An anthranilic acid-responsive transcriptional regulator controls the physiology and pathogenicity of Ralstonia solanacearum.

Quorum sensing (QS) is widely employed by bacterial cells to control gene expression in a cell density-dependent manner. A previous study revealed that anthranilic acid from Ralstonia solanacearum plays a vital role in regulating the physiology and pathogenicity of R. solanacearum. We reported here that anthranilic acid controls the important biological functions and virulence of R. solanacearum through the receptor protein RaaR, which contains helix-turn-helix (HTH) and LysR substrate binding (LysR_substrate) domains. RaaR regulates the same processes as anthranilic acid, and both are present in various bacterial species. In addition, anthranilic acid-deficient mutant phenotypes were rescued by in trans expression of RaaR. Intriguingly, we found that anthranilic acid binds to the LysR_substrate domain of RaaR with high affinity, induces allosteric conformational changes, and then enhances the binding of RaaR to the promoter DNA regions of target genes. These findings indicate that the components of the anthranilic acid signaling system are distinguished from those of the typical QS systems. Together, our work presents a unique and widely conserved signaling system that might be an important new type of cell-to-cell communication system in bacteria.

Introduction

Quorum sensing (QS) is a cell-cell communication mechanism used by many bacteria to coordinate group behaviors in response to cell density [1-3]. When the cell density of the microbial population in the environment reaches a threshold, the QS signal activates a responsive regulator to control target gene expression and regulate physiological characteristics. To date, a variety of QS signaling molecules have been discovered, and the first well-characterized QS signaling molecules used by many gram-negative bacteria are the N-acyl homoserine lactones (AHLs) [2,4-15]. As QS system usually regulates the important biological functions and virulence of pathogenic bacteria, many studies have demonstrated that interfering with the QS system is a new strategy to prevent the infection by pathogenic bacteria [16-19].

Bacterial wilt caused by thousands of genetically distinct strains within the Ralstonia solanacearum species complex (RSSC) is one of the most important plant diseases in tropical, subtropical, and some warm regions [20]. R. solanacearum can infect hundreds of plants belonging to 54 families, causing very large economic losses to global agricultural production [21,22]. There are two QS systems that were previously identified in R. solanacearum, the sol system and the phc system. The phc QS system plays a vital role in the regulation of virulence factors, including extracellular polysaccharides (EPS), cell wall-degrading enzymes, and motility [4,21,23-25]. A methyltransferase of R. solanacearum named PhcB synthesizes either methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3-OH PAME) as the signal of the phc QS system [23]. The signals phosphorylate the response regulator PhcR by activating the histidine kinase PhcS and then finally control the target genes and the sol system [4,26-28]. The sol system is a typical QS system based on acyl-homoserine lactone (AHL) signals, but does not regulate virulence factor production in R. solanacearum AW1 strain [4,23,28].

Our previous study showed that R. solanacearum GMI1000 utilizes anthranilic acid to regulate important biological functions and the synthesis of QS signals, including 3-OH MAME and AHL signals [29]. In this study, we identified a receptor responsive to anthranilic acid, RaaR (Regulator of anthranilic acid of alstonia solanacearum). RaaR is a transcriptional regulator with two domains, an HTH domain and a LysR_substrate domain. Perception of anthranilic acid by the LysR_substrate domain enhances the binding of RaaR to the target gene promoter and then controls the physiology and pathogenicity of R. solanacearum. We also found that homologs of both the anthranilic acid synthase TrpEG and the regulator RaaR are present in diverse bacteria, suggesting that the anthranilic acid-type signaling system is widespread.

Results

RaaR is a downstream component of the anthranilic acid signaling system in R. solanacearum

Our previous studies showed that anthranilic acid regulates the important biological functions and virulence of R. solanacearum [29]. As anthranilic acid is a precursor for the biosynthesis of 4-hydroxy-2-heptylquinoline (HHQ) and the Pseudomonas quinolone signal (PQS) in Pseudomonas aeruginosa, we then searched for homologs of MvfR, which is the receptor of both HHQ and PQS signals in P. aeruginosa [30-32], in the genome of R. solanacearum GMI1000 to identify the potential downstream component of the anthranilic acid signaling system by using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). We found 10 potential homologs of MvfR sharing 15.18% to 33.5% identity with MvfR (S1 Table). We in trans expressed each of all the homologs in the anthranilic acid deficient mutant ΔtrpEG and measured the motility activity of these overexpression strains. The results showed that only in trans expression of RSp0912 rescued the defective motility phenotype of the ΔtrpEG strain (Fig 1A and 1B). Then, RSp0912 was selected for further investigation. We found that in trans expression of RSp0912 restored all the tested phenotypes of ΔtrpEG to the wild-type strain levels, including biofilm formation, EPS production and cellulase production, which are all regulated by the anthranilic acid signaling system (Fig 1C, 1D and 1E). We then measured the gene expression levels of RSp0912 in the wild-type strain and the trpEG deletion mutant. The results indicated that deletion of trpEG caused a significant decrease in the gene expression level of RSp0912, and exogenous addition of anthranilic acid could almost restore the expression level of RSp0912 (S1 Fig). As RSp0912 has a helix-turn-helix (HTH) domain and a LysR_substrate domain (Fig 1B), we hypothesized that it might be a downstream component of the anthranilic acid signaling system and thus named it the regulator of anthranilic acid of Ralstonia solanacearum (RaaR).

Fig 1

Influence of raaR on the virulence-related phenotypes in the trpEG mutant strains.

(A) Effects of the homologs of MvfR in R. solanacearum on the motility activity of the trpEG-deficient mutant of R. solanacearum GMI1000. (B) Genomic organization of the raaR region in R. solanacearum GMI1000 (top). Domain structure analysis of RaaR (bottom). The RaaR protein has two domains: an HTH domain and a LysR_substrate domain. In trans expression of RaaR restored biofilm formation (C), EPS production (D) and cellulase production (E) in the TrpEG-deficient mutant. The data are the means ± standard deviations of three independent experiments. ***p < 0.001 (unpaired t-test).

RaaR is a key regulator of the anthranilic acid signaling system

As in trans expression of raaR fully restored the defective phenotypes of the anthranilic acid deficient mutant ΔtrpEG, we then continued to investigate the role of RaaR in regulating these important biological functions. An in-frame deletion mutant of raaR was generated, we found that deletion of raaR caused the same phenotypic changes as observed with the anthranilic acid mutant ΔtrpEG, including reduced motility activity (Fig 2A), biofilm formation (Fig 2B), EPS production (Fig 2C), and cellulase production (Fig 2D), but did not affect the growth rate of the bacterial cells in either nutrient-rich or nutrient-poor medium (S2 Fig). And the addition of exogenous anthranilic acid didn’t restore these phenotypes of both the raaR deletion mutant and the trpEG and raaR double deletion mutant to wild-type strain levels (Fig 2). From this, we concluded that RaaR is a key downstream regulator of the anthranilic acid signaling system in R. solanacearum.

