Introduction

Salmonella enterica is a human enteric pathogen that causes disease in approximately 1.2 million Americans annually (CDC). Numerous multi-state outbreaks of salmonellosis have occurred from the consumption of fresh produce every year since 2000 (National Outbreak Reporting System (NORS;1,2,3,4)). S. enterica survives on multiple agricultural crops after root or leaf immigration5,6,7, and the bacteria persist in soil for months6,8,9. Despite the continued isolation of S. enterica from the agricultural environments associated with outbreaks, S. enterica has relatively poor fitness in the plant environment. Bacterial populations steadily decline in the phyllosphere, or above-ground parts of plants8,9.

The rapidly fluctuating conditions on the leaf surface provide a harsh environment for epiphytic bacteria, and survival depends on the ability to adapt to abrupt changes in UV irradiation and the availability of water and nutrients. Upon arrival to the leaf surface, S. enterica migrates to protected sites like trichomes, stomates, hydathodes, and epidermal cell wall junctions7,10,11,12. These locations are often associated with a local increase in nutrient abundance due to cracks or leakage through the cuticular layer and may provide protection from desiccation or UV stress10,13. The accessibility of these sites likely explains both how S. enterica persists on leaves over long periods of time and why eventually populations decline. This failure to replicate faster than cellular death is most likely because S. enterica cannot access the nutrient-rich interior of plant tissues on its own.

Recent studies involving S. enterica colonization of plants have centered on the identification and characterization of biomultipliers, factors that lead to increased S. enterica survival. S. enterica exploits changes to the plant environment imparted by other organisms, including infestation with phytophagous insects and infection with bacterial phytopathogens14,15,16,17,18,19,20,21,22. Plants infected with Xanthomonas spp. lead to increased persistence of S. enterica on tomato (Solanum lycopersicum) leaves17,18. Four lineages of Xanthomonas cause bacterial spot of tomato: X. hortorum pv. gardneri (hereafter referred to as Xhg), X. euvesicatoria pv. euvesicatoria, X. euvesicatoria pv. perforans, and X. vesicatoria23,24,25. These xanthomonads colonize tomato leaf surfaces as epiphytes and enter plant tissue through natural openings to circumvent the epidermis, the first layer of plant defense. Once inside, Xanthomonas colonizes the apoplast where it multiplies and eventually spreads to neighboring leaves, and ultimately other plants. Bacterial spot disease manifests as lesions on leaves, fruit, and stems of tomato plants and ultimately leads to loss of leaves and damaged fruit, resulting in substantial agricultural impact23,24.

Although four Xanthomonas lineages cause the same disease on tomato, only a subset of these lineages has a beneficial effect on S. enterica populations. In plants colonized by Xhg, X. euvesicatoria pv. euvesicatoria, or, to a lesser extent, X. euvesicatoria pv. perforans, S. enterica populations on tomato leaves remain steady or even increase over time17. Contrastingly, S. enterica populations decline in the presence of X. vesicatoria or on healthy plants17. Core Type III secretion system (T3SS) effectors are shared among the four Xanthomonas species, yet each species also has unique effectors. Of particular interest for this study, Xhg has a transcription activator-like effector (TALE) AvrHah1 that is not found in X. vesicatoria26. TALEs induce host transcription by directly binding to the promoter region of target genes using a modular, central repeat domain that recognizes specific host effector binding elements 27,28. Xhg AvrHah1 has over 4,000 potential, predicted binding sites in tomato29.

AvrHah1 induces a water soaking phenotype during disease29,30, which is characterized by dark green lesions on leaves that reflect an influx of fluid into the apoplast. This water soaking is not observed in X. vesicatoria-infected tissues26. In tomato, AvrHah1 is required for the direct induction of two beta helix loop helix (bHLH) transcription factors, bHLH3 and bHLH629. The spike in transcription for these two regulators leads to indirect induction of at least two additional downstream genes, including a pectate lyase (PL) and pectinesterase (PE)29. The current model suggests that PL alters hygroscopicity in the plant cell wall by releasing oligosaccharides, resulting in an influx of fluid through breaks in the epidermis and the observed water-soaked phenotype29. Because the Xhg avrHah1 mutant reaches the same population level as wildtype Xhg in planta, it is thought that AvrHah1 is involved in pathogen dispersal or entry into leaf tissue as opposed to acquisition of nutrients during infection29.

In this study, we tested the hypothesis that AvrHah1 defines the differential ability between Xhg and X. vesicatoria to enable S. enterica persistence in the phyllosphere. We showed that lack of this effector reduced the ability of Xhg to promote S. enterica persistence on tomato leaves. Correspondingly, the addition of avrHah1 to X. vesicatoria allowed this phytopathogen to promote S. enterica survival. The data presented here provide evidence that AvrHah1-dependent water soaking may be the mechanism by which the phytopathogenic bacterium benefits the human enteric pathogen.

