Salmonella virulence in animals depends on effectors injected by Type III Secretion Systems (T3SSs). In this report we demonstrate that Salmonella mutants that are unable to deliver effectors are also compromised in infection of Arabidopsis thaliana plants. Transcriptome analysis revealed that in contrast to wild type bacteria, T3SS mutants of Salmonella are compromised in suppressing highly conserved Arabidopsis genes that play a prominent role during Salmonella infection of animals. We also found that Salmonella originating from infected plants are equally virulent for human cells and mice. These results indicate a high degree of conservation in the defense and infection mechanism of animal and plant hosts during Salmonella infection.
Citation: Schikora A, Virlogeux-Payant I, Bueso E, Garcia AV, Nilau T, Charrier A, et al. (2011) Conservation of Salmonella Infection Mechanisms in Plants and Animals. PLoS ONE6(9): e24112. https://doi.org/10.1371/journal.pone.0024112
Editor: Michael Hensel, University of Osnabrueck, Germany
Received: April 27, 2011; Accepted: July 31, 2011; Published: September 6, 2011
Copyright: © 2011 Schikora et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: AS was supported by the CNRS post-doc fellowship. AC was supported by the grant from Ecole Doctorale Des Genomes Aux Organismes (G.A.O) at University Evry-Val d'Essone. EB was founded by Spanish Ministry of Science and Technology. AVG was founded by the INRA-ANR Project ERASYSBIO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Different serotypes of Salmonella, together with Escherichia coli O157:H7 and Campylobacter spp. are the most prominent causes of food poisoning worldwide . Disease develops after consumption of contaminated food and the severity of symptoms correlates with the ability of Salmonella to enter epithelial cells of the intestine . Although a typical infection leads to self-limiting gastroenteritis, in susceptible hosts Salmonella causes systemic infections by invading spleen, liver and other organs. Studies of the infection mechanisms in animals have shown that Salmonella actively remodels the host cell's physiology and architecture, and suppresses the host immune system by injecting a cocktail of effectors delivered by Type III Secretion System (T3SS). Salmonella enterica subsp. enterica ser. Typhimurium (S. Typhimurium) has two distinct T3SSs, T3SS-1 and T3SS-2, encoded by the Salmonella Pathogenicity Islands (SPI) SPI-1 and SPI-2 respectively , . T3SS-1 secretes at least 14 proteins of which 6 were shown to interact with the host signaling cascades and the cytoskeleton. T3SS-2 secretes at least 19 Salmonella enterica specific effector proteins that are involved in survival and multiplication within the Salmonella containing vesicle (SCV) , . Some of the effectors can be translocated by both T3SSs, reviewed in .
Until recently, little was known about the infection mechanisms of Salmonella in the plant kingdom . Nonetheless, studies revealed that 25% of food poisoning outbreaks in the US could be associated with the consumption of contaminated vegetables or fruits . Generally, it was believed that Salmonella rather survives on or in plant tissues after accidental contact with contaminated water or animal products. However, a growing body of evidence points to an active process in which Salmonella infects plant organs and uses them as a viable host , , , , , , , , , . Besides Salmonella, other human-pathogenic bacteria can infect and cause severe diseases on different plant species. Staphylococcus aureus, Klebsiella pneumoniae and E. coli O157:H7 are able to proliferate on/in plants , . Furthermore, the Gram-positive Listeria monocytogenes was shown to grow and persist on Arabidopsis plants , , .
Salmonellosis linked to contaminated vegetables raises the question whether similar mechanisms are used in animal and plant infection. Saggers et al. suggest that Salmonella actively attaches to plant tissues for successful colonization . A large screen identified 20 out of 6000 Salmonella serotype Newport mutants with lower attachment ability to alfalfa sprouts . Interestingly, some of the identified genes also play central roles in the pathogenicity toward animals (e.g. rpoS and agfD). In another study, two previously uncharacterized genes (STM0278 and STM0650) were characterized as important factors for the infection of alfalfa sprouts, due to their essential role in biofilm formation and swarming . Light-dependent chemotaxis was shown to be involved in the internalization of Salmonella to lettuce leaves via open stomata . The same group reported recently that internalization of Salmonella depends on the host plant . On the other hand, the plant immune system seems to play an important role in preventing Salmonella infection. Arabidopsis npr1 mutant, which is impaired in salicylic acid (SA) signaling, or plants over-expressing the bacterial NahG gene encoding the SA-metabolizing enzyme salicylate hydroxylase, are more susceptible to Salmonella infection . Additionally, mutations in either mpk6 or mkk3 render plants more susceptible toward Salmonella, pointing to the important role of MAPK signaling cascades . Interestingly, the response to Salmonella seems to depend on the bacterial serotype. Bacterial strains belonging to the serogroup E4 (O:1, 3, 19) were reported to induce chlorosis and wilting in Arabidopsis .