Fig 2

Effects of raaR on the anthranilic acid-regulated motility (A), biofilm formation (B), EPS production (C) and cellulase production (D) in R. solanacearum GMI1000. The data are the means ± standard deviations of three independent experiments. ***p < 0.001 (unpaired t-test).

RaaR regulates target gene expression by directly binding to promoters

Our previous study demonstrated that anthranilic acid positively regulates both the phc and sol QS systems in R. solanacearum [29]. We then studied whether RaaR also regulates the QS systems. We constructed PphcB-lacZ and PsolI-lacZ reporter systems in a raaR mutant and a trpEG and raaR double deletion mutant, as both of the target genes are positively controlled by anthranilic acid [29]. Deletion of raaR resulted in reduced expression levels of both phcB and solI (Fig 3A and 3B), which are the signal molecule synthase-encoding genes of the phc and sol QS systems, respectively. Expression levels of both phcB and solI in the trpEG and raaR double deletion mutant also decreased to raaR deletion mutant levels. Therefore, we measured and compared the production of 3-OH MAME, C8-AHL and C10-AHL in the wild-type, raaR mutant, trpEG and raaR double deletion mutant and complemented strains. The production of 3-OH MAME, C8-AHL and C10-AHL was reduced in the raaR mutant strain and the trpEG and raaR double deletion mutant strain, and in trans expression of raaR almost fully rescued signal production (Fig 3C).

Fig 3

Influence of raaR on the QS signal production of R. solanacearum.

Effects of raaR on the gene expression levels of phcB (A) and solI (B) and on QS signal production (C) in R. solanacearum GMI1000. We used promoter activity assays to quantify gene expression. The data are the means ± standard deviations of three independent experiments. ***p < 0.001 (unpaired t-test). EMSA analysis of the in vitro binding of RaaR to the promoters of phcB (D) and solI (E); biotin-labeled 327-bp phcB and 324-bp solI promoter DNA probes were used for the protein binding assay. A protein-DNA complex, represented by a band shift, was formed when different concentrations of RaaR protein were incubated with the probe at room temperature for 30 min.

RaaR is a key downstream regulator of the anthranilic acid signaling system and contains an HTH domain that is predicted to be closely related to DNA binding. Therefore, we tested whether the transcriptional regulation of phcB and solI is achieved by direct binding of RaaR to their promoters by performing electrophoretic mobility shift assays (EMSAs). A 327-bp DNA fragment from the phcB promoter and a 324-bp DNA fragment from the solI promoter were PCR-amplified and used as probes. As shown in Fig 3, the phcB and solI promoter DNA fragments formed stable DNA-protein complexes with RaaR and migrated slower than unbound probes. The amount of labeled probe that bound to RaaR increased with increasing amounts of RaaR but decreased in the presence of a 100-fold greater concentration of the unlabeled probe (Fig 3D and 3E). We also selected a housekeeping gene gyrB, which encodes DNA topoisomerase (ATP-hydrolyzing) subunit B, and used it as a negative control. Deletion of raaR didn’t affect the expression level of gyrB, and RaaR didn’t bind to the gyrB promoter DNA (S3 Fig).

Anthranilic acid is a signal ligand of RaaR

Since RaaR regulates the same processes as anthranilic acid signal and has a LysR_substrate domain, we hypothesized that anthranilic acid may influence the activity of RaaR through ligand-protein interactions [33]. To confirm this hypothesis, we performed isothermal titration calorimetry (ITC) analysis to test whether RaaR binds anthranilic acid. RaaR, which contained 313 aa with a calculated molecular weight of 35.59 kDa, was purified to homogeneity using affinity chromatography and characterized for interactions with anthranilic acid. Anthranilic acid binds strongly to the purified RaaR protein (Fig 4). Fitting the binding isotherm data showed that RaaR binds to anthranilic acid in a 1:1 stoichiometry with an estimated dissociation constant (Kd) of 3.63 μM. In addition, we purified a polypeptide containing only the LysR_substrate domain of RaaR and tested it for anthranilic acid binding. ITC analyses revealed that the LysR_substrate domain is the anthranilic acid binding motif (S4 Fig).

Fig 4

ITC analysis of the binding of anthranilic acid to RaaR protein.

(A) SDS-PAGE of the purified RaaR protein. (B) ITC titration of 250 μM anthranilic acid in PBS buffer at 25°C. (C) ITC titration of 20 μM RaaR with 250 μΜ anthranilic acid in PBS buffer at 25°C.

To study how anthranilic acid influences the binding of RaaR to promoter DNA, we used circular dichroism (CD) spectroscopy to study anthranilic acid-RaaR interactions and detect secondary structural changes in the RaaR protein when it bound anthranilic acid. The CD spectrum of α-helixes is characterized by two negative bands of almost equal intensities at 222 and 208 nm along with a strong positive band at approximately 192 nm. The β-sheet spectrum is characterized by a negative band at 216 nm along with a strong positive band at approximately 195 nm [34]. As shown in S5 Fig, both the α-helix and β-sheet spectra of RaaR significantly changed when it was supplemented with 10 μM anthranilic acid (S5 Fig). These results are consistent with the ITC results, indicating that the RaaR protein binds to anthranilic acid signals and the binding significantly changes the secondary structure of the RaaR protein.

Anthranilic acid enhances RaaR binding to target promoter DNA

To determine how the binding of anthranilic acid to RaaR might affect the activity of RaaR, we then examined the effects of anthranilic acid on the binding of RaaR to the phcB and solI promoters by EMSA. As shown in Fig 5, the binding of RaaR to the phcB and solI promoter probes was enhanced when anthranilic acid was present in the reaction mixtures. The amounts of the probes that bound to RaaR increased with increasing amounts of anthranilic acid, suggesting that the addition of anthranilic acid increased the ability of RaaR to bind to the target promoter DNA (Fig 5). To further determine whether the effect of anthranilic acid on RaaR was specific. We evaluated the effect of one of the analogs of anthranilic acid, HHQ, on the ability of RaaR to bind to the target promoter DNA. The results showed that HHQ did not affect the RaaR binding to the target promoter DNA even at a final concentration of 100 μM (S6 Fig).

