Preliminary Studies Of Specific Interaction Between Phytophthora Parasitica And Host Plant

Oomycetes represent a group of eukaryotic microorganisms similar to true fungi, but related to diatoms and brown algae, causing diseases to plants and animals. Among the group, Phytophthora is the best-studied genus that includes many destructive plant pathogens. Due to the unique physiological and biochemical characteristics, most fungicides are ineffective in controlling oomycete diseases. In recent years, tremendous progress has been achieved in understanding diversity, evolution and lifestyles of oomycete plant pathogens, as well as the genetic and molecular basis of oomycete–plant interactions. However, most research has been focused on a few species, particularly the narrow host Phytophthora infestans and P. sojae. P. parasitica Dastur(syn. P. nicotianae Breda de Haan) is a soilborne pathogen with a wide range of host plants and represents most species in the genus Phytophthora. The emerged model pathogen will facilitate improved understanding of oomycete biology and pathology that are crucial to the development of novel disease-control strategies and improved diseasecontrol measures.In this study, we use P. parasitica as a model to explore mechanisms of oomycete pathogenicity. On one hand, we examined in details the phenotypic variation in the interaction Arabidopsis thaliana and P. parasitica and determined the presence of a potentially broadspectrum disease resistance locus, which provided a useful resource for investigating molecular process in the interaction between A. thaliana and P. parasitica. On the other hand, we attempted to search candidate proteins that play key roles in the pathogen infection by employing a high-throughput Agrobacterium tumefaciens-mediated transient expression assay on tobacco leaves to screen a c DNA library derived from infected tissues. We further investigated one candidate gene encoding a typical protein disulfide isormerase(Pp PDI1) by analyzing the cell death induction activities, expression pattern, gene silencing, and overexpression transformants. The main results are as follows:1. Detailed examination of the phenotypic variations between A. thaliana and P. parasitica in the interactions, with respect to the extent of host colonization and sporulation by the pathogen, and the relative degree of visible host response, showed the presence of a natural variation in host specificity between A. thaliana accessions and P. parasitica strains in both root and detached leaf inoculation assays. Of all the accessions tested, A. thaliana Zurich(Zu-1) exhibits high level of resistance to a set of 20 diverse P. parasitica strains with leaf inoculation method. Microscopic characterization showed that rapid and severe hypersensitive response(HR) at the primary infected epidermal cells is associated with pathogen restriction and disease resistance.2. Genetic analysis indicated that the resistance in Zu-1 to P. parasitica is semi-dominant. Crosses between the resistant accession Zu-1 and the susceptible Landsberg(Ler) showed the F1 plants exhibited moderate resistant phenotype with yellowish characters macroscopically and restricted lesion development, and the F2 populations were observed with a segregation(N: Y: H) ratio very close to 1: 2: 1(χ2=0.307, P=0.8578), indicating that the resistance in Zu-1 to P. parasitica is semi-dominant, as shown by infection assays of F1 progenies, and is likely conferred by a single locus, defined as RPPA1Zu-1(for Resistance to Phytophthora parasitica 1), as shown by analysis of F2 segregating populations. By employing specificlocus amplified fragment sequencing(SLAF-seq) strategy to identify molecular markers potentially linked to the locus, the strongest associated region was determined to be located between 7.1-11.2 Mb in chromosome IV.3. Functional screening using A. tumefaciens-mediated transient expression assay on tobacco leaves was employed to identify genes involved in plant-pathogen interaction. To screen for cell death-inducing factors, a high quality c DNA library was constructed using m RNAs derived from tobacco leaves infected with P. parasitica, and screened using a highthroughput A. tumefaciens-mediated transient expression assay on Nicotiana tobacum and N. benthamiana leaves. A screen of 16,000 c DNAs led to the identification of 24 candidate proteins that have cell death-inducing activities. Of all the candidate proteins, a typical protein disulfide isomerase gene from P. parasitica(Pp PDI1) was further confirmed to induce strong cell death on N. benthamiana leaves. Pp PDI1 contains the main structural building block with TRX-like domains and is conserved in eukaryotes. Deletion mutant analyses showed that the first CGHC motif in the active domain of Pp PDI1 is essential for inducing cell death.4. Genetic and molecular manipulation of Pp PDI1 preliminarily indicated its role as a virulence factor in P. parasitica, which contributes to plant infection. Pp PDI1 is expressed in all stages of P. parasitica life cycle, and is up-regulated during the late infection stage(60hpi) compared to germinating cyst(GC) stage and the early infection stage(36hpi), suggesting a role it may play during late plant infection stage(60hpi). To confirm the potential virulence role of Pp PDI1 in P. parasitica, we obtained gene silencing and overexpression transformants, respectively, using P. parasitica transformation method. The results showed that the silencing efficiency was very low, and Pp PDI1 expression was recovered in all the three examined Pp PDI1-silencing transformants after subculturing for three weeks, suggesting that Pp PDI1 expression is under strong selection and may play an essential role in P. parasitica development. Translational fusion to the enhanced green fluorescent protein(EGFP) in stable P. parasitica transformants showed that Pp PDI1 is highly accumulated in haustoria during pathogen infection. Compared with the control transformant 1121 that stably expresses GFP, the Pp PDI1-EGFP-expressing transformants produced more haustoria-like structures. Furthermore, real-time PCR analyses confirmed more colonization of N. benthamiana by the Pp PDI1-EGFP-expressing transformants. All these results suggest that Pp PDI1 is a virulence factor of P. parasitica and contributes to plant infection.