Function Analysis Of PdOs2, A Mitogen-activated Protein Kinase Gene From Penicillium Digitatum

P. digitatum is the most devastating pathogen of citrus fruit, being responsible for about 90%of production losses during post-harvest handling. Fludioxonil, a phenylpyrrole fungicide, recently received federal registration for postharvest use on citrus in the US. But its antifungal mechanism is unclear. Related research from mode fungi and certain plant pathogenic fungi showed that fludioxonil is extremely likely to interfere with the two-component system-high osmolarity glycerol response pathway (HK-Hogl) Therefore, in an attempt to study the antifungal mechanism of fludioxonil in P. digitatum, we cloned the critical gene PdOs2 of the Hogl signaling pathways and analysis its function.The sequence (4247bp) including the PdOs2 coding region, as well as its upstream and donstream was cloned by using the method of degenerate primer followed by Tail-PCR. Sequence analysis indicated that the PdOs2 contained an open reading frame of 1076 bp, interrupted by 8 introns of 86bp,73bp,52bp,55bp,54bp,60bp,60bp, and 57bp, located at positions between 56bp to 143bp,190bp to 264bp,393bp to 446bp,489bp to 545bp,644bp to 699bp,1055bp to 1116bp,1194bp to 1255bp, and 1438bp to 1496bp, respectively. The deduced PdOs2p consisted of 368 amino acids, with an estimated molecular mass of 42kDa and a pl of 5.16. Alignment of putative amino acide showed that PdOs2 was highly identity to the sequences of Botrytis cinerea BcSAK1 (90%), Magnaporthe grisea Osml(92%)and Alternaria alternate AaHOG1 (91%). Moreover, sequence comparison of PdOs2 with other MAP kinases revealed the presence of all characteristic conserved subdomains, including the dual phosphorylation site TGY at positions 171to 173.In order to study the function of PdOs2, the PdOs2 disrupted mutants and its complement mutants were created by the method of Agrobacterium tumefaciens-mediated transformation (ATMT), and their growth, sprolation, sensitivity to high osmatic stress and fungicide iprodione and fludioxonil, as well as pathogenicity were compared with their parental isolate PdOl. The results indicated that the mycilial growth was impaired slightly, whereas its spoliation was blocked extremely as PdOs2 was disrupted. The PdOs2 disruption mutants (â–³Os2) exhibited increased sensitivity to NaCl, KCl and D-sorbitol, and such increased sensitivity was restored by re introducing the PdOs2 gene intoâ–³Os2. In comparison to Pd01,â–³Os2 exhibited decreased sensitivity to fungicides iprodione and fludioxonil. In addition, the pathogenicity of P. digitatum Pd01 was impaired slightly once PdOs2 was disprupted. These results indicated that PdOs2 is involved in the regulation of vegetative growth, differentiation, virulence, adaptation to hyperosmotic stress. And the fungicides iprodione and fludioxonil most likely exhibite fungicidal effects through hyperactivation of a fungal Hogl signal transduction pathway.In addition, the critical genes HK-Hogl pathway, hybrid two histidine kinase NikA, MAPK kinase kinase Ssk2, Transcriptional regulatory factors AtfA, in Hogl signaling pathways were cloned and their deletion mutants were constructed in this study. Such works would be facilite to the function study of these genes.