In order to analyze the Salmonella infection mechanism of plants and animals we considered three topics. First, because different physiological conditions exist in plant tissues and in warm-blooded vertebrates, we asked whether plant-originated Salmonella retain their virulence toward animals. Second, we wondered whether T3SS-dependent delivery of effectors, necessary for Salmonella infection, is of equal importance in plants as in animals? Third, we questioned whether the plant genes that are employed in response to Salmonella infection are different from those of animals?
Proliferation in plants does not alter the virulence for mammalian cells
We examined the virulence potential of Salmonella enterica subsp. enterica ser. Typhimurium strain 14028s (S. Typhimurium) extracted from infected Arabidopsis thaliana leaves on the human enterocyte cell line Caco-2  (ATCC HTB-37). Bacteria were collected from plants 2 days after infection and immediately used to infect Caco-2 cells with different multiplicity of infection (moi) (Supplementary Fig. S1A). The internalization of S. Typhimurium into Caco-2 cells (2 h time point) was similar for bacteria originating from plants or the LB medium (2×102 or 5×102 for plant and LB medium, respectively) (Fig. 1A). Similar colony forming unit (cfu) numbers were recovered after 4 h of incubation. However, we observed a strong increase in the number of cfu inside Caco-2 cells at 20 hours post infection (hpi): 1,7×103 for LB- and 4,4×103 for plant-grown bacteria. Statistical analysis of variance in the recovered populations using F-test suggests that the rates at which plant- and LB-originated bacteria proliferate in epithelial cells are comparable (p≥0.05) (Fig. 1A, Supplementary Fig. S1B).
A; Infection and proliferation of plant-grown S. Typhimurium in Caco-2 epithelial cells. Caco-2 cells were infected for 1 h with bacteria originating from LB medium or Arabidopsis (plant), then washed and incubated for an additional 2, 4 or 20 h in the presence of gentamicin. Bacteria were harvested from lysed epithelial cells. Bacterial colony forming unit (cfu) numbers were normalized to the multiplicity of infection (moi) number; number of bacteria used for infection divided by the number of epithelial cells. Variance analysis of cfu numbers was performed using the ANOVA F-test. No differences between plant and LB grown bacteria were found (p>0.05). B–D; Mice infection assays. C57BL/6 mice were infected with wild-type S. Typhimurium 14028 s. Systemic infection in spleens was verified 3 and 6 days post infection (dpi) (B) or 4 dpi (C). B; Spleen infection in mice populations fed with Arabidopsis leaves (dark gray) is similar to infection in control population (black). C; Systemic infection with plant-originated Salmonella. Mice were infected via gastric injection and fed with Arabidopsis leaves (dark grey), fed with an infected leaves homogenate (gray) or fed with whole infected leaf (light gray). D; Survival of mice: fed with an infected Arabidopsis leaf (stars), fed with homogenate from infected leaves (triangles), infected with TSB-grown bacteria and fed with an Arabidopsis leaf (squares) or fed with non infected Arabidopsis leaf (diamonds). n = 10 mice in each group, observations were carried out for 21 dpi.
Arabidopsis-grown Salmonella infect mice
In order to test whether Salmonella bacteria proliferating inside of plants are pathogenic to animals, we infected A. thaliana leaves with S. Typhimurium and fed these leaves to C57BL/6 mice. Systemic infection was then followed by examination of the Salmonella cfu in spleens.
First, it was necessary to determine whether the consumption of Arabidopsis leaves would affect the infection process. Therefore, cfu numbers in spleens were compared between individuals, which were fed or not fed with Arabidopsis leaves, before oral inoculation with Salmonella grown on TSB medium. As shown in Figure 1B, no significant differences between these two cases were observed at 3 or 6 days post infection (dpi) (Fig. 1B).
In a second step the virulence of plant-grown Salmonella was assessed. For this purpose, Salmonella were allowed to grow in planta for two days before leaf discs were collected. Different variations of the infection protocol were then compared with each other: (i) mice feeding on intact non-infected leaf discs and subsequent gastric injection of TSB-grown Salmonella (“gastric injection+leaf” group); (ii) mice inoculated via gastric infection of a homogenate prepared from Salmonella-infected leaf discs (“infected leaf homogenate” group); or (iii) mice feeding on intact Salmonella-infected leaf discs (“infected leaf” group). Salmonella cfu in spleens were calculated at 4 dpi (Fig. 1C). All tested infection variants were successful and resulted in systemic infection. No significant differences were observed between the groups receiving gastric injection of Salmonella grown on TSB medium (gastric injection+ leaf group) or extracted from leaves (infected leaf homogenate group) (107 and 2×106 cfu/g spleen, respectively) (Fig. 1C). Similarly, feeding the mice with infected leaf discs caused systemic infection (Fig. 1C). However, the cfu recovered from the spleens of mice that were fed with intact Salmonella-infected leaf discs were much lower (103 cfu/g spleen), indicating that bacterial infection of mice by this route is less effective (Fig. 1C, Supplementary Fig. S2).
