Cloning And Functional Analysis Of Molecular Genes Encoding Phytophthora Capsici

The oomycete Phytophthora capsici Leonian is a broad-host-range hemibiotrophic pathogen that often attacks solanaceous, legume, and most cucurbit hosts. Phytophthora species are phylogenetically distinct from fungi and most of the existing fungicides are not effective against plant Phytophthora blight. In addition, the utilization of resistant varieties to control plant Phytophthora blight is unsatisfactory. Therefore, there have not been effective methods to control the plant diseases caused by P. capsici. In addition to the traditional control measures, understanding of the molecular mechanism underlying Phytophthora-plant interaction would provide valuable guidance to develop new target sites of fungicides and to design novel disease control strategies.Firstly, P. capsici genes differentially expressed during the pre-infection stages were screened. By the transcriptomic sequencing of the three key pre-infection stages (mycelium, zoospore and germinating cyst) and bioinformatic analysis,98 putative effector genes were identified, including 25 RXLR,9 CRN and 16 Elicitin genes.Semi-quantitative RT-PCR was utilized to examine the transcriptional levels of the predicted genes during the stages of Nicotiana benthamiana infection (1.5,3,6,12,24,36,72 h post-inoculation) and pathogen development (mycelium, sporangium, zoospore and germinating cyst). It was found that differentially expressed during infection stages of P. capsici were 35 genes, including 12 RXLR,5 CRN,13 Elicitin,2 PcF/SCR and 3 NLP genes. And, the transcriptional patterns of different gene category during the infection process are different from each other, and those of different genes within the same category were also different.The aforementioned 35 genes were PCR amplified using high-fidelity DNA polymerase and individually cloned into a binary Potato virus X (PVX) vector pGR107. The Agrobacterium-mediated transient expression assays were conducted to analysis whether these genes could cause plant cell death (PCD) in N. benthamiana. The results revealed that 4 Elicitin,1 NLP and 1 RXLR effectors induced PCD in N. benthamiana. To define the functional motifs of the RXLR effector Pc129113required for PCD inducing,13 mutants were constructed by deletion and substitution mutation. Transient expression results showed that the first and second W-Y-L motifs are necessary for PCD triggering, and the region of position 81-312 aa is important for cell death triggering.Except the aforementioned 6 effectors, others cannot trigger PCD. The abilities of RXLR and CRN effectors to suppress PCD was tested in N. benthamiana to analyze if they possess the virulence function. The selected effectors inducing PCD in N. benthamiana include INF1, BAX, PsCRN63, PsojNIP, PsAvh241 and the combination of R3a and AVR3a. It was revealed that the majority effectors (15/17) possess the virulence function of inhibiting PCD. Among the tested effectors,64.7% of them can suppress PCD induced by INF1,82.4% can conquer PCD induced by Bax,76.5% can inhibit PCD induced by PsCRN63,88.2% can inhibit PCD induced by PsAvh241 and PsojNIP, while only 17.6% can inhibit PCD induced by AVR3a/R3a combination. The RXLR effector Pc503142 that is predicted to contain the nuclear localization signal (NLS) can suppress PCD in N. benthamiana. To define the functional domains of this effector,4 mutants were made by deletion and substitution mutation. Transient expression showed that the NLS deletion resulted in the loss of PCD suppression, indicating that this effector may function in the host cell nucleus. Besides, N. benthamiana plants expressing effectors were inoculated with P. capsici to investigate whether effectors could enhance pathogen virulence. Surprisingly, ectopic expression of CRN effectors Pc563418 and Pc559084 in N. benthamiana significantly promoted P. capsici to infect the plants.To elucidate the host selection pressure faced by effectors during evolution, sequence polymorphisms of RXLR and PcF/SCR effectors were examined in a collection of 36 P. capsici isolates from different areas in China. Three RXLR effectors (Pc107349, Pc503142 and Pc129113) are highly diverse in sequence, and the family of PcF/SCR effector Pc96045 consists of at least four members. These suggest that these effectors may play an important role during host infection, and face great host selection pressure in the evolutionary process.To further analyze the functions of P. capsici effectors, gene silencing was conducted to investigate whether the effectors were important for the pathogenicity and the biological functions of P. capsici. The gene silencing of each of three RXLR effectors (Pc107349, Pc503142 and Pc129113) resulted in the decline of virulence of P. capsici on N. benthamiana. Moreover, Pc503142-silenced transformants were more sensitive to active oxygen than control isolates, implying that Pc503142 may play a role in the inhibition of reactive oxygen species (ROS) pathway related to disease resistance of N. benthamiana. Similarly, gene silencing results indicated that PcF/SCR effector Pc96045 is important for the pathogenicity of P. capsici. In summary, gene silencing results showed that RXLR and PcF/SCR effectors may play important roles during the interaction process of P. capsici with host plants.In this study, P.capsici 35 effector genes with different transcriptional patterns during the developmental and infection stages were obtained by transcriptome sequencing and RT-PCR analysis. By transient expression analysis using Agrobacterium infiltration method in N. benthamiana, it was revealed that some effectors may possess virulence function of suppressing plant immune, and some may possess avirulence functions of triggering the plant immune response. By means of CaCl2-PEG-mediated transformation and dsRNA-mediated transient gene silencing, the function of 4 effectors (Pc107349, Pc503142, Pc129113 and Pc96045) were found to play important roles during host plant infection of P. capsici. In this dissertation, by screening, cloning and functional analysis of differentially expressed genes, the roles of some important effectors in plant infection process of P. capsici were elucidated in detail, providing a solid foundation for comprehensive understanding of the pathogenicity mechanism of P. capsici and for effective control of the plant diseases caused by the pathogen.