Results

AvrHah1 is both necessary and sufficient for Xhg-dependent effects on S. enterica persistence

Previously, we had shown that infection with Xhg promotes survival of S. enterica on the leaves of tomato plants19. To determine the importance of AvrHah1 for Xhg-dependent effects on S. enterica, we examined the effects of co-inoculation with the Xhg AvrHah1 DNA-binding domain mutant avrHah1ΔDBD 30. Bacterial populations were monitored on tomato plants dip-inoculated with S. enterica in the presence or absence of wildtype Xhg or the Xhg avrHah1ΔDBD mutant. The presence of wildtype Xhg led to approximately ten-fold higher S. enterica populations than the S. enterica alone treatment by 6 dpi (Fig. 1a; P = 0.0058). Conversely, co-inoculation of S. enterica with the Xhg avrHah1ΔDBD mutant had no significant impact on S. enterica (Fig. 1a; P = 0.889). Xanthomonas populations were monitored over time, and there were no significant differences between treatments (Fig. 1b; P > 0.556 for all comparisons).

Figure 1
figure 1

TALE AvrHah1 is required for Xhg-dependent effects on S. enterica persistence. Bacterial populations (S. enterica, (a); Xanthomonas (b)) on tomato leaves were monitored following treatment with S. enterica (Se; black circles), Xhg (Xhg; open green triangles), Xhg avrHah1ΔDBD mutant (Xhg ΔAvr; open orange diamonds), S. enterica + Xhg (SeXhg; closed green triangles), or S. enterica + Xhg avrHah1ΔDBD mutant (SeXhg ΔAvr; closed orange diamonds). All data points from three independent experiments are presented as log CFU/cm2. Lines (black, S. enterica, Se; dashed green, Xhg, Xhg; dashed orange, Xhg avrHah1ΔDBD mutant, Xhg ΔAvr; solid green, S. enterica + Xhg, SeXhg; solid orange, S. enterica + Xhg avrHah1ΔDBD mutant, SeXhg ΔAvr) correspond to a linear regression model with 95% confidence intervals represented by the shaded areas. Letters denote significant differences between treatments for the regression lines according to the linear regression model (P < 0.01). Combining three independent experiments, n = 9 plants per treatment per time point.

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To determine if avrHah1 is sufficient for Xanthomonas-mediated effects on S. enterica persistence, X. vesicatoria, which does not impact S. enterica persistence, was transformed with a plasmid carrying the avrHah1 gene (pUFR034 (avrHah1)) and tested for its effects on S. enterica. Bacterial populations were determined in tomato plants dip-inoculated with S. enterica in the presence or absence of Xhg, X. vesicatoria + pUFR034 (vector alone control), or X. vesicatoria + pUFR034 (avrHah1). Co-inoculation of Xhg or X. vesicatoria + pUFR034 (avrHah1) with S. enterica resulted in approximately ten-fold higher S. enterica populations at 6 dpi than in plants co-inoculated with X. vesicatoria + pURF034 and S. enterica or inoculated with S. enterica alone (Fig. 2a; Xhg P = 6.40 × 10−7 and 1.74 × 10−6, respectively; X. vesicatoria + pUFR034 (avrHah1) P = 8.17 × 10−5 and 0.00019, respectively). There was no significant difference between the X. vesicatoria + pURF034 and S. enterica alone treatments (P = 0.8203). Xanthomonas populations were monitored over time, and there were no significant differences between treatments (Fig. 2b; P > 0.97). These data demonstrate that the presence of AvrHah1 in Xanthomonas spp. can increase the persistence of S. enterica in co-inoculated leaves.

Figure 2
figure 2

AvrHah1 is sufficient for Xanthomonas-dependent increases in S. enterica populations. Bacterial populations (S. enterica, (a); Xanthomonas (b)) on tomato leaves were monitored following treatment with S. enterica (Se; black circles), Xhg (Xhg; open green triangles), X. vesicatoria pUFR034 (Xv; open blue squares), X. vesicatoria pUFR034 (avrHah1) (Xv Avr; open cyan squares), S. enterica + Xhg (SeXhg; closed green triangles), S. enterica + X. vesicatoria pUFR034 (SeXv; closed blue squares), or S. enterica + X. vesicatoria pUFR034 (avrHah1) (SeXv Avr; closed cyan squares). All data points from three independent experiments are presented as log CFU/cm2. Lines (black, S. enterica, Se; dashed green, Xhg, Xhg; dashed blue, X. vesicatoria pUFR034, Xv; dashed cyan, X. vesicatoria pUFR034 (avrHah1), Xv Avr; solid green, S. enterica + Xhg, SeXhg; solid blue, S. enterica + X. vesicatoria, SeXv; solid cyan, S. enterica + X. vesicatoria pUFR034 (avrHah1), SeXv Avr) correspond to a linear regression model with 95% confidence intervals represented by the shaded areas. Letters denote significant differences between treatments for the regression lines according to the linear regression model (P < 0.01). Combining three independent experiments, n = 9 plants per treatment per time point.