To further analyze this system, the lethality of mice infected with Salmonella via Arabidopsis feeding was observed during 21 dpi (Fig. 1D). Mice fed only with uninfected Arabidopsis leaves (“leaf”) survived the entire 21 days of observation. In contrast, mice from the “gastric injection+leaf” or “infected leaf homogenate” groups did not survive past 9 dpi, confirming our previous observations on systemic infection. In the third group of mice fed with intact Salmonella-infected leaf discs (“infected leaf” group), a considerable number of animals survived 21 dpi and showed no symptoms of salmonellosis (70% survival rate) (Fig. 1D).
Salmonella virulence for plants depends on T3SS-dependent delivery of effectors
For infection of animal cells, S. Typhimurium uses two different T3SSs, encoded by SPI-1 and SPI-2 , , . To investigate whether Salmonella require these secretion apparatuses to infect plants, we tested the in planta performance of Salmonella mutants that are unable to assemble a functional T3SS. In the first set of experiments, we chose prgH− (encoded by SPI-1) and ssaV− (encoded by SPI-2) isogenic mutants of virulent S. Typhimurium 14028s , , , . Leaves of A. thaliana were syringe-infiltrated with Salmonella. The number of cfu was determined during 3 dpi from leaf discs of infiltrated leaves. When compared to wild type S. Typhimurium 14028s, both tested mutants (prgH− and ssaV−) showed reduced proliferation in plants (Fig. 2A, B). In order to verify these results, we tested an independent pair of mutants: invA− (encoded by SPI-1) and ssaJ− (encoded by SPI-2) , . Mutants in invA and ssaJ were constructed using the Lambda-Red recombination system  and tested for their proliferation in planta. Similar to the prgH− and ssaV− mutants, the proliferation rate of invA− and ssaJ− in plant are lower (Fig. 2A, B). To test the possibility that the lower proliferation of bacterial mutants is a result of different multiplication rates, bacteria were grown in LB medium. No differences between the growth rates of wild type and the four tested mutants were observed (Supplementary Fig. S3A–C).
Four-week old Arabidopsis plants were syringe-infiltrated with bacterial solutions at OD600 = 0.1 of wild-type S. Typhimurium 14028 s or T3SS mutants. A; In planta proliferation of mutants in structural elements of SPI-1 encoded T3SS. B; In planta proliferation of mutants in structural proteins of SPI-2 encoded T3SS. Leaf discs (0.7 cm3) were harvested at 1–72 hours post infection (hpi). Serial dilutions were plated for cfu determination. * represents p<0.05. C; Symptoms on Arabidopsis leaves infiltrated with Salmonella mutants. Leaves were infiltrated with bacterial solution at OD600 = 0.1, photos were taken 2 days after infiltration. D; Quantification of symptoms provoked by infiltration with T3SS mutants or wild type 14028 s bacteria. Analysis is performed with a color-based algorithm as described in .
Furthermore, in addition to the lower proliferation rates observed in planta, symptoms caused by the prgH− and ssaV− mutants are more apparent in Arabidopsis plants (Fig. 2C, D). The hypersensitive response (HR) is an induced, localized cell death, which limits the spread of pathogens. HR is repressed by successful biotrophic and semi-biotrophic pathogens. Therefore, the enhanced symptoms caused by T3SS mutants may suggest the inability of Salmonella mutants to suppress an HR, due to the lack of an intact secretion system enabling the bacteria to inject their effector repertoire. However, activation of Mitogen-Activated Protein Kinase 6 (AtMPK6) similarly activated in response to wild type 14028 s and T3SS mutants (Supplementary Fig. S3D) suggests that HR-suppressing Salmonella effectors act downstream or independently of MAPK activation.
