This work reports the in vitro expression of Citrus leprosis virus C (CiLV-C) putative coat protein (p29) and the production of a polyclonal antibody to be used as a tool for serological diagnosis of citrus leprosis. The ORF2/RNA1, corresponding to p29, was cloned in pET28a and transformed into Escherichia coli cells (BL21). Expression of p29 was induced in vitro and the protein was purified and used for immunization of rabbits to produce the polyclonal antibody. The anti p29 serum was shown to be highly specific to CiLV-C detection by immunological methods (Western blot, PTA-ELISA, tissue blot and in situ immunolocalization), without cross reaction with healthy citrus plants or other cytoplasmic and nuclear viruses transmitted by Brevipalpus mites. These results demonstrate that the antibody against CiLV-C p29 protein is highly specific for CiLV-C detection. In situ immunogold labeling assays on thin sections of sweet orange leaf cells infected by CiLV-C demonstrated that short, bacilliform particles present within cisternae of the endoplasmic reticulum were specifically labeled, confirming their viral nature. The dense cytoplasmic viroplasm was also heavily labeled indicating that it represents a site of p29 accumulation.

In silico expression profiles, of the discovered 3,103 citrus ESTs putatively encoding for PR protein families (PR-1 to PR-17), were evaluated using the Brazil citrus genome EST CitEST/database. Hierarchical clustering was displayed to identify similarities in expression patterns among citrus PR-like gene families (PRlgf) in 33 selected cDNA libraries. In this way, PRlgf preferentially expressed by organ and citrus species, and library conditions were highlighted. Changes in expression profiles of clusters for each of the 17 PRlgf expressed in organs infected by pathogens or drought-stressed citrus species were displayed for relative suppression or induction gene expression in relation to the counterpart control. Overall, few PRlgf showed expression 2-fold higher in pathogen-infected than in uninfected organs, even though the differential expression profiles displayed have been quite diverse among studied species and organs. Furthermore, an insight into some contigs from four PRlgf pointed out putative members of multigene families. They appear to be evolutionarily conserved within citrus species and/or organ- or stress-specifically expressed. Our results represent a starting point regarding the extent of expression pattern differences underlying PRlgf expression and reveal genes that may prove to be useful in studies regarding biotechnological approaches or citrus resistance markers.