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The AvrHah1 targets bHLH3, bHLH6, PL, and PE are transcribed to similar levels in plants infected with wildtype Xhg or the Xhg avrHah1 ΔDBD mutant

Previous work has shown that AvrHah1 activates expression of multiple tomato genes, including two direct targets bHLH3 and bHLH6 and two indirect targets PL and PE29. These targets provide the foundation for the current model of AvrHah1 water soaking. To examine whether these targets play a role in the mechanism by which Xhg enhances S. enterica persistence, we monitored transcription of the genes in leaf samples after inoculation with S. enterica in the presence or absence of wildtype Xhg or the Xhg avrHah1ΔDBD mutant. Under our experimental conditions, plants infected with the Xhg avrHah1ΔDBD mutant had the same or higher levels of the four targets compared to plants infected with wildtype Xhg at 1, 3, and 6 dpi (Table 1). In contrast, published results show that wildtype Xhg Xg153 significantly induces transcription of these genes (~ 90–1,300-fold) compared to a corresponding avrHah1ΔDBD mutant when infiltrated into tomato Heinz 1706 leaves for 48 hours29. To determine the effect of inoculation method (dip-inoculation vs infiltration) on our differing results, we infiltrated MoneyMaker tomato leaves with Xhg 444 wildtype and avrHah1ΔDBD mutant following published protocols29. Leaf samples were collected 48 h post-infiltration, and plant gene expression was measured using quantitative PCR (qRTPCR). As with the dip-inoculation experiments, there were no significant reductions in plant gene expression in plants inoculated with the Xhg avrHah1ΔDBD mutant compared to plants inoculated with wildtype Xhg for bHLH3, PL, or PE (Table 1). The only observed reduction in expression in plants inoculated with the Xhg avrHah1ΔDBD mutant was a ~ 25-fold reduction in bHLH6 transcription compared to plants inoculated with wildtype Xhg (Table 1). These data indicate that AvrHah1 may target other host genes in the mechanism that leads to increased S. enterica persistence in this system.

Table 1 Transcription of AvrHah1 targets in tomato leaves after infection with the Xhg avrHah1ΔDBD mutant.
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Xhg and X. vesicatoria elicit different immune responses in tomato leaves, which are further affected by co-inoculation with S. enterica

To test the hypothesis that infection with Xhg alters the plant immune response to the benefit of S. enterica, plant defense gene expression was monitored over time in tomato leaves inoculated with S. enterica, Xhg, X. vesicatoria, or a combination of each xanthomonad with S. enterica. The SA-inducible pathogenesis related protein gene pr1a1 and the JA-inducible proteinase inhibitor gene pin1 were used as established markers31,32 to indirectly monitor these two plant defense pathways with qRTPCR.

At 1 dpi, there were few differences in pr1a1 expression between treatments (Fig. 3a). The Xhg with S. enterica co-inoculation treatment was significantly different from the negative control (plants treated with water) (P = 0.0018), but all other treatments were statistically the same as the negative control at this time point (Fig. 3a). Compared to the negative control, tomatoes that were inoculated with Xhg, Xhg and S. enterica, or X. vesicatoria showed significant increases in pr1a1 expression by 3 dpi (Fig. 3a; P = 0.0205, 0.0005, and 0.0032, respectively). Contrastingly, tomato plants treated with X. vesicatoria and S. enterica or S. enterica alone had no change in pr1a1 expression compared to the water control at 3 dpi (Fig. 3a; P = 0.901 and 0.889, respectively). By 6 dpi, pr1a1 levels had increased in plants inoculated with Xhg, Xhg and S. enterica, or X. vesicatoria with changes reaching approximately 100–10,000-fold compared to water controls (Fig. 3a; P = 5 × 10−7, < 1 × 10−7, and 7.46 × 10−5, respectively). Plants treated with X. vesicatoria and S. enterica or S. enterica alone had no change in pr1a1 expression compared to the water control at 6 dpi (Fig. 3a; P = 0.244 and 1.00, respectively). Plants inoculated with X. vesicatoria and S. enterica had pr1a1 levels that were statistically the same as both the water control and the X. vesicatoria alone treatment (Fig. 3a; P = 0.244 and 0.084, respectively).

Figure 3
figure 3

Xanthomonas species differentially impact the plant immune responses. Plant gene expression was quantified at days 1, 3, and 6 dpi with water (purple circles), S. enterica (Se; black circles), Xhg (Xhg; green, open triangles), Xhg + S. enterica (SeXhg; green, closed triangles), X. vesicatoria (Xv; blue, open squares), X. vesicatoria + S. enterica (SeXv; blue, closed squares), Xhg avrHah1ΔDBD mutant (Xhg ΔAvr; open, orange diamonds), S. enterica + Xhg avrHah1ΔDBD mutant (SeXhg ΔAvr; closed, orange diamonds), X. vesicatoria pUFR034 (avrHah1) (Xv pAvr; open, cyan squares), or S. enterica + X. vesicatoria pUFR034 (avrHah1) (SeXv pAvr; closed, cyan squares). Data for the SA-inducible pr1a1 (a,c,d) and JA-inducible pin1 (b) are displayed as log-transformed relative expression ratios (RER) using water-treated plants as the calibrator. Each symbol represents transcription levels in one tomato plant, and the dashed lines indicate a two-fold change relative to the water control. Means for each treatment at each time point are depicted with horizontal black lines. Transcription was measured in plants from three biological replicates, sampling from three plants per treatment per timepoint for each replicate. Letters denote significant differences between treatments within a single time point using Tukey’s HSD test (P < 0.05). Combining three independent experiments, n = 9 plants per treatment per time point.

