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Open Access

Peer-reviewed

Research Article

The AltR transcription factor responds to plant thiosulfinates to regulate gene expression in a bacterial pathogen of onion

  • Hsiao-Hsuan Jan,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

    Affiliation University of Georgia, Department of Plant Pathology, Athens, Georgia, United States of America

    ⨯
  • Feng Kong,

    Roles Data curation, Formal analysis, Visualization, Writing – review & editing

    Affiliation University of Georgia, Department of Plant Pathology, Athens, Georgia, United States of America

    ⨯
  • Michelle P. MacLellan,

    Roles Data curation, Writing – review & editing

    Affiliation Valdosta State University, Department of Biology, Valdosta, Georgia, United states of America

    ⨯
  • Li Yang,

    Roles Supervision, Writing – review & editing

    Affiliation University of Georgia, Department of Plant Pathology, Athens, Georgia, United States of America

    ⨯
  • Bhabesh Dutta,

    Roles Funding acquisition, Supervision, Writing – review & editing

    Affiliation University of Georgia, Department of Plant Pathology, Tifton, Georgia, United States of America

    ⨯
  • Brian H. Kvitko

    Roles Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing

    bkvitko@uga.edu

    Affiliation University of Georgia, Department of Plant Pathology, Athens, Georgia, United States of America

    ⨯
?

This is an uncorrected proof.

Abstract

Pantoea ananatis, the causative agent of onion center rot, encounters potent antimicrobial thiosulfinates, volatile organosulfur compounds released from damaged Allium tissue during pathogen-induced necrosis. The allicin tolerance (alt) gene cluster allows P. ananatis to overcome this chemical barrier. We demonstrate that AltR, a TetR-family transcriptional repressor, specifically regulates expression of the alt cluster and thus thiosulfinate tolerance in vitro and fitness in vivo. We identified a putative AltR binding box both in the altR promoter and elsewhere in the alt cluster, show that AltR-mediated repression is relieved in response to thiosulfinates. Using cysteine to serine substitutions, we demonstrate that AltR Cys100 is essential for thiosulfinate-responsive de-repression, while other AltR cysteine residues tune responsivity. Strains expressing AltR alleles with reduced thiosulfinate responsivity have reduced fitness in planta. Our findings uncover a regulatory mechanism by which a plant antimicrobial secondary metabolite acts as an environmental cue to modulate bacterial gene expression, enabling pathogen survival and virulence.

Author summary

The bacterial pathogen Pantoea ananatis, causes onion center rot, a disease first reported in Georgia in 1997. P. ananatis encodes genes conferring tolerance to the natural antimicrobial sulfur compounds released by onion cells when they are damaged. These thiosulfinate chemicals, are produced by onion and garlic and are also associated with their distinct odor and flavors. We determined that the gene altR regulates the ability of Pantoea ananatis to tolerate thiosulfinates. When a single conserved cysteine amino acid of the AltR protein is changed, the bacterium no longer properly responds to thiosulfinates and the pathogen becomes more sensitive to thiosulfinates both in laboratory tests and less fit during onion infection. By showing how this pathogen monitors and responds to onion chemical defenses, our work may guide future strategies to manage or reduce onion center rot and other onion bacterial diseases.

Citation: Jan H-H, Kong F, MacLellan MP, Yang L, Dutta B, Kvitko BH (2026) The AltR transcription factor responds to plant thiosulfinates to regulate gene expression in a bacterial pathogen of onion. PLoS Pathog 22(4): e1014198. https://doi.org/10.1371/journal.ppat.1014198

Editor: David Mackey, The Ohio State University, UNITED STATES OF AMERICA

Received: December 3, 2025; Accepted: April 25, 2026; Published: April 30, 2026

Copyright: © 2026 Jan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Raw sequencing reads are archives at NCBI BioProject PRJNA1374235.

Funding: This work was supported the US Department of Agriculture (USDA-NIFA-OREI 2023-51300-40913 to BD and BHK) and US Department of Agriculture (HATCH 7002999 to BK). https://www.nifa.usda.gov/ The Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Onions (Allium cepa L.) are among the most economically important vegetable crop in the United States of America, valued at approximately 1 billion (USD) annually between 2018 and 2022 [1]. Onion center rot (OCR), caused by several Pantoea species including Pantoea ananatis in the southeastern U.S., is a major bacterial disease, particularly in the Vidalia onion-growing region of Georgia [2,3]. OCR typically begins as water-soaked lesions on leaves that progress to blighting and tissue collapse. Bulb symptoms may remain latent at harvest and develop during storage [4–6]. No onion center rot resistant onion cultivars are currently available, and disease management relies primarily on copper bactericides and insecticides targeting thrips which can serve as vectors [7–10].

Unlike many plant-pathogenic bacteria, P. ananatis lacks the canonical virulence-associated type II and type III secretion systems commonly required for plant infection [11–13]. Instead, P. ananatis strains that cause OCR produce the phosphonate phytotoxin pantaphos, which is required for necrosis and symptom development in onion [14–17]. Although the mode of action for pantaphos remains unclear, exposure of onion tissue to pantaphos leads to cell death within days [18].

Upon cell death and membrane disruption, onions release thiosulfinates, which are organic compounds that are thiol-reactive, sulfur-containing phytoavengins [19–21]. In intact cells, non-toxic S-alk(en)yl cysteine sulfoxides (CSOs) accumulate in the cytoplasm, while alliinase (CSO lyase) resides in the vacuole. Upon tissue disruption, CSOs mix with alliinase, producing sulfenic acid intermediates that spontaneously condense into thiosulfinates. These volatile compounds contribute to characteristic aroma of Allium species and exhibit strong antimicrobial activity by forming mixed-disulfides with cellular thiols, including cysteine residues of protein, which can inactivate critical enzymes [22–29].