Plants respond to Salmonella attack by the induction of defense genes
To study the responses of plants to Salmonella attack in more detail, we performed a global transcriptome analysis of 14-day-old A. thaliana plants infected with S. Typhimurium 14028 s for 2 and 24 hours post infection (hpi). In response to S. Typhimurium 14028 s challenge, at 2 hpi Arabidopsis shows differential expression of 249 genes (Bonferroni p-value<5%), of which 226 were up- and 23 down-regulated (Fig. 3A). Among the 249 Salmonella-responsive genes, the largest group encodes kinases and phosphatases (24 genes), followed by genes encoding transcription factors and reactive oxygen species (ROS)-responsive proteins (18 and 16 genes, respectively) (Fig. 3B, Supplementary Data Set S1). In addition, a significant number of ubiquitin ligases and different receptors are differentially expressed upon Salmonella treatment (Supplementary Data Set S1). In the second step, we compared Arabidopsis responses to the 14028 s strain at 2 and 24 hpi. From the 249 genes differentially expressed at 2 hpi, 44% (114 genes) are similarly modified at 24 hpi (Fig. 3A). Moreover, 1204 additional genes were up- or down-regulated at this latter time point, suggesting a major transcriptional reprogramming in response to Salmonella infection (Fig. 3A). The function of a great number of genes differentially expressed at both time points is unknown. Among the up-regulated genes at 24 hpi with known or predicted function, the biggest group encodes for plasma membrane associated proteins (14.2%, corrected p = 2.7E-3), and the most abundant encoded protein domain is the protein kinase core domain (5.9% of up-regulated genes, corrected p = 4.6E-1). This is in line with the calculated enrichment factors (DAVID Bioinformatics Recourses 6.7 , ); the statistically most overrepresented groups are genes involved in response to different stresses and signaling (enrichment factors of 5.2 and 3.9), as well as cell wall associated genes (enrichment factor 3.86) (Fig. 3B–C). Remarkably, the biggest groups of differentially expressed genes at 24 hpi are down-regulated genes encoding for proteins involved in protein synthesis (enrichment factor of 105.46, corrected p<1E-150) and RNA synthesis (enrichment factor of 12.62, corrected p<1E-17) (Fig. 3B–C).
Fourteen-day old in vitro-grown Arabidopsis seedlings were placed in MS/2 liquid medium and inoculated with bacteria. Transcriptional analysis was performed at 2 or 24 h after inoculation using CATMA chips. Two biological replicates were hybridized and each hybridization was repeated with a dye-swap. Genes were considered to be differentially expressed only if they showed the same pattern in biological replicates, dye-swap and had p values<0.05. For a complete list of differentially expressed genes see Supp. Data Set S1. A; Overlap between genes differentially expressed at 2 and at 24 hours of infection. B–C; Functional GO Term groups of genes differentially expressed at 2 h (B) and at 24 h (C) after infection. D–E; Transcriptional analysis of plant responses to different bacteria. Fourteen-day old Arabidopsis seedlings were infected with S. Typhimurium strain 14028 s, E. coli DH5α or P. syringae DC3000 for 2 h. D; up-regulated genes. E; down-regulated genes.
Transcriptional response to Salmonella includes genes common in response to pathogenic and non-pathogenic bacteria, as well as a set of Salmonella-specific genes
To better understand the differences between the interaction of plants with Salmonella and other bacteria, we compared the global transcriptome response of Arabidopsis thaliana Col-0 upon S. Typhimurium 14028 s challenge, to infection with Pseudomonas syringae pv. tomato strain DC3000 (P. syringae) as a representative plant pathogen and E. coli K12 strain DH5α (E. coli) as a non-pathogen. The transcriptional analysis was performed on 14-day old plants at 2 hpi. As shown above, in response to S. Typhimurium 14028 s challenge, Arabidopsis shows differential expression of 249 genes (Fig. 3D, E, Supplementary Data Set S1). A total of 283 genes respond to E. coli (270 up- and 13 down-regulated), whereas 2283 genes responded to treatment with P. syringae (1358 up- and 925 down-regulated) (Fig. 3D–E). In this experimental setup, 24 genes were induced and 11 genes were down regulated specifically in response to Salmonella (Fig. 3D–E). These genes consist of F-box proteins, an LRR receptor and many cytoskeleton- and ER-associated proteins (Supplementary Data Set S1). The transcriptional regulation of cytoskeleton and ER proteins is particularly interesting, as it seems to reflect the physiology of Salmonella as an intracellular pathogen and its impact on the host cell.
A strong overlap of 164 differentially expressed plant genes is seen upon challenge with any of the three bacteria (Fig. 3D–E, Supplementary Data Set S1), probably reflecting a basic plant response to the presence of bacteria and including known pathogen-responsive genes, such as the transcription factors WRKY22, WRKY33, WRKY40, WRKY48, WRKY53 and bZIP5, bZIP60 , , ,  and a number of protein kinases and phosphatases (Supplementary Data Set S1).
These findings show that Arabidopsis reacts to Salmonella attack with the induction of a large standard set of defense genes that respond similarly to pathogenic and non-pathogenic bacteria, but also that Arabidopsis responds to Salmonella infection by differential regulation of a specific group of genes.
