•Type III effector proteins (T3Es) of many Gram-negative pathogenic bacteria manipulate highly conserved cellular processes, indicating conservation in virulence mechanisms during the infection of hosts of divergent evolutionary origin.
•In order to identify conserved effector functions, we used a cross-kingdom approach in which we expressed selected T3Es from the mammalian pathogen Salmonella enterica in leaves of Nicotiana benthamiana and searched for possible virulence or avirulence phenotypes.
•We show that the T3E SseF of S. enterica triggers hypersensitive response (HR)-like symptoms, a hallmark of effector-triggered immunity in plants, either when transiently expressed in leaves of N. benthamiana by Agrobacterium tumefaciens infiltration or when delivered by Xanthomonas campestris pv vesicatoria (Xcv) through the type III secretion system. The ability of SseF to elicit HR-like symptoms was lost upon silencing of suppressor of G2 allele of skp1 (SGT1), indicating that the S. enterica T3E is probably recognized by an R protein in N. benthamiana. Xcv translocating an AvrRpt2–SseF fusion protein was restricted in multiplication within leaves of N. benthamiana. Bacterial growth was not impaired but symptom development was rather accelerated in a compatible interaction with susceptible pepper (Capsicum annuum) plants.
•We conclude that the S. enterica T3E SseF is probably recognized by the plant immune system in N. benthamiana, resulting in effector-triggered immunity.
Many Gram-negative bacteria that are pathogens or symbionts of mammals, plants and insects use a type III secretion system (T3SS) to ‘inject’ (translocate) bacterial proteins into the cytoplasm of their eukaryotic host cells. The T3SS is a complex apparatus that spans both bacterial membranes and the periplasmic space. It is encoded by 20 or more genes, many of which are highly conserved among animal and plant pathogens (Galán & Wolf-Watz, 2006). The translocated proteins are termed type III effectors (T3Es), as they are virulence factors that effect the changes in the host cells, allowing the invading pathogen to colonize, multiply, and in some cases chronically persist in the host (Galán & Wolf-Watz, 2006). A theme that has emerged over the last few years is that T3Es exert their function by mimicking activities of endogenous eukaryotic proteins without having obvious sequence similarity to their eukaryotic protein counterpart (Stebbins & Galán, 2001). Common effector target processes and structures in both animal and plant host cells include the cytoskeleton, defence and hormonal signalling, ubiquitination, gene expression, and vesicle trafficking (Grant et al., 2006; Orth, 2007; Galán, 2009; Spallek et al., 2009; Broberg & Orth, 2010). The effectors are most often distinct, having unique functions suited to a particular pathogens’s virulence strategy. However, the fact that plant and animal pathogens appear to have evolved similar mechanisms to attack different hosts makes it tempting to speculate that commonalities exist among the disease mechanisms of these fundamentally different host systems (Staskawicz et al., 2001; Guttman, 2004; Nürnberger et al., 2004). Indeed, effector homologues also exist among different T3SS-possessing bacteria. One of the most diverse and widely distributed families of T3Es is the Yersinia pestis YopJ family of cysteine proteases (Hotson & Mudgett, 2004; Lewis et al., 2011). Members of this large family of T3SEs are found among both plant and animal pathogens, raising the possibility that they may target conserved eukaryotic substrates.
In plant cells, T3SEs can also betray the bacteria to the plant host by activating effector-triggered immunity (ETI) (Dangl & Jones, 2001). ETI is a branch of plant immunity in which resistance (R) proteins recognize specific effector proteins, resulting in an effective immune response which is often accompanied by rapid, localized cell death, termed the hypersensitive response (HR), which eventually restricts bacterial spread. The predominant structural motifs found in R proteins are a nucleotide-binding (NB-ARC; nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain and a leucine-rich repeat (LRR) domain. In addition to the NBS-LRR domains, the N-terminal region is usually a coiled-coil (CC) domain or a Toll/Interleukin-1 receptor (TIR) domain, which was named on the basis of its similarity to the Drosophila Toll and mammalian interleukin-1 receptors. Intriguingly, these structures are reminiscent of the architecture of metazoan NACHT or nucleotide binding oligomerization domain (NOD) proteins which trigger innate immune responses in animal cells (Maekawa et al., 2011).
Although many pathogens cause disease in a single or limited number of host species, there is growing evidence for the existence of several universal bacterial virulence mechanisms highly conserved across phylogeny (Rahme et al., 1995, 1997, 2000). In fact, a number of bacterial pathogens can attack both plants and animals (van Baarlen et al., 2007). For instance, Agrobacterium tumefaciens and Erwinia spp., two well-characterized plant pathogens, have been shown to act as pathogens of humans, although mainly in immunocompromised patients (Cao et al., 2001; Paphitou & Rolston, 2003). An example of a particularly well-studied cross-kingdom pathogen is Pseudomonas aeruginosa, which is the major cause of mortality in humans with cystic fibrosis. In several laboratory studies, clinical isolates of P. aeruginosa displayed cross-kingdom pathogenicity by successfully infecting the plant species Arabidopsis thaliana, as well as tobacco (Nicotiana tabacum), lettuce (Lactuca sativa), the nematode Cenorhabditis elegans, and the insect Drosophila melanogaster (Rahme et al., 1995, 1997, 2000; Mahajan-Miklos et al., 2000; D’Argenio et al., 2001). Remarkably, several bacterial mutants have been identified that displayed reduced pathogenicity in mice as well as in A. thaliana. This argues for conservation of the virulence mechanisms used by P. aeruginosa to infect hosts of divergent evolutionary origins (Rahme et al., 2000).