Successful P. ananatis OCR strains encode the alt (allicin tolerance) thiosulfinate tolerance gene (TTG) cluster, which enables colonization of necrotic onion tissue [30]. The alt cluster confers tolerance to both endogenous onion thiosulfinates and the garlic derived thiosulfinate allicin [31]. Homologous TTG clusters have been identified in other onion-associated Pantoea species, and similar clusters have been identified in several onion-pathogenic Burkholderia species and the garlic saprophyte Pseudomonas fluorescens PfAR-1 [32–35].

Among the alt genes, several encoded enzymes are predicted to detoxify or reduce thiosulfinate stress [30,35]. A single transcriptional regulator, AltR, is conserved across characterized alt clusters and is predicted to encode a TetR-family repressor [30,31]. TetR-family repressors are known to be self-regulating homodimeric repressors controlling genes involved in secondary metabolism, antibiotic resistance, or stress responses, including the canonical TetR that confers tetracycline resistance [36]. These regulators feature an N-terminal helix-turn-helix (HTH) DNA-binding domain and a C terminal ligand-binding/dimerization domain [37,38]. In the Escherichia coli bleach responsive repressor NemR (an AltR homolog), one conserved critical redox-active cysteine was suggested as a key residue that controls responsiveness [39].

In this study, we provide genetic evidence delineating the regulatory role of P. ananatis AltR in thiosulfinate tolerance. We show that AltR represses its own promoter dependent on an inverted repeat operator sequence and that thiosulfinate exposure causes promoter de-repression dependent on the conserved cysteine residue Cys100. We further demonstrated that AltR controls transcription of other alt genes and that mutations impairing AtlR de-repression reduce bacterial colonization on onion scales, highlighting the importance of AltR-mediated regulation in the adaptation of P. ananatis to onion-derived chemical defenses.

Results

AltR represses its own promoter and is de-repressed in response to allicin

AltR is predicted to be a TetR-family repressor and related to NemR [30,31]. NemR repressed its own expression and the co-cistronic nemA reductase gene by binding to an inverted repeat operator in the nemR promoter region [39]. Inspection of the region upstream of altR in P. ananatis PNA 97-1R revealed a perfect inverted repeat motif (CAATCTAC [N6] GTAGATTG) partially overlapping the predicted -10 box and ribosome-binding site (RBS) of a putative σ70 promoter (Fig 1A). To build an altR promoter reporter, we inserted the full intergenic sequence between altR and altG (PaltR) from PNA 97-1R to drive an autobioluminescence Lux gene cassette (Fig 1A) which was introduced into various PNA 97-1R genetic backgrounds via site-specific Tn7 transposition [40].

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Fig 1. AltR represses its own promoter and the thiosulfinate-tolerance (alt) gene cluster in Pantoea ananatis.

(A) Schematic of the altR promoter bioluminescent reporter construct. The intergenic region between altG and altR was cloned as the altR promoter (PaltR) upstream of the luxCDABE operon. Predicted AltR binding boxes and the inverted-repeat 2 [IR2] motif, the σ⁷⁰ promoter region (–35/ –10), and the ribosome-binding site (RBS) are indicated. Alignment of the predicted AltR and IR2 boxes is shown next to the alt cluster diagram, with AltR box colored in blue and IR2 box colored in orange in both the diagram and the sequence alignment. (B) Zone-of-inhibition (ZOI) assay showing PaltRLuxK6 activity in P. ananatis PNA 97-1R wild-type (WT), Δalt, and ΔaltR strains. The WT strain displays a distinct ring of bioluminescence at the ZOI border, indicating AltR de-repression in response to allicin exposure. (C) Quantification of relative ZOI area among WT, Δalt, and ΔaltR strains. Values represent mean ± SD of three biological replicates each with three technical replicates of the ratio of the ZOI area relative to WT. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (p < 0.001). The Δalt mutant exhibited a significantly larger inhibition zone than WT, whereas ΔaltR did not differ significantly from WT. (D) Volcano plot of differential gene expression from RNA-seq comparing WT and ΔaltR strains (three biological replicates each). Genes that are up-regulated are highlighted in red, while down regulated genes are blue, with specific significant genes labelled. Deletion of altR resulted in strong up-regulation of altA–altJ and down-regulation of the YchH-like gene (B9Q16_RS12865). (E) Normalized RNA-seq read-coverage maps across the alt gene cluster in WT (red) and ΔaltR (blue). Plots show mean log-scaled coverage for three biological replicates, with individual replicate tracks below. In ΔaltR, coverage increases uniformly across altA–altJ, confirming AltR-mediated repression of the gene cluster.

https://doi.org/10.1371/journal.ppat.1014198.g001

To test whether AltR regulates its own promoter, the bioluminescent reporter (PaltRLuxK6) was introduced into wild type, Δalt, and ΔaltR backgrounds. Promoter activity was visualized in an allicin zone-of-inhibition (ZOI) assay (Fig 1B). Both Δalt and ΔaltR strains of PNA 97-1R displayed constitutive bioluminescence across the entire plate, consistent with loss of altR-mediated repression. In contrast, the wild-type strain exhibited a distinct ring of bioluminescence at the periphery of the inhibition zone, indicating promoter de-repression in response to allicin.

Quantification of inhibition zones (Fig 1C) showed that the Δalt mutant had a significantly larger ZOI than either the wild type or ΔaltR, confirming increased allicin sensitivity when the alt cluster is deleted. The ΔaltR strain displayed ZOI diameters similar to wild type, demonstrating that altR deletion alone does not alter allicin sensitivity. The difference in Δalt ZOI diameter also roughly correlated with the region of bioluminescence de-repression in the WT strain, indicating the AltR de-repression being correlated with allicin tolerance. Overall, these observations indicate that AltR represses its own promoter but is de-repressed during allicin exposure.

AltR represses the thiosulfinate tolerance (alt) cluster

To identify genes regulated by AltR, we performed RNA-seq analysis comparing the wild type and ΔaltR strains of P. ananatis PNA 97-1R grown under rich medium conditions (Figs 1D - 1E). Differential-expression analysis revealed that deletion of altR resulted in strong, coordinated upregulation of the alt cluster genes. The genes altA, altB, altC, altD, and altJ showed the highest induction, with log₂ fold-changes ranging from +5 to +10 (≈ 30–1000-fold increases).

