Rhizobacteria Bacillus subtilis restricts foliar pathogen entry through stomata

Authors

  • Amutha Sampath Kumar,

    1. Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA
    2. Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711, USA
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    • These authors contributed equally to this work.

  • Venkatachalam Lakshmanan,

    1. Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA
    2. Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711, USA
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    • These authors contributed equally to this work.

  • Jeffrey L. Caplan,

    1. Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711, USA
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  • Deborah Powell,

    1. Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711, USA
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  • Kirk J. Czymmek,

    1. Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711, USA
    2. Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA
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  • Delphis F. Levia,

    1. Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA
    2. Department of Geography, University of Delaware, Newark, DE 19716, USA
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  • Harsh P. Bais

    Corresponding author
    1. Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA
    2. Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711, USA
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(e-mail hbais@udel.edu).

Summary

Plants exist in a complex multitrophic environment, where they interact with and compete for resources with other plants, microbes and animals. Plants have a complex array of defense mechanisms, such as the cell wall being covered with a waxy cuticle serving as a potent physical barrier. Although some pathogenic fungi infect plants by penetrating through the cell wall, many bacterial pathogens invade plants primarily through stomata on the leaf surface. Entry of the foliar pathogen, Pseudomonas syringae pathovar tomato DC3000 (hereafter PstDC3000), into the plant corpus occurs through stomatal openings, and consequently a key plant innate immune response is the transient closure of stomata, which delays disease progression. Here, we present evidence that the root colonization of the rhizobacteria Bacillus subtilis FB17 (hereafter FB17) restricts the stomata-mediated pathogen entry of PstDC3000 in Arabidopsis thaliana. Root binding of FB17 invokes abscisic acid (ABA) and salicylic acid (SA) signaling pathways to close light-adapted stomata. These results emphasize the importance of rhizospheric processes and environmental conditions as an integral part of the plant innate immune system against foliar bacterial infections.

Introduction

Plants grow in close association with large communities of microbes, yet very little is known about the diversity of these microbes and how they interact and affect performance and crop yields. Specific microbes have been demonstrated to provide beneficial effects to plants, such as the well-known symbiotic associations in legumes (Long, 2001). In addition, soil microbes play a major role in nutrient composition and uptake, and confer immunity against a wide range of foliar diseases by activating plant defenses through the classical induced systemic defense response (ISR) (Schroth and Hancock, 1982; van Loon, 1997). Although the effect of beneficial microbes on plants is known, the mechanisms of their combinatorial interactions and their influence on plant performance and disease evasion are poorly understood.

The effects of plant root exudates on microbial populations in the soil or ‘rhizosphere’ have already been demonstrated (Bais et al., 2006; Rudrappa et al., 2008), and many important beneficial (Ryu et al., 2004) and harmful interactions (Lugtenberg et al., 2001; Timmusk et al., 2005) have been documented. We have shown in Arabidopsis thaliana that plants participate in an active positive feedback system by recruiting specific beneficial microbes to ward off pathogens (Rudrappa et al., 2008). A. thaliana leaves infected with the virulent pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter PstDC3000) induced AtALMT1 (A. thaliana aluminum-activated malate transporter 1) expression, leading to root secretion of malate (l-malic acid). Secretion of malate by the roots in turn attracted the beneficial FB17 strain of Bacillus subtilis (hereafter FB17), leading to ISR against PstDC3000, although it was unclear whether FB17 colonization of A. thaliana roots changed the infection strategy of PstDC3000 at the leaf surface.

Pathogen entry into hosts is a critical step before the onset of the infection and disease progression. Phyllobacteria associated with plant leaf surfaces orchestrate a wide range of colonization strategies, which include leaf habitat modification, aggregation, ingression and egression (Beattie and Lindow, 1999). There is evidence indicating that the bacteria adapt to the leaf surfaces for their entry and exit into the host tissue. Recent studies by Melotto et al. (2008) indicated that some foliar pathogens have evolved an active mechanism to enter plant tissues through stomata, and that A. thaliana stomata close in response to live bacteria and purified microbe-associated molecular patterns (MAMPs). Besides microbes, other physiological and genetic events have been shown to regulate stomatal opening, including an early flowering (elf3) and the FLOWERING LOCUS T (FT) genes (Kinoshita et al., 2011).

Bacteria-induced stomatal closure is mediated through MAMP signaling, and requires a homeostasis of the defense hormone salicylic acid (SA), and is upstream of signaling regulated by abscisic acid (ABA) in the guard cell. It was reported that the stomatal response requires both SA and ABA, as the aba3-1 mutants failed to close the stomata in response to the known MAMPs, flagellin (flg22) or lipopolysaccharide (LPS). Likewise, SA-deficient A. thaliana plants failed to respond to MAMPs, thus indicating the requirement of SA for stomatal responses. These studies also indicated that there exists a complex association between the MAMP, SA and ABA signaling networks to mediate stomatal closure (Melotto et al., 2006, 2008). Stomatal guard cells sense MAMPs through cognate receptors, such as the leucine rich repeat-FLS2 receptor or EFR (Gómez-Gómez and Boller, 2000; Chinchilla et al., 2007). To overcome host defenses, PstDC3000 has evolved two virulence mechanisms, hrc/hrp-encoded type-III secretion systems (TTSSs; He et al., 2004; Grant et al., 2006) and the phytotoxin coronatine (hereafter COR; Bent and Mackey, 2007), which suppresses basic stomatal defenses. Although basal-level bacterial entry occurs through open stomata that are non-responsive or dead, PstDC3000 reopens the closed stomata, thereby increasing bacterial entry points (Boureau et al., 2002), and upon entry, suppresses local and systemic defense responses (Kunkel and Brooks, 2002; Melotto et al., 2006; Underwood et al., 2007).