Salmonella enterica is a food-borne bacterial pathogen that is able to infect a wide range of animals and causes various diseases, ranging from enteritis to typhoid fever (Haraga et al., 2008). These bacteria are invasive, facultative intracellular pathogens and rely on type III secretion for pathogenesis and the colonization of host cells. The bacterium comprises two distinct virulence-associated T3SSs within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2, respectively) which function at different times during infection. Whereas SPI1 is active on contact with the host cell, the SPI2 T3SS is expressed within the salmonella-containing vacuole (SCV) and translocates effectors across the vacuolar membranes. The cellular function of a subfraction of type III effectors translocated by either SPI1 or SPI2 is known, although the mode of action remains elusive for most of the type III effectors (Gal-Mor & Finlay, 2006; Haraga et al., 2008). In recent years, it became evident that serovars of S. enterica are not only able to attach to and epiphytically proliferate on the surface of plant tissues but can also colonize plant tissues to grow endophytically (Dong et al., 2003; Iniguez et al., 2005; Klerks et al., 2007a,b; Kroupitski et al., 2009). However, the molecular mechanisms involved in the interaction of Salmonellae with plants are not well understood. Evidence suggests that plant defence responses might restrict endophytic colonization, as infection of A. thaliana with S. enterica serovar Thyphimurium strain 14028 induces a range of basal defence responses (Iniguez et al., 2005). Schikora et al. (2008) were able to show that S. enterica can overcome plant defences and enter and proliferate inside various A. thaliana tissues, causing wilting and chlorosis as disease symptoms. Serotypes of Salmonella behave differently with respect to symptom development on A. thaliana leaves. Wilting and chlorosis were only observed when strains belonging to serogroup E4 (O:1, 3, 19) were used for infection (Berger et al., 2011). Recently, it was shown that functional T3SS machinery is necessary for Salmonella to infect A. thaliana leaf tissue. Bacterial mutants lacking either a functional SPI1 or a functional SPI2 showed reduced proliferation in leaves, indicating a role of type III secretion in colonization (Schikora et al., 2011). In addition, these mutants caused enhanced symptom development, including an HR-like response. From these data, the authors concluded that T3Es may be involved in suppression of defence responses. Consistent with this hypothesis, it was shown that wild-type bacteria, but not the T3SS mutant invA−, were able to suppress the oxidative burst and the increase of extracellular pH after inoculation of a tobacco cell culture (Shirron & Yaron, 2011). This further argues for an involvement of T3Es in Samonella–plant interactions.
The above-mentioned studies indicate that plants can represent a useful tool with which to study the pathogenesis of human pathogenic bacteria. There are several benefits of using plants to model pathogenesis, such as the ease of genetic manipulation, which is not possible for most animal systems, and the ease of growing and maintaining the host, which has advantages in terms of cost-effectiveness.
In this study, we selected a range of Salmonella T3SEs either based on their similarity to T3SEs from plant pathogenic bacteria, or because they have as yet unknown target proteins in mammalian cells. Agrobacterium tumefaciens-mediated transient gene transfer was used to express those proteins in leaves of Nicotiana benthamiana. Subsequently, Salmonella T3SE-expressing leaves were, as a first approximation of a possible in planta activity, visually scored for phenotypic changes that might be associated with either virulence or an avirulence function of the proteins in plant cells. Here we show that the Salmonella T3E SseF triggers an HR-like response when expressed in leaves by A. tumefaciens-mediated infiltration or when translocated into leaves of N. benthamiana in a T3SS-dependent manner. Recognition of SseF inside plant cells was dependent on components of R-protein-mediated signal transduction. Our results show that a type III effector of the nonadapted mammalian pathogen S. enterica can probably be recognized by the plant’s immune system.
Materials and Methods
Plant material and growth conditions
Tomato (Solanum lycopersicum cv Moneymaker), pepper (Capsicum annuum cv ECW-10R) and tobacco (Nicotiana benthamiana Domin.) plants were grown in soil in a glasshouse with daily watering, and subjected to a 16 h light : 8 h dark cycle (25°C : 21°C) at 300 μmol m−2 s−1 light and 75% relative humidity.
Construction of Salmonella enterica T3E expression plasmids
Salmonella enterica T3E expression constructs were generated using Gateway™ cloning technology (Invitrogen, Karlsruhe, Germany). To this end, the entire open reading frame of the respective effector protein was amplified by PCR using gene-specific primers (Supporting Information Table S2) with S. enterica (NCTC 12023) genomic DNA as template. The resulting fragments were inserted into the pENTR-D/TOPO vector according to the manufacturer’s instructions (Invitrogen). Subsequently, the effector-encoding fragment was recombined into the plant expression vector pRB35S-3xmyc (Bartetzko et al., 2009), using L/R-Clonase (Invitrogen).
For infiltration of N. benthamiana leaves, A. tumefaciens C58C1 was infiltrated into the abaxial air space of 4- to 6-wk-old plants, using a needleless 2-ml syringe. Agrobacteria were cultivated overnight at 28°C in the presence of appropriate antibiotics. The cultures were harvested by centrifugation, and the pellet was resuspended in sterile water to a final optical density (OD600) of 1.0. The cells were used for the infiltration directly after resuspension. Infiltrated plants were further cultivated in the glasshouse with daily watering, and subjected to a 16 h light : 8 h dark cycle (25°C : 21°C) at 300 μmol m−2 s−1 light and 75% relative humidity.
Leaf material was homogenized in sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (100 mM Tris-HCl, pH 6.8, 9%β-mercapto ethanol, 40% glycerol, 0.0005% bromophenol blue and 4% SDS) and, after heating for 10 min at 95°C, subjected to gel electrophoresis. Separated proteins were transferred onto nitrocellulose membrane (Porablot; Machery und Nagel, Düren, Germany). Immunodetection was carried out using the ECL kit (GE Healthcare, Freiburg, Germany) according to the manufacturer’s instructions, using a primary anti-myc antibody (dilution 1 : 1000) from rabbit (Santa Cruz Biotechnology, Heidelberg, Germany) and peroxidase-conjugated secondary anti-rabbit antibody (Pierce, Rockford, IL, USA).