Interestingly, a single gene outside the alt cluster, B9Q16_RS12865, encoding a predicted YchH-like protein, was the most strongly downregulated transcript in the ΔaltR mutant (Fig 1D). Homologs of YchH in E. coli have been associated with stress-response modulation and redox balance [41–43], potentially suggesting that AltR may also influence broader oxidative-stress network in P. ananatis. No other loci showed substantial transcriptional changes, indicating that AltR primarily functions as an alt cluster-specific repressor rather than as a global regulator.

Visualization of normalized read coverage across the alt locus (Fig 1E) further confirmed these transcriptional patterns. In wild-type cells, RNA-seq reads across altA–altJ were limited, consistent with promoter repression, whereas in ΔaltR they increased uniformly across the cluster, reflecting de-repression of the cluster. Coverage dropped sharply within the altR coding region itself, validating the deletion.

Together, these data demonstrate that AltR primarily functions as a cluster-specific transcriptional repressor that governs expression of the thiosulfinate-tolerance (alt) gene cluster. The strong downregulation of the YchH-like gene in the ΔaltR mutant suggests that AltR activity may also indirectly influence additional stress-response pathways.

Thiosulfinates specifically mediate de-repression of the altR promoter

Thiosulfinates are highly reactive organosulfur compounds that oxidize protein thiols mimicking certain aspects of oxidative stress [20]. To test whether AltR responds broadly to thiol-reactive or oxidizing compounds, or specifically to thiosulfinates, we performed zone-of inhibition (ZOI) assays using the PaltRLuxK6 reporter strain exposed to various electrophilic chemicals (Figs 2A-2G).

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Fig 2. Thiosulfinates specifically trigger de-repression of the altR promoter in Pantoea ananatis.

(A–B) Zone-of-inhibition (ZOI) assays showing PaltR::lux reporter activity in Pantoea ananatis PNA 97-1R following treatment with the thiosulfinates allicin (A) and dimethyl thiosulfinate (“methicin”) (B). Both compounds induced a distinct ring of bioluminescence at the periphery of the inhibition zone, indicating AltR de-repression under sub-inhibitory concentrations. (C–D) Treatment with other thiol-reactive or oxidative compounds, including N-ethylmaleimide (NEM) (C) and diamide (D), no luminescent induction was observed, showing that AltR is unresponsive to these generic thiol oxidants. (E–G) The disulfide precursors diallyl disulfide (DADS) (E) and dimethyl disulfide (DMDS) (G), as well as hydrogen peroxide (H₂O₂) (F), also failed to trigger AltR de-repression. The absence of a luminescent ring in these treatments confirms that AltR activation is specific to thiosulfinates and not to disulfides or general oxidative stress.

https://doi.org/10.1371/journal.ppat.1014198.g002

As observed previously, allicin, the major thiosulfinate produced by garlic, induced a distinct ring of bioluminescence around the periphery of the inhibition zone (Fig 2A). A second thiosulfinate, dimethyl thiosulfinate (“methicin”), which is produced by both Allium and Brassica species [44,45], also triggered AltR de-repression, although the luminescence pattern appeared broader with less defined borders (Fig 2B).

In contrast, no promoter induction was observed in the presence of other thiol-reactive or oxidizing agents, indicating N-ethylmaleimide (NEM) and diamide, both known activators of the related NemR regulator in E. coli [39,46] (Figs 2C-2D). Likewise, hydrogen peroxide (H₂O₂), a catalyst used in thiosulfinate synthesis, and the disulfide precursors diallyl disulfide (DADS) and dimethyl disulfide (DMDS) failed to elicit de-repression (Figs 2E–2G).

Together, these results demonstrate that AltR specifically responds to thiosulfinates such as allicin and methicin, but not generally to oxidants or disulfides, confirming that AltR functions as a selective sensor of plant-derived thiosulfinates rather than a broad redox-stress regulator.

AltR promoter de-repression coincides with tissue necrosis during onion infection

To investigate AltR-mediated regulation during onion tissue infection, we inoculated red onion scales with the P. ananatis PNA 97-1R PaltRLuxK6 reporter strain to monitor both symptom development and bioluminescence over time (Fig 3). At 1 day post inoculation (dpi), a faint bioluminescence signal was detected at the inoculation site, corresponding to de-repression consistent with wound-based release of thiosulfinates. By 2 dpi, no visible bioluminescence was observed, suggesting limited atlR promoter activity during early asymptomatic colonization. At 3 dpi, the onset of necrotic lesion formation coincided with a pronounced increase in bioluminescence intensity at the infection site, indicating strong altR de-repression. By 4 dpi, as necrosis expanded, bioluminescence spread outward from the original inoculation point, mirroring the advancing zone of tissue collapse. These results demonstrate that altR promoter activation, and by extension, alt cluster expression, is tightly associated with host tissue necrosis, consistent with thiosulfinate production and bacterial adaptation to the environment of necrotic onion tissue.

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Fig 3. altR promoter de-repression coincides with necrosis during onion infection.

Red onion scale assays were performed using bioluminescently labeled Pantoea ananatis PNA 97-1R PaltRLuxK6 to visualize altR promoter activity during infection. Necrotic symptoms and corresponding bioluminescence (colored yellow) became evident at 3 days post-inoculation (dpi) and intensified at 4 dpi, coinciding with tissue collapse. The luminescence signal remained confined within necrotic tissue, consistent with thiosulfinate production from damaged onion cells. Note that signal intensities are not normalized across time points because images were captured independently on different days.

