In response to the changing environment, plants have the ability to sense and respond to single or multiple stimuli via activation of unique signalling cascades. The two-component systems in bacteria and yeast act as unique sensing and signalling systems responding to osmotic (Forst & Roberts, 1994; Posas et al., 1996; Paithoonrangsarid et al., 2004), temperature (Aguilar et al., 2001; Suzuki et al., 2005) and oxidative stimuli (Singh, 2000; Kanesaki et al., 2007). More recently, some plant HKs have been shown to function in response to environmental stimuli such as drought/osmotic (AtHK1, AHK2, AHK3 and AHK4) (Urao et al., 1999; Tran et al., 2007, 2010) and salinity stress (AHK2, AHK3 and ETR1) (Zhao & Schaller, 2004; Tran et al., 2007; Wang et al., 2008; Tran et al., 2010). Additionally, a role for AHK2 and AHK3 in modulating disease resistance in response to infection with PstDC3000 has been reported (Choi et al., 2010). Previously, our laboratory has shown that AHK5 mediates stomatal responses to endogenous and environmental cues (Desikan et al., 2008). Here we demonstrate the ability of AHK5 to function in response to both abiotic and biotic stimuli to affect the growth and survival of Arabidopsis.
AHK5 regulates tolerance to salinity
The present study shows that, of the various abiotic stresses tested, AHK5 positively regulates growth inhibition to salinity stress. This is the first demonstration of a function for AHK5 in salinity-induced growth responses in Arabidopsis. ABA is known to play a role in drought and salinity stress tolerance (Zhu, 2002). However, the finding that AHK5 negatively regulates ABA-induced root growth inhibition (Iwama et al., 2007) is hard to reconcile with the finding that it positively regulates salt-induced root growth inhibition. It is possible that ABA concentrations induced following salt stress are insufficient to enable AHK5-mediated signal transduction to act in an inhibitory manner. This possibility is supported by the promotion of root growth seen with low concentrations of ABA in both wild-type and ahk5-1 mutant plants (Ghassemian et al. (2000) and Fig. S8). In addition, there is evidence suggesting that ABA may function to maintain rather than to inhibit root growth under conditions of stress (Sharp & LeNoble, 2002).
Salinity stress imposes both osmotic and ionic stress on the plant (Munns, 2002; Munns & Tester, 2008). Here, we have obtained evidence to suggest that the ahk5-1 mutant is less sensitive to the ionic component of salt stress. Under salt stress, the increased uptake of Na+ disrupts homeostasis of ions and inhibition of cellular processes, through toxicity to cellular enzymes and disruption of K+ uptake by the plant (Munns, 2002; Zhu, 2002). Tolerance to salt stress in Arabidopsis is mediated in part through efflux of Na+ by the Na+/H+ antiporter SOS1 (SALT OVERLY SENSITIVE1) at the plasma membrane, which is activated by the myristolated calcium-binding protein SOS3 and the Ser/Thr kinase SOS2 as part of the SOS pathway (Zhu, 2002, 2003). The phenotype we observed in the ahk5-1 mutant appeared to be specific to the presence of Na+ (Figs 1a, S3), raising the possibility that AHK5 may interact with the SOS pathway. Whether AHK5 directly regulates uptake of Na+/K+ is not yet known. However, a link with the SOS pathway via ROS is plausible.
ROS are known to accumulate to varying concentrations in response to salinity treatment in different subcellular compartments (Miller et al., 2010) and the SOS1 protein has been shown to interact with RCD1 (RADICAL-INDUCED CELL DEATH1), a regulator of oxidative stress responses, to regulate ROS concentrations (Katiyar-Agarwal et al., 2006). Preliminary observations indicate that ahk5-1 seedlings also accumulate higher concentrations of H2O2 following salinity stress than treated wild-type Col-0 seedlings (2.3-fold and 1.4-fold increases over untreated controls, respectively). Given the previously identified function for AHK5 in regulating H2O2 concentrations (Desikan et al., 2008), it is possible that AHK5 acts to regulate redox balance following salinity stress challenge to mediate growth responses.
Loss of AHK5 function increases susceptibility to the bacterial pathogen PstDC3000
Stomata form an active part of plant defences against bacterial pathogens (Melotto et al., 2006; Underwood et al., 2007; Zeng et al., 2010) and previous work has shown that stomata of the ahk5-1 mutant are unresponsive to the presence of PstDC3000 on the leaf surface (Desikan et al., 2008). Here, we report that the ahk5-1 mutant is more susceptible to bacterial infection, when surface-inoculated with PstDC3000. These observations were similar to those reported by Zipfel et al. (2004) with the fls2 (flagellin sensitive2) mutant, which is defective in the perception of bacterial flagellin and does not close stomata in response to either flg22 or PstDC3000 at the leaf surface (Melotto et al., 2006; Zeng & He, 2010). In addition to PstDC3000, the ahk5-1 mutant is also defective in stomatal closure in response to flg22, ethylene, H2O2 and darkness (Desikan et al., 2008). AHK5 may therefore act to integrate responses at the leaf surface to diverse exogenous stimuli.