RNA extraction and expression analysis
Total RNA was isolated from leaf material as described previously (Logemann et al., 1987) and then treated with RNAse-free DNase (Fermentas, St. Leon-Roth, Germany) to degrade any remaining DNA. First-strand cDNA synthesis was performed from 2 μg of total RNA using a random hexamer using Revert-Aid reverse transcriptase (Fermentas). For RT-PCR, cDNAs were amplified using Taq polymerase (New England Biolabs, Frankfurt, Germany) and gene-specific primers (Table S2). The number of amplification cycles was reduced from 35 to 25 to evaluate and quantify any differences among transcript levels before the levels reached saturation. PCR products were separated on a 1% (w/v) agarose gel containing ethidium bromide and visualized by UV light. For quantitative realtime RT-PCR, the cDNAs were amplified using Brilliant II SYBR Green QPCR Mastermix (Stratagene, Heidelberg, Germany) in an MX3000P real-time PCR instrument (Stratagene). PCR was optimized, and reactions were performed in triplicate. The transcript level was standardized based on cDNA amplification of 18S rRNA as a reference. Relative gene expression data were generated using plants transiently expressing free GFP as a calibrator. Fold induction values of target genes were calculated with the ΔΔCP equation according to Pfaffl (2001). Statistical analysis was performed using a two-tailed Student’s t-test. Primers used for RT-PCR and quantitative real-time PCR, respectively, are listed in Table S2.
Ion leakage measurements
For electrolyte leakage experiments, triplicates of 1.76 cm2 infected leaf material were taken at 24 and 48 h post infiltration (hpi). Leaf discs were placed on the bottom of a 15-ml tube. Eight millilitres of deionized water was added to each tube. After 24 h of incubation in a rotary shaker at 4°C, conductivity was determined with a conductometer. To measure the maximum conductivity of the entire sample, conductivity was determined after boiling the samples for 30 min (Stall et al., 1974). For time-course experiments, ion leakage measurements were performed as described previously (Coll et al., 2010).
Virus-induced gene silencing
Virus-induced gene silencing in N. benthamiana was essentially carried out as described previously (Liu et al., 2002a,b). In brief, a fragment of N. benthamiana NDR1 was amplified by PCR using the primers indicated in Table S2 and cloned into pTRV2-Gateway (Liu et al., 2002a) using the Gateway™ recombination system (Invitrogen) as described in the section ‘Construction of expression plasmids’. pTRV2-NbSGT1 and pTRV2-NbEDS1 (Liu et al., 2002b) were obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org). The plasmids were transformed into A. tumefaciens C58C1. A lower leaf of a 4-wk-old N. benthamiana plant was co-infiltrated with a mixture of agrobacteria carrying either pTRV1 or pTRV2 containing the target sequence as described previously (Liu et al., 2002b). Silenced plants were analysed 14 d post infiltration.
Translocation of AvrRpt2–SseF-HA by Xanthomonas campestris pv vesicatoria
To construct the AvrRpt2–SseF fusion protein, a synthetically synthesized fragment containing the promoter and amino acids 1–100 of AvrRpt2 was translationally fused to a fragment of SseF comprising amino acids 26–260 and an HA tag in the broad host range vector pBBR1-MCS5 (Kovach et al., 1995). The resulting construct AvrRpt2(1–100)–SseF(26–260)-HA was introduced into X. campestris pv vesicatoria 85-10 (Xcv) by triparental mating (Huguet et al., 1998). HR assays in N. benthamiana were carried out by leaf infiltration with a bacterial density of 2 × 108 cfu ml−1. The HR was scored 48 hpi. For bacterial growth assays, Xcv (EV) and Xcv (AvrRpt21–100–SseF26–260) were diluted to 1 × 105 (pepper and tomato infections) or to 1 × 106 (N. benthamiana infection). In planta growth assays were performed as described previously (Kocal et al., 2008).
Generation of SseF deletion variants
The SseF deletion variants were constructed by PCR using the plasmids described in Abrahams et al. (2006) as a template and the primers indicated in Table S2. The fragments were inserted into pRB35S-3xmyc using Gateway™ (Invitrogen) cloning as described in the section ‘Construction of expression plasmids’.
Infection of plants with S. typhimurium
For infection experiments, bacteria were cultured as described previously (Yu et al., 2010). All infections were performed using a bacterial solution with a density of 2 × 108 cfu ml−1. Plants were infiltrated with a bacterial solution in 1 mM MgCl2.
Agrobacterium tumefaciens-mediated transient expression of selected S. enterica T3SEs in leaves of N. benthamiana
Individual S. enterica T3SEs were selected (AvrA, SseF, SseG, PipB2, SseJ, SopD2, SopB and SseL) either because they belong to effector families that also have representatives in plant pathogens (e.g. AvrA) or because their target molecule(s) in mammalian host cells is unknown (e.g. SseF) (summarized in Table S1). As a first approximation to assess the ability of S. enterica T3SEs to elicit a macroscopically visible response that might be linked to a possible virulence or avirulence function in plant cells, we used A. tumefaciens infiltration to transiently express the effector proteins in leaves of N. benthamiana. To this end, the entire coding region of the respective T3SEs was inserted into a binary vector under control of the Cauliflower mosaic virus (CaMV) 35S promoter mediating strong constitutive expression in plant cells. To facilitate protein detection, a triple myc-tag was introduced at the C-terminus of the effector proteins. At 24 and 48 hpi, samples were taken from A. tumefaciens-infiltrated leaves and T3SE expression was monitored by immunoblotting. The results revealed that, of the eight T3SEs tested, six (the exceptions being AvrA and SseJ) were expressed in leaves of N. benthamiana, albeit to different levels and with different kinetics (Fig. S1A). Analysis of the transformed A. tumefaciens strains verified that T3E expression was indeed mediated solely by plant cells and not by the bacteria (Fig. S1A). In the case of SseL, a signal migrating slightly above the expected molecular weight of the effector-myc fusion was detected in A. tumefaciens cultures; however, in plants a weak but specific band was only detected at 48 hpi, not at 24 hpi, which argues for protein expression from the plant cell. A visual inspection of S. enterica T3SE-expressing leaves 48 hpi revealed no obvious phenotypic alteration for five of the six effector proteins expressed (Fig. S1B). In the case of SseF only, agro-infiltrated leaves were severely damaged, showing tissue collapse and necrosis at 48 hpi (Fig. S1B). The phenotype of SseF-expressing leaves 48 hpi resembled features of an HR, a form of localized cell death that is usually associated with R-protein-mediated immunity triggered upon recognition of a pathogen-derived avirulence (Avr) protein. Therefore, we decided to investigate this phenomenon in more detail and performed a set of experiments designed to reveal some of the molecular and cellular events that SseF is able to trigger in plant cells.
