The Ferredoxin-Like Protein FerR Regulates PrbP Activity in Liberibacter asiaticus

ABSTRACT

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro. The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbP_Las), mRNA levels of ferR of L. asiaticus (ferR_Las) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbP_Las is modulated via interactions with FerR_Las. The induction of ferR_Las expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticus.

IMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbP_Las) and a ferredoxin-like protein (FerR) in L. asiaticus, FerR_Las. We show that the presence of FerR stabilizes and augments the activity of PrbP_Las. In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens. Altogether, these results suggest that FerR_Las and PrbP_Las may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.