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At 1 dpi, pin1 expression levels showed similar trends as pr1a1. There were few differences from the water control except for the X. vesicatoria with S. enterica treatment which showed tenfold higher levels of pin1 expression (Fig. 3b; P < 1 × 10−7). By 3 dpi, treatment with Xhg led to reduced levels of pin1 while treatment with X. vesicatoria and S. enterica resulted in increased pin1 expression compared to the water control (Fig. 3b; P = 0.0011 and 0.00097, respectively). Although inoculation with X. vesicatoria or S. enterica alone had no change in pin1 expression compared to the water-inoculated plants at 6 dpi (Fig. 3b; P = 0.071 and 0.939, respectively), inoculation with Xhg either with or without S. enterica led to a 1–3 log decrease in pin1 levels (Fig. 3b; P < 1 × 10−7 and 3 × 10−7, respectively). Co-inoculation with X. vesicatoria and S. enterica led to a tenfold increase in pin1 expression at 6 dpi (Fig. 3b, P = 0.00048). Taken together, these data show that the two Xanthomonas spp. induce different plant immune responses.

To determine if there was a connection between AvrHah1 and the observed induction of pr1a1 transcription, we monitored pr1a1 levels at 6 dpi in the Xhg avrHah1ΔDBD mutant. Wildtype Xhg infection led to increased pr1a1expression (Fig. 3c; P = 1 × 10−7 and 5 × 10−7). Inoculation with the Xhg avrHah1ΔDBD mutant also induced pr1a1 expression compared to the water control (Fig. 3c; P < 1 × 10−7). Further, we measured both free and conjugated forms of SA in leaf tissue at 1 and 3 dpi after dip-inoculation with water, S. enterica, Xhg, Xhg avrHah1ΔDBD, X. vesicatoria, or S. enterica with each xanthomonad. SA levels between the treatments were not significantly different (P > 0.05; Fig. S1). These data demonstrate that avrHah1 is not required for the induction of pr1a1 during Xhg infection.

To examine the impact of AvrHah1 on plant gene expression in response to X. vesicatoria, leaf samples were taken at 6 dpi and examined for pr1a1 expression. Compared to the negative control, tomatoes that were inoculated with Xhg, Xhg and S. enterica, X. vesicatoria + pURF034, X. vesicatoria + pUFR034 (avrHah1), X. vesicatoria + pUFR034 (avrHah1) and S. enterica showed significant increases in pr1a1 expression by 6 dpi (Fig. 3d; P = 1 × 10−7, 8 × 10−7, 0.00048, 1.1 × 10−5, and 7 × 10−7, respectively). Contrastingly, tomato plants treated with X. vesicatoria + pURF034 and S. enterica or S. enterica alone had no change in pr1a1 expression compared to the water control at 6 dpi (Fig. 3d; P = 0.165 and 0.174, respectively). Thus, addition of avrHah1 to X. vesicatoria alters the immune response to resemble the host response to Xhg.

AvrHah1 is not required for electrolyte leakage in Xhg-infected tomato leaves

Previous work has demonstrated that Xhg infection leads to more cellular damage in tomato leaves, as measured through electrolyte leakage, than X. vesicatoria infection17. From those data, it was hypothesized that this increase in cellular damage led to a resulting increase in S. enterica persistence in the phyllosphere17. Separately, it was shown that AvrHah1 is required for induction of electrolyte leakage in pepper leaves that have been infiltrated with Xhg30. To determine if AvrHah1 is linked to cellular damage and the resulting increases in S. enterica populations in tomato, plants were dip-inoculated with water, S. enterica, Xhg, Xhg avrHah1ΔDBD, X. vesicatoria, or S. enterica with each xanthomonad. Leaf samples were collected at 6 dpi. Plants treated with wildtype Xhg and S. enterica or X. vesicatoria with or without S. enterica had higher levels of electrolyte leakage than the water or S. enterica controls (P < 0.01, Fig. S2). The Xhg avrHah1ΔDBD treatment resulted in intermediate electrolyte leakage levels that were statistically similar to the water and S. enterica controls and both the wildtype Xhg and X. vesicatoria treatments (P > 0.01; Fig. S2). The Xhg avrHah1ΔDBD mutant and S. enterica treatment gave similar results as the avrHah1ΔDBD mutant treatment except that it had statistically lower levels of electrolyte leakage than the two X. vesicatoria treatments (P < 0.01; Fig. S2). These results indicate that specific Xanthomonads cause different levels of electrolyte leakage, but these differences do not correlate with increased S. enterica persistence.

Discussion

As foliar pathogens, Xanthomonas species survive on both the leaf surface and in the leaf interior. To succeed on the surface, epiphytic bacteria tolerate varying degrees of stress, including UV exposure, desiccation, and nutrient limitation. By actively moving to the leaf interior, xanthomonads can avoid these stresses and thrive. Two goals of this work were to identify which aspect(s) of the Xhg disease cycle benefit S. enterica and to characterize the mechanism that explains the observed phenotypic differences between S. enterica populations on Xhg- and X. vesicatoria-infected plants.