Further analysis of metabolic changes following PstDC3000 challenge revealed quantitative differences between wild-type and ahk5-1 mutant plants in the hormones SA, ABA and JA. To our knowledge, this is the first report of changes in plant hormones following surface inoculation of bacteria onto Arabidopsis leaves, with the timing of hormone changes correlating with the spread of symptoms. Concentrations of all hormones were maintained at low levels following infection of ahk5-1 leaves. It is clear from recent studies that the balance between the concentrations of the different hormones influences the outcome of disease (Block et al., 2005; de Torres Zabala et al., 2009). During the Arabidopsis–PstDC3000 interaction, antagonism of SA defences through manipulation of ABA production promotes virulence of the bacterium (de Torres Zabala et al., 2007, 2009). In this study, AHK5 differentially affected the temporal accumulation of SA, JA and ABA in response to PstDC3000. In the absence of a stomatal closure response to bacteria, as in ahk5-1, PstDC3000 would enter at an accelerated rate into the apoplastic space and multiply to higher numbers than that seen in wild-type plants. This is likely to suppress basal defence more efficiently, and have prolonged effects on hormonal imbalance, as seen in ahk5-1, throughout the course of the infection.
Another obvious cause for increased virulence in the mutant could be increased production of the bacterial phytotoxin coronatine, also known to suppress basal defences and to manipulate hormone signalling to promote disease (Brooks et al., 2005; Uppalapati et al., 2007; Ishiga et al., 2010; Zeng et al., 2010). Surprisingly, however, PstDC3000 produced lower concentrations of coronatine in ahk5-1 plants. This might result from host defences (e.g., hormones) already being suppressed to a large extent, thereby not necessitating the production of coronatine by PstDC3000. In support of this, Block et al. (2005) suggest that coronatine concentrations do not necessarily correlate with the growth of bacteria. Rather, PstDC3000 might be reallocating its resources to other virulence mechanisms, such as expression of the type-three secretion system and effector proteins, and expression of genes involved in nutrient assimilation or adaptation to the apoplastic environment (Boch et al., 2002; Rico & Preston, 2008).
An alternative explanation for the reduced synthesis of coronatine in ahk5-1 plants is that AHK5 indirectly regulates coronatine biosynthesis. There have been some early reports in the literature of plant-derived factors from the shikimate pathway regulating coronatine biosynthesis in PstDC3000 (Li et al., 1998). In this study, we observed a metabolite unique to ahk5-1 mutant leaves which appeared to be independent of bacterial infection (Fig. S9). Preliminary mass spectrometry analysis is indicative of this metabolite belonging to the indolic class of compounds (part of the shikimate pathway). Is it possible that AHK5 normally suppresses the synthesis of this unique metabolite, and lack of AHK5 removes this suppression, leading to altered susceptibility to PstDC3000? Further work to identify this compound will address this question.
AHK5 contributes to resistance to the necrotrophic fungal pathogen B. cinerea
In addition to increased susceptibility to the hemibiotroph PstDC3000, the ahk5-1 mutant was also strikingly susceptible to the necrotrophic fungal pathogen B. cinerea. Although differences in lesion formation between wild-type and mutant plants were not apparent until 3 dpi in response to B. cinerea, close inspection of infected leaves revealed an increase in fungal growth in ahk5-1 as early as 8 h after contact of the fungal spores with the leaf surface. The cause of this early difference in fungal growth could be due to differences in the properties of the leaf surface structure and composition and/or compounds exuded onto the leaf surface affecting fungal growth (Rossall et al., 1977; Doss et al., 1993; Calo et al., 2006; Bessire et al., 2007; Chassot et al., 2008; Curvers et al., 2010). DAB staining of leaves at early time-points showed more fungal structures being stained, and from the images obtained it appeared that individual hyphae on the mutant showed more DAB staining (Fig. S5). Although there is an increase in fungal hyphal length in the mutant, the experiments here cannot reveal clearly whether or not the mutant leaves somehow enhance fungal production of ROS, thereby enhancing fungal growth. As our data also reveal a requirement for ROS in in vitro hyphal growth, this is a strong possibility.
In addition to the early increase in fungal growth observed on the ahk5-1 mutant, in planta ROS production in ahk5-1 leaf cells was significantly lower than that in wild-type tissue at 24 hpi at the site of inoculation. Early ROS production in tomato (Solanum lycopersicum) and bean (Phaseolus vulgaris) has previously been linked to resistance to infection with B. cinerea (Unger et al., 2005; Asselbergh et al., 2007), and the early attenuation in ROS production by the ahk5-1 mutant may be linked to a dampened immune response resulting in more severe disease symptoms seen in this mutant. However, plant-derived ROS is also required for virulence of the fungus, as ROS-triggered cell death, although effective against biotrophic pathogens in the form of the hypersensitive response (HR), enhances infection by Botrytis cinerea (Govrin & Levine, 2000). This is in agreement with the DPI inhibition of lesions seen here at 3 dpi (Fig. 5a). Moreover, after 24 hpi, the presence of ROS correlated to the area covered by the lesions appearing later (i.e. there appeared to be more extensive ROS production in ahk5-1 leaves as a consequence of larger lesions). Clearly, the role of ROS in the outcome of necrotroph–plant interactions is complex and is determined by factors such as the timing of induction, and the cellular location in the host and the pathogen, as well as the source and concentration of ROS generated. In response to infection with B. cinerea, whereas early ROS production regulated by AHK5 contributes to triggering defence mechanisms against B. cinerea infection, at the later stages of infection, loss of AHK5 function results in increased ROS production associated with cell death, which further facilitates disease progression.
