| Clubroot is an important soil-borne disease caused by Plasmodiophora brassicae,which occurs on cruciferous vegetables and crops.P.brassicae is an obligate parasitic pathogen,stable genetic transformation system has not been established,and the pathogenic mechanism is not clear.Comprehensive understanding the biological characteristics of P.brassicae,especially its pathogenic mechanism,is very important for the development of new prevention and control technologies.In our lab,de novo sequencing was performed on the genome of P.brassicae ZJ-1.Two peptidase inhibitors,PbPI16-1 and PbPI16-2 were screened by multi-omics techniques,such as genomics,transcriptomics and secretome.This study conducted a preliminary study on the biological functions of these two peptidase inhibitors,and the main results are as follows:1.Preliminary study on the biological functions of PbPI16-1.The full-length cDNA of PbPI16-1 contained an open reading frame of 1206 nucleotides encoding a peptide of 402 amino acid residues with the predicated molecular mass of 44 kD.The protein contains a signal peptide and a CAP domain.PbPI16-1 can inhibit tobacco cell deaths caused by a death-promoting member Bax.The subcellular localization of PbPI16-1 was observed in the cell membrane,cytoplasm,nucleus and apoplast space;if the signal peptide was removed,it lost secretory function and aggregated into clumps in the cytoplasm.Expression pattern of PbPI16-1 in theinfection stages were conducted by qPCR,PbPI16-1 was expressed inall infection processes,and the highest expression level was in the cortex infection stage.Three transgenic plants overexpressing PbPI16-1 were obtained by the dipping method,and the biocontrol effects of L18,L19 and L21 on clubroot is 38.45 %,32.10 % and 24.55 %,respectively.The results suggested that transgenic plants overexpressing PbPI16-1had enhanced defense against P.brassicae.Used flg22 treated with transgenic plants,the expression levels of SA signaling pathway related genes were indueced.These results suggested that PbPI16-1 may play an important role in the infection process of P.brassicae.2.Preliminary study on the biological functions of PbPI16-2.The full-length cDNA of PbPI16-2 contained an open reading frame of 1038 nucleotides encoding a peptide of 345 amino acid residues with the predicated molecular mass of 348 kD.The protein contains a signal peptide and a CAP domain.PbPI16-2 can inhibit tobacco cell deaths triggered by Bax.The subcellular localization of PbPI16-2 was observed in the cell membrane,cytoplasm,nucleus and apoplast space;it lost secretory function and aggregated into clumps in the cytoplasm without signal peptide.Expression pattern of PbPI16-2 in theinfection stages were conducted by qPCR,PbPI16-2 was expressed in all infection processes,and the highest expression level was in the cortex infection stage.Two transgenic plants overexpressing PbPI16-2were obtained by the dipping method,and the biocontrol effects of L26 and L27 on clubroot is 39.02 % and 32.35 %,respectively.The results suggested that transgenic plants had enhanced defense against P.brassicae.Used flg22 treated with transgenic plants,the expression levels of SA signaling pathway related genes were indueced.And MAPK activation were analyzed by western blotting,transgenic plants L27 can induced MAPK activation compare to wild type plants.322 candidate proteins that may interact with PbPI16-2 were screened by IP-MS technology,selected 33 candidate proteins and PbPI16-2 co-transformated to yeast cells,used yeast two-hybrid assays to test protein-protein interactions.Only two proteins GLL23 and JAL23 can interact with PbPI16-2 in yeast cells.The results of luciferase complementation assays indicated that GLL23 and JAL23 can interact with PbPI16-2in tobacco cells,respectively. |