Fig 5

The effects of anthranilic acid on the binding of RaaR to the promoters of phcB (A) and solI (B) were assessed by performing EMSA in vitro. A protein-DNA complex was formed when the protein was incubated with the probe, and different concentrations of anthranilic acid had an effect on the formation of the complex at room temperature for 30 min.

RaaR contributes to R. solanacearum pathogenicity

Previous studies characterized the attenuated virulence of an anthranilic acid synthase-negative mutant of R. solanacearum [29]. We evaluated the effect of RaaR on the ability of R. solanacearum to infect host plants. As shown in Fig 6A, compared with the plants infected by the R. solanacearum wild-type strain and complemented strains, tomato plants infected by the R. solanacearum raaR mutant strain were significantly reduced in wilting symptoms. After 14 days of inoculation, the disease indexes of tomato plants inoculated with wild-type and complemented strains of R. solanacearum were 3.7 and 3.5, respectively, while that of ΔraaR was 1.8, which was 51.4% lower than that of the wild-type strain (Fig 6B).

Fig 6

Influence of raaR on the pathogenicity of R. solanacearum in tomato plants.

(A) Analysis of R. solanacearum virulence in tomato plants at 14 days after inoculation of R. solanacearum. (B) The effects of raaR on the virulence of R. solanacearum were measured by assessing the disease index of bacterial wilt in tomato. **p < 0.01; ***p < 0.001 (unpaired ANOVA test). The number of CFUs of the R. solanacearum wild-type, raaR mutant and complemented strains in the roots (C) and stems (D) of tomato plants. The data are the means ± standard deviations of three independent experiments. **p < 0.01; ***p < 0.001 (unpaired t-test).

We further quantified the colony-forming units (CFU) of R. solanacearum strains in both the roots and stems of the tomato plants. The numbers of CFUs recorded for the R. solanacearum wild-type, ΔraaR mutant, and complemented strains were 4.27 × 108, 0.53 × 108, and 3.73 × 108 per gram of root tissue at 7 d postinoculation, respectively (Fig 6C). A similar result was observed for tomato stems, in which the numbers of CFUs of the three strains were 6.83 × 108, 0.91 × 108, and 6.03 × 108 per gram of stem tissue at 7 d postinoculation, respectively (Fig 6D). These results suggest that RaaR plays an important role in the pathogenesis of R. solanacearum.

RaaR controls the expression of a wide range of genes

To further study the regulatory roles of RaaR in controlling bacterial physiology, we analyzed and compared the transcriptomes of the wild-type strain and the ΔtrpEG and ΔraaR mutants by using RNA sequencing (RNA-Seq). Differential gene expression analysis showed that 41 genes were increased and 145 genes were decreased in the ΔraaR mutant compared with their expression in the wild-type strain (Log2 fold-change ≥1.5), whereas 227 genes were increased and 278 genes were decreased in the ΔtrpEG mutant (S7 Fig and S2 Table). These differentially expressed genes are associated with a range of biological functions, including motility, virulence, regulation, transport and signal transduction (S7 Fig and S2 Table). We also compared the transcriptome profiles of the ΔtrpEG mutant and the ΔraaR mutant and found an overlap in their target genes (S7B and S7C Fig).

The anthranilic acid signaling system is unique and widespread in bacteria

In P. aeruginosa, anthranilic acid is a precursor of the QS signal PQS. The PQS signal is sensed by the receptor protein MvfR to regulate the biological functions and virulence of P. aeruginosa [15,30-32]. To verify whether the mvfR gene of P. aeruginosa could functionally replace raaR, the coding region of mvfR was cloned in an expression vector and conjugated to the raaR deletion mutant. As shown in S8 Fig, the expression of mvfR in the ΔraaR mutant does not restore the phenotypic changes, indicating that mvfR is not a functional homolog of raaR. We then expressed and purified the MvfR protein by affinity chromatography and examined its interaction with anthranilic acid by ITC. The results showed that the MvfR protein did not bind to anthranilic acid (S9 Fig). We also found that the RaaR protein did not bind to PQS, HHQ or 2,4-dihydroxyquinoline (DHQ) (S10 Fig). Taken together, these results suggested that anthranilic acid specifically binds to RaaR and that the anthranilic acid signaling system of R. solanacearum is completely different from the pqs system in P. aeruginosa. To investigate whether the anthranilic acid signaling system is widely present in bacteria, both trpEG and raaR homologs were sought in the genome database by BLAST. It was revealed that the anthranilic acid signaling system likely operates in many different bacterial species, including in the bacterial species of Burkholderiales, Caballeronia, Trinickia, Pandoraea, and Cupriavidus (S3 Table).

From the results of this study, we can build a model for how the anthranilic acid-mediated signaling system controls gene expression in R. solanacearum. At high cell densities, when the concentration of anthranilic acid reaches a threshold, it binds to RaaR to cause allosteric conformational changes and enhance the RaaR ability to bind target gene promoters (S11 Fig).

Discussion

Our previous study found that anthranilic acid controls important biological functions and virulence in R. solanacearum [29]. In this study, we identified that the transcriptional regulator RSp0912, which we named RaaR, is a receptor protein of anthranilic acid. RaaR regulates the same processes as anthranilic acid, including motility, biofilm formation, QS signal production and virulence (Figs 2, 3 and 6). In addition, the anthranilic acid mutant phenotypes were rescued by trans expression of RaaR (Fig 1). RaaR has an HTH domain and a LysR_substrate domain (Fig 1B). Anthranilic acid was shown to bind to the LysR_substrate domain of RaaR with high affinity and enhanced RaaR binding to target promoter DNA through induction of allosteric conformational changes (Figs 4 and 5, S4 and S5). A previous study showed that 9 site-specific substitution mutations affected MvfR activity [32]. To identify the critical residues in RaaR, we compared RaaR with MvfR using amino acid sequence alignments. Analysis of the two proteins identified four conserved amino acid residues at positions A171, L191, L192 and I249 that might be critical for the interaction between RaaR and anthranilic acid. We then generated four single point mutants (RaaRA171F, RaaRL191A, RaaRL192A and RaaRI249A). ITC analysis showed that mutations at L191 and L192 abolished the binding between RaaR and anthranilic acid (S12 Fig). Our results suggest that RaaR is a specific receptor protein of anthranilic acid and represents a new one-component signaling system.