For this study, we hypothesized that one or more modifications to the leaf caused by Xhg infection benefit other leaf community members and lead to increased S. enterica survival. The infection process includes aggregation on the leaf surface, entry into the leaf interior, promotion of a water-soaked apoplast, and suppression of host immunity (Fig. 4). Initial S. enterica attachment to the leaf surface appears to be independent of Xhg as there are no statistical differences between treatments for early S. enterica populations (Fig. 1a; 1 and 3 dpi). Primary lesions develop approximately three dpi and appear as small, circular lesions just visible on the underside of leaves and become more numerous with a water-soaked appearance at four dpi. Based on the rapid appearance of primary lesions, Xhg quickly modifies the leaf environment to create the macroscopically visible water-soaked lesions. The prevalence of the primary lesions and water-soaked areas continue to increase through six dpi, the final time point sampled in these experiments, but there is little to no growth in individual lesion size at that time. By six dpi, Xhg-treated plants have ten-fold more S. enterica than plants treated with S. enterica alone (Fig. 1a). The connection between the observed differences in S. enterica populations with the timing of disease progression suggests that the appearance or increased frequency of the water-soaked lesions resulted in increased S. enterica survival (Fig. 4). Alternatively, or concurrently, the SA-inducible pr1a1 gene begins to show signs of induction as early as 1 dpi with Xhg (Fig. 3a). The rapid impact of Xhg on the plant immune response could manifest as increased S. enterica survival six days later. Regardless of the mechanism, it takes less than a week in the Xhg infection process to significantly affect the persistence of S. enterica in this environment.

Figure 4
figure 4

Model for Xhg enhancement of S. enterica persistence in water-soaked leaf tissue. Depiction of the tomato leaf surface (a) shows the location of bacterial cells (Xanthomonas, orange; S. enterica, cyan). Xhg tends to cluster at stomates (pores in the leaf surface) while S. enterica has a more random distribution with some cells at cell junctions (wavy lines), stomates (large ovals), and trichomes (not shown). Cross sections of the leaf (b,c) indicate entry of Xanthomonas bacterial cells (wildtype Xhg, b; Xhg avrHah1ΔDBD mutant, c) into the apoplastic space through stomates and some of the resulting consequences to leaf physiology. Water soaking seen during infection by wildtype Xhg is depicted by the darker green plant cells and the increased amount of water and/or nutrients (purple) in the apoplast (b). This figure was adapted from “Leaf Surface Structure” and “Leaf Anatomy”, by BioRender (2021). Retrieved from https://app.biorender.com/biorender-templates.

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As Xhg impacts S. enterica in the early stages of disease (Fig. 1), the T3SS effectors that have been linked to the acquisition of nutrients and the suppression or evasion of the plant immune response (for review, see33) could be factors that affect this process. We chose to focus on AvrHah1 because this effector has been linked to the water soaking phenotype in Xhg and is absent from the X. vesicatoria genome26,30. Thus, its specificity in Xhg made it a potential factor that could explain the different effects of these two species on S. enterica survival. As shown in this work, AvrHah1 is both necessary and sufficient for Xanthomonas-dependent increases in S. enterica persistence. The Xhg avrHah1ΔDBD mutant is no longer beneficial towards S. enterica survival (Fig. 1), and X. vesicatoria carrying a plasmid-borne copy of avrHah1 demonstrates a gain of function for this phenotype (Fig. 2). To characterize the mechanism by which AvrHah1 leads to increased S. enterica persistence, we examined downstream host targets of AvrHah1. Following dip-inoculation, we saw no significant changes in gene expression for bHLH3, bHLH6, PL, or PE (Table 1). This result contrasted with previous work demonstrating that infiltration of wildtype Xhg induced significantly higher transcription of these four genes compared to tomato plants that were infiltrated with the Xhg avrHah1ΔDBD mutant29. The two studies differ in several aspects of experimental design, including Xhg strain, tomato genotype, and inoculation method (dip-inoculation vs infiltration). We found that infiltration of wildtype Xhg mildly induced expression of only one of the four genes, bHLH6, compared to the Xhg avrHah1ΔDBD mutant (Table 1). An alignment of the predicted AvrHah1-binding sites upstream of bHLH3 and bHLH6 showed that the two tomato varieties (Heinz 1706 and MoneyMaker) are 100% identical in that region (data not shown). While these results do not completely preclude a role for tomato variety, they support the idea that bacterial strain likely impacts the observed differences in expression between experiments (Table 1; 29). Regardless, these data demonstrate that AvrHah1 plays a key role in the ability of Xhg to increase S. enterica persistence. In this system, AvrHah1 may have different targets that may influence the leaf environment. Schwartz et al. identified 4,106 potential AvrHah1 binding sites in tomato29. Future characterization of potential targets could provide a more detailed mechanism for AvrHah1-mediated increases in S. enterica persistence.