Although evidence exists that root-associated beneficial microbes induce a systemic defense response in plants against pathogens (Lugtenberg and Kamilova, 2009), to date there have been no reports that indicate that the association of beneficial microbes with plants change physiological conditions leading to restricted entry of foliar pathogens through stomata. Having shown that A. thaliana plants under foliar attack from PstDC3000 recruit FB17 and facilitate its multiplication (Rudrappa et al., 2008), we evaluated the functional significance of FB17 root binding on the modulation of stomatal defense in A. thaliana. Here, we report that FB17 binding to the Col-0 roots effectively closed stomata, limited apoplastic entry of PstDC3000 and lowered the endogenous titers of PstDC3000. Strikingly, our data revealed that of the different rhizobacterial species tested, only Bacillus spp. (FB17, BS168 and GB03), and not Pseudomonas fluorescens (Pf0-1), decreased stomatal aperture sizes and prolonged the closure of the stomata in Col-0, suggesting that a specific component of B. subtilis was involved in modulating the stomatal defense in A. thaliana plants after infection with PstDC3000. Finally, to elucidate the in planta genetic mechanism for FB17-mediated stomatal closure, we showed that FB17 triggered an ABA/SA-dependent and jasmonic acid (JA)-independent pathway to close stomata. These experiments revealed a previously unknown feature of the beneficial rhizobacterial association in response to pathogen attack.

Results

Bacillus subtilis FB17 treatment modifies stomatal aperture in Arabidopsis thaliana

Our previous studies showed that root-associated FB17 induces resistance against foliar pathogens, and that there is complex root-to-shoot intraplant signaling in the event of distress (Rudrappa et al., 2008). To determine if stomatal closure contributes to FB17-mediated defense, roots of A. thaliana (Col-0) were root inoculated with FB17 and stomatal apertures were measured. We evaluated two esta-blished methods to measure stomatal aperture: light microscopy of epidermal peels and cryo-scanning electron microscopy (cryo-SEM) of the frozen leaf pieces (van Gardingen et al., 1989). Cryo-SEM was chosen as the preferred, established method because it was possible to examine and capture the effect of the signals generated during the early time points of tritrophic interactions, in the context of the entire plant. FB17 root inoculation reduced the average stomatal aperture size at both 1 and 3 h after FB17 inoculation, compared with the mock inoculation control (Figure 1a and b). Using images obtained through the cryo-SEM technique, three different measurements were averaged per stomata, and overall aperture size per treatment was calculated (Figure S1a). Besides reducing the aperture sizes, FB17 inoculation significantly decreased the percentage of open stomata on the abaxial leaf surface at 1 h (9.8%) and at 3 h (43%), compared with the untreated control (56%) (Figure S1b). Taken together, these data showed that root inoculation of FB17 can modulate stomatal aperture.

Figure 1.

 Root inoculation with Bacillus subtilis FB17 causes stomatal closure in Arabidopsis thaliana Col-0 plants. (a) Stomatal aperture in leaves of Col-0 plants root inoculated with or without FB17 (∼0.5 OD600). Data represent means ± SEMs, (n = 100 stomata) **P ≤ 0.01 by Student’s two-tailed t-test. (b) Cryo-SEM image of stomata in leaves of Col-0 plants inoculated with or without FB17 (scale bar: 10 μm). (c) Stomatal aperture in leaves of Col-0 plants inoculated with PstDC3000 (∼0.1 OD600) or Coronatine (COR) (0.05 μm), or co-inoculated with FB17. The results shown as means ± SEMs (n = 100 stomata). Different letters denote significantly different means as analyzed by Duncan's Multiple Range Test (DMRT). Means of common letters are not significantly different at ≤ 0.05.

PstDC3000 induces an MAMP-triggered immune response in plants that leads to stomatal closure at 1 h post inoculation (Melotto et al., 2006). Our data replicates the PstDC3000 MAMP-triggered stomatal closure after 1 h of PstDC3000 inoculation (Figure 1c). To gain entry, PstDC3000 uses the phytotoxin COR to reopen stomata by 3 h post-inoculation (Figure 1c). As both FB17 and PstDC3000 modify stomatal apertures, we co-inoculated Col-0 plants by inoculating FB17 specifically to the roots and dipping the aerial rosette in PstDC3000. Stomatal apertures were subsequently measured at 1 and 3 h after co-inoculation. At 1 h, FB17 and PstDC3000 had an additive effect that further reduced the average stomatal apertures compared with FB17 or PstDC3000 inoculation alone (Figure 1c). More importantly, at 3 h after co-inoculation, FB17 dramatically disrupted the ability of PstDC3000 to reopen stomata, thereby increasing the PstDC3000 population on the leaf surface (Figure S2a and b). As PstDC3000 uses COR to reopen stomata, we examined whether FB17 was preventing the reopening of stomata by disrupting the function of COR. Indeed, at 3 h, plants co-inoculated with COR (0.05 μm) and FB17 had lower stomatal aperture sizes than COR alone (Figure 1c). The ability of FB17 to close the stomata after leaf dip with COR was concentration dependent. At all lower COR concentrations tested, (≤1.0 μm), stomatal apertures increased at 3 h (Figure S1c), but FB17 showed a reduction in the stomatal aperture (Figure S1d). This suggests that FB17 may regulate stomatal aperture by suppressing the ability of PstDC3000 to reopen stomata with COR. However, at concentrations ≥ 5 μm of COR, FB17 did not reduce the stomatal aperture (Figure S1d).

