Preliminary Functional Characterization Of An Atypical Plasma Membrane H~+-Atpase Gene PnPMA1in Phytophthora Parasitica

Species in the genus Phytophthora, which belong to a group of fungus-like eukaryotic microorganisms called Oomycetes, includes many destructive plant pathogens. They can infect many agriculturally and ornamentally important plants, causing substantial yield losses worldwide. Phylogenetic analyses revealed that Oomycetes are phylogenetically distant from true fungi. As a consequence, most fungicides are ineffective in controlling diseases caused by oomycete pathogens. In order to provide insights into the molecular mechanisms underlying Phytophthora asexual development which is required for the development of novel disease control strategies, we use P. parasitica as a model for the studies of oomycete biology and pathology.Oomycetes can reproduce asexually by forming multinucleate sporangia. Sporangia usually undergo cytoplasmic cleavage to produce uninucleate zoospores. The motile zoospores are considered to be the major infective agent to initiate plant diseases for most Phytophthora species. Zoospores are able to swim and actively target host tissues. Once reaching the host, they encyst and subsequently germinate. The germ tubes emerging from cysts often develop appressorium-like structures that facilitate plant penetration. However, the molecular mechanisms that control zoospore development and behavior are still largely unknown. The P. parasitica PnPMA1gene encodes an atypical plasma membrane H+-ATPase and was shown to be highly expressed in zoospores and germinated cysts in previous studies. The PnPMA1protein contains an insertion of-155amino acid residues at the C terminus which is absent in homologous proteins from other organisms. In the present study, we aimed at elucidating the role of PnPMA1in asexual development and pathogenicity by subcellular localization, topology characterization, and gene silencing approaches. In addition, we selected PnPMA1as an endogenous target, GFP as a reporter, to test the effect of transgenic expression of dsRNAs on the expression of homologous genes in the invading and colonizing oomycete pathogen, using Arabidopsis thaliana-P. parasitica as a model biotrophic pathosystem. The main results are as follows:1. PnPMA1::GFP fusion plasmids were constructed by fusion PCR method, using native regulatory sequences of PnPMA1, and were introduced into P. parasitica by genetic transformation. Analysis of generated P. parasitica transformants showed that PnPMAl is localized in the plasma membrane.2. The C-terminal insertion loop of PnPMAl protein is predicted to be located in the extracellular space using homology modeling method. The determined crystal structure of the plasma membrane proton pump from Arabidopsis, AHA2, was used as a template for homology modeling of PnPMAl structure. We further employed lacZ gene fusion-based biochemical approach and confirmed in yeast that the C-terminal insertion loop in PnPMAl is localized in the extracellular space by analyzing β-Gal activities.3. Stable gene silencing transformants were obtained by transformation of P. parasitica with antisense and hairpin constructs, respectively. Northern blot analyses were performed to detect accumulation of siRNAs in PnPMA1-silenced transformants and quantitative RT-PCR was conducted to measure the PnPMA1expression levels. A negative correlation between siRNA accumulation and PnPMA1expression was confirmed, indicating that siRNAs are involved in gene silencing in P. parasitica. The accumulation of PnPMA1transcripts in the most silenced transformant T77was shown to be reduced by92%.4. High level of PnPMA1silencing resulted in production of non-motile zoospores. The zoospores quickly encysted after release from sporangia, and poorly germinated. Moreover, the heavily PnPMA1-silenced transformants produced large aberrant zoospores. These results indicate that PnPMAl is required for zoospore development.5. We selected GFP as a reporttor, and PnPMA1as an endogenous target to test whether the host induced gene silencing functions in the oomycete pathogen. Our results showed that production of dsRNA sequences in the host plant had no effect on the expression of homologous target genes in the colonizing P. parasitica, indicating that oomycete pathogens may lack genetic machinery required for uptake of silencing signals or that the translocated, if possible, silencing signals are not sufficient to initiate gene silencing. Therefore, the engineered resistance against oomycete pathogens through the production of homologous dsRNAs in the host plants would not be feasible.