Pathogens, such as Xhg, have restricted access to the leaf interior through natural openings such as stomates, hydathodes, and wounds. Entry through stomates brings the bacteria to the apoplast, an air-filled, intercellular space34,35. During infection with wildtype Xhg, the apoplast is inundated with host cellular constituents, creating the water-soaked lesions seen in infected tomato plants (Fig. 4; 30). The water soaking phenotype is absent in plants infected with the Xhg avrHah1ΔDBD mutant. A water-soaked apoplast leads to higher Xanthomonas populations, spread of pathogens into host tissues, suppression of host defense responses, and potentially promotes availability of nutrients36. As part of the defense response, plants cause local desiccation to limit pathogen replication (reviewed in37), and the AvrHah1-dependent water soaking may benefit S. enterica by reducing desiccation stress in this niche. In Arabidopsis thaliana, virulent P. syringae pv tomato DC3000 causes water potentials that promote pathogen growth while avirulent DC3000 are associated with higher levels of desiccation that inhibit bacterial growth38,39. In that study, desiccation was measured using a reporter fusion to the proU promoter, which was induced under low water potentials. Utilizing a reporter to monitor water potentials in future experiments could prove to be informative regarding the role of AvrHah1-mediated water soaking in resulting water stress levels.

In addition to desiccation, epiphytic bacteria also experience nutrient limitation on the leaf surface due to the cuticle that restricts diffusion of nutrients from inside the leaf40. Migration to the leaf interior may provide access to the many nutrients (sugars, amino acids, etc.) that are transported through the apoplast and into phloem tissue. Although carbon sources such as glucose, fructose, GABA, succinate, and others accumulate in the apoplast, they are typically sequestered in forms that are bound in the cell wall or within cellular vacuoles41. Thus, we hypothesized that Xhg water soaking could release cellular constituents, nutrients, and electrolytes in an accessible form due to changes in host cell membrane permeability (Fig. 4). This result would not be the first example where S. enterica utilizes host-derived molecules made available by other bacteria. Plant cell wall breakdown by the soft-rotting pathogen Pectobacterium carotovorum produces oligosaccharides that are thought to be scavenged by S. enterica16,21. There are also several examples of phytopathogens altering host cell membrane permeability for growth. Pantoea stewartii subsp. stewartii causes water-soaked lesions on corn leaves by damaging cell membranes to release water and nutrients42. Water soaking caused by the endophytic pathogen P. syringae pv. phaseolicola releases Ca2+, Fe2/3+, and Mg2+ into the apoplast of bean leaves43. Our conductance data suggests that electrolyte leakage does not correlate with increased S. enterica persistence. Plants co-inoculated with either Xhg or X. vesicatoria and S. enterica have increased electrolyte leakage compared to the water and S. enterica controls (Fig. S2). However, unlike Xhg, X. vesicatoria infection has no impact on S. enterica populations (Fig. 2). Thus, gross changes in electrolyte leakage cannot explain the X-gardneri specific effects on S. enterica survival. Although overall differences in electrolyte leakage do not appear to be correlated with Xhg-mediated increases in S. enterica survival, these experiments could have missed changes in the levels of specific electrolytes. Fluctuations in ions of low abundance, like iron, wouldn’t have been detected using this approach. Iron has repeatedly been identified as a host-limited nutrient, and pathogens, including S. enterica, have multiple mechanisms to scavenge iron from their environment44,45,46. An increase in iron availability could indicate that host manipulations by Xhg release this critical factor for S. enterica growth. Future experiments examining the release of specific ions could elucidate more details of this process.

Invading microorganisms face a number of plant defense responses (antimicrobials, reactive oxygen species (ROS), ‘pathogenesis related’ (PR) proteins, etc.) that must be overcome to survive in intercellular spaces. The differential ability of S. enterica to colonize a range of tomato cultivars suggests an active plant response to the enteric pathogen7. Leaf discs treated with the S. enterica flagellar epitope flg22 showed increased levels of ROS47, and dip-inoculation of tomatoes with S. enterica resulted in a transient induction of the SA-inducible pr1a1 defense gene14. Other work has demonstrated that the presence of a virulent pathogen can protect another non-pathogenic epiphyte from host immunity. A nonpathogenic X. euvesicatoria T3SS mutant regains the ability to grow in planta if co-inoculated with pathogenic X. euvesicatoria48. The authors showed that the pathogenic X. euvesicatoria suppressed host immunity, prevented recognition of LPS from both bacterial strains, and inhibited host defense responses48. Further, manipulation of the immune system has been linked to water soaking and T3SS in other phytopathogens. P. syringae pv tomato induces water soaking and suppresses SA-dependent responses in A. thaliana using two T3SS effectors HopM1 and AvrE49,50. Recent work also demonstrated that HopM1 and AvrE induce abscisic acid (ABA) production to close stomata and produce water soaked lesions51,52. We hypothesized that Xhg may similarly manipulate the immune system, providing protection for S. enterica on tomato leaves. Co-inoculation of S. enterica with Xhg resulted in sustained induction of pr1a1 and repression of the JA-inducible pin1 (Fig. 3). As induction of pr1a1 contrasts with suppression or no change in expression of pr1 genes by other pathogens in wheat or A. thaliana50,53, respectively, our results suggest that Xhg host immune manipulation may occur through a different mechanism. Contrastingly to Xhg, co-inoculation of S. enterica with X. vesicatoria showed no changes in pr1a1 transcription and induced expression of pin1 (Fig. 3). Despite these differences in pr1a1 gene expression between treatments, there was no effect on levels of free or conjugated SA at one and three dpi for any treatment (Fig. S1). Induction of the SA pathway typically results in a spike in hormone levels followed by a rapid return to basal levels54. Thus, it is possible that we missed a transient increase in SA that could have led to pr1a1 induction in the Xhg-treated plants. Even a short burst of SA production could have beneficial effects on S. enterica. Although SA is normally considered to be a mediator of defense responses against bacteria, increased SA levels can result in increased S. enterica antibiotic resistance55 which could enhance S. enterica resistance to stresses in this niche. Alternatively, SA levels may remain constant, and pr1a1 may be induced by a different signaling pathway, such as ethylene. However, because the Xhg avrHah1ΔDBD mutant induced pr1a1 expression to the same extent as wildtype Xhg, we concluded that pr1a1 induction does not contribute to the role of AvrHah1 in increasing S. enterica persistence. Although AvrHah1 is not involved, the importance of SA and other plant hormones in S. enterica survival requires further study as there were still significant differences between Xhg and X. vesicatoria in these initial experiments.

