'CandidatusLiberibacter asiaticus' putative effectors:in silicoanalysis and gene expression in citrus leaves displaying distinct huanglongbing symptoms

Huanglongbing (HLB) is considered the most devastating bacterial disease in citriculture. It is caused by the phloem-limited alpha-proteobacterium 'CandidatusLiberibacter asiaticus' (CLas). All citrus varieties are susceptible to HLB and many studies have been conducted in an attempt to identify potential targets involved in bacterial virulence and pathogenicity mechanisms. One of the most important virulence factors of bacterial pathogens is the effector molecule that can modify host cell structures, facilitate infection, and/or trigger host defense responses. In this study, 14 CLas candidates for secreted hypothetical proteins were predicted by a bioinformatics pipeline, one of which (NSEC01) was predicted to carry a nuclear localization sequence (NLS). Putative effectors HLBSP04 and HLBSP21 were conserved in all genomes evaluated, whereas HLBSP01, HLBSP19, and HLBSP30 were exclusively found in CLas. Expression analysis of candidate effector genes was performed in citrus leaves exhibiting five distinct HLB symptoms (asymptomatic, blotchy mottle, zinc deficiency, yellowed veins, and vein corky) and used as a guide for the selection of effectors for further functional studies. Genes coding for candidate effectors HLBSP13, HLBSP20, HLBSP28, and NSEC01 had high expression levels in blotchy mottle leaves, suggesting they could be used as marker genes that may facilitate detection of the pathogen in trees with this HLB symptom. As NSEC01 was exclusively found in this work, it was selected for transient expression inNicotiana benthamianaleaves, but further investigations need to be carried out. Also, other candidate effectors predicted in this work still need to be functionally characterized. This study allowed discussing and proposing hypotheses about CLas classification according to virulence and lifestyle, which can help understand the citrus-CLas interaction.