In P. aeruginosa, anthranilic acid is a precursor of the QS signal PQS. The PQS signal is sensed by the receptor protein MvfR to regulate the phenotypes and virulence of P. aeruginosa [15,30-32]. Consistent with the results of our previous study, the findings in this study also suggest that the anthranilic acid signaling system is distinguished from the pqs QS system in P. aeruginosa. The addition of PQS, HHQ or DHQ resulted in no restoration of the anthranilic acid mutant phenotypes, and trans expression of mvfR showed no effect on the defective phenotypes of the ΔraaR mutant (S8 Fig). We also revealed that the MvfR protein binds the PQS signal but not anthranilic acid (S9 Fig). In addition, the RaaR protein does not bind PQS, HHQ or DHQ (S10 Fig). Collectively, our findings in this study support that the anthranilic acid/RaaR system is different from the PQS/MvfR system in P. aeruginosa.

Previous studies have demonstrated that the phc system regulates the solIR system [4,26,28]. Our recent study showed that the expression levels of both phcB and solI and the production of 3-OH MAME and AHL signals were reduced in the trpEG deletion mutant [29]. The findings in this study showed that the anthranilic acid signaling system regulates the transcriptional expression levels of phcB and solI and the production of AHL and 3-OH MAME signals through the RaaR receptor (Fig 3). We also found that RaaR directly binds to the promoters of phcB and solI and that exogenous addition of anthranilic acid enhances the binding (Figs 3 and 5). Interestingly, RaaR can also regulate the transcriptional expression levels of epsA by directly binding to the promoter of epsA (S13 Fig), suggesting a complicated hierarchy of signaling systems in R. solanacearum. To further study the binding site of the RaaR protein in the phcB, solI and epsA promoters, we compared and analyzed the promoter sequences of the three genes and identified the potential binding site sequence as GCGGGTGCG. EMSA analysis showed that no DNA–protein complexes formed when the GCGGGTGCG fragment was deleted from the promoter regions of phcB, solI and epsA (S14 Fig), suggesting that this fragment is essential for the binding of RaaR to the phcB, solI and epsA promoters.

In addition, a BLAST search with the TrpEG/RaaR homologs revealed that both the signal synthase gene and sensor gene are widely present in many bacterial species, including in other RSSC phylotypes like R. solanacearum RS476 (phylotype I), R. solanacearum CFBP2957 (phylotype IIA), R. solanacearum UW551 (phylotype IIB), R. solanacearum CMR15 (phylotype III) and R. solanacearum PSI07 (phylotype VI), B. cepacia, P. piptadeniae, C. calidae, T. symbiotica, P. thiooxydans, and C. gilardii (S3 Table). Intriguingly, some bacteria present only one homolog of TrpEG or RaaR (S3 Table), suggesting that anthranilic acid could be used in both intraspecies signaling and cross-talking, or as a common metabolic product. Together, these findings will trigger further investigation of the roles and mechanisms of this signaling system in diverse bacterial genera.

Materials and methods

Bacterial strains and growth conditions

The bacterial strains and plasmids used in this work were listed in S4 Table. R. solanacearum GMI1000 (ATCCBAA-1114) was obtained from the American Type Culture Collection (ATCC) and maintained at 28°C in casamino acid-peptone-glucose (CPG medium) [29,35]. The following antibiotics were added when necessary: ampicillin and kanamycin, 100 μg mL-1; tetracycline, 10 μg mL-1; and gentamicin, 10 μg mL-1. Bacterial growth was determined by measuring the optical density at 600 nm.

Protein expression and purification

The coding regions of raaR and mvfR were amplified with the primers listed in S5 Table with HindIII and EcoRI restriction sites and fused to the expression vector pMAL-C5X. The fusion gene constructs were transformed into E. coli strain BL21. Affinity purification of RaaR and MvfR proteins was performed following a method described previously [36]. E. coli BL21 cells expressing RaaR and MvfR were sonicated in a solution containing 20 mM Na-HEPES pH 8.1, 1M NaCl, and 5 mM DTT (solution A). The cleared lysate was applied to a maltose binding protein (MBP) affinity column. The MBP fusion RaaR or MvfR was eluted by solution A supplemented with 40 mM maltose, and the MBP tag was then cleaved by tobacco etch virus protease. The RaaR and MvfR proteins were pooled and concentrated, then shock-frozen in liquid nitrogen and stored at −80°C for further assay.

Electrophoretic mobility shift assay

The DNA probes used for the EMSA were prepared by PCR amplification using the primer pairs listed in S5 Table [37,38]. The purified PCR products were 3-end-labeled with biotin following the manufacturer’s instructions (Thermo). The DNA-protein binding reactions were performed according to the manufacturer’s instructions (Thermo). A 5% polyacrylamide gel was used to separate the DNA-protein complexes. After UV cross-linking, the biotin-labeled probes were detected in the membrane using a Biotin luminescent detection kit (Thermo).

ITC and CD spectroscopy analysis

ITC measurements were performed using an ITC-200 microcalorimeter following the manufacturer’s protocol (MicroCal, Northampton, MA) [37]. In brief, titrations began with one injection of 2 μL of anthranilic acid (250 μM) solution into a sample cell containing 350 μL of RaaR solution (20 μM) in the MicroCal ITC-200 microcalorimeter. The heat changes accompanying injections were recorded. The titration experiment was repeated at least three times, and the data were calibrated with the final injections and fitted with a one-site model to determine the binding constant (Kd) using MicroCal ORIGIN version 7 software. Circular dichroism (CD) analysis of RaaR was carried out on a Chirascan spectropolarimeter (Applied Photophysics, UK) as previously described [33]. RaaR and anthranilic acid solutions were mixed at room temperature for 1 h at a final concentration of 10 μM.

Construction of in-frame deletion mutants and complementation

R. solanacearum GMI1000 was used as the parental strain for the generation of in-frame deletion mutants following methods described previously [29,39]. The primers used to generate the upstream and downstream regions flanking raaR are listed in S5 Table. For complementation analysis, the coding regions of the regulator genes were amplified and cloned into the pBBR1 MCS-2 plasmid. The resulting constructs were introduced into R. solanacearum GMI1000 deletion mutants using electroporation.

Phenotype assays

For analysis of biofilm formation [40], a single colony of each strain was inoculated and grown overnight at 28°C with agitation in 5 mL of CPG liquid medium. Bacterial cells were inoculated 1:100 in CPG medium in 96-well polystyrene plates. After incubation at 28°C for 24 h, the cells were stained with 0.1% crystal violet (CV) for 30 min. The planktonic cells were removed by several rinses with H2O. The CV-stained bound cells were air dried for 1 h and then dissolved in 95% ethanol, and the optical density at 570 nm (OD570) of the solution was measured to quantify biofilm formation.

