Functional Analysis Of CPK1 Protein Kinase In Fusarium Graminearum And Its Regulation Of DON

Fusarium head blight(FHB), caused by Fusarium graminearum, is the second economically devastating plant disease on wheat, just following stripe rust, for not only does it can cause significant reduction in seed yields and quality, but also the infested seeds often contaminated with deoxynivalenol and zearalenon, which pose a serious threat to human and animal health. As a result of the huge destruction of FHB, the difficulty to breed resistant cultivars, the scarcity of resistance resources, it is very urgent for us to understand the molecular mechanism of pathogenicityof Fusarium head blight. Like many other filamentous ascomycetes, Fusarium graminearum contains two genes named CPK1 and CPK2 that encode the catalytic subunits of cyclic AMP(cAMP)-dependent protein kinase A(PKA). In this study, to determine the role of cAMP signaling in pathogenesis and development in F. graminearum, we functionally characterized these two genes by using spilt-PCR to knock out them and then screened the positive transformants via southern blotting. In addition, we generated and characterized fac1 adenylate cyclase(AC) gene deletion mutants. At last, the functions of CPK1, CPK2 and AC can be determined by observing the phenotypes defects in those mutants.The cpk1 and fac1 mutants were significantly reduced in vegetative growth, conidiation, deoxynivalenol production as well as pathogenicity to wheat, while the cpk2 mutant has no defective in those aspects. To confirm the phenotypes defects in cpk1 mutant were caused by CPK1 deletion, we re-introduced CPK1 tagged with GFP fusion protein into the cpk1 mutant and got the CPK1 complement strain cpk1-C. By fluorescence observation, we found the subcellular localization of CPK1 is in cytoplasm. And we found that all the defective in the cpk1 mutant can be returned to normal. This indicated that CPK1 plays an important role in vegetative growth, conidiation, deoxynivalenol production and pathogenicity. In the respect of self-cross, both cpk1, cpk2 and fac1 mutants were able to produce normal perithecia. However, in comparison with the wild type and the cpk2 mutant, only a few perithecia formed by the cpk1 and fac1 mutant, which produced diminished cirrhi that appeared as small, pale drops on top of perithecia. Under the same conditions, the wild-type, cpk2 mutant can produce extensive, yellowish cirrhi. These results indicateed that the fertility of the cpk1 mutant was reduced.When cultured at 35℃, in the wild type, conidium germination was rarely observed, under the same conditions, conidium germination and germ tube growth were observed in the cpk1 mutant. And the expression level of three heat shock protein HSP30,HSP70 and GSY2 in the cpk1 mutant were significantly higher than that in the wild type strain. This results the cpk1 mutant increased tolerance to elevated temperatures and CPK1 plays a negative role in the regulation of heat shock.In infection assays with flowering wheat heads, the wild type and the cpk2 mutant could cause typical head blight symptoms in the inoculated kernels and spread to other spikelets. On wheat heads inoculated with the cpk1 and fac1 mutant, only the inoculated floret developed scab symptoms 14 days postinoculation(dpi). Themfailed to spread to nearby spikelets. And the cpk1 and fac1 mutant were defective in the production of penetration branches on plant surfaces, colonization of wheat rachises, and spreading in flowering wheat heads.The DON production in the inoculated floret inoculated with the cpk1 mutant were significantly reduced while in the fac1 mutant, the DON production was barely detectable. We also assayed the expression levels of the TRI5, TRI6, and TRI12 genes in DON-producing cultures containing 5mM arginine by quantitative reverse-transcriptase polymerase chain reaction(qRT-PCR). In comparison with that of the wild type, the expression level of TRI5, TRI6, or TRI12 was reduced approximately fourfold in the cpk1 mutant. In the fac1 mutant, however, all of these three TRI genes were significantly reduced in expression and their transcripts were barely detectable. In order to determine the subcellular localization of TRI1, TRI4 and TRI5 in the wild type as well as the cpk1, fac1 mutants, we tagged TRI1, TRI4 with GFP and RFP, and TRI5 with GFP fusion protein, respectively. The GFP fluorescence in the wild type was much brighter and more mycelium contain fluorescence than that in the cpk1 mutant. But the fluorescence can not be observed in all the mycelium both in the wild type and the cpk1 mutant, this may demonstrate that not all mycelium participated in DON production. We also assyed the expression level of TRI1 in both the cpk1, fac1 mutant and the wild type via western blotting. Consistant with the results of qRT-PCR, the expression level of TRI1 was largely reduced in the cpk1 mutant while in the fac1 mutant, it was barely detectable.These results indicated that CPK1 is the major PKA catalytic subunit gene and that the c AMP-PKA pathway plays critical roles in hyphal growth, conidiation, ascosporogenesis, and plant infection in F. graminearum.