Stomatal apertures are modified by MAMPs and Bacillus subtilis species

The non-pathogenic Escherichia coli OP50 (hereafter OP50) does not provide enhanced resistance to pathogens (Rudrappa et al., 2008). If FB17 does confer resistance by closing stomata, then OP50 should have no effect on stomatal closure. Surprisingly, OP50 root inoculation closed stomata at 1 h (Figure 2a), but at 3 h OP50 failed to keep the stomata closed, implying that the initial closing at 1 h is not specific to FB17. Similarly, co-inoculation of OP50 with PstDC3000 did not reduce the stomatal aperture sizes at 3 h, suggesting that it is unable to keep stomata closed in the presence of PstDC3000 (Figure S3a). Stomatal closure at 1 h by FB17 and OP50 was reminiscent of an MAMP-triggered response in leaves (Melotto et al., 2006). To show that this MAMP-triggered response can occur in roots to indirectly close stomata in leaves, two well-characterized MAMPs, lipopolysaccharide (LPS) and flagellin (flg22), were applied directly to the roots. Similar to FB17 and OP50, both LPS (100 μg ml−1) and flg22 (1 μm) were able to reduce stomatal apertures at 3 h after treatment compared with the control (Figure 2b). However, flg22 caused a rapid reduction in the aperture when compared with LPS at both 1 and 3 h (Figure 2b). In the presence of PstDC3000 the addition of flg22 to the roots reduced stomatal apertures, with nearly complete closure, perhaps because of the additive effect of flg22 (1 μm) and PstDC3000 (Figure S3b). Both LPS and flg22 exerted a dose-dependent effect on the stomatal aperture sizes when added to the roots (Figure S3c and d). These data show that keeping stomata closed at 3 h post-inoculation was not only specific to FB17, but was also actuated by MAMPs in a dose-dependent manner. To further corroborate the marked reduction in the stomatal aperture with flg22 addition to the roots, the FLS2 mutant (fls2) was root inoculated with FB17 and OP50. Root inoculation of fls2 plants did not change the stomatal aperture sizes (Figure 2c), implying the need for FLS2 for signal perception in rhizobacteria-mediated stomatal closure in plants. The role of FLS2 was further supported by the elevated expression of FLS2 in FB17 root-inoculated Col-0 plants, compared with the control and Pf0-1/OP50-inoculated plants (Figure S3e). It is known that FLS2 expression increases during an flg22-dependent MAMP-triggered response (Mersmann et al., 2010). To determine whether stomatal closure was a common attribute of beneficial microbes, we examined three strains of B. subtilis (FB17, BS168 and GB03) and one P. fluorescens strain (Pf0-1). At 1 h post-inoculation, the MAMP-triggered immune response actively reduced stomatal apertures for all of the tested bacterial strains added to the roots (Figure 2d). Albeit similar to OP50, Pf0-1 at 3 h after root inoculation caused no reduction in stomatal apertures, but at 1 h after root inoculation the stomatal apertures were higher when compared with other B. subtilis strains. Strikingly, at 3 h after root inoculation, only the beneficial rhizobacterial strains of B. subtilis (FB17, BS168 and GB03) prolonged the closure of the stomata (Figure 2d), suggesting that a specific component of B. subtilis may modulate stomatal closure in A. thaliana plants.

Figure 2.

 Stomatal defense by MAMPs and Bacillus species. (a) Stomatal aperture in leaves of Col-0 plants root inoculated with FB17 or Escherichia coli OP50 (∼0.5 OD600). (b) Aperture sizes in Col-0 plants root inoculated with the MAMPs lipopolysaccharide (LPS; 100 μg ml−1) or flagellin (flg22; 1 μm); *P ≤ 0.05, ***P ≤ 0.001, Student’s two-tailed t-test. (c) Stomatal aperture in leaves of fls2 plants, root inoculated with FB17 or OP50. (d) Stomatal apertures in leaves of Col-0 plants, root inoculated with FB17 or BS168 (∼0.5 OD600), GB03 (∼0.5 OD600) or Pf0-1 (∼0.5 OD600). The results represent means ± SEMs, n = 40 stomata. Different letters denote significantly different means as analyzed by DMRT. Means of common letters are not significantly different at ≤ 0.05 in (a), (c) and (d).

