Location: Subtropical Plant Pathology Research2012 Annual Report
1a. Objectives (from AD-416):
Characterize the hypl gene and determine its effects on insect transmission and/or virulence in host plants. The objectives of the original proposal include: 1) determine the cellular localization of the Hypl protein within insect or plant cells and the efficacy of the two putative NLSs coded by the hypl gene; 2) determine the expression dynamics of hypl both in psyllids and plants (citrus and periwinkle) and elucidate its activity with respect to effector function; 3) develop new techniques for more sensitive detection and differentiation of Las bacteria based on the unique sequence and antigenicity of the hypl gene, and 4) develop transgenic citrus resistant to HLB by expressing plantibody against the Hypl protein.
1b. Approach (from AD-416):
Transient expression with alternative expression systems and RT-qPCR, etc., will be used to elucidate the function of the hypl gene of Las and shed the light on the molecular mechanism of this "phase variation" phenomenon and thereby develop a novel control strategy for citrus HLB. In addition, antibodies and probes along with standardized protocols, developed during this project can be applied for better detection and differentiation of the HLB bacteria.
3. Progress Report:
This research is related to inhouse project objective 1a. Characterize the etiology, molecular biology and genetics of ‘Candidatus Liberibacter asiaticus (Las),’ the bacterium associated with citrus huanglongbing (HLB). The hyvI and hyvII within two Candidatus Liberibacter asiaticus (Las) prophages were further characterized and some of the results were published in Applied Environmental Microbiology 77:6663-6673, 2011. "Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont "Candidatus Liberibacter asiaticus" as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats". We have developed an improved real-time polymerase chain reaction (PCR) using SYBR Green 1 (LJ900fr) and TaqMan® (LJ900fpr) protocols with primers and probe targeting the nearly identical tandem repeats of 100bp hyvI and hyvII. The results were published in Molecular and Cellular probes, 26:90-98, 2012. Monoclonal antibodies against the partial HyvI protein (only one repeat) were generated, and their sensitivity and specificity were evaluated for the detection of HyvI protein expressed in E. coli and huanglongbing (HLB)-infected citrus and psyllids. All antibodies were able to recognize the E. coli expressed HyvI antigen, but were not able to detect the HyvI antigen from HLB-infected plants and psyllids. To determine the cellular localization of the HyvI protein in plant cells and the role of the two putative Nuclear localization signals (NLSs) in hyvI gene, full-length hyvI and C-terminal region including two putative NLSs were amplified and cloned into pCX-DG vector with green fluorescent protein (GFP) driven by CaMV35S promoter. The results indicate that the HyvI protein did not target in plant nucleus but located in cytoplasm (possible in organelle) when transient expression in tobacco. We determined that HyvI targeted tobacco mitochondria by transient expression and MitoTracker Mitochondria-selective probe staining. When the full length of hyvI gene was cloned, and replicated in heterologous hosts, the repeat number of hyvI gene remained the same in E. coli, but varied in Xanthomonas citri (X. citri). Clones of X. citri containing the hyvI gene displayed different degree of growth retardation, indicating potential toxic effect of hyvI gene to X. citri. HyvI C- terminus and full length HyvII were fused to GFP and expressed in E. coli driven by T7 promoter. Confocal microscopy results show both proteins are localized to the bacterial poles. Protein localization, sequence analysis and protein structure prediction suggest both protein belong to autotransporter family. HyvI and II are unique autotransporters because the non-conserved translocator domain can export both its natural passanger domain and also the GFP fusion domain to E.coli's cell surface. The protein was determined to localize at cell surface by dot blot, and furthermore the protein surface localization was proved by protease treatment of intact bacterial cells. The immunofluorescence assay to confirm the surface localization of the HyvI protein was unsuccessful. The proteins appear to be on the cell surface but are not folded properly.