrophobic medium, respectively. The expression of both genes at the early stage of spore germination was significantly down-regulated. With the germination process, the expression level gradually increased, and the second peak appeared at the appressorial formation stage, and then the expression level was down again. However, in the whole process, the highest expression level of StH-PDE appeared in the appressorial formation period, reaching nearly 7-fold and 2-fold of that in the mycelia and the conidia, respectively, and the expression level of StL-PDE was always lower than that in conidia. During the interaction of S. turcica with the susceptible host, the expression of StH-PDE and StL-PDE in the process of pathogen infection was consistent with that under the induction of artificial hydrophobic material. The expression of both genes in the early stage of spore germination was significantly down-regulated. With the germination process, the expression level gradually increased. The expression of StH-PDE was down-regulated in 18 and 24 h post-inoculation beyond the early stage of spore germination. 【Conclusion】StH-PDE and StL-PDE showed a down-regulation-up-regulated-down-regulated expression pattern in the development of pathogen infection. The relative expression level of StH-PDE was the highest during the appressorial formation, and the expression level of StL-PDE in conidia was the highest.

Key words: Setosphaeria turcica, cAMP phosphodiesterase, quantitative real-time PCR, gene expression