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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.
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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.