https://doi.org/10.1371/journal.ppat.1014198.g003

The predicted AltR binding box is required for altR promotor repression

To determine whether AltR directly requires the predicted altR box inverted repeat (IR) sequence upstream of altR, we generated site-directed mutations within the putative operator region. Two mutant promoter constructs were designed: mod1, which introduced transversion mutations in one altR box half-site, and mod2, which altered both altR box half-sites. The resulting reporter strains (PaltRmod1LuxK6 and PaltRmod2LuxK6) were evaluated using ZOI assays. ZOI areas of the two mod mutants box was calculated and compared with WT (S1 Fig). Both mutants lost the repression phenotype and exhibited display constitutive bioluminescence across the plate, indicating that integrity of the altR box sequence is required for AltR-mediated repression (Fig 4). This supports the prediction that the predicted altR box is required for AltR to regulate promoter activity. Interestingly, this specific IR motif (CAATCTAC[N6]GTAGATTG) is located only upstream of altR, altG, altH and altJ; but absent upstream of altA and altD, two of the most strongly upregulated genes in the ΔaltR RNA-seq dataset (Figs 1A, 1D and 1E). We identified an additonal inverted repeat (TTACCAAC[N6]GTTGGTAA), which we designated IR2, in the intergenic regions between altA - altD, altG - altH, and upstream altI. Given the strong upregulation of these genes in the absence of AltR, IR2 may represent an additional AltR operator site or a secondary cis-element involved in indirect regulation. Further biochemical studies are needed to determine whether AltR binds IR2 directly or through cooperative mechanisms involving other transcriptional regulators.

AltR cysteine residues are crucial for thiosulfinate-mediated de-repression

Because thiosulfinates are known to react directly with protein cysteine thiols [26], we hypothesized that AltR cysteines would be crucial for sensing and responding to these compounds. The P. ananatis PNA 97-1R AltR protein contains three cysteine residues: Cys100, Cys101, and Cys159. To test their roles in regulation, we constructed a complete panel of seven cysteine-to-serine substitution mutations, encompassing all combinations. In these mutants, serine replaces cysteine’s reactive thiol with a hydroxyl group. Each mutant allele, designated according to residue substitution (e.g., AltRSCC = C100S, C101, C159; AltRCSS = Cys100, C101S, C159S,) was introduced into the alt cluster via allelic exchange. In allicin ZOI assays, the four alleles carrying the C100S substitution lost the allicin-induced de-repression phenotype (Fig 5A) and exhibited larger inhibition zones relative to wild type (Fig 5B), indicating increased sensitivity to allicin. Consistent results were observed with methicin treatments (Fig 5C-D). Mutations at Cys101 or Cys159 impacted basal or de-repressed bioluminescence levels, suggesting that these residues fine-tune AltR repression strength or inducibility rather than acting as the primary sensing site. Together, these findings indicate that Cys100 is essential for AltR-mediated thiosulfinate sensing, while Cys101 and Cys159 modulate regulation.

Quantification of de-repression and reduced repression in response to thiosulfinates

To quantitatively assess AltR-mediated promoter activity, we used a Newton 7.0 bioluminescence detection system (Vilber Smart Imaging) capable of photon-count quantification. ZOI assay images were analyzed by six regions of interest (ROIs) within both the inhibition zone and the background region, and the maximum photon counts were recorded for each ZOI (Fig 6A).

Bioluminescence from assays treated with allicin and methicin were quantified and plotted separately (Fig 6B-6C). All strains retaining Cys100 exhibited significantly higher mean maximum photon counts within the inhibition zone compared to background regions, confirming AltR de-repression upon thiosulfinate exposure. In contrast, mean maximum photon counts of C100S mutant alleles are not higher than their background, consistent with their loss of responsiveness to thiosulfinates. Notably, several AltR alleles exhibited distinct regulatory behaviors. The CSS allele showed elevated background bioluminescence, indicating reduced repression, while the CCS allele displayed stronger de-repression than wild type, suggesting enhanced promoter induction. Conversely, the CSC allele exhibited reduced de-repression compared to wild type, correlating with its weaker resistance phenotype observed in ZOI assays. These quantitative measurements reinforce that Cys100 is essential for thiosulfinate sensing, while Cys101 and Cys159 modulate AltR sensitivity and basal repression strength.

AltR Cys mutations alter bacterial proliferation in onion tissue

To determine how altR cysteine mutants affect bacterial fitness during host colonization, we performed bacterial load assays on red onion scales using the complete set of altR cysteine-to-serine substitution strains. The altR cysteine-to-serine substitution strains displayed similar growth under rich media conditions (S2 Fig). In addition all strain were capable of producing necrotic lesions consistent with their intact HiVir gene clusters for pantaphos biosynthesis (S3 Fig). Four days post inoculation, both the CCS and CSS mutants reached bacterial population levels comparable to wild type, while the CSC mutant exhibited significantly lower population, similar to the Δalt strain (Fig 7A). This result parallels the in vitro inhibition zone assays (Fig 5), where the CSC mutant displayed increased sensitivity to thiosulfinate compared with WT, CCS, and CSS. All four C100S-containing mutants exhibited markedly reduced bacterial loads relative to WT yet still achieved higher population than the Δalt strain (Fig 7B). These findings demonstrate that Cys100 is critical for AltR function and thiosulfinate tolerance in vivo, and that specific cysteine combinations modulate pathogen proliferation within onion tissue.

Discussion

In this study we present evidence that AltR functions in an analogous manner to the TetR-family repressor NemR in that it (1) represses its own transcription and (2) the transcription of corresponding stress tolerance factors and (3) is derepressed by the offending chemical stressor. In addition, AltR regulation requires an inverted repeat operator and is dependent on a key conserved cysteine residue [39]. The deletion of altR results in increased transcription of the ten other genes in the PNA 97–1 alt gene cluster. The AltR-mediated repression of PaltR is relaxed by exposure to either allicin or methicin as well as in vivo in necrotic onion tissues where thiosulfinates would be natively produced. AltR de-repression is driven by thiosulfinates but not by other oxidizers nor disulfide derivatives. Lastly, mutational evidence supports Cys100 as the key cysteine residue required for thiosulfinate-mediated de-repression, while Cys101 and Cys159 contribute to fine-tuning AltR-mediated regulation, for instance the AltR C159S mutant has higher inducibility than WT AltR. The expression of the alt gene cluster is tightly controlled such that tolerance-associated gene products are produced only upon exposure to the toxic compounds produced by the host [36]. From an applied perspective, this tightly regulated system presents an attractive target for exploitation, particularly in the development of strategies aimed at blocking altR de-repression as a means for improved disease control.