FB17-mediated stomatal closure in Arabidopsis thaliana is transient

As FB17 can specifically keep stomata closed, we root inoculated FB17 and examined stomatal apertures over a 120-h time course. Surprisingly, stomatal closure induced by FB17 root inoculation was only a transient response by the plant. There was gradual increase in the stomatal aperture of the FB17-inoculated plants after 48 h (Figure 3a). At 120 h, stomatal apertures were similar to the sizes found in the control. The simultaneous colonization of FB17 and PstDC3000 is probably a rare event in nature. Rather, if FB17 provides beneficial protection against incoming PstDC3000 infection, the root colonization is initiated naturally or when applied as a biocontrol agent. Our previous studies showed that the priming of plants with FB17 leads to biofilm formation on roots (Rudrappa et al., 2008, 2010). FB17 root inoculation of Col-0 plants 3 h prior to foliar PstDC3000 inoculation impeded PstDC3000 entry through the stomata, compared with the uninoculated control (Video Clips S1 and S2). Plants root inoculated with FB17 for 24–72 h showed reduced stomatal apertures when compared with the uninoculated control (Figure 3a). These plants had low PstDC3000 titers when infected with the pathogen, which may be attributed to increased FB17 root colonization (Figure S4a and b). However, at 3 h the average stomatal aperture (0.5 μm) was less in the FB17 root-inoculated plants (treated for 120 h) infected with PstDC3000 compared with FB17 uninoculated plants (1.9 μm), indicating that ISR is still in effect (Figure 3b). As abaxial stomatal pore sizes were reduced in FB17-inoculated plants, we measured the stomatal conductance and transpiration rates in plants after 24 h. The overall stomatal conductance and transpiration rate decreased in FB17-inoculated plants compared with the control (Figure S5a and b).

Figure 3.

 FB17-mediated stomatal closure in Arabidopsis thaliana is transient. (a) Stomatal aperture in the Col-0 seedlings after FB17 root inoculation over a time period of 120 h. Data represent means ± SEMs for n = 40 stomata. Asterisks denote significant differences as analyzed by Student’s t test. **P ≤ 0.01, *P ≤ 0.05, Student’s two-tailed t-test (ns-not significant). (b) Stomatal apertures in the 120-h FB17 root-inoculated plants and uninoculated plants at 1 and 3 h after PstDC3000 foliar infection. Data represent means ± SEMs (n = 40 stomata). **P ≤ 0.01, Student’s two-tailed t-test.

Abscisic acid levels in planta depend on the mode of bacterial entry

Abscisic acid is a central regulator of stomatal closure (Acharya and Assmann, 2009), and therefore we measured the ABA content in the leaves and roots of plants 1, 3 and 24 h after root inoculation with FB17. FB17 root inoculation caused a significant increase in ABA titers in the leaves (Figure 4a), and a slight decrease in the roots (Figure 4b). The precise mechanism by which this rhizobacteria induces ABA in leaves remains to be shown. However, the increase in ABA correlated with an increase in transcript levels of ABA biosynthetic genes, nced2 and aba1, in plants inoculated with B. subtilis strains FB17, GB03 or BS168 (Figure S6). As stomatal functions are sensitive to light and dark conditions, we ascertained the levels of the cca1 and toc1 genes that make up the proposed central circadian loop in A. thaliana (Somers et al., 1998; Wang and Tobin, 1998). Plants root-inoculated with FB17 showed no change in transcript expressions of cca1 and toc1 in time-course experiments (Figure S6), implying that FB17 closure of stomata is not mediated by the regulatory components of the photomorphogenetic pathway in A. thaliana.

Figure 4.

 Endogenous ABA levels in roots and leaves. (a, b) Endogenous total ABA content in the shoots (a) and in the roots (b) of Col-0 plants, root inoculated with FB17. (c, d) Total ABA content in the shoots (c) and in the roots (d) of Col-0 plants after root inoculation with FB17 and leaf infiltration/dip inoculation with PstDC3000. Control plants refer to mock leaf-dipped/infiltrated plants. Data represent means ± SEMs, n = 3. Different letters denote significantly different means as analyzed by DMRT. Means of common letters are not significantly different at ≤ 0.05 in (a), (b), (c) and (d).

It is known that infiltrating plants with PstDC3000 alone can induce ABA biosynthetic components (deTorres-Zabala et al., 2007). As we also observed an endogenous increase in ABA titer with FB17, we examined whether the method of inoculation of PstDC3000 manipulated in planta ABA levels. Hence, we measured the ABA content in plants that were leaf-infiltrated or leaf-dipped with PstDC3000. Our results showed that the plants that were leaf-infiltrated with PstDC3000 showed a substantial increase in endogenous ABA titers in leaves (Figure 4c). In contrast, the ABA titers in plants that were leaf-dipped with PstDC3000 showed no changes in the ABA content 1 h post-inoculation, and significantly less of an increase 3 h post-inoculation, compared with leaf-infiltration. However, when leaf-dipped with COR, at 1 h, ABA levels increased in roots rather than in the shoots (Figure 4d). These results suggest that the mode of pathogen entry may alter ABA levels in planta in Arabidopsis.