The prgH− mutant is unable to suppress plant immune responses
In order to test the hypothesis, that T3SS-delivered effectors might modify the plant immune response, we analyzed the global transcriptome response of Arabidopsis to infection with the Salmonella prgH− mutant in comparison to S. Typhimurium 14028 s wild type, at 24 hpi. Of the more than 1300 (14028 s) or 1600 (prgH−) differentially expressed genes, 14028 s and the prgH− commonly regulate a group of 944 genes (Fig. 4A). However, a group of 649 genes appears to be specifically regulated upon infection with prgH− (Fig. 4A, Table 1). GO term enrichment analysis (AmiGO version 1,7)  of these 649 prgH−-specific genes revealed an overrepresentation of genes related to responses to biotic stress, relations with other organisms and defense mechanisms (Supplementary Data Set S1). We used the MapMan algorithm , which clusters differentially regulated genes to known physiological pathways or functional groups, to better characterize those genes. The majority of the prgH−-specific genes cluster with pathways related to pathogen responses and ubiquitin-mediated protein degradation (Fig. 4B and Supplementary Fig. S4). Interestingly, this group includes BAK1, BIK1, WRKY18 and WRKY33, EIN3, PR4 and PUB23, all of which are marker genes that are up-regulated upon pathogen infections (Table 1).
A; Comparison of transcriptional profiles of Arabidopsis plants after infection with 14028 s wild-type strain or the prgH− Salmonella mutant. The overlap between 14028 s- and prgH−-regulated genes subsists of 944 genes. 649 (33% of all differentially expressed) genes are specifically regulated by prgH− mutant. B; MapMan  -based representation of the biggest group within the 649 prgH− specific genes: response to biotic stress. Graphic represents functional groups in which prgH− upregulated genes were identified. Each red square represent one upregulated gene in particular pathway or group. For the entire list of genes up-regulated during infection with prgH− mutant see Supp. Data Set S1.
A more general analysis of the transcriptional profiles shows that more than 2000 genes have higher expression levels after infection with prgH− than upon infection with 14028 s wild type, although the vast majority of these genes lies only close to the statistical significance level (p<0.05; Supplementary Data Set S1). Enrichment analysis (DAVID) ,  of these genes classifies the up-regulated genes into cell wall, defense response and WRKY clusters. On the other hand, the down-regulated genes fall into the protein synthesis cluster (Supplementary Fig. S4D). Genes with the highest differences in expression levels between infection with wild type and prgH− mutant, encode for protease inhibitors, extensins, glutathione transferases, Ca2+-binding, wound-responsive and LRR proteins as well as chitinase and PR4 (Supplementary Tab. S2).
Salmonella from plants retain virulence toward animals
Using epithelial cells and in vivo mice assays, we demonstrate in this report, that Salmonella originating from Arabidopsis leaves are as virulent as Salmonella grown in standard media (Fig. 1). When using Salmonella-infected leaf homogenates as a source for bacteria, no differences in Salmonella infection rates in cells or in mice were observed when compared with Salmonella grown in vitro, suggesting that proliferation in planta has no impact on Salmonella virulence for animals. Interestingly, whole leaves were clearly less infective than when Salmonella were derived from homogenized leaves in the feeding experiments with mice (Fig. 1C–D). Although several factors could be responsible for this phenomenon, a likely explanation is that the incomplete chewing and gastric/intestinal extraction of Salmonella from leaf tissues by mice may lower infection levels by hindering Salmonella access to the mouse intestinal epithelial target cells. These findings have important consequences for food protection and risk assessment associated with the consumption of Salmonella-contaminated plant products. The fact that Salmonella extracted from leaves show similar virulence levels to animal cells and mice as bacteria grown on standard media, suggests that Salmonella infected plant material possesses a considerable risk in food security and processing. The fact that Salmonella and other Enterobacteriaceae can actively use plants as hosts  strongly suggests that plants might represent a much larger reservoir of animal pathogens than so far estimated.
