Characterization of orf6 gene in conserved effector locus and translocon genes involved in type III secretion system of Pseudomonas syringae pv. averrhoi
| Autor: | Hsueh-Wen Hu, 胡學文 |
|---|---|
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 97 Pseudomonas syringae pv. averrhoi (Pav) is a gram-negative, rod-shaped bacterium, and it causes bacterial spot of carambola in Taiwan. Pav possesses a Hrp pathogenicity island (Pai) responsible for eliciting pathogenicity on susceptible host and hypersensitive response (HR) on nonhost plants. The Hrp Pai contains three regions, including an exchangeable effector locus (EEL), a conserved effector locus (CEL), and a hrp/hrc cluster which encodes a type III secretion system (T3SS) to facilitate the translocation of effectors into host cell. The orf6 is located in the Pai CEL. In previous studies, it was shown that orf6 gene is highly expressed during bacterial colonization on dry leaf surfaces, and it appears not to be a member of the hrp-regulon. In this study, the Pav HL1 native or N-terminal 188 a.a Orf6 protein was expressed in trans, which suppressed the hypersensitive response (HR) on nonhost tobacco. The growth of Pav strains overproducing Orf6 protein was reduced in in host carambola. Meanwhile, overexpression of Orf6 in Pav triggered more callose deposition on plant cell walls. Using CyaA as a translocation reporter, Orf6-CyaA fusion protein was neither translocated into plant cells nor secreted into HMM inducing medium. Those results suggest that Orf6 may act on bacterial surface, as a the pathogen-associated molecular pattern (PAMP) that could induce PAMP triggered immunity (PTI) in plants.Besides, plant pathogens secret a group of specific pore-forming proteins called translocon to help T3SS structure crossing the plant plasma membrane. In Pav HL1, there are four translocon genes, hrpK1, hrpZ1, hrpW1, and hopAK1. HopAK1 amino acid sequence consists of the harpin domain in the N-terminus and the pectate lyase domain in the C-terminus. The crude extract HopAK1 protein was able to elicit HR on tobacco by using T7-RNA polymerase dependent expression system, and in pectate lyase activity assay, it revealed that pectate lyase domain has enzyme activity. To test whether HopAK1 is secreted via T3SS, the protein was fused with a FLAG-CTC tag which can be detected by the M2 monoclonal antibody in immunoblotting. A western blot analysis revealed that HopAK1-FLAG fusion protein was secreted via T3SS into HMM. The functional domains of HrpW1Pav are similar to HopAK1, however, hrpW1Pav gene is truncated. Western blot analysis of the HrpW1Pav protein with HrpW1Pto antiserum revealed that HrpW1Pav was not detected in Pav HL1 and PA5. Therefore, we predicted hrpW1Pav might be a pseudogene. serially diluted Pav HL1 translocon mutant strains were infiltrated into tobacco leaves and found the mutant strains can be classified into three groups based on the HR phenotypes: (i) The △hopAK1 and △hrpZ1 single mutants and the △hopAK1/hrpZ1 double mutant strains showed wild type-like HR; (ii) The △hrpK1 single mutant had a moderate HR elicitation ability at the inoculum concentration of 5×107 CFU/ml; (iii) The △hrpK1/hopAK1, △hrpK1/hrpZ1 double mutants and △hrpK1/hopAK1/hrpZ1 triple mutant failed to elicit the HR even at a high inoculum concentration of 1×108 CFU/ml. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|