FB17 treatment triggers signaling through ABA and SA to close stomata

To further verify the possible involvment of ABA in FB17-mediated stomatal closure, we measured stomatal apertures in the ABA-deficient mutants root inoculated with FB17. The ABA biosynthetic mutant aba2-1 is deficient in ABA production (Schwartz et al., 1997) and has larger stomatal apertures (average aperture size, 2.37 μm; Figure 5a), compared with stomata of wild-type plants (average aperture size, 0.8 μm). Similarly, stomata of aba1 and aba3-1 mutants were larger (average aperture size: aba1, 2.83 μm; aba3-1, 1.43 μm; Figure S7a); however, these mutants can still biosynthesize ABA from precursors (Léon-Kloosterziel et al., 1996). The addition of FB17 to the roots of aba1 and aba3-1 resulted in only a partial disruption of FB17-mediated stomatal closure (Figure S7a). However, the FB17-mediated closure of stomata at 3 h after root inoculation was completely disrupted in the aba2-1 mutant, suggesting that FB17 may mediate stomatal closure through ABA (Figure 5a). These results showed that by knocking out ABA2 function, a critical step in ABA biosynthesis was affected, and that insufficient endogenous ABA titers negate the ability of FB17 to close stomata. As ABA played a vital role during stomatal closure, we also tested the effect of the addition of extraneous ABA to the roots of the Col-0 plants. The addition of ABA (100 nm) to the roots reduced the stomatal aperture sizes at both 1 and 3 h (Figure 5b), compared with the mock-treated plants. In addition, supplementation of ABA to Col-0 roots also led to a significant reduction in the PstDC3000 titers (Figure 5f).

Figure 5.

Bacillus subtilis FB17 mediates the closure of stomata through ABA and SA pathways. (a) Stomatal aperture in leaves of Col-0, aba2-1, ics1 and NahG plants inoculated with water or FB17 (∼0.5 OD600 ; n = 40 stomata). (b) Stomatal aperture in Col-0 plants inoculated with water, 0.5 mm 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.6), ABA (100 nm) or SA (100 nm in MES buffer) to the roots (n = 40 stomata). (c) Stomatal aperture in the leaves of ost1-1, ein3-1 and coi1 plants after root inoculation with water or FB17 (∼0.5 OD600; n = 40 stomata). (d) Stomatal aperture in ost1-1 plants after root treatment with ABA or SA (n = 40 stomata). (e) Stomatal aperture at 3 h in Col-0 and ost1-1 plants after foliar PstDC3000 inoculation and SA or ABA root treatment (n = 40 stomata). (f) PstDC3000 titers in the Col-0 and ost1-1 plants after root treatment with SA or ABA. Data represent means ± SEMs, n = 12. Means of common letters are not significantly different at ≤ 0.05 in (a), (b), (c), (d), (e) and (f).

During a MAMP-triggered response to PstDC3000, the defense signaling component, SA, functions upstream of ABA to signal stomatal closure (Zeng and He, 2010). To determine whether SA is also involved in FB17-mediated stomatal closure in A. thaliana, we analyzed stomatal aperture in ics1 and transgenic NahG lines after root inoculation with FB17. Both ics1 and NahG plants showed a disruption of FB17-mediated stomatal closure at 3 h after root inoculation with FB17 (Figure 5a). Only NahG plants had disrupted stomatal closure at 1 h, suggesting that an isochorismate synthase (ICS)-independent source of SA functions during the non-specific response at 1 h. These data suggested that, in addition to ABA, FB17-induced stomatal closure requires SA. However, in SA signaling mutant npr1-1, FB17 caused stomatal closure at 1 h, indicating that SA-mediated signaling is not required for stomatal closure at 1 h (Figure S7b). This data is further supported by the lack of PR1 expression in Col-0 plants at 1 h (Figure S8a).

Directly downstream of ABA perception by the PYR1 receptor, SnRK2 serine/threonine protein kinases are activated and specifically expressed in guard cells, and are required for guard cell closure (Mustilli et al., 2002; Fujita et al., 2009). Therefore, we measured stomatal apertures in ost1-1 plants. Indeed, FB17-mediated stomatal closure was completely disrupted in OST1 mutants at 1 and 3 h after FB17 root inoculation (Figure 5c), further supporting a key role of ABA. To determine the exact stage at which both SA and ABA interact, we treated the roots of Col-0, ost1-1 and aba2-1 plants with SA (100 nm) and/or ABA (100 nm). The stomatal apertures in the Col-0 plants showed a reduction in aperture sizes at 3 h (Figure 5b), whereas ost1-1 root-treated plants revealed apertures similar to those observed in the controls (Figure 5d) in the presence of SA or ABA. This scenario changed when ost1-1 plants were infected with PstDC3000 in the presence of SA or ABA in the roots. In the presence of SA in the roots, PstDC3000-infected ost1-1 plants showed reduced aperture sizes compared with ABA root treatments (Figure 5e), indicating that SA mediates stomatal closure in the absence of OST1, which does not require PR1 (Figure S8b). However, neither SA nor ABA reduced PstDC3000 titers in ost1-1 plants as opposed to the Col-0 plants (Figures 5f and S9a). The addition of SA and/or ABA neither reduced stomatal apertures (Figure S9b) nor PstDC3000 titers (Figure S9c) in aba2-1 plants. This suggests that FB17-mediated protection against PstDC3000 involves both SA and ABA. When PstDC3000 colony-forming units (CFUs) were measured in mutants/plants that negated stomatal closure (aba2-1, ost1-1, ics1 and NahG), at 48 h after PstDC3000 inoculation there was no reduction in the CFUs in these mutants (Figure S10a). However, in the ABA-deficient aba1 and aba3-1 plants, significant reductions in the PstDC3000 titers were observed (Figure S10b).

