Xanthomonas campestris sensor kinase HpaS co‐opts the orphan response regulator VemR to form a branched two‐component system that regulates motility

Abstract

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two‐hybrid and protein pull‐down assays revealed that HpaS physically interacts with VemR. Phos‐tag SDS‐PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection.

The RavA/VemR two‐component system plays vital regulatory roles in the motility and virulence of Xanthomonas campestris

“…The HK autophosphorylates its histidine residues via ATP hydrolysis and then transfers the phosphate group to the aspartate residues of the cognate RR to trigger adaptive responses (Stock et al, 2000). In Xanthomonas , five TCSs, namely, RpfC/RpfG (Stock et al, 2000), RavA/RavR (Tao et al, 2014), HpaS/HrpG (Li et al, 2014), HpaS/HpaR2 (Li et al, 2014), and HpaS/VemR (Li, Wang, et al, 2020), have been identified. RpfC/RpfG positively regulates the expression of extracellular enzymes and EPS, and negatively regulates intercellular c‐di‐GMP signal transduction (Stock et al, 2000).…”

“…Deletion of hpaS or hrpG eliminates bacterial virulence and the hypersensitive response (HR) (Li et al, 2014). Similar to HpaS/HrpG, hpaS deletion reduces the phosphorylation level of the orphan RR VemR in vivo, and the HpaS/VemR TCS controls the swimming behaviour of Xcc (Li, Wang, et al, 2020). According to these studies, HpaS phosphorylates at least three RRs (HrpG, VemR, and HpaR2), but how and when HpaS interacts with these RRs needs to be determined.…”

A HU‐like protein is required for full virulence in Xanthomonas campestris pv. campestris

“…The minimal inhibitory concentration (MIC) method (Li et al, 2020 ) was employed to test the sensitivity of the Xcc strains to several environmental stresses. Xcc strains were cultured overnight and diluted to an OD 600 of 0.1.…”

“…EPS and extracellular enzyme assays were performed as previously described (Li et al, 2020 ; Tang et al, 1991 ). To estimate EPS production, Xcc strains were inoculated into 100 ml NY liquid medium containing glucose (2% wt/vol) at 28 °C, 200 rpm for 3 days.…”

“…Cell motility was detected as previously described (Li et al, 2020 ). To test swimming motility, two microlitres of bacterial suspension (10 9 cfu/ml) was stabbed into 0.28% agar plates composed of 0.03% Bacto peptone and 0.03% yeast extract followed by incubation at 28 °C for 4 days.…”