| Phytophthora capsici is found worldwide attacking solanaceous and cucurbit hosts, which include pepper, tomato, eggplant, cucumber, pumpkin, melon and so on. In 1922, Leon Leonian describrd P. capsici after observing a blight of peppers at the New Mexico Station. The success of pathogen depends on their ablility to overcome the extensive denfences of their hosts, as well as to gain nutrition and proliferate. This ability in turn depends on specific physiological functions in the pathogens. Both plant and animals host have complex multifaced defense systems. Thus, the pathogen must be able to suppress or avoid a sufficient number of these machnisms to be successful. Effector molecules are a key weapon uesed to combat their hosts'defense systems. Effectors are defined as any molecules released by the pathogen that have a function to modify the physiology of the host to promote the success of the pathogen which includes to suppress or neutralize some component of the host defense system. NPP, known as necrosis-inducing Phytophthora proteins, is also called NLP (Nep-1 like proteins). Dicot plants treated with NPP proteins have been shown many characteristic defence responses, such as defence-related gene expression, calcium accumulation, phytoalexins release, active oxygen emission and mitogen-actived protein (MAP) kinase activity increase. Genes encoding NPPs, or the proteins themselves have been detected in superfamily in eukaryotic and prokaryotic organisms. The NPPs are not found in plants, animals, but they often occur in fungi, bacteria, and also in oomycetes, particularly many Phytophthora species. However, the reason why Phytophthora species have a large NPPs gene family and the roles these genes play during infection process are still unclear.In this work, we showed the pathogenesis and functinal analysis of NPPs gene family from P. casici. The focus of the present study was to elucidate in vitro and in vivo the function of NPPs gene family in P. casici by stable gene silencing and agroinfection for the first time. See detailed content as follows:(1) We showed the presence of NPPs in a high-virulent P. capsici strain SD33 isolated from China.Genome-wide identification of NPPs was performed in the released P. capsici genome sequence. A series of primers were designed using the released P. capsici genome sequence (JGI). The PCR product of expected length was cloned and then sequenced in company. And a database search in BLAST confirmed that they were NPP genes. Sequnences alignment was performed using DNAman software. The result showed that all these 18 genes had conserved'GHRHDWE'motif and a relative conserved bexakis-residue'QDLIMW'at the C-terminal.(2) Eighteen Pcnpp genes expression patern analysis in hyphe growth and phylogenetic analysis. Twelve genes appeared to be inactive in hypha growth by RT-PCR analysis. The tree was constructed on the basis of alignment of the selected NPP genes predicted protein sequences. Phylogenetic trees were generated by neighbor-joining, as implemented in PAUP﹡ 4.0 Beta. All oomycetes genes were clustered together, while fungal sequences were grouped into two separate clades. This result suggested that NPPs family was a character of oomycete organisms, and was suitable for oomycetes classification.(3) Transient expression of 12 Pcnpp genes in tobacco and pepper plants showed function diversity of different genes. We observed that diverse changes when Pcnpp 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. These results indicated that paralogous in Pcnpp gene family had distinct biological functions in different plant species.(4) For further analysis function of Pcnpp1, site-directed mutation of Pcnpp1 was performed, and then PVX recombinants were constructed to complete transient expression in tobacco and pepper plants. The four sites were individually exchanged for alanine using overlap PCR. Also substitution of four sites by alanine simultaneously was carried out to further investigate gene functions as previously described. When we tested their ability to induce cell death by agroinfection assay on tobacco plants, results showed that all the five mutation genes failed to trigger any hypersensitive response in plant leaves. These results showed that NPP protein from P. capsici may have enzyme activity, and that NPPs maybe represent a kind of effector with enzymatic activity capable of causing membrane damage. However, the substrate is still unknown.(5) Expression pattern of 12 members in NPP gene family was analyzed during pathogenesis process. Twelve candidate genes were selected for functional analysis. Real-time quantitative PCR analysis in pepper leaves infected with P. capsici demonstrated the 12 genes to be differently expressed. Five genes were shown to be expressed obviously, especially reached the highest expression levels at 3 dpi. It is valuable to note that six members displayed similar expression patterns, in which expression levels were increasing during the infection process, peaking at 7dpi. The results indicated that Pcnpp genes played important roles in infection process.(6) We got stable transformation lines for Pcnpp genes using PEG-mediated protoplast gene-silencing mathod. We assessed the expression levels of other members in the gene family by RT-PCR and Real-time RT-PCR. Seven transformants were selected. Unexpectedly, individual genes expression levels varied in different silenced lines as a target gene silenced would cause other members in the family repression simultaneously. All the selected genes except Pcnpp6 and Pcnpp8 were silenced at a degree above 80%. Phenotypes of the seven mutants'colonies were examined and no differences were observed between controls and the mutants.(7) The virulence of silenced lines is impaired. To determine the effect of Pcnpp silencing 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 smaller spots at the same time point. Generally, silenced strains showed obviously reduction of virulence or pathogenicity.We evaluated Pcnpp gene family roles for inducing hypersensitive response in different plant species, which documented the avirulence function and distinct biological functions. Moreover, we inferred that Pcnpp proteins possessed enzymic activity based on in vitro mutation, which will be beneficial to further evaluation of the characteristic of Pcnpp on protein level. Pcnpp genes expression deficiency could decrease virulence, which suggested the probable roles in the process of plant pathogen interaction. This is the first report that directly demonstrates virulence functions of NPPs from P. capsici. These results provide further information in comprehending the NPPs pathgenicity.
|
PDF Full Text Request