SseF induces the expression of HR marker genes
SseF has been shown to contain two hydrophobic transmembrane domains (Abrahams et al., 2006) and its ectopic expression is toxic to yeast (Aleman et al., 2009) and HeLa cells (Kuhle et al., 2004). Thus, the phenotype observed in SseF-expressing leaves of N. benthamiana could easily be an unspecific effect of SseF toxicity rather than a defence response of the plant. To distinguish between these two possibilities, the induction of marker genes that have previously been shown to be strongly correlated with HR was investigated in SseF-expressing N. benthamiana leaves using quantitative real-time RT-PCR (qPCR). In parallel, phenotype development and protein expression in leaves agro-infiltrated with the 35S-SseF-myc construct as compared with control leaves transiently expressing the green fluorescent protein (GFP) or 35S-HopZ1a-myc, serving as a positive control for HR elicitation (Ma et al., 2006), were monitored. Expression of SseF as well as of HopZ1a led to the necrotic phenotype 48 hpi for SseF and 60–72 hpi for HopZ1a, respectively, while GFP expression induced no macroscopically visible changes in agro-infiltrated leaves (Fig. 1a). Western blot analyses showed that all proteins were expressed at the time-points investigated (Fig. 1b). However, we observed some variation of SseF expression at 48 hpi. Depending on the degree of cell death progression and tissue collapse, the SseF signal was lost at this time-point (e.g. Fig S1A). Slight differences in the kinetics of SseF expression and elicitation of HR-like symptoms on an experiment-to-experiment basis probably account for the differences in expression at 48 hpi.
qPCR revealed that all three genes tested, harpin-induced 1(HIN1), Hsr201, and Hsr203J (Yoshihiro et al., 2004), were strongly induced in SseF-expressing leaves as compared with tissue infiltrated with A. tumefaciens harbouring the control construct (Fig. 1c). Induction of HIN1 remained high at both time-points tested (24 and 48 hpi) while expression of Hsr201 and Hsr203J was highest at 24 hpi and subsequently dropped to control levels or even below at 48 hpi, when the tissue was already severely necrotic. These results strongly support the notion that the phenotype observed in SseF-expressing N. benthamiana leaves 48 hpi is a form of pathogen-associated programmed host cell death rather than the mere killing of cells, as would be expected in the case of simple SseF toxicity.
Plant defence associated with the recognition of bacterial effector proteins not only triggers HR but also leads to the activation of basal defence responses which usually are linked to pathogen associated molecular pattern (PAMP)-triggered immunity (PTI) (Jones & Dangl, 2006). Thus, we examined whether transient expression of SseF alters the mRNA levels of previously described PTI marker genes (Nguyen et al., 2010). The expression levels of all three reporter genes tested, Pti5, Gras2 and Acre31, were significantly induced in leaves transiently expressing SseF compared with those expressing the control construct 35S-GFP (Fig. S2). Expression of Pti5, Gras2 and Acre31 was highest at 24 hpi and decreased at 48 hpi, probably as a consequence of the tissue collapse. Taking these findings together, we conclude that SseF expression in leaves of N. benthamiana triggers responses associated with both ETI and PTI.
HR is often preceded by an increase in electrolyte leakage in dying cells, and measurement of electrolyte leakage caused by membrane damage is a quantitative measure of HR-associated cell death (Mackey et al., 2003). SseF expression led to strong electrolyte leakage already at 24 hpi compared with tissue infiltrated with an A. tumefaciens strain harbouring the GFP control construct (Fig. 1d). At this time-point, no visible signs of HR were observable in SseF-expressing leaves. Ion leakage slightly increased until 48 hpi when full HR symptoms were visible (Fig. 1d). This again argues for SseF as a trigger of HR-like symptoms in N. benthamiana.
SseF-mediated cell death requires signalling components of R-protein-mediated resistance
The results obtained so far suggest that leaf necrosis observed upon SseF expression involves an HR-like process and thus might be a consequence of R-protein-mediated recognition of the S. enterica effector protein by the plant. Defence signalling by R proteins requires further signalling components such as SGT1 (suppressor of G2 allele of skp1) which, in N. benthamiana, was found to be required for responses mediated by a diverse range of R proteins against various pathogens (Peart et al., 2002). Based on these findings, we predicted that the SseF-elicited HR-like symptoms would only be compromised by silencing of SGT1 if it was a bona fide resistance response. A toxic response, which for instance was observed for Nep1 (for necrosis and ethylene-inducing peptide1)-like proteins (NLPs) that trigger leaf necrosis and immunity-associated responses in various plants, would not be compromised by SGT1 silencing (Qutob et al., 2006). To test whether SGT1 is required for SseF-induced cell death, we used virus-induced gene silencing (VIGS) with Tobacco rattle virus (TRV), followed by agro-infiltration assays. For this purpose, young N. benthamiana plants (at the five-leaf stage) were infiltrated with a mixture of A. tumefaciens strains of pTRV1 (CaMV 35S-driven TRV RNA1) and pTRV2-SGT1 (TRV RNA2 containing the target sequence), or pTRV-GFP (serving as a control for infection symptoms). Two weeks after TRV inoculation, silencing of SGT1 was confirmed by RT-PCR (Fig. 2c). Subsequently, plants were infiltrated with A. tumefaciens containing SseF-3xmyc and infiltrated leaves were evaluated at 48 hpi. Silencing of SGT1 abolished the development of macroscopically visible HR symptoms when compared with the TRV:GFPsil control (Fig. 2a), although immunoblot analysis revealed SseF protein expression to similar levels in both plants at 48 hpi (Fig. 2b).
