| Phytophthora blight of peppers is a worldwide disease, which impacted on agriculture almost in every country, and caused enormous losses on economy. It can infect pepper, tomato, eggplant, cucumber, pumpkin, melon and so on. In1922, Leon Leonian described Phytophthora capsici after observing a blight of peppers at the New Mexico Station. The oospores of P. capsici are capable of surviving in the soil for several years, germinating in favorable environmental conditions. After germinating, it can cause stem rot, stem wilt and root rot in a wild host range. This has caused huge economic losses to our country and the worldwide agricultural production. At present, the pepper blight was controlled mainly by chemical methods and breeding resisitant cultivars. However, strong variability in pathogenicity of P. capsici caused the instability of resistant breeds. For a very long time, the chemical methods were the most effective and rapid method to prevent and congtrol P. capsici with the consequence of pesticide residues and enviromental pollution. So it is very important to study the disease control in phytophthora blight of peppers by carrying out resistance identification and disease-resistance breeding via investigating the infection mechanism, resistance biochemistry mechanism, resistance genetics, and quantitative trait loci and genetic engineering as well, so as to the resistance identification and application in breeding for disease resistance.The success infection of pathogens depend on their ability to overcome the extensive defences of their plant hosts and this ability in turn depends on specific physiological functions in the pathogens. During the infection the pathogens could secret a series of cell wall degrading enzymes (CWDE) or pathogenicity-related enzymes, including cutinase, pectinase, cellulase, hemicellulase, and protease. Pectate lyase, also known as pectate transeliminases, exhibits a P-elimination mechanism in the cleavage of α-1,4-glycosidic bonds of polygalacturonic acid which results in the formation of a double bond between C4and C5at the non-reducing end via E2elimination mechanism and an elimination of CO2It is one of the most important CWDEs, it can be secreted by many fungi, bacteria and oomycete. There are many PEL genes in the genome sequence of Phytophthora spp, however, the reason why Phytophthora species have a large PELs gene family and the roles these genes play during infection process are still unclear.In this work, we carried out the pathogenesis and functional analysis of PELs gene family from P. casici. The focus of the present study was to elucidate in vitro and in vivo the function of PELs gene family in P. casici by stable gene silencing, over-expression and agroinfection for the first time. The details are as follows:(1) We analyzed the presence of PELs in a high-virulent P. capsici strain SD33isolated from China and carried out the bioinformatics analysis. Genome-wide identification of PELs was performed in the released P. capsici genome sequence. A series of primers were designed according to the released P. capsici genome sequence (JGI). The PCR products of expected length were cloned and then sequenced in company. There are9sequences accepted with the primers. And a database search in BLAST confirmed that they were PEL genes. Sequences alignment was performed using DNAMAN software. The results showed that all these22genes were classfied into two clades, but remain had conserved motif and a relative conserved bexakis-residue. Most genes have signal peptide and N-glycosylation sites. The tree was constructed on the basis of alignment of the selected PEL genes predicted protein sequences. Phylogenetic trees were generated by neighbor-joining, as implemented in PAUP’4.0Beta. At last we chosed twelve PELs for the founction research.(2)The expression pattern analysis of Pcpel genes in the process of pathogen-host interaction. Twelve Pcpel genes expression pattern analysis in infection. The leaves of peppers with4-6weeks fully expanded were inoculated with zoospores of SD33and then the total RNA of infected leaves were isolated from the1th,3th,5th and7th day, and cDNA was synthesized. Specific primers were designed based on the sequence of twelve Pcpel genes and the expression of Pcpel genes in different pepper leaves was detected by Quantitative Polymerase Chain Reaction (QRT-PCR). The results indicated that Pcpel genes could express in infected peppers respectively and the expressed level became stronger with the time prolong after inoculation, and then reached the peak on the7th day. But different genes have different levels. The results indicated that Pcpel genes played important roles in infection process.(3) The Pcpel genes’function in plants. Agrobacterium mediated transient expression of twelve Pcpel genes in tobacco and pepper plants showed function diversity of different genes. We observed that diverse changes when Pcpel genes were expressed in pepper and tobacco leaves. These symptoms including necrosis, chlorisis in varying degrees, wrinkle and roll, appeared on different plants corresponding to individual genes at different time points. In pepper, all the genes have more visible symptoms in varying degrees then in tobacco. Three genes (Pcpell, Pcpel16and Pcpel20)have heavier symptoms than other genes. These results indicated that paralogous in Pcpel gene family had distinct biological functions in different plant species.(4) Silenced transformants construct and analysis. We got stable transformation lines for Pcpel genes using PEG-mediated protoplast gene-silencing mathod. We assessed the expression levels of other members in the gene family by Real-time RT-PCR. Twelve transformants were selected. Individual genes expression levels didn’t affect other genes expression level. All the selected genes except Pcpel15, Pcpel18and Pcpel20were silenced at a degree above70%. Phenotypes of the twelve transformants’colonies were examined and no differences were observed between controls and the transformants.(5) Over-expressing transformants and analysis. We also got stable over-expressing lines for Pcpel genes using PEG-mediated protoplast gene-silencing method. We assessed the expression levels of other members in the gene family by Real-time RT-PCR. Twelve transformants were selected. Individual genes expression levels didn’t affect other genes expression level. All the selected genes except Pcpel17and Pcpel19were over-expressing at a degree above25fold. Phenotypes of the twelve transformants’colonies were examined and no differences were observed between controls and the transformants.(6) Transformants pathogenicity testing. The virulence of silenced and over-expressing lines is tested with pepper leaves. To determine the effect of Pcpel gene silencing and over-expression on virulence, we used the induced zoospores inoculation assay to measure virulence in the susceptible pepper cultivar. Most leaves inoculated with the silenced lines showed significantly different spots at the same time point. Most genes have the same or smaller spots with the wild type and the CK, only3genes have obviously small spots. Generally, silenced strains showed obviously different reduction of virulence or pathogenicity. The same to the over-expressing lines, most lines didn’t showed significantly biger spots at the same time point, only Pcpel16and Pcpel20over-expressing lines indicated the bigger spots. The over-expressing line of Pcpel1inoculated area but smaller, indicating that it may be the key gene in the pathogenic process. Generally, not all the over-expressing strains showed obviously increase of virulence or pathogenicity. These results indicated that paralogous in Pcpel gene family had distinct biological functions with different genes.We evaluated Pcpel gene family roles for inducing hypersensitive response in different plant species, which documented the avirulence function and distinct biological functions. Pcpel genes expression deficiency could decrease virulence, genes expression increase could enhance virulence which suggested the probable roles in the process of plant pathogen interaction. This is the first report that directly demonstrates virulence functions of PELs from P. capsici. These results provide further information in comprehending the PELs pathgenicity. |
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