T3SSs are required for virulence for animals and plants
Although it is accepted that Salmonella can infect plants and animals, it is not known whether the bacteria use different mechanisms for different hosts. Recent reports showed that Salmonella actively attach to plant tissues . Among 20 mutants with lower attachment ability to alfalfa sprouts identified by Barak et al, some carry mutations in genes playing central roles in the pathogenicity toward animals (e.g. rpoS and agfD) . Swarming and the formation of biofilms, both important factors in pathogenicity, also play a crucial role in the infection of alfalfa sprouts . Previous studies showed that Salmonella actively access the interior of plant tissues using stomata as natural openings, but also root hairs and trichomes as points of infection , , , . In order to answer the question whether Salmonella relies on intact T3SSs for infection of plants we used two different types of T3SS mutants that are unable to inject effector proteins into host cells and are therefore not virulent for animal hosts , . Although these mutant strains had normal proliferation rates when grown in LB medium, the proliferation of these mutants in plants were strongly reduced (Fig. 2), indicating that both SPI-1- and SPI-2-encoded apparatuses are necessary to establish robust infection. Moreover, symptoms caused by these mutants were more pronounced (Fig. 2C, D), suggesting that plants can react to Salmonella infection with an enhanced HR rate, and/or that T3SS mutants are unable to restrain the induced HR response. Comparable results were presented very recently after tobacco infection with the wild type S. Typhimurium strain . Wild type bacteria, but not the T3SS mutant invA−, were able to suppress the oxidative burst and the increase of extracellular pH after inoculation. Consequently the authors concluded that Salmonella actively suppresses plant defense mechanisms using the SPI-1 encoded T3SS . Comparison of transcriptome responses to infection with Salmonella wild type and prgH− mutant, revealed a large number of genes with higher expression levels upon infection with the prgH− mutant (Fig. 4A, Supplementary Table S1, S2). The fact that this up-regulation is observed only during infection with the prgH− mutant but not with wild type Salmonella may reflect the ability of wild type bacteria to repress the plant defense machinery (Fig. 4). Although a direct search for bacterial effectors with known targets in plants like AvrPto, AvrPtoB, HopF2 or TALs, did not reveal homologous sequences in the Salmonella genome, such functions might be performed by yet uncharacterized effectors. Moreover, the situation may vary between plant species. Iniguez et al. (2005) showed enhanced growth of the T3SS Salmonella spa− mutant in Medicago sativa roots. Additionally, distinct Salmonella enterica serotypes might have different pathogenicity toward plants. Klerks et al. reported differential growth of five S. enterica serotypes in lettuce . Interestingly, the authors pointed out that lettuce cultivars vary in their resistance/susceptibility toward Salmonella infection . Similar results were reported by Barak et al. (2011), showing diverse resistance/susceptible phenotypes against Salmonella in different tomato cultivars . Also internalization into plant leaves seems to be plant species-specific. Lettuce and arugula were recently reported to be easily colonized by Salmonella, while parsley and tomato were significantly more resistant against internalization of bacteria into their leaves .
Taken together, these observations suggest that Salmonella uses T3SS-delivered effector proteins to suppress the plant immune system. How Salmonella achieves the delivery of effectors across plant cell walls and plant plasma membranes remains unclear. However, numerous phytopathogenic bacteria (like Pseudomonas, Erwinia and Xanthomonas spp.) deliver their effectors via T3SSs, indicating that the plant cell wall is not a sufficient barrier to prevent bacteria from effector delivery . T3SSs present in animal and plant pathogens are broadly conserved but show some degree of specialization in the extracellular machinery due to the adaptation to different host cell barriers. The strategy, used by Salmonella to overcome the mechanical and chemical barriers of plants remains to be clarified. Furthermore, the transcription of T3SSs is generally induced by specific signals coming from their animal and plant hosts and therefore another open question is how the transcriptional induction of SPI-1 and SPI-2 operons occurs during infection of host plants. The T3SS of Pseudomonas syringae is induced when the bacteria reaches the leaf mesophyll by plant-cell derived small and soluble compounds , . Whether these compounds play a similar role in the induction of Salmonella SPIs or whether other molecules are involved, needs to be investigated in the future.
Functional conservation of genes implicated in Salmonella infection of animals and plants
Our previous report and this work demonstrates that Arabidopsis reacts to Salmonella attack by inducing plant defense responses  (Fig. 3). Transcriptome analysis 2 h after the infection revealed 249 Salmonella-regulated genes of which 12% (34 genes) respond exclusively to Salmonella. Several of the 34 Salmonella-specific responsive genes encode for proteins whose functions are expected to be involved in the intracellular part of the life cycle of Salmonella (ER quality control, ubiquitination, ROS homeostasis and a cytoplasmic TIR-NBS-LRR protein) (Supplementary Data Set S1). BiP2 and BiP3 (up-regulated upon Salmonella infection) are chaperones present in the lumen of the ER and are thought to play a role in the secretion of PR proteins and the assembly of the ERF receptor , . E3 ubiquitin ligases are essential factors in plant immunity and 6 members of this gene family are specifically up-regulated upon Salmonella challenge . Finally, the TIR-NBS-LRR family includes receptors (R-genes) for bacterial effectors (e.g. RPS2) that activate effector-triggered immunity (ETI). Interestingly, 66% of Salmonella-responsive genes react also to treatment with P. syringae and E. coli (Fig. 3D–E). This list includes known PAMP-responsive genes as the WRKY transcription factors WRKY22, WRKY33 and WRKY53. A very similar reaction was observed after infection with the pathogenic E. coli strain O157:H7 , suggesting that the regulation of these genes represents a general PAMP-triggered response to bacteria.
