Preliminary Functional Characterization Of PnGR1, Glycine-rich Repeat Containing Protein1in Phytophthora Parasitica

Oomycetes represent a unique class of eukaryotic microorganisms that resemble fungimorphologically but are phylogenetic cousins of diatoms and brown algae within the kingdomStramenopiles. Phytophthora de Bary is one of the most important genera of oomycetes andcaused epidemic and devastating plant diseases called pestilence. Phytophthora specieshave several distinct asexual developmental stages, such as vegetative growth, sporulation,zoospores, cysts and geminated cysts. The infection cycle of Phytophthora generally startswith zoospores, followed by a series of processes including attachment to the host surface,encystment and adhesion, spore germination and host penetration, plant colonization andnutrient acquisition, and sporulation.PnGR1(P. nicotianae glycine-rich repeat containing protein1) gene was identified sinceit was highly up-regulated in geminated cysts of P. nicotianae (P. parasitica). It belongs to agene family with at least7members showing high homology to each other. In the presentstudy, we analyzed the function of PnGR1in asexual development and pathogenesis by anintegrated approaches, including bioinformatic analysis, subcellular localization, geneover-expression and the achieved main results include:1. PnGR1protein was shown to share high sequence similarity with PITG19548andmoderate sequence similarity with elastin. PnGR1was predicted to be secreted protein richin repeat motif of GVAGVGVGVP and possesses O-glycosylation sites.2. Blast search led to the identification of22homologous proteins all from oomycetes.They were named as GR proteins, that is glycine-rich repeat containing proteins.Phylogenetic analysis showed that PnGR1is closer to1PpGR genes of P. parasitica and7PcGR genes of P. infestans. The homogenizing force based on concerted evolution may bepresent in the evolution of GR proteins.3. The PnGR1-GFP fusion construct using Hsp70gene promoter and Ham34geneterminator of Bremia lactucae was generated and introduced into P. parasitica via PEG/CaCl2mediated protoplast transformation. A total of625transformants were obtained and13transformants were identified to be mitotically stable during vegetative mycelial growth andasexual sporulation. The PnGR1-GFP fusion gene is expressed constitutively.Microscopic characterization of transformants showed that the green fluorescence was observed to be accumulated in the cell wall of vegetative hyphae, chlamydospores andoospores, and the mastoids of the sporangia. Green fluorescence was accumulated in thetwo vertices of cysts and the tip of germ-tubes of germinated cysts. In addition, unusualmassive accumulation of green fluorescence was observed at several sites in mycelium wherehaustoria-like structures developed.4. The PnGR1-GFP transformants showed little difference on the germination rate butobvious increase in the surface adhesion compared with the wild-type strain. In addition,inoculation of tobacco leaves with these transformants, both zoospores and mycelia, showedthat expression of the PnGR1-GFP fusion gene enabled P. parasitica more virulent.5. A total of274transformants were obtained by transformation of P. parasitica with ahairpin construct to target PnGR1. The accumulation of PnGR1siRNAs was checked byNorthern blotting to screen the silencing transformants. However, no clear siRNAs weredetected for all the51transformants examined. Direct analyses of37transformants for thegermination rate and adhesion, and26transformants for pathogenicity on N. benthamiana, ledto the identification of transformant3-9with reduced adhesion capacity and transformant2-22with reduced pathogenicity, respectively.