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Citrus bacterial canker is a disease caused by Xanthomonas citri subsp. citri (Xcc). The bacterial type III secretion system (TTSS) consist of an extracellular filamentous structure mediating transfer of bacterial proteins into the plant cytosols. The main part of the pilus is pilin protein (HrpE) that is encoded by the HrpE gene. Production of specific antibodies against the pilin protein can be implemented for detection of infected plants as well as to develop a source of genetic resistance against disease. Inhibition of HrpE protein through antibody therapy leads to suppression of disease in the plant. The objective of the present study was to isolate, clone and express the HrpE gene. To do this, the HrpE gene was amplified by PCR using gene-specific primers and cloned to the pTZ57R/T vector. Then, it was cloned to the pET28a(+) bacterial expression vector and expressed in the E. coli strain Rosetta as host. The protein production procedure was optimized for incubation time and temperature as well as the concentration of inducer. The greatest amount of the recombinant protein was yielded at 1 mM IPTG at 30° C for 16 h. Purification of HrpE recombinant protein was done via metal affinity chromatography. The results of both SDS-PAGE and Western blotting assays confirmed the expression accuracy and purity of recombinant pilin protein. The recombinant protein can be used as an antigen to develop monoclonal and polyclonal antibodies.

The citrus bacterial canker is among the most important diseases in Mexican lime gardens in southern area of Iran. The disease is caused by Xanthomonas citri subsp. citri (Xcc). The PthA bacterial protein, as an effector protein, is crucial in pathogenesis pathway. Inhibition of its functions through antibody could lead to suppression of disease in plant. The present study was performed for isolation and expression of pthA gene and purification of PthA recombinant protein. For this aim the specific primers were designed for isolation of 606 bp of hydroxyl terminal of this gene followed by PCR amplification and cloning into pTZ57R/T vector. The coding sequence of truncated pthA was inserted to pET28a bacterial expression vector as a C-terminal fusion to 6XHis tag. Production of recombinant protein was performed in BL21(DE3) strain of E. coli. The purification was done under native condition through affinity chromatography on nickel column. Total yield was assessed by SDS-PAGE and western blotting analysis. The results confirmed production of PthA recombinant protein with molecular weight around 26 kDa. The purified recombinant protein could be used as an antigen for production of recombinant monoclonal antibodies.

Asiatic citrus canker caused by Xanthomonas citri subsp. citri is a bacterial disease with economic importance in tropical and subtropical citrus-producing areas. One of the potential reaction of plants to pathogens attack is an increased levels of pathogenesis-related proteins such as chitinase and β - 1,3 -glucanase. The objective of present study was to investigate the expression of the PR proteins; β-1,3-glucanase and chitinase in Washington Navel orange (Citrus sinensis) inoculated with Xanthomonas citri sub sp.citri. Quantitative reverse transcription-polymerase chain reaction was used to quantify the mRNA level of chitinase and ß- 1,3 -glucanase genes in early stage of plant defense response. For this purpose, total RNA was extracted from inoculated and non inoculated citrus plants at different times post-inoculation. Higher amounts of transcripts were detected 24 hours after pathogen inoculation of citrus leaves and four-fold more than the control citrus plant. The transcript levels of the two genes were reduced with the time after inoculation, and decreased significantly at 96 hours post inoculation. This study indicated that the role of chitinase gene is more than glucanase in early stage of citrus canker disease.