Cloning And Function Analysis Of Pdpg1, An Endopolygalacturonase Gene From Penicillium Digitatum

Pectinases secreted by pathogenic fungi are a group of cell wall degrading enzymes (CWDEs) that could breakout the mechanical defense system of host plant, thereby the pathogen could invade and grow saprophytically in pant tissue. Polygalacturonases (PGs), consisting of a subset of pectinases and using homologous galactose polysaccharide as the substrate, are verified to be related to the virulence in some fungi-host interaction systems. However, not all the pg deletion mutants of phytopathogenic fungi show reduced virulence. Penicillium digitatum is the most important pathogen that causing green mold decay of postharvest. The interaction system of P. digitatum and citrus differs from other pathogen-host interaction systems, since the pathogen could only infect from wound of mature fruit; the host (citrus fruit) had been separated from the tree, and was lack of the importion of carbohydrate and was gradually senescing. The symptom of water-soaking and soft rot implicated that the CWEDs secreted by P. digitatum must involve in the pathogenesis although there has been no experimental evidence proofed.To explore whether PGs participate in the pathogenesis of P. digitatum-citrus interaction, an endoPG gene Pdpgl was cloned from P. digitatum based on the reported sequence (ID AB015286).Nucleotide sequence alignment showed that Pdpgl was highly identity to the sequences of other PG1 genes from Penicillum olsonii (79%) and Claviceps purpurea (85%). The ORF of Pdpgl contained 1101bp, with 3 exons and 2 introns. There was a putative 18 amino acid N-terminal signal peptides. Expression analysis showed that Pdpgl was a constituted expression gene that could not be repressed by glucose. Qualitative real-time RT-PCR analysis indicated that Pdpgl was an acid expression gene. The expression level at pH4.0 was 15 times higher than that at pH 7.0.To verify the function of Pdpgl, deletion and overexpression vectorsâ–³Pdpgl and EX-vector were constructed and introduced into Agrobacterium tumefaciens strain EHA-105 respectively, then transformed into P. digitatum strain Pdw03 using agrobacterium mediated transformation system (ATMT). Two deletion mutants (â–³Pdpgl-10 andâ–³Pdpgl-38), an overexpression mutant (EX-3) and an ectopic insertion mutant (Etc-2) were selected and verified by PCR and Southern blot analysis. On the medium PDA all types of mutants showed the similar characterics in colony morphology, growth rate, and sporulation with those of parental strain Pdw03. In contrast, the deletion mutants grew obviously slower than Pdw03 in the medium that pectin was the only carbon resource. Comparing with parental strain Pdw03, the pectinase activity was decreased 26% for the deletion mutants, and increased 26% for the overexpression mutant. However, the pathogenicity test demonstrated that the virulence was not decreased for theâ–³Pdpgl and wan not increased for the overexpression mutant, suggesting that a single PG is not obslutely necessary for virulence in P. digitatum.