Gene Cloning, Expression And Pathogenicity Of Endo-polygalacturonase From Rhizoctonia Solani Kühn

Sheath blight of corn, caused by Rhizoctonia solani, is a very serious disease in the Corn Belt around the world, which has become the most important disease in China in recent years. A variety of cell wall degrading enzymes are produced and released in vivo. Phytopathogenic fungi often produce a variety of enzymes active against plant-wall constituents such as polygalacturonase (PG),polymethylgalacturonase (PMG) and cellulose. Pectin is the main components of middle layer structure of plant cell wall. It can form a tough barrier, together with cellulose and hemicellulose, to block the outside world. The polygalacturonase (PG) is the main pathogenic factor, playing an important role in the pathogenicity of plant pathogenic fungi.The object of this study was Rhizoctonia solani Kühn AG1-IA. Degenerate primers designed on the conserved domain of other reported endo-polygalacturonases, and three cDNA fragments encoding the endo-polygalacturonases genes from Rhizoctonia solani Kühn AG1-IA were obtained through RT-PCR. The RACE and TAIL were used to generate full-length cDNA clones. The full length of pg1 cDNA gene is 1086bp, which contained an ORF of 344 amino acids. The cDNA sequence of gene pg1 has been registered in GenBank with accession number HQ413152. The full length of pg2 DNA gene is 1414 bp, which contained six introns. The full length of pg2 cDNA gene is 1086 bp, which contained an ORF of 344 amino acids. The DNA and cDNA sequence of gene pg2 has been registered in GenBank with accession number HQ156225 and HQ156226 respectively. The full length of pg3 DNA gene is 1195 bp, which contained five introns. The full length of pg3 cDNA gene is 927 bp, which contained an ORF of 309 amino acids. The DNA and cDNA sequence of gene pg3 has been registered in GenBank with accession number HQ413151 and HQ413150 respectively. Sequence analysis of the deduced amino acid sequence revealed high homology with the catalytic domains of the Glycoside hydrolase family 28.The gene of endo-polygalacturonase from R. solani and expression vector pPIC9K were digested ligated in vitro to construct eukaryocyte expression plasmid pPIC9K/pg1,pPIC9K/pg2 and pPIC9K/pg3. The recombinant vectors pPIC9K/pg1 and pPIC9K/pg3 were transformed to Pichia pastoris GS115 competent cell. The recombinant P. pastoris GS-PG1-4 and GS-PG3-18 were obtained and the expressed endo-polygalacturonase pg1 and pg3 were purified. Maize was inocubated with the purified protein, and was inocubated with inactive enzyme as a control. The result showed that leaves inoculated by pg1 and pg3 were diseased after 24h, however, the leaves inoculated by inactive enzyme was no symptom. This indicates that the pg1 and pg3 may be pathogenic genes of the gene family encoding polygalacturonase from Rhizoctonia solani AG1-IA. The result showed that leaves inoculated by Rhizoctonia solani Kühn AG1-IA were diseased after 24h. The target bands were obtioned by three primers of pg1,pg2 and pg3, which were said that endo-polygalacturonase plays an important role of the pathogenic process. Rhizoctonia solani AG1-IA grows in the medium of pectin and glucose as substrate respectively. The genes of endo-polygalacturonase were expressed in the pectin medium, however, which were not expressed in the glucose medium. The result showed that endo-polygalacturonase has strong substrate specificity.