Previous work supported AltR functioning as a repressor of thiosulfinate tolerance [31]. AltR repressed expression from the PaltR Lux reporter, but de-repression was observed at the border of allicin thiosulfinate inhibition zones (Fig 1B). Notably, the Δalt strain had increased thiosulfinate sensitivity in the ZOI assay with the increased diameter correlating with the PaltR Lux “induction ring” of de-repression in the WT. This suggested that AltR-mediated de-repression coincides with increased phenotypic thiosulfinate tolerance. We also observed that altR promoter de-repression occurs in vivo, correlating with the onset of necrotic symptom where dead onion cells release thiosulfinates (Fig 3). We identified a perfect inverted repeat (CAATCTAC [N6] GTAGATTG) in the altR promoter region as a potential altR box operator as well as two additional imperfect altR box operators in the alt cluster (Fig 1A). We also identified a second inverted repeat (TTACCAAC [N6] GTTGGTAA) present in alt gene intergenic regions which we named Inverted Repeat 2 (IR2) also with two others imperfect IR2 boxes found in the alt cluster (Fig 1A). We provided genetic evidence that the predicted altR box within the altR promotor region is required for AltR-mediated repression of the PaltR Lux reporter. Mutating the sequence of the predicted AltR binding box results in loss of repression (Fig 4).

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Fig 4. The predicted altR box is required for promoter repression.

(A) Diagram of the altR promoter region showing the predicted altR box (CAATCTAC[N6]GTAGATTG). Inverted repeat (IR) half-sites are highlighted, with transversion mutations introduced in mod1 (one half-site) and mod2 (both half-sites). (B) Zone-of-inhibition (ZOI) assays of P. ananatis PNA 97-1R strains carrying PaltRmod1LuxK6 and PaltRmod2LuxK6 reporter constructs. Mutation of one or both IR half-sites led to constitutive bioluminescence across the plate, indicating complete loss of AltR-mediated repression.

https://doi.org/10.1371/journal.ppat.1014198.g004

In the absence of altR, all ten remaining alt genes were upregulated consistent with AltR regulating thiosulfinate tolerance. The altA, altB, altC, altD, and altJ genes were some of the most up-regulated genes (Fig 1D). However, only the altJ gene has a predicted upstream altR box. The expression of altA, altB, altC, and altD, may be mediated via the intergenic IR2 box either through direct AltR recognition of IR2 as a second operator or indirectly through AltR-regulation of an additional transcription factor. While no other annotated transcription factors were identified as upregulated in the altR mutant, a truncated ORF (B9Q16_23175) predicted to encode a HtH motif downstream of altB was upregulated. The consensus sequence of the altR and IR2 boxes does constitute an inverted repeat YWAYCWAC [N6] GTWGRTWR with 4/8 conserved bases per half site and palindromic nucleotide degeneracies. Thus, AltR may in fact recognize both operator sequences (Fig 1A). Determining AltR DNA binding specificity will be an important area for future study and may provide more insights into the unexpected behavior of the AltR CSC allele. Among the RNA seq results, a single gene was strongly down regulated, locus number B9Q16_RS12865. This gene is predicted to be a homolog of an E coli ychH, which is a gene of unknown function identified from one study to be regulated in response of hydrogen peroxide, cadmium, and acid stress [43].

Previous studies demonstrated that the TTG clusters of Pseudomonas fluorescens strain PfAR-1 confer tolerance to synthesized allicin but not to other oxidant compounds like H2O2, or NEM [32]. NemR responds to reactive electrophile compounds, e.g., NEM, bleach, and diamide, to regulate bacterial tolerance to this class of compounds. Thus, we reasoned that AltR would specifically respond to thiosulfinates in a similar manner. We tested the responsiveness of AltR against several oxidative compounds. NEM and diamide were chosen to test because NemR responds to both NEM and diamide, and diamide, like thiosulfinates, oxidizes thiols (Figs 2C and 2D) [32,39]. AltR de-repression occurred in response to allicin and methicin but not to NEM, diamide, H2O2 or the allyl-disulfide or methyl-disulfide precursors used for thiosulfinate synthesis (Figs 2E-2G). Thus, AltR has some degree of specificity for thiosulfinates.

Thiosulfinates vary based on their side chains [24,47,48] although they share the same functional group and can participate in similar chemical reactions. Allicin is the characteristic thiosulfinate of garlic and functionally there is little to no production of allicin by damaged onion cells. Onions produced a mixure of different kinds of symmetric and asymmetric thiosulfinates derived from either isoalliin, methiin, or propiin [20]. This is why we included synthesized methicin which can be produced by both onions and garlic as well as by Brassica spp. We observed similar overall responses to both allicin and methicin exposure.

PNA 97–1 AltR encodes three cysteines, Cys100 Cys101, and Cys159. However, in a sequence alignment of the Pseudomonas and Pantoea AltR, only Cys100 was conserved. We generated Cys to Ser mutants for all possible combinations of the Cysteine residues to see how the activity of the AltR repressor was impacted. After creating the 7 alleles of Cys to Ser and performing inhibition zone assays, we learned that Cys100 is, indeed, the most critical for thiosulfinate-mediated de-repression as only strains with this residue retain the thiosulfinate induction ring phenotype. Strains without this C100S mutation also all show increased sensitivity towards thiosulfinates comparable to a WT strain, implying AltR with the C100S stays bound to the altR box even in the presence of thiosulfinates. This behavior was consistent result for both allicin and methicin (Fig 5).

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Fig 5. Cysteine residues of AltR are required for thiosulfinate-dependent de-repression.