To further investigate whether silencing SGT1 would also prevent electrolyte leakage of SseF-expressing cells, ion leakage was measured at 48 hpi in SseF-expressing TRV:SGT1 plants as compared with TRV:GFPsil plants. TRV:SGT1 or TRV:GFPsil plants without any additional agro-infiltration were included in the analysis to exclude an effect of TRV infection on electrolyte leakage (Fig. 2d). The analyses revealed that expression of SseF caused a significant increase in ion leakage in TRV:GFPsil plants but not in TRV:SGT1 plants (Fig. 2d), indicating that SGT1 silencing abrogates SseF-associated electrolyte leakage.
Collectively, these results demonstrate that SseF requires SGT1 to elicit cell death in N. benthamiana. It is likely, therefore, that the SseF-triggered HR-like phenotype is not attributable to mere toxicity of the effector but could rather represent an R-protein-mediated response.
R proteins differ in their requirement for signalling components downstream of SGT1. NBS-LRR proteins with amino-terminal Toll and interleukin-1 receptor homology (TIR domain) use EDS1, whereas those with CC domains signal through NDR1 (Aarts et al., 1998). To provide the first insights into the nature of a possible R protein involved in SseF-triggered HR in N. benthamiana, VIGS directed against EDS1 and NDR1 was used. Efficient down-regulation of EDS1 and NDR1 could be confirmed 2 wk after TRV inoculation of EDS1 and NDR1 by RT-PCR (Fig. 3b). Silencing of NDR1 resulted in a clear and consistent decrease in HR-like symptom development upon transient SseF expression, while plants with reduced EDS1 expression showed no apparent phenotypical differences when compared with the TRV:GFPsil control (Fig. 3a). Immunoblot analysis revealed similar SseF protein levels in all VIGS plants (Fig. 3c). To further investigate whether silencing of NDR1 would also result in a decrease in electrolyte leakage of SseF-expressing cells, ion leakage was measured at 48 hpi in SseF-expressing TRV:NDR1 and TRV:EDS1 plants as compared with TRV:GFPsil plants. TRV:NDR1, TRV:EDS1 or TRV:GFPsil plants without any additional agro-infiltration were included in the analysis to exclude an effect of TRV infection on electrolyte leakage (Fig. 3d). Expression of 35S-GFP served as an additional control to exclude an effect of agro-infiltration per se. Consistent with the observed phenotype, a significant decrease in ion leakage following SseF expression was evident in TRV:NDR1 plants but not in TRV:EDS1 and TRV:GFPsil plants, respectively (Fig. 3d). This opens the possibility that a putative R protein mediating the response to SseF is a member of the CC-NBS-LRR class.
SseF also triggers an HR in N. benthamiana when translocated into plant cells in a T3SS-dependent manner
We next sought to examine whether SseF would also trigger HR-like symptoms in N. benthamiana when translocated into plant cells through a T3SS. To this end, we engineered Xcv, the causal agent of bacterial speck disease on tomato and pepper, to deliver SseF. We chose Xcv because it does not inherently induce localized cell death in N. benthamiana (Fig. 4a) in addition to its promiscuous secretion of proteins from a variety of pathogens through the T3SS (Rossier et al., 1999; Mudgett et al., 2000). In S. enterica, T3SS-dependent translocation of SseF requires its chaperone, SscB (Dai & Zhou, 2004). Thus, it is likely that the SseF T3S signal is not recognized by Xcv. In order to circumvent this problem, we constructed an N-terminal deletion of SseF to remove its S. enterica T3S signal sequence (generally contained within the first 50 amino acids) and replaced it with a functional T3S signal sequence (contained within amino acids 1–100) from AvrRpt2 derived from Pseudomonas syringae (Guttman & Greenberg, 2001). Agrobacterium tumefaciens-mediated transient expression of the AvrRpt21–100–SseF26–260 fusion protein did not impair induction of HR-like symptoms (Fig. S3). This finding indicated that the fusion partner did not affect SseF functionality in this assay.
We next determined whether Xcv expressing the AvrRpt21–100–SseF26–260 fusion under control of the native AvrRpt2 promoter could elicit an HR-like response in N. benthamiana leaves. As shown in Fig. 4(a), the Xcv wild type did not elicit an HR in N. benthamiana leaves at 48 hpi while two independent Xcv clones expressing the AvrRpt2–SseF hybrid protein induced a strong SseF-dependent HR-like response at 48 hpi. As a positive control, we generated an Xcv strain carrying the T3SE HopZ1a, which was previously described to induce an HR in N. benthamiana (Ma et al., 2006). Infiltration of N. benthamiana with two independent Xcv clones expressing HopZ1a resulted in a HopZ1a-dependent HR at 48 hpi, which is comparable to the kinetics of the SseF-mediated HR-like symptom development (Fig. S4). To confirm that the AvrRpt2–SseF fusion protein is effectively secreted in an hrp-dependent manner into the plant cell, we introduced the AvrRpt2–SseF fusion construct into Xcv 85-10 ΔhrpB1, lacking the accessory T3SS protein HrpB1. This Xcv mutant is no longer able to secret T3Es (Rossier et al., 2000). For in vivo translocation assays, bacteria were inoculated into N. benthamiana leaves. AvrRpt2–SseF was not able to induce HR-like symptoms when expressed by the Xcv 85-10 ΔhrpB1 strain, suggesting that no translocation occurred in the absence of HrpB1 (Fig. 4b). Differences in the observed phenotypes were not the result of different protein amounts, because similar protein levels of AvrRpt2–SseF were detected in both Xcv strains harbouring the fusion construct (Fig. 4c). Next we sought to investigate whether the SseF-triggered HR-like response in translocation assays is also dependent on SGT1. For this purpose, N. benthamiana silenced for SGT1 expression using VIGS was infiltrated with Xcv (AvrRpt21–100–SseF26–260) with a high bacterial titre (2 × 108 cfu ml−1). Silencing of SGT1 abolished the development of macroscopically visible HR-like symptoms when compared with the TRV:GFPsil control (Fig. 4d), suggesting that the SseF-triggered HR-like response is a consequence of R-protein-mediated recognition when delivered by Xcv into N. benthamiana plants. These data demonstrate that the AvrRpt2 T3S signal sequence can target SseF into N. benthamiana cells via the Xcv T3S apparatus. Furthermore, elicitation of the HR-like response by SseF in leaves of N. benthamiana requires SGT1 and is not an artifact caused by gross overexpression of the effector protein, as might be the case in agro-infiltration assays.