Cellulase production was determined on carboxymethylcellulose sodium (CMS) solid medium (1 liter contained 1 g of CMS, 3.8 g of Na3PO4, and 8.0 g of agar, pH 7.0) [41]. The CMS medium was added to a culture dish (Φ = 9 cm). An overnight culture was diluted to an OD600 of approximately 0.1, and 2 μL of the bacterial suspension was inoculated into the center of the CMS plates. The plates were incubated at 28°C for 48 h. The plates were stained with 0.5% Congo red for 30 min. The plates were rinsed three times with 1 M NaCl. Then, a transparent ring was apparent, and its size was measured.

Swarming motility was determined on semisolid agar (0.3%) [42]. Bacteria were inoculated into the center of plates containing 1% tryptone (Becton, Dickinson and Company, Maryland, USA) and 0.3% agar (Becton, Dickinson and Company, Maryland, USA). The plates were incubated at 28°C for 48 h before the diameter of the resulting colonies was measured.

For quantification of EPS production, bacteria were inoculated into sucrose and peptone (SP) liquid medium (1 liter contained 5 g of peptone, 20 g of sucrose, 0.5 g of KH2PO4, and 0.25 g of MgSO4, pH 7.2) [43]. A 100-mL aliquot of the culture (OD600 = 3.0) was collected and centrifuged at 12,000 rpm for 20 min. The supernatants were filtered through a 0.22-μm membrane. The collected supernatants were mixed with 4 volumes of absolute ethanol, and the mixture was incubated at 4°C overnight. The precipitated EPS were isolated by centrifugation and dried overnight at 55°C before the dry weight was determined.

Bacterial growth analysis

An overnight bacterial culture in CPG liquid medium was washed twice in fresh CPG liquid medium or MP liquid minimal medium and inoculated to an OD600 of 0.01 in fresh CPG medium and of 0.1 in fresh MP minimal medium. A 200-μL cell suspension was grown in each well at 28°C with low-intensity shaking using the Bioscreen C automated growth curve analysis system. CPG medium or MP minimal medium was used as the negative control. MP minimal medium (1 liter): FeSO4·7H2O, 1.25×10−4 g; (NH4)2SO4, 0.5 g; MgSO4·7H2O, 0.05 g; and KH2PO4, 3.4 g. The pH was adjusted to 7, and 20 mM glutamate was added [29,44,45].

Construction of reporter strains and measurement of β-Galactosidase activity

We used promoter activity assays to quantify gene expression. The promoters of phcB, solI and raaR were amplified using the primer pairs listed in S5 Table. The resulting products were inserted upstream of the promoterless lacZ gene in the vector pME2-lacZ. Transconjugants were then selected on CPG agar plates supplemented with tetracycline and X-gal. Measurement of β-galactosidase activities was performed following previously described methods [19,38].

LC-MS analysis

R. solanacearum cells were cultured in CPG liquid medium for 48 h, and one liter of culture supernatant was collected by centrifugation and extracted with equal volume of ethyl acetate. The culture supernatants were extracted twice with ethyl acetate, the solvent was evaporated, and the residue was dissolved in methanol. Analyses were performed by using a Waters ACQUITY UPLC/Xevo G2 QTOF system (Waters, Milford- Massachusetts, USA), which consisted of an ACQUITY UPLC system and a Waters Q-TOF Premier high-resolution mass spectrometer in negative electrospray ionization mode interfaced with an ACQUITY UPLC BEH C18 column (2.1×50 mm). Elution was performed via a gradient of 5–100% CH3OH in water supplemented with 0.01% formic acid at a flow rate of 0.4 mL/min for 10 min. Then, 100% CH3OH was used for 2 min, and 5% CH3OH was used for 3 min. The entire column eluate was introduced to the Q-TOF mass spectrometer according to the manufacturer’s instructions [29].

Virulence assays in tomato plants

Analysis of the virulence of the R. solanacearum WT, ΔraaR and complemented strains was performed in an AIRKINS greenhouse (28°C, light 16 h and dark 8 h). A mixture including field soil, sand, and compost (1.25:1.25:0.5) was prepared and autoclaved at 121°C for 20 min. Tomato seeds (Jinfeng 1) were surface-sterilized in 2% NaClO for 3 min and 75% ethanol for 2 min, rinsed 3 times in sterile water, and then planted in soil. The disease status of tomato plants was assessed daily by scoring the disease index on a scale of 0 to 4 as previously described [35]. All plants were monitored for disease index analysis, and the following scale was used: 0, no symptoms; 1, 1–25% wilted leaves; 2, 26–50% wilted leaves; 3, 51–75% wilted leaves; and 4, 76–100% wilted leaves. R. solanacearum cells were grown in CPG medium to OD600 = 1.0. Aliquots of 5 mL of the R. solanacearum WT, ΔraaR, and ΔraaR(raaR) CPG liquid medium were inoculated onto the tomato seedlings. Each treatment included 20 replicates [29,35,39].

Analysis of R. solanacearum cell numbers in plants

One-gram samples of plant roots and stems were collected and milled in 9 mL of sterilized water for 20 min. The suspensions were serially diluted and spread on CPG plates. The plates were incubated at 28°C for 48 h. Then, the numbers of R. solanacearum cells were counted [29,39].

Statistical analysis

Statistical analyses were performed with GraphPad Prism 8. The data are presented as the means ± standard deviations. Asterisks in figures indicate corresponding statistical significance as it follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

The effect of trpEG on the expression level of raaR was evaluated by assessing the β-galactosidase activity of raaR-lacZ transcriptional fusions in the wild-type, the trpEG mutant strains and the trpEG deletion mutant strain supplemented with anthranilic acid.

The data are the means ± standard deviations of three independent experiments. **p < 0.01; ***p < 0.001 (unpaired t-test).

(DOCX)

Click here for additional data file.

Effects of raaR on the growth curve of R. solanacearum GMI1000 in CPG medium (A) and MP minimal medium (B). The cells were inoculated at 28°C in three replicates with low-intensity shaking in the Bioscreen-C automated growth curve analysis system. The experiment was started at initial OD600 values of 0.01 in CPG medium and 0.1 in MP minimal medium. The data are the means ± standard deviations of three independent experiments.

(DOCX)

Click here for additional data file.

Influence of raaR on the expression level of housekeeping gene gyrB.

(A) Effect of raaR on the gene expression levels of gyrB in R. solanacearum GMI1000. We used promoter activity assays to quantify gene expression. (B) EMSA analysis of the in vitro binding of RaaR to the promoters of gyrB.

(DOCX)

Click here for additional data file.