(A) Zone-of-inhibition (ZOI) assays showing bioluminescence from PNA 97-1R PaltRLuxK6 strains carrying different altR cysteine-to-serine substitution alleles following treatment with synthesized allicin. Strains retaining Cys100 (e.g., CCC, CSS, CSC, CCS) displayed a luminescent ring at the edge of the inhibition zone, whereas all Cys100Ser (C100S) mutants lacked a luminescent ring, indicating loss of AltR responsiveness to allicin. (B) Quantitative analysis of relative ZOI area for wild-type and mutant strains in response to allicin. Data represents three biological replicates, each with three technical replicates of the ratio of the ZOI area relative to WT, analyzed by one-way ANOVA followed by Tukey’s multiple-comparison test (p < 0.001). (C) ZOI assays of the same altR cysteine mutants exposed to methicin (dimethyl thiosulfinate). As with allicin, only strains retaining Cys100 exhibited promoter induction, confirming that this residue mediates AltR redox sensing of thiosulfinates. (D) Relative ZOI area measurements for methicin assays analyzed as described in (B).

https://doi.org/10.1371/journal.ppat.1014198.g005

Quantitative photon counts for the bioluminescence signal in the induction ring and background showed similar results although also demonstrated that Cys101 and Cys 159 tune both in the intensity of de-repression and the strength of repression (Fig 6). For instance, the C159S allele has exaggerated de-repression resulting in a brighter induction ring than that produced by the WT AltR allele, whereas the C101S/C159S double mutant has an elevated background Lux expression consistent with reduced AltR repression. It is also interesting to note that the CSC mutant has higher thiosulfinate sensitivity compared to other C100S strains (Fig 5B and 5D) and reduced bacterial load in onion scales after 4 days post inoculation (Fig 7). We are, as of yet, unclear how Cys101 mediates a more dramatic impact on bacterial fitness in onion tissue than the four thiosulfinate non-responsive C100S alleles.

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Fig 6. Quantitative analysis of cysteine-to-serine substitutions on AltR-mediated de-repression and reduced repression in response to thiosulfinates.

(A) Zone-of-inhibition (ZOI) assay imaged using the Newton 7.0 (Vilber Smart Imaging) bioluminescence detection system. Six regions of interest (ROIs) were selected from both the induction ring (wt1 ~ wt6) and the background region (wt1b~wt6b) to measure photon counts, as illustrated. (B) Quantification of bioluminescence for P. ananatis PaltRLuxK6 altR cysteine mutants exposed to allicin. Mean maximum photon counts (blue bars) within the inhibition zone and mean background photon counts (orange bars) are shown for each allele. Strains retaining Cys100 exhibited significant de-repression compared to their background levels, while C100S mutant alleles did not. (C) Quantification of bioluminescence for the same mutant panel exposed to methicin. Similar trends were observed, confirming that Cys100 is essential for thiosulfinate-dependent induction. Error bars represent standard deviation of three biological replicates, each with three technical replicates. Significance was determined by two-sample t-tests: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

https://doi.org/10.1371/journal.ppat.1014198.g006

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Fig 7. AltR cysteine mutations alter bacterial proliferation in onion tissue.

(A) Bacterial population sizes of PNA 97-1R PaltRLuxK6 strains carrying non-C100S altR alleles (CCC, CSS, CSC, CCS) on red onion scales at 4 days post-inoculation (dpi). The CSC mutant exhibited significantly reduced bacterial loads, comparable to the Δalt strain. (B) Bacterial populations of C100S mutant strains (SCC, SCS, SSC, SSS) at 4 dpi. All C100S alleles showed decreased proliferation relative to wild type but maintained higher populations than Δalt. Each data point represents a single biological replicate (six biological replicates, five technical repeats per biological replicates). Data were log₁₀-transformed prior to analysis. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple-comparison test (p < 0.001). Pairwise comparisons against the WT control were evaluated by t-test.

https://doi.org/10.1371/journal.ppat.1014198.g007

Although we have made structurally conservative cysteine to serine mutations and used allelic replacement to express the altR mutant alleles from their native location and promoter, variations in expression or protein stability might also be considered as possible explanations for some observed phenotypes. For instance, the higher background bioluminescence associated with the C101S/C159S (CSS) double mutant might be associated with reduced AltR protein levels.

In conclusion, this study provides new mechanistic insights into the regulation of the allicin tolerance gene cluster. As failure of AltR to derepress is associated with reduced bacterial loads in onion tissue, identification of antagonist compounds that can interfere with the capacity of AltR to respond to thiosulfinates may have utility both for management of onion center rot disease and potentially other diseases of onion caused by bacteria.

Materials and methods

Bacterial strains and cultural conditions

Overnight cultures of Escherichia coli and Pantoea ananatis were routinely cultured from single colonies recovered on LB parent plates, and were grown in 5 mL of LB medium in 14 mL culture tubes placed in incubator of 28°C (P. ananatis) or 37°C (E. coli) with 200rpm shaking for 18–22 h. See S1 Table for list of strains and plasmids. Reference genome for the main Pantoea strain in this study (PNA97-1R) can be found on GenBank: NZ_CP020943.2, NZ_CP020944.2, NZ_CP020945.2 [30].

Bioluminescence signal imaging

Imaging was performed with the analyticJena UVChemStudio imager (Upland, CA, USA). For the strong bioluminescence signal strains (Mostly Δalt strains), 2-minute exposure time was used; for the weak bioluminescence strains, 5-minute exposure time was used. Strong signal strains and weak signal strains were not imaged together because the weak signal strains were overshadowed. An image under white light was also taken for each bioluminescence image for generating image overlays with ImageJ, with the non-bioluminescence background colored blue and the bioluminescence signal colored yellow.

Construction of the pTn7PaltRLuxK6 vector

The bacterial promoter PaltR, intergenic region of P. ananatis altG and altR, was synthesized as a dsDNA gblock (S3 Table) by IDT (Coralville, Iowa, USA) and cloned into the StuI and DraIII double-digested backbone of pTn5/7LuxK6 [40] via Gibson assembly (New England Biolab). The insertion of the PaltR promoter was confirmed by sequencing.