FB17-mediated stomatal closure is jasmonic acid (JA) and ethylene (ET) independent

To determine whether JA and ET are required for FB17-mediated stomatal closure, we measured stomatal apertures in respective mutants. We initiated the experiments with the coi1 plants, which are insensitive to the biologically active JA derivative methyl jasmonate (MeJA), and mediate the interplay between ABA and MeJA in guard cells (Xie et al., 1998; Li et al., 2004; Munemasa et al., 2007). In coi1 plants there was a marked reduction in the stomatal aperture at 1 h after root inoculation of FB17, suggesting COI1 may play a role early on during the non-specific response at 1 h. However, FB17-mediated stomatal closure was not disrupted at 3 h after root inoculation (Figure 5c), negating the involvement of MeJA during FB17-mediated stomatal closure at 3 h.

To analyze the requirement of ET, we measured the average stomatal pore sizes of the ET mutants root inoculated with FB17. At 1 h after root inoculation the ET-insensitive mutant, etr1-1, had a partial loss of stomatal closure, and the constitutive ET mutant, ctr1-1, had enhanced stomatal closure, compared with the Col-0 control. At 3 h post-inoculation, ctr1-1 had less of an effect on FB17-mediated stomatal closure compared with etr1-1 (Figure S10c). We also examined the ein3-1 mutant that has reduced ET sensitivity and is far downstream of ET perception by ETR1 and CTR1 (Roman et al., 1995). In contrast, ein3-1 mutants exhibited normal FB17-mediated stomatal closure at 1 h, but had a partial loss of FB17-mediated stomatal closure at 3 h compared with the Col-0 control (Figure 5c). Both the ein3-1 and coi1 plants showed reduced PstDC3000 titers after 48 h (Figure S10a).

Discussion

In this study, we report that the beneficial association of the rhizobacteria B. subtilis FB17 with A. thaliana roots restricts stomatal entry, protecting the host from the foliar pathogen PstDC3000. Rapid cryo-immobilization and cryo-SEM were employed to capture the complex interaction between FB17, A. thaliana and PstDC3000 in intact leaves. The study revealed that although both E. coli and FB17 can induce stomatal closure at 1 h, only rhizobacteria FB17 protected the plants at 3 h by preventing PstDC3000 from reopening stomata. Using mutants of ABA, SA, JA and ET signal transduction pathways, we found that the addition of the rhizobacteria FB17 mediated stomatal closure mainly through SA and ABA. This study shows that the beneficial association of rhizobacteria plays an important role in preventing pathogen entry and plant defense response.

Bacillus subtilis and MAMPs cause stomatal closure in Arabidopsis thaliana

Previous studies showed that the application of bacteria to isolated epidermal peels induced a MAMP-triggered immune response that closed stomata (Melotto et al., 2006; Zeng et al., 2010). Our data show that root inoculation of bacteria can trigger long-distance signals that can lead to stomatal closure. The rapid stomatal closure at 1 h after root inoculation was a common phenomenon to both FB17 and E. coli OP50, which suggests that the response was most likely related to a MAMP-triggered immune response (Zeng et al., 2010). A similar response was observed when known MAMPs, such as LPS and flg22, were added to the Col-0 roots. Melotto et al. (2006) showed that flg22 and LPS close stomata in the isolated leaf epidermal peels of Col-0 plants, whereas our data show that root inoculation with FB17 as well as MAMPs (flg22 and LPS) caused a similar response of drastic reduction in the stomatal apertures at selective concentrations. These observations were further supported by the expression of fls2 in the Col-0 plants and the lack of stomatal closure in fls2 plants root inoculated with FB17 and OP50. This supports the previous findings of Melotto et al. (2006) where flagellin perception was considered a key modulator in aerial stomatal defense. However, through the current study it is evident that flagellin perception could also be mediated through long-distance root-to-shoot signaling to regulate stomatal defense, as FLS2 is universally expressed in all parts of the plants (Gómez-Gómez and Boller, 2000).

FB17-mediated stomatal closure leads to enhanced resistance against PstDC3000

Stomata have been shown to possess an innate capacity to sense and close in the presence of human and plant pathogenic bacteria (Melotto et al., 2006). Using the leaf epidermal peels it was shown that the virulent plant pathogen PstDC3000 transiently reduces stomatal aperture sizes and reopens stomata with the help of the phytotoxin, COR. The reopening of the stomata has been shown to be associated with higher bacterial titers in the infected plants (Melotto et al., 2006). However, in the present study, root inoculation of FB17 together with foliar infection with PstDC3000 has been shown to significantly reduce stomatal apertures compared with lone treatments. This implies that there is an additive effect of root/leaf-initiated stomatal closure, which was also observed when COR was used in place of the pathogen during the leaf-dip experiments. At 3 h post-inoculation, both PstDC3000 and COR can reopen stomata; however, the ability of FB17 to continue to keep the stomata closed at 3 h post-inoculation, even in the presence of PstDC3000 or COR, was striking. At high concentrations of COR (≥ 5 μm), FB17 root inoculation could not reduce stomatal aperture sizes, indicating that FB17 has a threshold to keep the stomata closed. FB17 root inoculation together with PstDC3000 in the leaves resulted in the overall reduction of the PstDC3000 titers after 48 h. This supports the hypothesis that FB17-mediated stomatal closure prevents PstDC3000 entry by minimizing the bacterial ports of entry.