ITC analysis of the binding between anthranilic acid and the LysR_substrate domain of the RaaR protein.

(A) SDS-PAGE of the purified LysR_substrate domain of RaaR protein. (B) ITC titration of 20 μM LysR_substrate domain of RaaR with 250 μM anthranilic acid in PBS buffer at 25°C.

(DOCX)

Click here for additional data file.

CD spectroscopy of anthranilic acid binding to RaaR protein.

The α-helix and β-sheet spectra of the RaaR protein were changed after RaaR protein was supplemented with anthranilic acid at a final concentration of 10 μM.

(DOCX)

Click here for additional data file.

The effects of HHQ on the binding of RaaR to the promoters of phcB (A) and solI (B) were assessed by performing EMSA in vitro.

A protein-DNA complex was formed when the protein was incubated with the probes, and different concentrations of HHQ showed no effect on the formation of the complex at room temperature for 30 min.

(DOCX)

Click here for additional data file.

Differential gene expression profiles between R. solanacearum wild-type strain GMI1000, trpEG and raaR mutants as measured by RNA-Seq (Log2 fold-change ≥1.5).

(A) GO term enrichment analysis of differentially expressed genes between the ΔraaR and wild-type strains. Venn diagrams showing the overlap of genes with (B) upregulated or (C) downregulated expression on different mutant backgrounds. Divergently regulated genes are not depicted in these Venn diagrams but are found in S2 Table.

(DOCX)

Click here for additional data file.

Effects of mvfR of P. aeruginosa on the RaaR-regulated motility (A), biofilm formation (B), EPS production (C) and cellulase production (D) in the R. solanacearum raaR deletion mutant strain. The data are the means ± standard deviations of three independent experiments. **p < 0.01; ***p < 0.001 (unpaired t-test).

(DOCX)

Click here for additional data file.

ITC analysis of the binding of PQS or anthranilic acid to MvfR protein.

(A) SDS-PAGE of the purified MvfR protein. (B) ITC titration of 20 μM MvfR protein with 250 μM PQS in PBS buffer at 25°C. (C) ITC titration of 20 μM MvfR with 250 μΜ anthranilic acid in PBS buffer at 25°C.

(DOCX)

Click here for additional data file.

ITC analysis of the binding between (A) PQS, (B) HHQ and (C) DHQ and RaaR protein. ITC titration of 20 μM RaaR protein with 250 μM PQS, HHQ and DHQ in PBS buffer at 25°C.

(DOCX)

Click here for additional data file.

Schematic representation of the regulatory network of the anthranilic acid/RaaR signaling system in R. solanacearum.

RaaR is involved in sensing anthranilic acid signals and increases the expression levels of PhcB and SolI, which are required for the synthesis of 3-OH PAME/3-OH MAME and AHL signals, respectively. At the same time, the anthranilic acid/RaaR signaling system also directly controls the motility, biofilm formation, and virulence of R. solanacearum.

(DOCX)

Click here for additional data file.

Analysis of the binding of anthranilic acid to RaaR variants.

(A) SDS-PAGE of the purified RaaRA171F, RaaRL191A, RaaRL192A and RaaRI249A proteins. ITC analysis of the binding of anthranilic acid to (B) RaaRA171F, (C) RaaRL191A, (D) RaaRL192A and (E) RaaRI249A proteins.

(DOCX)

Click here for additional data file.

Influence of raaR on the epsA gene expression of R. solanacearum.

(A) Effects of raaR on the gene expression levels of epsA in R. solanacearum GMI1000. We used promoter activity assays to quantify gene expression. (B) EMSA analysis of the in vitro binding of RaaR to the promoter of epsA. The biotin-labeled 336-bp epsA promoter DNA probe was used for the protein binding assay. A protein-DNA complex, represented by a band shift, was formed when different concentrations of RaaR protein were incubated with the probe at room temperature for 30 min.

(DOCX)

Click here for additional data file.

Analysis of the RaaR binding sites in the promoter regions of target genes.

Analysis of the binding between RaaR and the mutated phcB (A), solI (B) and epsA (C) promoters with deletion of the RaaR binding sequence GCGGGTGCG. EMSA analysis was performed in vitro.

(DOCX)

Click here for additional data file.

Analysis of the homologs of MvfR in R. solanacearum.

(DOCX)

Click here for additional data file.

List of genes differentially expressed in the trpEG and raaR mutants compared to the wild-type strain (Log2-fold change ≥ 1.5).

Significantly differentially expressed genes were determined by Cufflinks after Benjamini-Hochberg correction. The fold-change is the ratio of the mutant FPKM to the wild-type FPKM.

(DOCX)

Click here for additional data file.

Analysis of the homologs of RaaR in various bacteria.

(DOCX)

Click here for additional data file.

Bacterial strains and plasmids used in this study.

(DOCX)

Click here for additional data file.

PCR primers used in this study.

(DOCX)

Click here for additional data file.

11 Jan 2022

Dear Dr. Deng,

Thank you very much for submitting your manuscript "A responsive transcriptional regulator controls Ralstonia solanacearum pathogenicity by sensing anthranilic acid" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

The three reviewers all commended the discovery of the novel sensor of anthranilic acid RaaR in Ralstonia solanacearum. The reviewers also identified some major issues that need to be addressed including to further corroborate the relationship between RaaR and anthranilic acid and EMSA experiments. Please also move the method section in the supplementary information to the main text. In addition, please conduct the experiment to restore ΔtrpEG with exogenous anthranilic acid if this has not been done in the past.

Please show images of infected and control plants corresponding to Fig. 6A in the supplementary

Please move Fig. S1 to the main text.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Nian Wang

Associate Editor

PLOS Pathogens

Wenbo Ma

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

The three reviewers all commended the discovery of the novel sensor of anthranilic acid RaaR in Ralstonia solanacearum. The reviewers also identified some major issues that need to be addressed including to further corroborate the relationship between RaaR and anthranilic acid and EMSA experiments. Please also move the method section in the supplementary information to the main text. In addition, please conduct the experiment to restore ΔtrpEG with exogenous anthranilic acid if this has not been done in the past.

Please show images of infected and control plants corresponding to Fig. 6A in the supplementary

Please move Fig. S1 to the main text.

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The manuscript titled "A responsive transcriptional regulator controls Ralstonia solanacearum pathogenicity" by Song et al. characterize RaaR, a novel transcriptional regulator that function upstream to the biosynthesis of Ralstonia solanacearum QS signals AHL and 3-OH MAME by directly controlling the expression of phcB and solI which results in reduction in exoenzyme activity, EPS production, motility and virulence. RaaR was identified to complement the anthranilic acid biosynthesis mutant trpGE and therefore was reported in the manuscript to act downstream to anthranilic acid.