Mini-Tn7 labelling of strains

E. coli RHO3 pTNS3, E. coli RHO5 pTn7PaltRLuxK6, and the target Pantoea strain were combined in a tri-parental mating [49,50]. 5 mL LB cultures of each strain were grown overnight. The following day 1 mL of each culture was pelleted and resuspended in 100 mL of fresh LB. 10 μL of each concentrated culture was added to a clean tube. LB plates with 400 μg/ml of diaminopimelic acid (DAP) were prepared and sterile nitrocellulose membranes were placed on the plates. 30 μL droplets of the mixed cultures, and independent strain controls were placed on the nitrocellulose membranes to dry. Following overnight incubation, the mixture was removed from the nitrocellulose membrane using a sterile loop and resuspended in 1 mL LB. 200 μL of the incubated mixed culture suspension was plated on LB selection plates with 50 μg/ml Kanamycin (Km) but no DAP. The following day Km resistant colonies were selected confirmed for luminescence with 2 m exposure settings on the analyticJena UVChemStudio imager.

Preparation of allicin and methicin stock solutions

Fresh allicin and methicin stock solutions were prepared for experiments by using a modified microscale synthesis protocol [51]. Solutions were stored at -20 C and used within three days of prep. For allicin synthesis, 5 μL of diallyl disulfide (DADS) 96% (Carbosynth), 25 μL of glacial acetic acid, and 15μL of 30% hydrogen peroxide were added to 4x 200μL PCR tubes. For methicin synthesis, dimethyl disulfide 99% (Sigma) was used in the same way. Tubes were agitated in a 28°C shaker for at least 4h. Following agitation, the reaction in all 4 tubes were quenched in 1 mL of methanol. This methanol allicin mixture was used as the stock synthesized allicin preparation and was used directly in ZOI assays. Controls for disulfide precursors testing (Fig 2) were made following the same protocol with the absence of hydrogen peroxide.

Zone of inhibition assay

Styrene Petri plates (100 x 15 mm) with 20 mL LB adding the required antibiotics were spread with 400μL of overnight bacterial culture using a sterile cotton swab. A 0.125 cm2 agar plug was removed from the center of the plate with a biopsy punch. 50 μL of treatment solution was added into the agar well. Treatment solutions that were used in this research include the allicin or methicin stocks, 30 mM NEM 99% (Thermoscientific) dissolved in ethanol, and 150 mM diamide 97% (Tokyo Chemical Industry) dissolved in DMSO. Plates were incubated for 24 h at RT and evaluated for a zone of inhibition (cm2) by measuring the diameter, calculating the relative inhibition area compared with mean value of WT for statistical analysis. Raw data was processed through Excel (Microsoft), and Rstudio (Package agricolae) was used for conducting one-way ANOVA analysis and t-tests, as well as drawing graphs.

Images of the plates were taken via the analyticJena UVChemStudio imager with 2 or 5 minutes exposure (depending on the strength of bioluminescence) for bioluminescence signals and a normal condition image under white light for overlapping, color coded bioluminescence as yellow and white light image as blue in the images.

Red onion scale necrosis assay

Consumer produce red onions (Allium cepa. L., red onion) were purchased, cut to approximately 3 cm wide scales, sterilized in a 3% household bleach solution for 20 seconds, then rinsed in dH2O for 4 ~ 5 times gently. Red onion scales at least 0.5 cm thick with a healthy undamaged appearance were used. Scales were placed in a potting tray (27 x 52 cm) containing two layers of paper towels pre-moistened with 90 mL of distilled water. The plastic removable portion of 20 μL pipette trays (sanitized before use) were positioned on top to prevent direct contact between the paper towels and onion scales. Individual onion scales were wounded cleanly through the scale with a sterile 20 μL pipette tip and inoculated with a 10 μL drop of bacterial culture of 0.3 OD600. Sterile deionized water was used as a negative control inoculum. The tray was covered with a plastic humidity dome and incubated at RT for 96 h; images were taken every 24 h with the analyticJena UVChemStudio imager to record the disease development.

Quantification of bacteria growth (cfu/mg) within onion scales was performed after red onion scales assays by sampling small chunks of tissues (approximately 50 ~ 100 mg) 1 cm away from the inoculation site. The sampled tissues were weighed and placed in plastic maceration tubes with 200 mL of sterile dH2O and three Xmm diameter high density zircon beads. The tubes beat at 4 m/s for 1 min using the Bead Ruptor Elite Bead Mill Homogenizer (Omni International, Kennesaw, GA, USA). Samples then underwent 10-fold serial dilution (20μl to 180 μl) with sterile water up to 10^-7 in a sterile 96 well plate. 10μl of the diluted suspensions were then plated on LB rifampicin (rif, 20 ~ 60 μg/ml) plates, incubated overnight in 28°C. Colonies were counted the next day, normalized by sample weight to quantify bacteria load. Raw data was processed through Excel (Microsoft), and Rstudio (Package agricolae) was used for conducting one-way ANOVA analysis and t-tests, as well as drawing graphs.

RNA seq analysis

Total RNA was extracted from overnight cultures of Pantoea ananatis PNA 97-1R PaltRLuxK6 and a ΔaltR mutant using the Qiagen miRNeasy Mini Kit according to the manufacturer’s instructions. Three biological replicates per strain were submitted to Azenta Life Sciences (South Plainfield, NJ, USA) for DNase treatment, library preparation, and next-generation sequencing. RNA-seq libraries were prepared using an rRNA depletion protocol and sequenced on an Illumina platform using paired-end 2 × 150 bp reads. Sequencing depth was comparable among samples, with approximately 13 million reads per sample, and mapping efficiencies were consistent across samples, with more than 94% of reads aligning to the Pantoea ananatis PNA 97-1R reference genome. Raw sequencing reads are available in the NCBI BioProject database with the ID PRJNA1374235.

Raw sequencing reads were assessed for quality using FastQC, and adapter sequences and low-quality bases were removed using Trimmomatic. Clean reads were aligned to the Pantoea ananatis PNA 97-1R reference genome using Bowtie2. Transcript assembly and quantification were performed using StringTie based on the reference genome annotation. Differential gene expression analysis was conducted using DESeq2 in R. Volcano plots were generated in R and read coverage across biological replicates was calculated using deepTools and BEDTools. Coverage visualization was performed using Integrative Genomics Viewer (IGV).