In addition to stomatal defense, root inoculation of FB17 also potentiates ISR and in planta signaling of defense hormones (Conrath et al., 2002). After the entry of the virulent pathogen into the plant, rhizobacteria-mediated stomatal closure becomes a pre-invasion defense strategy. Our studies indicate that PR1 expression occurs in FB17 root-inoculated plants as early as 3 h, which suggests that the ability of FB17 to keep the stomata closed at 3 h is dependent on ISR, and contributes towards pre-invasive defense. Our previous work and other studies support the findings that at later time points, FB17 root colonization reduces pathogen multiplication through ISR (Conrath et al., 2002; Ryu et al., 2004; Rudrappa et al., 2008, 2010). Stomatal closure was specifically observed in all B. subtilis strains tested, signifying that a critical component(s) from Bsubtilis could elicit this response.

FB17 root inoculation causes transient reduction in aperture sizes

The most vital function of guard cells is to carefully balance the opening of stomata for gaseous exchange during transpiration and the closure of stomata to conserve water (Acharya and Assmann, 2009). Our data shows that root inoculation with FB17 caused a reduction in stomatal aperture size as early as 1 h. When monitored for a 5-day time period, stomata remained closed for up to 3 days, but were restored back to the stomatal aperture sizes present in the control plants at 4–5 days. Thus, plants cannot keep their stomata closed for a considerably longer duration, as this may impede gaseous and water exchange (Acharya and Assmann, 2009). In addition, FB17 root inoculation also caused a 30% reduction in the overall stomatal conductance and the rate of transpiration at 24 h. The reduction did not cause a decrease in plant growth, but rather FB17 and other rhizobacteria were commercially used to promote plant growth and biomass (Lugtenberg and Kamilova, 2009). Hence, the overall decrease in stomatal conductance and transpiration rate at 24 h may only impose a temporary effect on plant growth and photosynthesis, which cannot be correlated with disease resistance. Besides rhizobacteria, bacterial exometabolites, such as homoserines and homoserine lactones, when added to the roots at nanomolar concentrations, were shown to increase stomatal conductance and transpiration rate in Phaseolus vulgaris. This proposes that other indirect plant–microbe interactions also affect stomatal responses in plants (Joseph and Phillips, 2003).

SA and ABA have a central role during FB17-mediated stomatal closure

Stomatal regulation and function are affected by abiotic, biotic and hormonal interactions, and typically ABA plays an overriding role during stomatal closure (Acharya and Assmann, 2009). Our study followed this paradigm. ABA was essential for FB17-mediated stomatal closure. The ABA biosynthetic mutants, aba1, aba2-1 and aba3-1, are all deficient in ABA production (Léon-Kloosterziel et al., 1996). However, both aba1 and aba3-1 are independent of ABA deficiency (Schwartz et al., 1997). Our analysis of these mutants also showed that because of the ability of aba1 and aba3-1 to produce ABA from other precursors, FB17-mediated stomatal closure occurred in these mutants and not in aba2-1. These data correlated with the increased expression of the ABA biosynthetic pathway genes and increased endogenous ABA titers after FB17 root inoculation. A significant increase in the ABA content may be caused by the production of cytokinins by the Bacillus spp., as reported in other gram-positive bacteria (Arkhipova et al., 2005).

Similarly, we found that by pressure-associated infiltration of the pathogen PstDC3000, there was a significant increase in the in planta ABA levels when compared with leaf-dip treatments. Previous P. syringae leaf infiltration studies indicate that Col-0 plants produced more ABA at 6 h post infection in response to the delivery of PstDC3000 type-III effectors (TTEs). The pathogen has been shown to overcome the ABA signaling pathway to cause infection and colonization (deTorres-Zabala et al., 2007, 2009). Hence, in general, the factors that trigger ABA accumulation in PstDC3000 leaf-infiltrated plants are not known. It may be speculated that higher ABA levels in planta after PstDC3000 infiltration could be caused by PAMPs and also by PstDC3000 multiplication. Although an increase in the ABA level occurs, COR produced by PstDC3000 inhibits ABA-induced stomatal closure through COI1. The phytotoxin COR is suspected to act downstream of nitric oxide (NO) to suppress ABA-mediated stomatal closure (Melotto et al., 2006). This possibly explains the reopening of the stomata by COR despite an increase in ABA levels.

By using SA-deficient ics1 and NahG plants, we identified that SA was also essential for FB17-mediated stomatal closure. SA from both PAL and ICS biosynthetic pathways are removed in NahG plants, whereas only SA from the ICS biosynthetic pathway is reduced in ics1 (Wildermuth et al., 2001). This suggests that SA from the PAL pathway may account for FB17-mediated stomatal closure at 1 h. However, at 3 h, SA from either biosynthetic pathway may induce stomatal closure. Zeng and He (2010) reported that at 3 h, SA acts upstream of both ABA and OST1 during bacterial- or MAMP-induced stomatal closure, which requires NPR1. In the present study, we observed that root inoculation of FB17 systemically induces stomatal closure through the same pathway at the similar time period. However, stomatal closure in npr1-1 mutants and lack of PR1 expression at 1 h in Col-0 plants suggests that SA-mediated NPR1 signaling is not required at 1 h.