Influence of SseF on compatible Xcv–host interactions
To investigate whether Xcv delivering the AvrRpt21–100–SseF26–260 hybrid protein would be affected in a compatible Xcv–host interaction, we examined disease progression and bacterial growth in susceptible ECW-10R pepper (C. annuum) and tomato (cv Moneymaker) plants infected with Xcv (AvrRpt21–100–SseF26–260). As shown in Fig. 5(a), Xcv (AvrRpt21–100–SseF26–260) did not elicit HR symptoms either in pepper or in tomato plants (Fig. S5B) when infiltrated with a high bacterial titre (2 × 108 cfu ml−1). However, Xcv expressing the AvrRpt21–100–SseF26–260 fusion protein caused faster symptom development in pepper plants in comparison to the Xcv harbouring the empty vector (EV). While the latter caused typical water-soaked lesions 4–5 d post-infiltration, the AvrRpt2–SseF translocating strain consistently caused lesion development already at 3 d post-infection (dpi) (Fig. 5a). The earlier symptom development in pepper correlated with slightly, but significantly elevated bacterial growth at 3 and 6 dpi, suggesting a contribution of SseF to virulence when translocated by Xcv 85-10 (Fig. 5b). Furthermore, disease progression in tomato plants was not altered in leaves infiltrated with Xcv (AvrRpt21–100–SseF26–260) relative to the Xcv (EV) strain (Fig. S5A). In addition, delivery of AvrRpt21–100–SseF26–260 also enhanced bacterial growth slightly but significantly in tomato at 3 dpi when compared with Xcv (EV) (Fig. S5C). Taken together, these data demonstrate that in the genotypes tested SseF does not render compatible Xcv–host interactions incompatible, indicating that SseF is not recognized inside pepper and tomato cells.
The delivery of AvrRpt21–100–SseF26–260 restricts Xcv growth in N. benthamiana
We next tested whether HR-like symptom elicitation in N. benthamiana by SseF is correlated with restricted bacterial growth after infiltration with low bacterial inocula. To this end, we measured bacterial populations of Xcv (EV) and Xcv (AvrRpt21–100–SseF26–260) in N. benthamiana at 0 and 6 dpi. In contrast to leaves infected with a high bacterial titre, a low bacterial titre did not trigger visible signs of HR-like symptom development at 6 dpi (data not shown). However, at 6 dpi there was a clear and significant reduction in bacterial growth of Xcv expressing the AvrRpt21–100–SseF26–260 hybrid protein compared with Xcv (EV) (Fig. 6a), indicating that the recognition of SseF by the resistance machinery in N. benthamiana limited in planta bacterial growth when delivered by Xcv 85-10.
Furthermore, we monitored changes in conductivity attributable to HR-dependent electrolyte leakage of N. benthamiana leaves infected with Xcv (AvrRpt21–00–SseF26–260). PstDC3000 and Xcv (HopZ1a) strains were included in this experiment, as a positive control for HR elicitation (Ma et al., 2006; Wei et al., 2007) and thus increased ion leakage in N. benthamiana. The measurements revealed that Xcv expressing AvrRpt2–SseF exhibited significantly increased ion leakage at 24 hpi compared with Xcv (EV), although no visible signs of HR were detectable on the leaves at that time-point. PstDC3000 and Xcv (HopZ1a) showed similar conductivity kinetics. Ion leakage of leaves infected with Xcv (AvrRpt21–00–SseF26–260), PstDC3000 and Xcv (HopZ1a) steadily increased until 48 hpi when the HR was fully developed (Fig. 6b).
SseF requires the second transmembrane domain for HR elicitation
The two hydrophobic transmembrane (TM) regions of SseF, named TM1 (amino acids 63–110) and TM2 (aminoacids 128–211) (Fig. 7a), are located in the amino and carboxy-terminal moieties of the protein, respectively. A functional dissection of SseF revealed that TM1 seems to be required for the secretion and translocation of the protein, whereas essential effector functions of the protein are located in the TM2 region (Abrahams et al., 2006). Therefore, we sought to investigate the roles of distinct regions of the SseF polypeptide in activating HR-like symptom development in N. benthamiana leaves using deletional analysis. Based on the SseF deletion mutants previously used to analyse effector function during Salmonella infection of HeLa cells (Abrahams et al., 2006), a series of in-frame myc epitope-tagged deletion variants consisting of one C-terminal deletion (SseFΔC4) and an internal deletion (SseFΔ7) was generated for in planta expression (see Fig. 7a for a schematic representation of the deletions). Both SseF variants alongside the full-length protein were transiently expressed in N. benthamiana leaves using agro-infiltration, and the development of HR-like symptoms was assessed at 48 hpi by visual inspection. In accordance with previous results, expression of full-length myc-tagged SseF triggered a strong HR-like response that was also observed upon expression of SseFΔC4 (Fig. 7b). Expression of SseFΔ7 did not lead to any visible symptoms (Fig. 7b). Western blot analysis with an antibody directed against the myc epitope revealed single bands of the predicted molecular weights for each of the deletion constructs (Fig. 7c). In accordance with the observed phenotypes, ion leakage in SseFΔC4-expressing leaves was comparable to that of the wild-type SseF-expressing leaves, while no increase in electrolyte leakage was observed in SseFΔ7-expressing leaves (Fig. 7d).
Taken together, these results indicate that the integrity of the hydrophobic TM2 domain of SseF is crucial for HR-like symptom elicitation in leaves of N. benthamiana. Interestingly, the same domain has been shown to be essential for a range of SseF effector functions in mammalian cells (Abrahams et al., 2006).