In summary, this work identifies one Xhg factor, AvrHah1, which is both necessary and sufficient for providing conditions that lead to increased S. enterica persistence. Although the mechanism by which AvrHah1 promotes S. enterica survival remains unclear, several potential explanations have been described and will require further exploration. Xhg could provide increased nutrient availability through water soaking and/or defense against the plant immune response. Interestingly, X. perforans strains carrying the avrHah1 gene have recently been identified in the southeastern United States, and these strains have gained the ability to cause water soaking in pepper leaves56. While X. perforans is typically found in the southeastern United States, Xhg is commonly isolated in the midwestern United States. The recent emergence of another species, with different geographic distribution, carrying avrHah1 suggests selective pressure for horizontal transfer of the avrHah1-containing extrachromosomal plasmid between species. Acquisition of avrHah1 may promote Xanthomonas disease and raises the concern that this genetic transfer could lead to an increased risk of S. enterica contamination of crops around the country. Continued research in this area will provide fundamental knowledge of how an inhospitable environment changes during infection, resulting in survival of human pathogens and an increased risk of human disease.

Materials and methods

Bacterial strains, media, and culture conditions

A kanamycin-resistant strain of S. enterica serovar Typhimurium 14028s and nalidixic acid strains of X. vesicatoria 1111 (ATCC 35937), and Xhg 444 (spontaneous Nal-resistant strain; this study) were used as wildtype strains in this study. S. enterica was made kanamycin resistant by inserting the aadA gene at the attTn7 site. The Xhg avrHah1ΔDBD mutant was obtained from J. Jones30 and the X. vesicatoria pUFR034 and pUFR034 (avrHah1) strains were made by transforming X. vesicatoria 1111 with the Kanamycin-resistant plasmids pUFR034 or pURF034 (avrHah1) (57 and Minsavage, G., unpublished). Bacterial cultures were grown in lysogeny broth (LB) for S. enterica at 37 °C or nutrient broth (NB) for Xanthomonas spp. at 28 °C with shaking at 200 rpm. The antibiotics nalidixic acid (Nal) and Kanamycin (Kan) were used at concentrations of 20 and 50 µg ml−1, respectively. Strains used in this study are shown in Table 2.

Table 2 List of Strains and Primers.
Full size table

Plant inoculation

Tomato cultivar MoneyMaker seeds were purchased commercially (Eden Brothers). No plant material was collected. This study complies with the relevant institutional, national, and international guidelines and legislation for experimental research on plants. Seedlings were cultivated in Professional Growing Mix (Sunshine Redi-earth) with a 16 h photoperiod at 24 °C for five weeks. For colonization assays, Xanthomonas bacterial cultures were grown for two days in NB (or NB with Kan for pUFR strains) at 28 °C, and S. enterica cultures were grown overnight in LB at 37 °C. Bacterial strains were normalized to an optical density at 600 nm (OD600) of 0.2 (for S. enterica and X. vesicatoria strains), 0.25 (for the Xhg avrHah1ΔDBD mutant), and 0.3 (for wildtype Xhg) in sterile water. These OD600 values correspond to a bacterial population level of ~ 108 CFU/ml for the respective strains. Normalized cultures were diluted 1:100 in sterile water for an inoculum level of ~ 106 CFU/ml. Treatments consisted of individual bacterial strains mixed with equal parts water (S. enterica, Xhg, X. vesicatoria, Xhg avrHah1ΔDBD mutant, X. vesicatoria pUFR034, or X. vesicatoria pUFR034 (avrHah1)), S. enterica mixed 1:1 with each individual Xanthomonas strain, or water alone. Prior to inoculation, 0.025% Sil-Wett was added to water or the bacterial inoculum. Tomato plants were dip-inoculated by inverting plants in either sterile water or the bacterial inoculum for 30 s with agitation to prevent bacterial cell settlement. Plants were incubated at high humidity for 48 h in lidded, plastic bins under grow lights with a 16 h photoperiod at room temperature (~ 26 °C). After 48 h, plants were exposed to low humidity conditions (bin lids were removed) during the day and high humidity conditions (bin lids were replaced) during the night. At multiple time points post-inoculation, leaf samples were taken using destructive sampling to determine bacterial populations or collect samples for tomato RNA extraction. For infiltration experiments, MoneyMaker tomato leaves were infiltrated with Xhg wildtype or the avrHah1ΔDBD mutant at OD600 = 0.25, following published protocols29. At 48 h post-infiltration, two 79 cm2 leaf discs were taken from each of two leaflets on middle leaves and combined for a total of four leaf discs per plant. Samples from four plants per treatment were collected and frozen at −80 °C for further processing, as described below.

