The Tad Pilus Apparatus of ‘Candidatus Liberibacter asiaticus’ and Its Regulation by VisNR

Abstract

Citrus huanglongbing (HLB) is one of the most destructive diseases affecting citrus plants. ‘Candidatus Liberibacter asiaticus’, an uncultivated α-proteobacteria, is the most widely spread causal agent of HLB and is transmitted by the Asian citrus psyllid Diaphorina citri. ‘Ca. L. asiaticus’ attachment to the psyllid midgut is believed to be critical to further infect other organs, including the salivary gland. In this study, the type IVc tight adherence (Tad) pilus locus encoded by ‘Ca. L. asiaticus’ was characterized. The Tad loci are conserved among members of Rhizobiaceae, including ‘Ca. L. asiaticus’ and Agrobacterium spp. Ectopic expression of the ‘Ca. L. asiaticus’ cpaF gene, an ATPase essential for the biogenesis and secretion of the Tad pilus, restored the adherence phenotype in cpaF mutant of A. tumefaciens, indicating CpaF of ‘Ca. L. asiaticus’ was functional and critical for bacterial adherence mediated by Tad pilus. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that ‘Ca. L. asiaticus’ Tad pilus-encoding genes and ‘Ca. L. asiaticus’ pilin gene flp3 were upregulated in psyllids compared with in planta. A bacterial one-hybrid assay showed that ‘Ca. L. asiaticus’ VisN and VisR, members of the LuxR transcriptional factor family, were bound to the flp3 promoter. VisNR regulate flp3. Negative regulation of the flp3 promoter by both VisN and VisR was demonstrated using a shuttle strategy, with analysis of the phenotypes and immunoblotting together with quantification of the expression of the flp3 promoter fused to the β-galactosidase reporter gene. Comparative expression analysis confirmed that ‘Ca. L. asiaticus’ visNR was less expressed in the psyllid than in the plant host. Further, motility and biofilm phenotypes of the visNR mutant of A. tumefaciens were fully complemented by expressing ‘Ca. L. asiaticus’ visNR together. The physical interaction between VisN and VisR was confirmed by pull-down and stability assays. The interaction of the flp3 promoter with VisR was verified by electrophoretic mobility shift assay. Taken together, the results revealed the contribution of the Tad pilus apparatus in the colonization of the insect vector by ‘Ca. L. asiaticus’ and shed light on the involvement of VisNR in regulation of the Tad locus.