| Puccinia helianthi is an important disease in sunflower production,which seriously affects the yield of sunflower and causes great economic losses to sunflower industry.In our earlier screening of secreted proteins from P.helianthi,we found that an extracellular metalloproteinase gene Mep had a very high expression level during urediospore germination.Therefore,we speculated that this protease might be involved in the pathogenesis of P.helianthi.In this study,protein characteristics and structure of this metalloproteinase secreted by P.helianthi were analyzed,and the phylogenetic tree was constructed.Moreover,treatments with several screened metalloproteinase inhibitors were carried out to verify the role of metalloproteinase in tne pathogenesis of P.helianthi.The Mep gene expression was measured during the infection process of P.helianthi in sunflower.The results laid a foundation for the study of the pathogenesis of Mep.The main results are as follows:1.Bioinformatics analysis showed the metalloproteinase had high homology with the extracellular metalloproteinase of Puccinia striiformis,and were closely related to protein kinases.Analysis of its amino acid sequence showed that it contained the HEXXH motif signature of zinc metalloproteinase and the fungal catalytic M36 peptidase domain.Three-dimensional structure analysis showed that there were zinc ion binding sites in the catalytic active region.It is speculated that the secreted protein belongs to metalloproteinase.2.The extracellular metalloproteinase had hydrolytic activity against azocasein.The optimal temperature and p H of pure enzyme were 50℃and 7.0,respectively,and remained stable at p H 5.0～9.0 and below 50℃.The protease was significantly inhibited by the inhibitors EDTA,EGTA and 1,10-phenanthroline.Ca2+,Zn2+,Mg2+could significantly increase the activity of the protease,indicating that the enzyme was a metalloproteinase.3.The expression of Mep in sunflower infected by rust was determined by q RT-PCR.The results showed that the gene expression reached its peak at 48 h after inoculation,and then decreased gradually,indicating that the protease participated in the early infection process of P.helianthi.4.The subcellular localization of the metalloproteinase indicated that it was localized on the cell wall or membrane of tobacco.Sunflower leaves were treated with metalloproteinase inhibitor and then inoculated with P.helianthi.The results showed that the disease of leaves treated with Mep was significantly earlier than that of the control,and the amount of sporulation increased after 10 days of inoculation,suggesting that the metalloproteinase was involved in the pathogenic process of rust bacteria on sunflower. |