Bacterial population sampling

Bacterial populations on leaves were determined as described14. Briefly, at indicated times, one 79 cm2 leaf disc was taken from a leaflet on middle leaves using a surface sterilized cork borer18. Samples from three plants per treatment per time point were individually homogenized in 500 µl of sterile water in microfuge tubes using a 4.8 V rotary tool (Dremel, Mt. Prospect, IL) with microcentrifuge tube sample pestle attachment (Fisher Scientific). Homogenates were diluted as needed and spiral plated (Autoplate 4000, Spiral Biotech, Norwood, MA) on LB Kan (for S. enterica) or NB Nal or NB Kan Nal (for Xanthomonas spp.) plates. Resulting colonies were counted after overnight incubation at 37 °C (for S. enterica) or after incubation for three days at 28 °C (for Xanthomonas spp.) to determine bacterial populations. Although not shown in the figures, bacterial samples were taken at Day 0 to confirm that all bacterial populations were equivalent at the beginning of each experiment. No statistical differences were noted between treatments for any experimental setup at Day 0 (P > 0.01). Experiments were performed with three biological replicates.

RNA isolation

Tomato gene expression levels were determined as described14. At indicated time points, two 79 cm2 leaf discs were taken from each of two leaflets on middle leaves and combined for a total of four leaf discs per plant. Samples from three plants per treatment per time point were collected and frozen at −80 °C for further processing. RNA was extracted using the Maxwell® RSC Plant RNA kit (Promega). Briefly, four leaf discs were homogenized with a mortar and pestle in the presence of liquid nitrogen. Ground tissue was transferred to a small weigh boat containing 450 µl homogenization solution, mixed with a pipette tip, and transferred to a microcentrifuge tube. Samples were then either stored at −80 °C until further processed or immediately processed according to manufacturer’s instructions using a Maxwell® RSC (Promega). RNA samples were eluted in 50 µl volume of RNase-free water and were quantified by NanoDrop (Thermo Scientific). RNA was isolated from three technical replicates for each of three biological replicates.

cDNA synthesis and real-time PCR

cDNA synthesis and real-time PCR were performed as described14,58,59. Briefly, cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). Real-time PCR primers for pin1, pr1a1, bHLH3, bHLH6, PL, and PE were designed with Beacon Designer software (Premier Biosoft International) avoiding template secondary structure (Table 2). Primer efficiencies (Table 2) were calculated using serial dilutions of MoneyMaker genomic DNA and CFX Manager 3.0 software (Bio-Rad). Reference transcripts and primers were chosen based on published works: act41 and ubi360. Stable expression between treatments was validated using the Best Keeper program and three independent RNA samples from each treatment per biological replicate. Real-time PCR experiments utilized the CFX96 Real-Time System, and data were analyzed with the CFX Manager 3.0 software (Bio-Rad). The mean Cq of each target transcript was normalized by the mean Cq of each reference gene using the formula: 2(−(Cq target-Cq reference)). As previously described60, we determined the relative expression ratio (RER) of the target gene by dividing the normalized target RNA by a calibrator consisting of the average of the normalized values of the control samples (expression after water treatment in most experiments; expression after infection with wildtype Xhg in the AvrHah1 target experiments (Table 1)).

Measurement of free and conjugated SA

Plants were inoculated with S. enterica, Xhg, X. vesicatoria, Xhg avrHah1ΔDBD mutant, with S. enterica mixed 1:1 with each individual Xanthomonas strain, or with water alone, as described above. Leaf tissue samples were collected at multiple time points post-inoculation, weighed, flash frozen in liquid nitrogen, and extracted in 600 µl of H2O:1-propanol:HCL (1:2:0.005) with 200 ng of d4-SA as an internal standard. For total SA, 2 µl of glacial HCL was added, and the sample boiled for 30 min as in 53. Both free and total SA were phase partitioned with 1 ml MeCl2 and the MeCl2:1-propanol layer was collected and derivatized with trimethylsilydiazomethane, collected by vapor phase extraction on SuperQ columns, eluted in MeCl2and analyzed using GC–MS as in61.

Electrolyte leakage

Plants were inoculated with S. enterica, Xhg, X. vesicatoria, Xhg avrHah1ΔDBD mutant, with S. enterica mixed 1:1 with each individual Xanthomonas strain, or with water alone, as described above. Leaf tissue samples were collected at 6 dpi for bacterial populations (as described above) and electrolyte leakage measurements. Three leaf discs taken from a leaflet from a middle leaf were pooled from each plant, and changes in electrolyte leakage (conductance) were quantified as previously described15.

Statistical analysis

All statistical analyses were performed using R software (version 2.14.1; R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria [http://www.R-project.org]) as described62. Briefly, three biological replicates were performed for each experiment, and samples taken from one replicate were considered as subsamples. Linear regression analysis was used to determine whether bacterial population results differed between treatments for S. enterica and Xanthomonas spp. For real-time PCR analysis, three samples were compared for each treatment at each time point using Tukey’s HSD test. Results were considered statistically significant at P < 0.01.