Salmonella typhimurium 14028 does not elicit HR-like symptoms in Nicotiana benthamiana
To test whether Salmonella is able to induce HR-like symptoms in N. benthamiana, leaves were infected with Salmonella typhimurium wild-type strain 14028. Besides normal growth medium (LB), minimal medium (pH 5.0) was used to cultivate Salmonella in order to facilitate the assembly of the T3SS (Yu et al., 2004). Furthermore, bacteria were exposed to a pH shift from 5.0 to 7.2 to promote secretion of effector proteins (Yu et al., 2010). The different treatments did not result in the development of any visible signs of HR-like symptoms 2 d after infection (Fig. S6). Thus, it appears that Salmonella, although harbouring SseF, does not elicit an HR-like response in N. benthamiana leaves.
The HR is a form of programmed cell death in plants commonly associated with disease resistance (Greenberg, 2003; Jones & Dangl, 2006). It occurs upon intracellular recognition of a pathogen effector by a corresponding R protein, usually an NB-LRR protein, in a process called ETI. This specific pathogen recognition has been associated with host as well nonhost resistance to a range of pathogens (Mysore & Ryu, 2004).
We show here that the Salmonella T3E SseF elicits cell death when expressed in leaves of N. benthamiana. Several lines of evidence suggest that this cell death is not merely a consequence of the potential toxicity of SseF for plant cells but shows characteristics of an HR as a hallmark of R-protein-mediated defence in plants. Agrobacterium tumefaciens-mediated SseF expression rapidly led to the induction of a subset of HR marker genes, in addition to causing membrane damage, as revealed by increased electrolyte leakage from SseF-expressing cells. Most strikingly, SseF-induced cell death was dependent on SGT1, which is required for resistance mediated by multiple R proteins recognizing a diverse set of pathogens. SGT1 has been shown to control the steady-state level of preactivated R proteins (Peart et al., 2002; Azevedo et al., 2006). Virus-induced silencing of SGT1 in N. benthamiana considerably reduced HR-like symptom development upon SseF expression, suggesting the involvement of R-protein-mediated signalling in this process.
Furthermore, the possibility can be excluded that HR-like symptom elicitation by SseF is attributable to strong CaMV 35S-mediated overexpression of the effector protein, as translocation of an AvrRpt2–SseF fusion protein into plant cells via the T3SS of Xcv also causes induction of an HR-like response in leaves of N. benthamiana in an SGT1-dependent manner. In this case, expression of the effector fusion protein is driven by the AvrRpt2 promoter and thus it can be assumed that protein levels are close to what is naturally found in an infection situation. Importantly, delivery of the AvrRpt2–SseF fusion protein via the T3SS of Xcv restricted bacterial multiplication in leaves of N. benthamiana, clearly showing that SseF is able to induce plant immunity. By contrast, delivery of AvrRpt2–SseF by Xcv does not negatively affect bacterial multiplication in susceptible genotypes of pepper and tomato, suggesting that the corresponding R gene is not present in the genotypes tested. This adds further weight to the conclusion that SseF recognition by N. benthamiana is a specific effect rather than a consequence of toxicity. The question of whether SseF is able to promote virulence in a compatible interaction is currently difficult to answer. The observation that Xcv translocating AvrRpt2–SseF show accelerated symptom development on susceptible pepper plants might argue for a promotion of virulence by SseF. However, the strain used for all infection experiments translocates the full complement of Xcv 85-10 effector proteins and thus might mask additional quantitative contributions to overall virulence imparted by AvrRpt2–SseF expression. A virulence function of SseF in Salmonella infecting epithelial cells has been reported as a loss of function mutation in this effector, rendering the bacterium less able to replicate (Abrahams et al., 2006). SseF helps to maintain the SCV in juxtanuclear, Golgi-network-associated localization, which is required for replication. However, it is currently not known by which mechanism SseF exerts its function or which host proteins it might target. Whether in plants SseF is able to promote virulence of an attenuated Xcv strain lacking particular effector proteins will be the subject of future investigations.
Taken together, our results suggest that the Salmonella T3E SseF is specifically recognized by a plant R protein in N. benthamiana and elicits a defence response closely resembling ETI.
Effector recognition by R proteins can either be directly by physical association or indirectly by detecting effector activity on other plant proteins that are ‘guarded’ by NB-LRR proteins. This guard model predicts that modification of the effector target results in activation of the R protein which than triggers disease resistance in the host (Dangl & Jones, 2001; van der Hoorn & Kamoun, 2008). The indirect effector perception mechanism postulated by the guard model explains how multiple effectors could be perceived by a single R protein, thus enabling a relatively small R gene repertoire to target the broad diversity of pathogens that attack plants. Only a few specific examples of direct recognition of effectors by cognate R proteins exist, and available evidence suggests that the vast majority of bacterial T3Es are detected indirectly by recognizing their activity on target proteins in the host cell (Dodds & Rathjen, 2010). The available data suggest that SseF is only weakly conserved in pathogenic bacteria other than Salmonella, with sequences having low similarity to SseF thus far only found in Edwardsiella tarda (Xie et al., 2010) and Chromobacterium violaceum (Brazilian National Genome Consortium, 2003). No similar sequences have so far been detected in plant pathogenic bacteria. Thus, evolution of an R protein specifically recognizing this effector in a direct manner in plant cells appears unlikely. Although the recognition mode of SseF in plant cells is currently unknown, the possibility of an indirect recognition according to the guard model raises the very intriguing possibility that T3E targets are conserved between diverse hosts such as animals and plants, although bacterial pathogens are adapted only to a particular host.
The postulated R protein that mediates recognition of SseF in N. benthamiana is undefined; however, it does not appear to be universal to plants, as susceptible tomato and pepper genotypes fail to recognize the AvrRpt2–SseF fusion protein following its delivery by the T3SS of Xcv. This would be in accordance with a model of SseF recognition comparable to what has been described as cultivar-specific resistance. This variant of race-specific resistance relies upon genetic variation within the host plant species or genus, and the production of proteins capable of altering the outcome of an otherwise compatible plant–pathogen interaction in only certain plant cultivars or species (Grant & Mansfield, 1999).