Aside from ROS, nitric oxide (NO) is also known to contribute to resistance to B. cinerea (Asai & Yoshioka, 2009). Additionally, a recent study found that NO produced in B. cinerea was able to diffuse into the surrounding growth medium, raising the possibility that fungal-derived NO may influence plant signalling (Turrion-Gomez & Benito, 2011). However, we found that pretreatment of leaves with the NO scavenger cPTIO did not significantly affect the size of lesions caused by B. cinerea in wild-type Col-0 (Fig. S7). Similarly, treatment of spores with cPTIO did not affect hyphal elongation in vitro (Fig. S10), suggesting that neither a defect in NO production in the ahk5-1 mutant nor alteration in NO production in B. cinerea contributes to the increased susceptibility of the ahk5-1 mutant.
Another factor that contributes to resistance to B. cinerea is the phytoalexin camalexin, which is known to accumulate at the site of infection (Denby et al., 2004; Kliebenstein et al., 2005). In this study, the increased susceptibility of the ahk5-1 mutant was not attributed to a defect in camalexin production, indicating that factors other than camalexin determine host susceptibility or that a mutation in AHK5 results in the secretion of other compounds that influence the sensitivity of B. cinerea to camalexin.
In response to necrotrophic pathogens such as B. cinerea, the plant hormones ethylene (ET), SA and JA are known to positively contribute to resistance (Thomma et al., 1998, 1999, 2000; Zimmerli et al., 2001; Ferrari et al., 2003; van Baarlen et al., 2007; Vicedo et al., 2009). Although the endogenous concentrations of the plant hormones measured were similar in ahk5-1 and wild-type leaf tissue, it would be interesting to see how concentrations of these hormones change over the course of infection with B. cinerea in the ahk5-1 mutant.
Interestingly, pretreatment of leaves with flg22 has been shown to increase resistance to infection with B. cinerea (Ferrari et al., 2007; Galletti et al., 2008), and the kinase Botrytis-induced kinase 1 (BIK1) which is involved in resistance to B. cinerea infection (Veronese et al., 2006) has also been shown to interact with the flagellin receptor FLS2 and BAK1 (BRI1 ASSOCIATED RECEPTOR KINASE1) to mediate flagellin responses (Lu et al., 2010). Given the link with AHK5 and flagellin signalling (Desikan et al., 2008), it is possible that AHK5 acts to integrate basal defence signalling pathways activated by diverse pathogens.
In conclusion, we have shown here that the hybrid histidine kinase AHK5 functions in salinity tolerance and resistance to PstDC3000 and B. cinerea. An underlying theme linking AHK5 to the signalling pathways of the responses tested here is phytohormones and ROS. It is possible that, depending on the stimulus, AHK5 functions differentially to perturb hormone accumulation/signalling and redox balance, resulting in the ahk5-1 mutant being more tolerant to salinity stress, yet less resistant to infection with bacterial and fungal pathogens. In previous studies, AHK2 and AHK3 were found to positively contribute to resistance to PstDC3000 but to negatively regulate cold, salinity and drought stress (Tran et al., 2007, 2010; Choi et al., 2010; Jeon et al., 2010). The opposing functions of both AHK2 and AHK3 in biotic and abiotic stress responses are similar to that found for AHK5 in this study, whereby AHK5 is required for full resistance to the plant pathogens PstDC3000 and B. cinerea but loss of function increases resistance to salinity stress. This study provides further evidence for the importance of hybrid HK function in regulation of growth and survival responses to both abiotic and biotic stresses. Further studies to identify AHK5 interactors will be important for determining the mechanism of AHK5 function. Other key genes which appear to integrate abiotic and biotic stress responses via redox changes and hormones include the DELLA proteins (Achard et al., 2008), UPS1 (UNDERINDUCER AFTER PATHOGEN AND STRESS1) (Denby et al., 2005), BOS1 (BOTRYTIS SUSCEPTIBLE1) (Mengiste et al., 2003) and ATAF1 (ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN2) (Wu et al., 2009). Thus, single genes controlling multiple stress responses do exist and are undoubtedly important for the phenomenon of cross-tolerance and acclimation to stress. Our novel findings highlight the importance of identifying key nodes in the signalling pathways that mediate multiple stress responses in plants.