Protein-protein interactions identified for effector proteins of the phytopathogen Xanthomonas axonopodis pv. citri

DUNGER, G.; PEREDA ROSA, M.C.; CHUCK FARAH; ORELLANO, E; JORGELINA OTTADO

Rosario

Congreso; V Congreso Argentino de Microbiología General; 2008

Sociedad Argentina de Microbiología General

Xanthomonas axonopodis pv. citri (Xac) cause citrus canker, a serious disease of the citrus genus that results in important losses in citriculture regions. Xac is a bacteria Gram (-) and uses secretion systems for translocation of pathogenicity and avirulence proteins to the plant cells. There are many genes characterized like elicitors of the host plants response and/or of hypersensitive response (HR) in non host plants. In the sequenced genome of Xac have been found genes that possibly be effectors proteins that exert their function in the plant cell, called XAC3090, avrXacE1 (XAC0286) and avrXacE2 (XAC3224). XAC3090 presents six domains of leucine rich repeats (LRR), while avrXacE1 and avrXacE2 show similarity with avirulence proteins from other plant pathogens. We isolated XAC3090, avrXacE1 and avrXacE2 of Xac and constructed mutants in this gene, using a suicide vector transferred by biparental mating. For disease symptoms assays, bacterial suspensions were infiltrated into leaves of host plant orange (Citrus sinensis). The results showed that there are not differences in the lesions by the infection with Xac-XAC3090 in host plants; while Xac-avrXacE1 and Xac-avrXacE2 showed different phenotype than Xac wild type. Xac-avrXacE1 produce a darkness lesion than Xac wild type; and inoculation with Xac-avrXacE2 generate a necrotic lesion in citrus leaves. The bacterial growth in citrus leaves observed for mutants were similar to the Xac wild type. Two-hybrids assay in yeasts was carried out to characterize the interaction of XAC3090, AvrXacE1 and AvrXacE2 with possible white sequences in a library of total genomic DNA from Xac. Shortly, XAC3090, avrXacE1 and avrXacE2 were cloned in the vector pOBD; and a library of total genomic DNA library (Xac chromosome plus plasmids) containing fragments of 500 to 3,000 bp cloned into the vector pOAD. Two-hybrid assays were performed by using simultaneous screening of two reporter genes under the control of different inducible promoters (GAL1-HIS3 and GAL2-ADE2). This simultaneous screening reduced the number of false positives. 192 positive clones were evaluated by sequenciation. 38 positive preys for AvrXacE1 were found, among this preys we found the molecular chaperone DnaK (XAC1522) and the avirulence protein Hbss3.0, 40 preys for XAC3090 where it presented interaction with a two component system sensor histidine kinase (XAC3643), and 22 preys for AvrXacE2 that revealed interaction with the enzyme cellulase (XAC2522). Among the hypothetical protein there are several preys that present domains involucrate in protein-protein interaction. These results suggest that these effector proteins could have different roles in the pathogenicity.