Functional Analysis Of Transcription Factors PacC,Crz1, And Protein Kinase SNF1in Penicillium Digitatum

Green mold caused by Penicillium digitatum (Pers.:Fr.) Sacc is the most destructive postharvest disease of citrus. It is responsible for about90%of the losses during postharvest citrus packing, storage, transporting and marketing. However, the knowledgement of molecular biological mechanisms involving in growth, reproduction, adaptation and pathogenesis in P. digitatum is limited. In this study, we characterized the functions of pH-response transcription factor PacC, Ca2+/calmodulin signaling pathway transcription factor Crzl, and sucrose non-fermenting protein kinase SNF1via Agrobacterium tumefaciens-mediated fungal transformation. The results are as follows.1. PdPacC is required for Na+stress, utilization of pectin and full virulence in P. digitatumSequence analysis indicated that the pH signaling cascade in P. digitatum is similar with that in A. nidulans. The deduced protein sequence of PdPacC contains641amino acids, and has three C2H2-type zinc finger motifs located at aa57to145, and a protease recognition region located at aa461to484. The nucleotide sequence upstream of the start codon (-222to-723) contains four repeats of characteristic PacC binding site (5’-GCCARG-3’). The gene expression analysis showed that alkaline conditions (pH>7), Na+and pectin could induce the expression of PdPacC. However, PdPacC was definitely up-regulated during primary stage of P. digitatum infection regardless of the low pH. Comparing the wild type P. digitatum grown in PDB, The expression of PdPacC was up-regulated8.4fold at48h post inoculation. Mycelial growth of the△PdPacC mutant was impaired on SM added pectin as the sole carbon source. The△PdPacC mutant was more sensitive to Na+and alkline stresses. Disruption of PdPacC also resulted in attenuated virulence to citrus fruits, and the lession diameters of the△PdPacC mutant infected citrus was56.3%to that of the wild type infected citrus. Reintroduction of a functon PdPacC into the △PdPacC mutant recovered the defect mentioned above. The△PdPacC mutant lost the up-regulation of PdPG2, a gene encoding polygalacturonase, and PdPNL1, a gene encoding pectin lyase, during infection. Furthermore, disruption of PdPG2also resulted in attenuated virulence to citrus fruits (30%). Collectively, the results indicated that PdPacC is an important transcription factor involving in the regulation of adaption to growth environment, as well as the full virulence of P. digitatum via regulation of the expressions of PdPG2and PdPNL1.2. PdCrzl is required for conidiation, virulence, cell wall integrity and DMIs resistanceZinc finger transcription factor Crz1is a downstream of Ca2+/calmodulin signaling pathway. The deduced protein sequence of PdCrz1contains760amino acids and has two C2H2-type zinc finger motifs located at aa522to574. The△PdCrz1mutant was impaired in conidiation, cell wall integrity, virulence, and was hypersensitive to stresses caused by Ca2+, H2O2, and14α-demethylation inhibitors (DMI) fungicides. The expression of cell wall synthase genes (CHS2, CHS3and FKS1), and P-type ATPases genes (PMR1and PMC1) were significantly reduced in the△PdCrz1mutant compared with the wild type. Calcineurin inhibitors increased the effect of imazalil on P. digitatum, and Ca2+(≤0.2mol.l-1) could alleviate the antifungal activity by imazalil. These results indicated that PdCrzl has important roles in conidation, virulence, and DMI resistance. In addition, this study also indicated that specific inhibitors of calcium signaling pathway could be potential to be developed as useful adjuvants of conventional DMI for the application of plant fungal disease control.3. PdSNF1is required for utilization of un-prefered carbon source, conidiation and virulence in P. digitatumSucrose non-fermenting protein kinase1gene (SNF1) regulates derepression of glucose-repressible genes in microorganisms. The deduced protein sequence of PdSNF1contains789amino acids and has a protein kinase domain at aa65to316. Growth of the△PdSNF1mutant was severely disturbed on media supplemented, respectively, with pectin and polygalacturonic acid as the only carbon source, and, a mild effect was observed when this mutant was grown on medium with either glucose or fructose as the carbon source. The appearance of disease symptoms on the△PdSNF1mutant-inoculated citrus fruits was significantly delayed as well. The expression levels of the cell wall degrading enzyme (CWDE) genes (e.g., XY1, PL1, PNL1and EXPG2) after pectin induction were up-regulated in wild type, but unchanged or less up-regulated in the APdSNF1mutant. During infection in citrus fruit, the up-regulation of XY1was delayed in the△PdSNFl mutant. Disruption of PdSNF1also resulted in impaired conidiation (10%of the wild type) and caused malformation of the conidiophore structures. In addition, the expression of Br1A, a gene that regulates conidiophore development, was significantly impaired in the△PdSNF1mutant. Collectively, our results demonstrate that the PdSNF1plays a role in adapting P. digitatum to alternative carbon sources. Its involvements in the virulence of P. digitatum is probably via regulation of the expression of CWDE genes; and it is also involved in conidiation, probably through activation of the conidiation signaling pathway.