Overall, our results suggest that Salmonella employs the same T3SS machinery for delivering effectors to succumb plants as well as animals as hosts. Several plant genes that become induced upon Salmonella infection encode highly conserved factors that were found to play essential roles during animal infection. Future studies will reveal whether these factors play similar roles in the infection cycle of plants. The conserved nature of the Salmonella infection strategy and the host response mechanisms of plants and animals could underlie the ability of Salmonella to transit between animal and plant species and should be of more than pure academic interest to future studies.
Materials and Methods
Animal experiments in this study were carried out in strict accordance with the French recommendations (number 2001-131 from 4.02.2001 and number 2001-464 from 29.05.2001). The protocol for this study was approved (N° CL2008-16) by the Ethics Regional Committee for Animal Research “Comité Régional d'Ethique pour l'expérimentation Animale” Centre Limousin, that is recognized by the French Ministry for Research and Education. All efforts were made to minimize suffering.
Epithelial cells assay
Wild type Salmonella enterica subsp. enterica ser. Typhimurium strain 14028 s was grown either in planta for two days, or in LB medium until early logarithmic phase. Bacteria were harvested and resuspended in PBS for infection. Human Caco-2 cells  (ATCC number HTB-37) were infected with different multiplicity of infection (moi) for 1 h at 37°C. Cells were subsequently washed in PBS supplemented with 100 µg/mL gentamicin and incubated in growth medium (with 10 µg/mL gentamicin) for an additional 2, 4 or 20 h at 37°C. Cells were lysed in 0.5% Triton X-100 and adequate dilutions were plated on LB plates in order to monitor the intracellular Salmonella population. Calculations were made on the basis of cfu recovered from lysed Caco-2 cells. cfu numbers were normalized to either moi number; number of bacteria used for infection divided by the number of epithelial cells or to bacterial input population, as input we defined the number of bacteria recovered after 2 h post infection. Statistical analysis was performed using the Analysis of Variance (ANOVA) F-test. For detailed descriptions, see Supplementary Material and Methods S1.
Mice infection assay
Eight-week-old C57BL/6 mice were orally inoculated with approximately 5×105 cfu of S. Typhimurium 14028 s provided in four different ways; (i) animals were inoculated by gavage with 0.2 mL of an inoculum, (ii) a piece of a healthy A. thaliana leaf (0.6 cm2) was given to each animal to eat after which animals were orally inoculated as the first group of mice, (iii) a piece of a leaf infected with S. Typhimurium 14028 (0.6 cm2) was provided as food to each animal, (iv) mice were inoculated by gavage with a homogenate of infected A. thaliana leaves. Spleen colonization was estimated at day 3 and 6 or day 4 post-inoculation. The course of survival was recorded for 21 days following inoculation. Animal care and handling were conducted in accordance with institutional guidelines. For detailed descriptions, see Supplementary Material and Methods S1.
In order to asses the Salmonella proliferation rate in plants, soil-grown, 4-week old Arabidopsis thaliana Col-0 plants were infiltrated with wild type S. Typhimurium strain 14028 s or prgH−, invA−, ssaV− and ssaJ− isogenic mutants, using syringe infiltration. Bacteria were grown until early log phase in LB medium, washed and resuspended in 10 mM MgCl2. Infiltration solution was adjusted to OD600 = 0.1, (1.7×108 bacteria/ml). Bacterial population was monitored during 3 days post infiltration as described in . The lesions were calculated as percentage of diseased regions on the base of whole leaf area using the “Salmonella Analyzer” software  based on color-differences detecting algorithm, 5 leaves per treatment were used for calculation, experiment was repeated 4 times.
For transcriptome analysis 14-day old Arabidopsis thaliana seedlings were submerged in sterile MS/2 medium without sucrose overnight at 24°C prior to bacterial treatment. Salmonella Typhimurium 14028 s or its prgH− mutant were grown until early log phase and washed with 10 mM MgCl2. Infections were performed by inoculation of the MS/2 media with bacteria at a final OD600 = 0.1 for 2 and 24 h. Treatments with E. coli strain DH5α and virulent Pseudomonas syringae DC3000 pathovar tomato (Pst) were performed in similar manner, for 2 hours.