The research subject is interesting, the manuscript is well written and the results are described in clear and organized manner. However, I do not think that the authors provided enough data to support that RaaR indeed function downstream and not in parallel to anthranilic acid and some key controls are missing in some of the experiments. Therefore, I suggests major revisions.

Reviewer #2: Summary of the Research.

This manuscript describes the discovery of the novel sensor of anthranilic acid RaaR in the economically important plant pathogen Ralstonia solanacearum (Rs). The authors build off a previous study which found that anthranilic acid can positively regulate a variety of important virulence traits in Rs, including motility, biofilm production, and the production of plant cell wall degrading enzymes. Based on structural similarity to the quorum sensing receptor MvfR in Pseudomonas aeruginosa, the authors studied the putative receptor RSp0912, which they named RaaR. Using both genetic and biochemical techniques, the authors demonstrate that RaaR is required for anthranilic acid signaling, physically binds both anthranilic acid and several key promoters, and contributes to Rs virulence. The authors end by proposing that RaaR and anthranilic acid contributes to the complex regulation of virulence factors in Rs. They further suggest that, because they can detect anthranilic acid in the tissues of some Rs hosts, anthranilic acid could be an interkingdom signal between host and pathogen.

Strengths

The authors admirably use a variety of means to show how RaaR interacts with anthranilic acid and gene expression. Their genetic methods use both RaaR mutants and RaaR overexpression constructs, reporters of putative RaaR-regulated genes, and complementation of RaaR mutants both with the gene itself and with exogenously applied anthranilic acid. They use EMSA, ITC, and CD to demonstrate that RaaR binds the promoters it regulates and to anthranilic acid. Combined, these multiple lines of evidence show very clearly and convincingly that RaaR functions as they suggest. They further make a successful effort to show the specificity of the RaaR-anthranilic acid interaction, and convincingly show that RaaR is not affected by other related QS molecules. Further, their result showing that virulence on tomato is lowered in an RaaR mutant shows that this is important for Rs, and that this regulatory pathway plays a significant role in an important plant pathogen.

Concerns

One area where this paper falls short is in examining the breadth of the R. solanacearum species complex. When they search for RaaR homologs in Table S2, it would be useful to show if RaaR is conserved across Rs phylotypes. They could include comparisons to strains like CFBP2957 (Phylotype II), UW551 (Phylotype II), CMR15 (Phylotype III), PSI07 (Phylotype VI), or other strains with available genome sequences. The authors also suggest in the discussion that because they can detect anthranilic acid in some Rs hosts but not in non-host maize or rice, anthranilic acid may help Rs with host sensing. However, the three hosts they test are all solanaceous crops (tomato, eggplant, and pepper) and do not capture the diversity of the Rs host range. Some individual strains, including GMI1000, can have extremely wide host ranges. It would be better to include a wider variety of Rs hosts in this test, including monocot hosts like banana and ginger. However, this experiment is not vital to the main focus of the manuscript and could be simply removed. If this supplemental figure is included, the authors should also provide more detail in how they sampled tissues from the tested crops.

Reviewer #3: In this manuscript “A responsive transcriptional regulator controls Ralstonia solanacearum pathogenicity by sensing anthranilic acid” (PPATHOGENS-D-21-02479) the authors identified a new signaling system formed by one-component system RaaR (LysR-type regulator) and signal molecule anthranilic acid. RaaR-anthranilic acid complex was verified by ITC and CD experiments. Also, it was suggested that anthranilic acid binding caused conformational changes in RaaR, which positively affected its interaction with target promoters. I have just a few suggestions to reinforce these interesting findings and clarify some parts in text.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. The major weakness of the paper is that it does not provide sufficient proof that RaaR act downstream to anthranilic acid and not in parallel to it. If RaaR regulate QS signal synthesis, its overexpression might rescue the trpGE phenotypes regardless if it act downstream to it or not. To clearly demonstrate that RaaR and RrpGE function in the same pathway, epistasis experiments in the presence and absence of anthranilic acid are required:

a. The authors should produce a raaR/trpGE double mutant and examine if their phenotypes (phcB and solI expression by LacZ promter fusions, motility, exoenzyme activity) are additive or not.

b. The authors should try to rescue these phenotypes by exogenous addition of anthranilic acid (as conducted in fig 6 in authors ISME paper): If indeed RaaR act downstream to TrpGE, exogenous addition of anthranilic acid will rescue the trpGE mutant but not the trpGE/raaR double mutant.

2. Some of the experiments described in the manuscript are lacking proper controls:

a. The authors demonstrated that RaaR binds to the phcB, solI and espA promoters in vitro by EMSA. Many transcriptional regulators harbors general affinity to DNA in in vitro, which results in high false positive rate in EMSA analysis when conducted without proper controls. As far as I see, all EMSA experiments used with RaaR in this study provided positive results, which make one wonder how specific is the binding of RaaR to these particular promoters. To address this issue the authors should repeat the EMSA experiments using promoters of genes that are NOT directly regulated by RaaR and show that RaaR demonstrate high affinity to phcB, solI and espA promoters and not others.

b. To make sure that RaaR does not harbor a pleiotropic effect on protein expression or LacZ activity, the authors should use a negative control (maybe a promotor of a housekeeping gene fused to LacZ) in their promoter LacZ fusion assays.

c. Figure 5. The authors should conduct their binding assays with one of the structural analogs of anthranilic acid that were used in fig S8 as a negative control.

Reviewer #2: The authors should comment on the presence and conservation of RaaR within the R. solanacearum species complex.

Reviewer #3: Major points:

1) As anthranilic acid is produced from chorismic acid and L-glutamine and this pathway is necessary to synthesis of L-tryptophan in microorganisms, it is easy to think that anthranilic acid is essential to bacterial survive in condition without tryptophan in the growth medium. Also, it seems that most bacterial species can synthesize anthranilic acid but they do not encode a RaaR homologs in their genomes (only a few groups of bacterial species that carry a raar homolog were cited in the text). In this case, most bacterial specie can synthesize anthranilic acid but may not encode for a RaaR homolog but still can communicate with other species that carries a raar homolog in their genomes (cross-talking). What the authors think about this point? Can it be better discussed in the manuscript?

2) In material and methods are lacking description of RaaR expression in E. coli and purification, ITC and CD experiments including concentration of purified protein and ligand as well as instruments that were used in these studies. Also, how the QS signal molecules were isolated and quantified and the disease Index was calculated in virulence assays. Please, add more details about these experiments in Material and Methods.