Based on the finding that silencing of NDR1 strongly reduces development of SseF-mediated HR-like symptoms, it might be assumed that the R protein associated with SseF-mediated HR-like responses belongs to the CC-NBS-LRR class (Aarts et al., 1998). Possibly, this R protein will recognize virulence factors from other pathogens which might have a better defined function than SseF and thus identification of the specific R protein recognizing SseF in plant cells could help to further elucidate its mode of action. Recently, the R protein recognizing the P. syringae T3E HopZ1a was identified in A. thaliana by screening knock-out lines lacking individual R genes (Lewis et al., 2010). A similar approach could be undertaken in N. benthamiana using VIGS, especially as a draft genome sequence of this model plant is now available (http://www.solgenomics.net), facilitating the analysis of its full R gene complement.
The SseF protein comprises two hydrophobic TM regions, with TM2 (amino acids 128–212) being indispensible for effector functions during the Salmonella infection process (Abrahams et al., 2006). Abrahams et al. (2006) showed that the region necessary for SseF functionality is linked to the amino acids 179–212 within TM2, as the respective deletion mutant failed to elicit SseF-associated phenotypes during infection of HeLa cells. Strikingly, expression of the same deletion mutant in leaves of N. benthamiana also failed to elicit an HR, which suggests that the same amino acid residues define the functional requirements of SseF in both processes.
Salmonella is not considered an established plant pathogen, although recent evidence suggest that it can infect various tissues of A. thaliana and proliferate in intracellular compartments (Schikora et al., 2008, 2011). In addition, Salmonella has been shown to endophytically colonize a range of plant hosts (Guo et al., 2001; Cooley et al., 2003; Dong et al., 2003; Kutter et al., 2006; Klerks et al., 2007a). This poses a considerable health risk for consumers, as endophytes cannot be effectively removed by surface sterilization and several outbreaks of salmonellosis have been associated with contaminated vegetables and fruits (Brandl, 2006). The extent of endophytic colonization by different Salmonellae is an active process that is regulated by plant defence and by the genetic background of both the bacterium and the plant (Dong et al., 2003; Iniguez et al., 2005; Klerks et al., 2007a; Schikora et al., 2011). Infection of A. thaliana protoplasts with Salmonella resulted in a rapid activation of the mitogen-activated protein kinases MPK3 and MPK6, both of which have been implicated in defence signalling during PAMP-triggered immunity (Schikora et al., 2008). In addition, A. thaliana infected by Salmonella initiates transcription of a number of defence genes, including PDF1.2, PR1, and PR2 (Iniguez et al., 2005; Schikora et al., 2008). Among the factors contributing to recognition of Salmonella by the host plant are bacterial PAMPs such as flagellin. A Salmonella mutant lacking both flagellin genes is able to hypercolonize A. thaliana, indicating that Salmonella flagellar components are specifically recognized and induce plant defences (Iniguez et al., 2005). However, components of the Salmonella SPI1 also appear to be recognized by host plants. For example, deletion of spaS, which encodes the structural subunit of the T3SS SPI-1 apparatus, and sipB, an effector protein and translocator, resulted in increased colonization of alfalfa (Medicago sativa) roots and wheat (Triticum aestivum) seedlings (Iniguez et al., 2005). By contrast, mutations in SPI-1 and SPI-2 have been shown to reduce bacterial proliferation in A. thaliana leaves, suggesting a role for T3Es in suppression of plant defence responses (Schikora et al., 2011). It is currently not known whether factors of the SPI2 are also recognized during endophytic colonization, but our data clearly indicate that the SPI2-encoded T3E SseF is sensed by the plant immune system when directly delivered into the plant cell or by a T3SS from a heterologous bacterium. However, no visible signs of HR-like symptom development were observed when leaves of N. benthamiana were infected with Salmonella. This suggests that either translocation of T3Es from the bacterium into the plant cell does not efficiently occur or that a response to SseF is suppressed by the action of other T3Es. The latter assumption would be in accordance with previous studies, demonstrating that Salmonella is able to suppress early immune responses in tobacco and also appears to suppress HR-like symptom development when infiltrated into tobacco leaves (Shirron & Yaron, 2011).
Further studies will need to address the question of to what extent type III secretion of effector proteins into plant cells, either from SPI1 or from SPI2, actually occurs during endophytic growth and how this contributes to the interaction.
In conclusion, our results demonstrate that plants constitute a useful experimental system in which to functionally analyse T3Es from mammalian pathogenic bacteria such as Salmonella. The use of model organisms as surrogate hosts has increasingly been recognized as an attractive strategy to study the function, localization, and host target of bacterial effectors (Alegado et al., 2003; Botham et al., 2008). In particular, the budding yeast Saccharomyces cerevisiae has successfully been used to analyse T3Es from a range of plant and animal pathogens, such as Shigella flexneri, Yersinia pestis, S. enterica, Legionella trachomatis, and P. syringae (Munkvold et al., 2008; Siggers & Lesser, 2008). Yeast growth inhibition as a result of the expression of bacterial proteins was employed as a sensitive and specific indicator of the activity of effector proteins that perturb conserved cellular processes. Although yeast is a genetically tractable organism that is easily accessible to manipulation, it does not encode an immune system. Thus, plants might offer another way in which to analyse effector function for diverse pathogens, as defence responses provide an additional read-out of effector action. In the present study we have focused our analyses on immune responses triggered by SseF and thus on its avirulence function. In order to also test possible virulence functions of Salmonella T3Es in plants, we will in future extend our investigations to the other Salmonella T3Es that showed expression upon agro-infiltration (e.g. SopD2, PipB2, SopB, SseG, and SseL) for their ability to interfere with basal defence responses during PTI (Nguyen et al., 2010; Shirron & Yaron, 2011).
In addition, the presence of R proteins in plants that can recognize Salmonella T3Es may potentially be exploited to breed plants for higher resistance to endophytic growth of Salmonella and thus help to reduce internal leaf contamination with this important human pathogen.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 796; Reprogramming of host cells by microbial effectors). We are also grateful to Hildegard Voll for excellent technical assistance and Prof. U. Bonas for providing Xcv strains.