The stomata closed when SA or ABA were applied to Col-0 roots, but remained open when SA or ABA was applied to the roots of ost1-1 plants. In the ost1-1 plants, titers of PstDC3000 were the same with and without SA or ABA treatments. Nevertheless, in the presence of PstDC3000, the addition of SA to the roots significantly reduced stomatal apertures, suggesting that the SA root treatment may trigger a similar response as the MAMP-triggered immune responses in the leaves, to close stomata. In ost1-1 plants, exogenous and endogenous SA or ABA levels could not reduce aperture sizes, indicating that SA may function upstream of ABA in regulating stomatal opening and closure (Zeng and He, 2010). This further suggests that OST1 is necessary for FB17-mediated stomatal closure when a plant encounters pathogens or MAMPs above or below ground. When SA and/or ABA were added to the roots of the ABA biosynthetic mutant aba2-1, the stomatal apertures were significantly reduced. Although the exogenous addition of SA and/or ABA causes a reduction in aperture sizes, neither of these purified compounds could cause a significant reduction in the PstDC3000 titers in planta, suggesting that FB17-induced stomatal defense along with ISR is required for disease resistance. Thus FB17-mediated stomatal defense involves both SA and ABA (Figure 6). A similar epistatic relationship between SA and ABA during stomatal defense was shown in A. thaliana during PstDC3000 leaf infection studies. However, the mechanism of disease resistance after gaining entry into the plant was shown to depend on a wide array of defense responses elicited within the plant in response to pathogen multiplication and the accumulation of other MAMPs (Zeng and He, 2010).

Figure 6.

 Interplay between SA and ABA during FB17-mediated stomatal defense. After root inoculation with FB17, SA through NPR1 leads to stomatal closure through unknown mechanisms. FB17-mediated root-to-shoot signaling suppresses COR-mediated stomatal reopening through an unknown mechanism. The FB17-mediated guard cell closure through OST1 requires ABA2. Rhizobacterial determinants such as volatile organic compounds (VOCs) affecting stomatal closure are yet to be identified. Dashed lines indicate unknown mechanisms.

Both JA and ET were previously shown to play key roles during ISR induced by rhizobacteria P. fluorescens WCS417 (Pieterse et al., 1998) and Pseudomonas putida LSW17S (Ahn et al., 2007). Although JA and ET are required for ISR, our analysis showed that neither pathway played a major role during FB17-mediated stomatal defense. In the COI1 mutants, stomatal closure was unaffected after FB17 root inoculation. FB17 root inoculation also reduced PstDC3000 titers in coi1 plants, which may be explained by the nho1 (non-host 1) gene expression in the absence of COI1 (Li et al., 2005). Thus, a reduction in the stomatal aperture sizes in coi1 plants along with NHO1 expression may primarily be responsible for lowering in planta PstDC3000 titers in the coi1 plants.

FB17 may mediate stomatal closure by parallel ABA and ET pathways. In accordance, a recent genetic epistatic analysis revealed that these two pathways (ABA and ET) act in parallel, at least in primary signal transduction pathways (Cheng et al., 2009). FB17-mediated stomatal closure occurred only in the ein3-1 mutant at 3 h, which may be explained by ABA signaling through EIN3 independent of ET, as EIN3 regulates stomatal closure through auxin and cytokinins involving ABA (Tanaka et al., 2006; Acharya and Assmann, 2009).

To our knowledge, these data on tritrophic interaction represent one of the interesting examples where a particular mechanism of action that leads to disease evasion is specific to the biocontrol species studied. All B. subtilis strains tested showed a reduction in stomatal apertures, suggesting a component of the genus Bacillus may play a critical role in defying pathogen entry. These results offer an improved explanation for the efficacy of biocontrol agents by indirectly targeting the pathogen’s mode of entry in hosts. A comprehensive understanding of the effects of soil rhizobacteria on crop plants will enable the development of agricultural technologies that exploit the natural alliances among microbes and plants, and provide alternative avenues to increase yields beyond conventional plant genetics and breeding.

Experimental procedures

Plant materials, pathogen inoculations and root treatment

Plant material and conditions for infection are described in Appendix S1.

Cryo-SEM and stomatal aperture measurements

Leaf pieces (0.5 cm2) were excised from inoculated plants after 5 h of exposure to light within the growth chamber and processed for the Cryo-SEM as described in Appendix S1. Aperture measurements were performed using ImageJ 1.38g (Figure S1a).

Confocal microscopy

Biofilm formation by FB17 on the root surface of A. thaliana Col-0 plants was observed by laser scanning confocal microscopy. Fixation and data acquisition conditions are described in Appendix S1.

Abscisic acid quantification

Abscisic acid (ABA) was quantified according to the method described by Zhang et al. (2008). The leaf porometer and conductance assay is described in Appendix S1.

RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from the seedlings at 1, 3, 24 and 48 h post-inoculation with FB17. RNA was extracted using PureLink RNA isolation buffer according to the manufacturer’s instructions. Further purification and PCR conditions for amplification can be found in Appendix S1.

Statistical analysis

The data were analyzed by a one-way analysis of variance (anova) using Microsoft Excel 2010® (Microsoft Corporation, http://www.microsoft.com), and post-hoc mean separations were performed by Duncan’s Multiple Range Test (DMRT) at P ≤ 0.05, by using spass 12.0 (Harter, 1960). A Student’s two-tailed t-test was used wherever necessary to compare two means.

Acknowledgements

HPB acknowledges support from the University of Delaware Research Foundation (UDRF), NSF-EPSCoR and NSF Award IOS-0814477. We are also thankful to two anonymous reviewers for their insightful comments. The authors thank Ms Courtney Siegert for initiating the porometer experiments.

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