Characterization Of Correlation Between Proline Metabolic Pathway And Virulence In Cryphonectria Parasitica

19973 Cryphonectria parasitica mutants were generated by Agrobacterium tumefaciens-mediated transformation(ATMT).The mutants demonstrated various phenotypes:I)812 mutants just grew on the surface of the PDA plate without aerial and substrate hyphae and seemed transparent;II)49 mutants produced deeper pigment and grew at normal speed;III)49 mutants produced less or no pigment and grew at normal speed;IV)72 mutants produced deeper pigment and grew slower significantly;V)38 mutants produced less or no pigment and grew slower significantly;VI)18953 mutants showed the same phenotype as EP155.Southern blot analysis showed that 79%of the ATMT mutants contained a single copy of T-DNA in the genome,and TAIL-PCR analysis indicated that T-DNA inserted randomly.The mutant 77HE12 showed reduced virulence and the T-DNA inserted in the second exon of Prodh,encoding proline dehydrogenase.The abnormal virulence of 77HE12 could be restored to like EP155 by re-introducing a copy of Prodh.The prodh null-mutants were constructed by homologous recombination and showed the same phenotype as 77HE12.The colony area of△prodh cultured for 7 days was 77%and 82%of the colony area of EP155 and△ku80,respectively.The sporulation of △prodh was 8.5%and 9.8%of the sporulation of EP155 and △ku80,respectively.△prodh showed obviously reduced virulence,when the canker area of △prodh was 18%and 20%of the canker area of EP155 and △ku80,respectively.In addition,P5Cdh,the downstream gene of Prodh and encoding△1-pyrroline-5-carboxylate(P5C)dehydrogenase,was deleted by homologous recombination.The phenotype of △p5cdh was not distinguished from △ku80 or EP155.But the virulence of △p5cdh reduced significantly.The canker area of△p5cdh was 16%and 18%of the canker area of EP155 and △ku80,respectively.Recombinant Prodh and P5Cdh proteins were expressed and purified from Escherichia coli and shown to have the Prodh and P5Cdh activity,respectively,by enzyme activity tests.In in vivo assays,Prodh and P5Cdh were able to complement the put1-and the put2-null mutants,respectively,to allow the proline utilized defect yeast mutants to grow with proline as the sole source of nitrogen.Both Aprodh and Ap5cdh couldn’t live on the minimal medium supplemented with proline as the sole nitrogen source.Even though △prodh and△p5cdh were cultured on the PDA plates supplied with proline,their growth,pigmentation and sporulation was suppressed significantly.It suggested that the intracellular proline levels and P5C levels were so high that they were toxic to the cells.Real-time PCR analysis showed that the transcript levels of Prodh and P5Cdh were down-regulated by hypovirus CHV1-EP713.Deletion of other genes(Carl,Car2,Prol,Pro2,Pro3,Put3 and Put4)whose products are involved in conversion of arginine to ornithine,ornithine or glutamate to P5C,and P5C to proline,did not seem to affect virulence.But the asexual sporulation was significantly decreased in Aprol and Apro2.The sporulation of Aprol was 5.6%and 6.5%of the sporulation of EP155 and Aku80,respectively.The sporulation of Apro2 was 0.5%and 0.6%of the sporulation of EP155 and Aku80,respectively.Moreover,△prol and Apro2 showed hypersensitive to osmotic stress and salt stress.Combined together,both Prodh and P5Cdh are essential for virulence,and some of the components of the proline metabolic pathway are essential for sporulation and may represent down-stream targets of a hypovirus regulation in C.parasitica.