| Phytophthora blight of peppers is a worldwide soil-borne disease, and it has become a serious threat to the agricultural production of China and other countries in the world, and result in enormous economic loss. The disease was caused by phytophthora capsici Leonian. P. capsici is a soilborn oomycete, and it can survive in the soil as oospores or chlamydospore for several months even longer, repeated cultivation of susceptible hosts results in a high density of oospores in siol. P. capsici can attack the host plant at any growth stage and causes seedling death, crown rot, foliar blight, ang fruit rot; the pathogen can also cause several crop lossed in pepper, other cucurbits, eggplant, and tomato and so on.At present, the phytophthora blight of peppers was controlled mainly by crop rotation, application of chemicals and breeding resisitant host. However, phytotoxicity and chemical residues may pose a serious threat to the environment, strong variability of physiological strains of P. capsici caused the instability of resistant host. So search the important pathogenic factors of P. capsici and tocontrol the phytophthora blight of peppers have become the focus of the phytopathologist. Many previous studies have shown that plant pathogens must breach cell walls before they can infect the hosts, in which plant pathogens can secrete an array of pectinases during plant-pathogen interactions. These enzymes, such as polygalacturonase, pectate lyase, and pectin methylesterase, appear to play a key role in pathogenicity in most pathogens for the decomposition of pectin, which is one of the main components in the plant cell wall, and make the host more susceptible to the pathogen. The pathogenicity of many plant pathogens is dependent upon the synthesis and secretion of numerous pectinases, and the maceration of the plant parenchymatous tissues result primarily from the enzymatic degradation of the pectic polysaccharides by pectinases in plant cell walls, which indicated that the pectinase activities were important factors in the maceration of the plant tissues, and pectinase activity is an important factor to determine the pathogenicity of microorganisms.Among these pectinases, pectin methylesterase is an important pathogenitic factor, and plays a major role in the pathogenisis of phytophthora blight. The study took the P. capsici as subject, analysis the polygalacturonase, pectate lyase, and pectin methylesterase activities of high pathogenic P. capsici SDPH-33. Five pme genes were obtained by screening genomic library of SDPH-33 through Pool PCR. Then PCPME1 fusion protein was acquired according to heterologous eukaryotic expression and purified by Ni-NTA resin, western blot was used to test whether the pcpme1 expressed in Pichia pastoris. purified PCPME1 was used to prepare antibodies in New Zealand white rabbits, then the antibody was used to tested the expression of pcpme1 in pepper. The effect of potential glycosylation site to the PCPME1 activity was analysised by deglycosylation through PNGase F. we predict the activity site of pcpme1 and analysis the function by site-directed mutagenesis, and got the purified mutational protein by eukaryotic expression and affinity chromatograph. At last, the expression level of pcpme1 in pepper tissues was investigated by RT-PCR and Northern Blot. The purified fusion protein, deglycosylated protein, and mutational protein were used to inoculate pepper leaves, PMEs activity varied in PMEC treated pepper leaves was consistent with symptom development in pepper leaves. transmission electron microscope was used to analysis the enzymatic degradation of the plant cell wall. Main researchs were as follows:The activities of PGs and PELs were determined with spectrophotometer, and PMEs activities were analysized by neutralization titration; the results indicated that there was a high correlation between activities of PGs, PMEs, and PELs and the pathogenicity of P. capsici. The genomic library of SDPH-33 was screened by Pool PCR, and 5 pcpmes were isolated from the library, and structural features of the 5 genes were analysized. Alignment of these amino acid sequences indicated that there were high similarities between the 5 pme genes, and the pcpmes contain 5 characteristic sequence segments and active sites(GxYxE,QDTL,QAVAL,DFIFG and LGRPW). All the amino acid sequences of the 5 pcpmes possess 3-8 potential N-linked glycosylation sites, and have a similar tertiary structure. The evolution tree based on the alignment of PME amino acid sequences with different sources showed that PMEs from oomycetes formed their own cluster.The leaves of peppers with 4-6 fully expanded leaves were inoculated with the zoospores suspension of SDPH-33 and then the total RNA of infected leaves was isolated and cDNA was synthesized. Specific primers were designed based on the sequences of 5 pcpmes and the expression of these pcpmes in pepper leaves was detected by RT-PCR. The results indicated that all these genes can express in infected peppers respectively and the expressed level became stronger with the time prolong after inoculation, and then reached the peak on 7th day, however, there was no expression of pcpmes in the healthy peppers. Based on the result of RT-PCR, the pcpme1 was determined to be the most important pathogenetic gene. The non-conservative fragment of pcpme1 was amplified with specific primers and then was used to synthesize probe and then the probe was used to determine the expression of pcpme1 in pepper leaves, the similar result to RT-PCR was obtained.Gene-specific primers were designed and synthesized to amplify the mature peptide of pcpme1according to known sequence. The amplified fragment was inserted into pPIC9K to construct expression plasmid pPIC9K/pcpme1, and then it was transformed into E. coli JM109 to obtain recombinated plasmid. The plasmid pPIC9K/pcpme1 was transformed into Pichia pastoris GS115 competent cell after linearized with restriction enzyme SacI. After screening by G418and PCR amplification of these transformants, several recombinant strains with pcpme1 inserted were obtained. The result of SDS-PAGE indicated that Heterologous expression of pcpme1 in Pichia pastoris produced a protein of 60 kDa that was not corresponded to the predicted mass of this protein. Western Blot with anti-His antibody showed that pcpme1 can express efficiently in Pichia pastoris. Expressed PCPME1 was purified through Ni-NTA system, and was used to prepare antibodies in New Zealand white rabbits, then the antibody was used to tested the expression of pcpme1 in pepper, the result indicated that pcpme1 can express in infected peppers and the expressed level became stronger with the time prolong after inoculation, and then reached the peak on 7th day, however, there was no expression of pcpmes in the healthy peppers. The purified N-linked wild type protein PCPME1 was treated with PNGase F, and the molecular mass of the deglycosylated PCPME1 was 37KDa, which was similar to the predicted mass of PCPME1. The activity of deglycosylated PCPME1 was similar to that of N-linked wild type PCPME1, which suggested that glycosylation does not play a major role in the activity of PCPME1. Site-directed mutagenesis of the activity sites of pcpme1 was carried out by over lap PCR amplification. Comparison of the activities between wild type and mutational PCPME1 showed our hypothesis was correct. The wild type, deglycosylated and mutational PCPME1 were used to inoculate the leaves of peppers with 4-6 fully expanded leaves, lesions appeared in wild type and deglycosylated PCPME1, and the lesions became larger with the time prolong after inoculation, while mutational PCPME1 and sterile water could not cause any lesion on the surface of pepper leaves. The cell walls of the leaves treated with wild type and deglycosylated PCPME1 were found degradated with different levels through transmission electron microscope, while those treated with mutational PCPME1 and sterile water kept almost integrity.The results of this study indicate that pcpme1 was one of the key pathogenetic genes that encode pectin methylesterase in the pathogenesis of P. capsici.
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