AltR box modification construction

Mutated variants of the altG and altR intergenic region were synthesized as dsDNA gblocks by IDT (S3 Table) and cloned into the StuI and DraIII double-digested backbone of pTn5/7LuxK6 [40] via Gibson assembly. The insertion of the two mod box promoter was confirmed by sequencing. After assembly of the plasmid, the modified altR box were inserted into PNA 97-1R with the mini-Tn7Lux labelling method mentioned in previous sections, making the two strains PNA 97-1R PaltRmod1LuxK6 and PNA 97-1R PaltRmod2LuxK6.

Construction of cysteine mutants of altR expression vectors and allelic exchange of cysteine to serine mutants

Cysteine residue mutants of altR were built in gateway expression vectors and later transformed in PNA 97-1R ΔaltR PaltRLuxK6 for plasmid-based complementation. The pDONR221::altR plasmid was made by PCR amplifying the altR gene region then insertion into the pDONR221 empty plasmid with BP cloning. See S2 Table for primer sequences. PCR was conducted under the following condition: 95℃ for 30s, then repeat 35 cycles of 95℃ denaturing for 30s, 60℃ annealing for 30s, 72℃ extension for 1m. Final extension at 72℃ for 4m. The altR mutants, SSS, CSS, SCS were synthesized by Genscript (Piscataway, NJ, USA) in the Gateway compatible plasmid pUC57 with an additional BspE1 site. The four other combinations of the cysteine mutants, CSC, CCS, SSC, SCC, were made by restriction enzyme BspE1 to swap the C-terminal portions including altR Cys159 from the altR sequence, then ligation with T4 DNA ligase (ThermoScientific) was performed.

The flanks around altR(100–159) were first synthesized along with a dual BbsI cutsite in the middle, sequence named attB-altRd100 ~ 159-flanks-BbsI (S3 Table), then was inserted into allelic exchange vector pR6KT2G via BP clonase recombination. The mutated AltR(100 ~ 159) regions were then amplified with PCR by using the expression vector mutants as templates under the following PCR condition: 95℃ for 30s, then repeat 35 cycles of 95℃ denaturing for 30s, 60℃ annealing for 30s, 72℃ extension for 1m. Final extension at 72℃ for 4m. Gibson assembly was then utilized to assemble 7 different allelic exchange plasmids that includes all 7 different combinations of cysteine to serine mutants (pR6KT2G_altRXXX). The 7 assembled plasmids were transformed into donor strain RHO5, then introduced into target strain PNA 97-1R ΔaltR(100–159) PaltRLuxK6 by bacterial mating on LB DAP plates. Merodiploids were recovered 24 hours later on LB plates with 10 μg/ml gentamicin (Gm). Two merodiploid colonies of the same mutant were then selected and added in a tube of 1mL sterile LB with 3mL sterile 1M Sucrose to grow for 24hr at 37°C. Counter selection was done the next day by plating 1x105 or 1x106 dilution of the culture on LB plates with 50μg/ml X-Gluc (5-Bromo-4-chloro-1H-indol-3-yl β-D-glucopyranosiduronic acid). Colonies that are shown blue has not lost the inserted gateway plasmid, therefore white colonies were patch plated to find loss of Gm resistant. Altered bioluminescence were also checked for the newly made strains. Further sequencing were performed to confirm the allelic exchanged mutants.

Photon quantification

Bioluminescence photon count data images were taken by the camera Newton 7.0 (Vilber Smart Imaging, Paris, France) under software auto recommendation settings. Images were then processed through the software Kaunt 2.5 (Vilber Smart Imaging), and areas of interest were selected through it (Fig 6A). The Max ph/s/cm²/sr data for regions of interests were then exported to Excel (Microsoft) and processed to draw graphs. Rstudio (Package agricolae) was used for conducting t-tests.

Bacteria growth curve

Overnight liquid culture of the P. ananatis strains were prepared in 5mL LB. Overnight liquid cultures were then standardized to OD600 0.2 with fresh LB and 400μl of normalized culture was added in each well of a 96 well plate. The OD600 from each well was measured by SpectraMax iD3 (Molecular devices, San Jose, CA, USA) every 30 minutes for 26 hours.

Supporting information

S1 Fig. ZOI comparison between the modified altR box strains.

Quantitative analysis of relative allicin ZOI area for wild-type (WT) and PaltRmod box strains. Data represents three biological replicates, each with three technical replicates, relatively compared with the average area of WT, analyzed by one-way ANOVA on ranks followed by Dunn’s multiple-comparison test (p < 0.01).

https://doi.org/10.1371/journal.ppat.1014198.s001

(DOCX)

S2 Fig. Growth curve of the PNA 97-1R Cys to Ser mutants in rich media.

Growth curve in LB media of altR Cys to Ser mutant strains used in Fig 5 to Fig 7. Overnight LB cultures were normalized to OD600 of 0.2 before incubating in a 96 well plate and kinetic measurement of OD600 every 30 minutes for 26 h with a SpectraMax iD3 plate reader (Molecular devices, San Jose, CA, USA).

https://doi.org/10.1371/journal.ppat.1014198.s002

(DOCX)

S3 Fig. Necrotic lesions on red onion scales caused by Cys to Ser mutants.

3 DPI onion scales necrotic symptoms for PNA 97–1 mutants.

https://doi.org/10.1371/journal.ppat.1014198.s003

(DOCX)

S1 Table. General cloning, deletion, and labeling strains and vectors used in this study.

https://doi.org/10.1371/journal.ppat.1014198.s004

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S2 Table. Primers listed in this study for PCR and cloning purposes.

https://doi.org/10.1371/journal.ppat.1014198.s005

(DOCX)

S3 Table. Synthesized sequences that were utilized for Gateway cloning and Gibson assembly in this study.

https://doi.org/10.1371/journal.ppat.1014198.s006

(DOCX)

Acknowledgments

We acknowledge Dr. Jovana Mijatovic for assistance with data analysis.

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