Phytopathogenic bacteria utilize host glucose as a signal to stimulate virulence through LuxR homologues

Abstract

Chemical signal-mediated biological communication is common within bacteria and between bacteria and their hosts. Many plant-associated bacteria respond to unknown plant compounds to regulate bacterial gene expression. However, the nature of the plant compounds that mediate such interkingdom communication and the underlying mechanisms remain poorly characterized. Xanthomonas campestris pv. campestris (Xcc) causes black rot disease on brassica vegetables. Xcc contains an orphan LuxR regulator (XccR) which senses a plant signal that was validated to be glucose by HPLC-MS. The glucose concentration increases in apoplast fluid after Xcc infection, which is caused by the enhanced activity of plant sugar transporters translocating sugar and cell-wall invertases releasing glucose from sucrose. XccR recruits glucose, but not fructose, sucrose, glucose 6-phosphate, and UDP-glucose, to activate pip expression. Deletion of the bacterial glucose transporter gene sglT impaired pathogen virulence and pip expression. Structural prediction showed that the N-terminal domain of XccR forms an alternative pocket neighbouring the AHL-binding pocket for glucose docking. Substitution of three residues affecting structural stability abolished the ability of XccR to bind to the luxXc box in the pip promoter. Several other XccR homologues from plant-associated bacteria can also form stable complexes with glucose, indicating that glucose may function as a common signal molecule for pathogen-plant interactions. The conservation of a glucose/XccR/pip-like system in plant-associated bacteria suggests that some phytopathogens have evolved the ability to utilize host compounds as virulence signals, indicating that LuxRs mediate an interkingdom signalling circuit.

Keywords: LuxR ligand; glucose; interkingdom signalling; quorum sensing.

FIGURE 1

Xanthomonas campestris pv. campestris (Xcc) infection induces sugar accumulation in host plants and glucose enhances XccR‐DNA binding. (a and b) Accumulation of glucose and fructose in Arabidopsis (a) and cabbage (b) leaves after Xcc infection. FW, fresh weight; n = 4. Error bars represent SEM. **p < 0.001, two‐tailed t test. (c) The binding ability of XccR to the target DNA sequence (the luxXc box) with increasing concentrations of glucose as determined by electrophoretic mobility shift assay. Each “+” represents 20 pmol XccR‐MBP; the right triangle represents the addition of glucose with a gradient of 0.005, 0.05, 0.5, and 5 μM. DNA probe is the [³²P]‐labelled luxXc box sequence. MBP, maltose‐binding protein.

FIGURE 2

Exogenous supply of glucose enhances pip expression. (a and b) Addition of 25 mM glucose in minimal medium (MM) increased the bacterial endogenous glucose content (a) and β‐glucuronidase (GUS) activity in Xcc 8008 (b). (c) GUS activity in Xanthomonas campestris pv. campestris (Xcc) 8008 and ∆sglT after 6 h of incubation with 200 μM 2‐deoxyglucose, glucose, or UDP‐glucose; water was used as a control. (d) The XccR‐bound glucose content in Xcc 8004 cultured to OD₆₀₀ = 0.3 in MM with 25 mM glucose. *p < 0.05, **p < 0.001, ***p < 0.0001, two‐tailed t test. Error bars represent SEM.

FIGURE 3

XccR recruits glucose as a signal. (a) A docking model of XccR with glucose. The top panel shows glucose in purple and XccR in cyan. The bottom panel shows the solvent‐accessible surface of XccR. (b) Essential residues of XccR for interaction with glucose. Amino acids forming hydrogen bonds with glucose (Cys21 and Tyr41) and the corresponding hydrogen bonds are shown as blue dashed lines; the amino acid (Gln60) forming hydrogen bonds with Cys21 and Tyr41 to stabilize the glucose‐recognizing interface of XccR and the corresponding hydrogen bonds are shown as green dashed lines. (c) XccR‐MBP binds glucose, as determined by microscale thermophoresis (MST) assays (n = 3) with maltose‐binding protein (MBP) as the negative control. Error bars represent SEM. (d and e) XccR‐SUMO binds glucose, as determined by MST assays, with the SUMO protein as the negative control. Isothermal titration calorimetry assay detecting the binding of XccR with glucose. Results shown are for XccR‐MBP (10 μM) titrates with 100 μM glucose.

FIGURE 4

AtSWEET2 and AtSWEET15 are required for sugar transportation after Xanthomonas campestris pv. campestris (Xcc) infection. (a) Relative expression levels of AtSWEET members in Arabidopsis leaves after Xcc infection. hpi, hours postinfection. (b) Xcc infection alters apoplastic glucose flux in wild‐type Arabidopsis (Col‐0) but not in AtSWEET2 or AtSWEET15 loss‐of‐function mutants (Atsweet2‐3‐2 or Atsweet15‐4‐7). FW, fresh weight; n = 5. (c and d) Reduced bacterial population (c) and virulence (d) of Xcc in AtSWEET2 or AtSWEET15 loss‐of‐function mutant leaves, n = 6. (e) Glucose restores pip promoter‐driven β‐glucuronidase (GUS) activities in Atsweet2 or Atsweet15 loss‐of‐function mutants, n = 3. (f) Effects of essential amino acids of XccR on pip expression. The pip promoter‐driven GUS activities were detected after infiltration of bacteria into plants, n = 9. GUS activities were normalized to the bacterial population before comparison between samples. *p < 0.05, **p < 0.001, two‐tailed t test. Error bars represent SEM. Scale bar in (d), 0.5 cm.

FIGURE 5

Conservation of the glucose‐binding pocket and glucose‐binding ability in LuxR homologues of several plant‐associated bacteria. (a–e) Glucose binding models of LuxR homologues: OryR (a), PsoR (b), PsyR (c), QscR (d), and XagR (e). Left, the presence of a predicted glucose‐binding pocket in each protein. The hydrogen bonds between essential residues and glucose are shown as blue dashed lines. Right, the relative binding curves of LuxR‐MBPs and sugars as determined by microscale thermophoresis assays.

FIGURE 6

Working model of glucose as a signal sensed by the bacterial transcription factor XccR. Xanthomonas campestris pv. campestris infection promotes the expression of host plant sugar transporters, which leads to sugar accumulation at the infection sites. The translocated sucrose is hydrolysed to glucose and fructose by cell‐wall invertase (Cw‐Inv). XccR senses and binds to glucose to induce expression of the virulence gene pip.