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.
This project is related to inhouse objective 1: Characterize ecology, biology, epidemiology, genetics and host interactions of domestic, exotic, newly emergent and re-emerging pathogens.
To determine the cellular localization of the HyvI (rename from HypI) 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, and transformed Agrobacterium tumefaciens strain GV 2660 with the pCX-DG-hyvI constructs. The results indicate that the HyvI protein did not target in plant nucleus but located in cytoplasm when it was transient expressed in tobacco. In addition, the results show that the hyvI gene did not induce a hypersensitive response on tobacco leaves. RT (Reverse transcriptase) polymerase chain reaction (PCR) confirmed the hyvI gene expression both in the host plant and psyllids though the HyvI protein was not detected with Western blot. The obstacles for detection of the antigen from huanglongbing (HLB)-infected plants remains to be further investigated.