3) It will be important the authors calculate and define the interaction parameter “Kd” for the RaaR-anthranilic acid complex. This result can help in the discussion about physiological concentration of anthranilic acid in bacterial cell and in host root/stem by affecting RaaR activation.

4) Have the authors identified any conserved cis-element in the RaaR target promoters? If so, it will be important to generate site mutants on this putative motif to confirm the RaaR specific binding by in vitro experiments.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1. The data provided in figure S2 suggest that RaaR is regulating its own transcription. Have the authors looked into this matter?

2. It will be nice if the virulence test conducted in Fig 6 will be supplemented with representative pictures.

3. Have the authors tested the expression or promoter activity of epsA in the raaR mutant? If not, what is the point of doing EMSA?

4. As far as I know PLOS pathogens don’t have word limitation. Therefore, it is unclear to me why the materials and methods section was split between the main text and the supplementary material.

Reviewer #2: -Line 65 – There are two more recent studies of phc regulation that may be worth citing here: Perrier et al. (2018), Microbial Pathogenesis (doi: 10.1016/j.micpath.2018.01.028) and Khokhani et al. (2017), mBio (doi: 10.1128/mBio.00895-17)

-Line 273 – The authors should clarify what they mean by “fermentation broth”. If it is not CPG or a water suspension, the authors should specify the components of fermentation broth

-Line 426 – Did the authors perform any statistical tests on the disease progress data in figure 6A? Possible tests to perform might include repeated measures ANOVA or AUDPC (see Schandry (2017), Front Plant Sci (doi: 10.3389/fpls.2017.00623))

-Supplementary Table 2 – The authors include in this table R. solanacearum GMI1000 and R. pseudosolanacearum RS 476. If the authors are using the division of the Ralstonia solanacearum species complex (RSSC) into three species (Prior et al. (2016), BMC Genomics (doi: 10.1186/s12864-016-2413-z)), GMI1000 should also be referred to as R. pseudosolanacearum, as it is also a Phylotype I strain. Alternatively, the authors should refer to RS 476 as R. solanacearum and include its Phylotype. The authors should also include a line in the introduction describing the RSSC and refer to GMI1000 throughout as R. pseudosolanacearum.

Reviewer #3: Minor points:

Line 29: RaaR regulates the same processes as anthranilic acid, and both are conserved in various bacterial species. In this sentence should be better to say that anthranilic acid is present and not conserved in bacterial species.

Line 30: Please, add ”a” before “anthranilic acid-deficient mutant phenotypes were rescued by in trans expression of RaaR” and explain in the text way a trans expression can recue mutant phenotypes without adding anthranilic acid. It will help to clarify this part for the readers.

Line 68: “The signals phosphorylate the response regulator PhcR by activating the histidine kinase” should be “The signals promote the phosphorylation of the response regulator PhcR

by…”

Line 128: Please, indicate the percentage of recue for the sentences in text that are described “almost fully rescued signal”.

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at .

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Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

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To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

11 Mar 2022

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

30 Mar 2022

Dear Dr. Deng,

Thank you very much for submitting your manuscript "An anthranilic acid-responsive transcriptional regulator controls the physiology and pathogenicity of Ralstonia solanacearum" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers commended the revisions and extra data as requested and appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Nian Wang

Associate Editor

PLOS Pathogens

Wenbo Ma

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have successfully addressed all the issues I raised in the first review cycle. The manuscript is well written and the data is solid and presented in a clear and organized manner. I suggest some minor text revisions that will improve the paper. Outside of that, I do not have any additional requests.

Reviewer #3: The authors have answered the reviewer´s concerns and added new data which reinforce their findings. The authors described changes in RaaR secondary structure caused by anthranilic acid. Although, I think the binding of anthranilic acid and RaaR should be better explored by using some mutant in critical residues of RaaR that are involved in this interaction. The lost of this interaction should be observed in ITC experiments.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: None

Reviewer #3: Identification of the RaaR critical residues involved in interaction with anthranilic acid should be accomplished to give the reader a complete understanding of this mechanism. Mutant in critical residues of RaaR generated by site direct mutagenesis may help to answer this important question.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Suggested minor text modifications (lines numbers in "track changes" draft):

*promoter fusion assays: throughout the manuscript, the authors used the term "gene expression" to describe their promoter fusions assays. In most cases, the term "gene expression" is referred to RNA accumulation (usually by RT-qPCR) and not reporter fusions analyses. The authors should clearly state in the text they used promoter activity assays to quantify gene expression.

*Line 118: a sentence should not start with "and"

*Line 203, 205,207, and 209: trpEG and raaR should be italicized

*Line 228: all the mentioned bacterial genera belong to the Burkholderiales order. This should be stated in the text.

*lines 260-265/Fig S13- The data presented here is more suited to be part of the result section and not the discussion section

*line 294-303 (Protein Expression and Purification) - MvfR was cloned to an expression vector and the protein was purified as well. This should be added to the M&M section.

*line 325 – citation is missing after "as described previously"

*Figure 1: the title "Overexpression of the trpEG mutant with raaR" is not clear. The authors should consider rephrasing it.

*Figure 1A: " Effects of the homologs…". Does not describe the presented experiment well. The authors should mention that the regulators are overexpressed and that the bacteria were assayed for plate motility.

Figures 3A and B/S3A/S11A: the authors should state that experiments present promoter activity analyses.

Figure 6A: the authors should state in the figure legend at which time after inoculation these pictures were taken.

Figure S7: ∆raaR should be italicized

Reviewer #3: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

28 Apr 2022

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

29 Apr 2022

Dear Dr. Deng,

We are pleased to inform you that your manuscript 'An anthranilic acid-responsive transcriptional regulator controls the physiology and pathogenicity of Ralstonia solanacearum' has been provisionally accepted for publication in PLOS Pathogens.

Please address some minor editorial issues during the proof stage.

For example:

Line 254. please add that before might.

Analysis of the two proteins identified four conserved amino acid residues at positions A171, L191, L192 and I249 that might be critical for the interaction between RaaR and anthranilic acid.

Line 312: 312 The coding regions

Line 315: should be was

Here the line numbers refer to the document tracking changes.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Nian Wang

Associate Editor

PLOS Pathogens

Wenbo Ma

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

23 May 2022

Dear Dr. Deng,

We are delighted to inform you that your manuscript, " An anthranilic acid-responsive transcriptional regulator controls the physiology and pathogenicity of Ralstonia solanacearum ," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064