Total RNA was extracted from A. thaliana Col-0 plants; 2 or 24 hours after the infection with bacteria, amplified and fluorochemically stained as described in . Two biological repetitions were performed. Hybridizations to CATMA chips ,  were repeated using the dye-swap technique. Transcriptome data were analyzed using the algorithm developed for CATMA chips, as described in . Expression levels were compared to a mock treatment (10 mM MgCl2). For detailed descriptions, see Supplementary Material and Methods S1.
Microarray data from this article were deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), accession Nb.: GSE20996, GSE23790 and GSE23791, and at CATdb (http://urgv.evry.inra.fr/CATdb/; Projects: Au07-07_Salmonella and AU10-06_Salmonella_EduardoB) according to the “Minimum Information About a Microarray Experiment” standards.
A: Multiplicity of infection (moi) used to infect Caco-2 cells. Bacteria were recovered from Arabidopsis leaves (squares) or LB medium (diamonds) and cfu numbers were calculated. Because of the experimental design, moi numbers (cfu number/Caco-2 cells) have been calculated only post factum from serial dilutions of bacterial solution used to infect epithelial cells. Bacteria recovered from plants or LB medium were used immediately. In the calculations of differences in proliferation between bacteria originated from plants and LB medium, only experiments where moi numbers were comparable between the two groups were taken into account. B: Infection and proliferation of plant-grown S. Typhimurium 14028 s in Caco-2 epithelial cells. Caco-2 cells were infected for 1 h with bacteria originating from LB or plants, then washed and incubated for an additional 2, 4 or 20 h in the presence of gentamicin (10 µg/mL). Bacteria were harvested from lysed epithelial cells and serial dilutions plated on LB agar. Bacterial cfu recovered from Caco-2 cells after 2 h incubation was used for normalization (bacterial invasion). In contrast to the normalization on moi base (Fig. 1A), this graph presents the ability of Salmonella to proliferate within Caco-2 cells. It is very striking that the plant-originated bacteria proliferate at a higher rate than those originated from LB medium. C–E: Localization of GFP-expressing S. Typhimurium grown in plant in the pre-nuclear region of an epithelial cell 20 h post infection. Caco-2 cells were fixed and labeled with rhodamine-phalloidin to visualize actin filaments. Observations were done using CLSM. C; Rhodamine channel; Ex 543, Em LP 590. D; GFP channel; Ex: 488, Em: BP 515–530. E; overlay. Arrows show GFP-expressing Salmonella, * nucleus.
Bacterial input used in mice infection experiments. S. Typhimurium 14028 s was infiltrated and allowed to grow in Arabidopsis leaves for two days. 0.6 cm2 leaf discs from infected leaves were cut off and provided as food directly (infected leaves) or pooled together (25 discs) and homogenized in PBS (leaf homogenate). Homogenate was force-fed to mice. cfu numbers present in different leaves and in homogenate were calculated on a base of serial dilutions made from discs cut out from the same leaf (infected leaves) or an aliquot of the prepared disc homogenate (leaf homogenate). Dilutions were prepared in sterile water and plated on LB agar plates.
A: Proliferation rates of Salmonella mutants in LB standard medium. 14028 s wild type and prgH−, invA−, ssaV−, ssaJ− isogenic mutants were grown in liquid LB medium at 37°C. Optical density (600 nm) was measured every 30 min. B: Duplication time was calculated on the base of logarithmic section of growing curves. C: Student's t-test results. Compared were the growing rates of the wild type 14028 s strain with growing rates of mutant strains, all strains have similar duplication times. C: MPK6 activity upon infection with the wild-type 14028 s Salmonella strain, or the prgH− and ssaV− mutants. Two weeks old A. thaliana seedlings were treated with either 10 mM MgCl2 or S. Typhimurium wild type or mutants for 20 min. Endogenous MPK6 was immunoprecipitated from total protein extraction. Myelin basic protein (MBP) was used as substrate to test the activity of MPK6 (activity). Protein amounts were detected by Western blotting with antibodies specific for AtMPK6 (MPK6).
A: k-means cluster analysis of the 1956 differentially expressed genes upon infection with wild type 14028 s Salmonella and the prgH− mutant. Expression levels 24 h after infection with prgH− or 14028 s wild type was calculated on the base of CATMA array hybridization. Each infection was compared independently to mock treatment (10 mM MgCl2). As differentially expressed genes we regarded genes with p value<5% (Bonferroni method). B–C: MapMan representations of the genes up regulated under infection with prgH− mutant in the ubiquitin-dependent protein degradation pathway. 47 genes are represented. RING and E2 ligases form the second largest cluster. Expression levels 24 h after infection with prgH− or 14028 s wild type was calculated on the base of CATMA array hybridization. Each infection was compared independently to mock treatment (10 mM MgCl2). Differentially expressed genes are presented in Fig. 4. The entire list of differentially expressed genes is presented in Supplementary Data Set S1. D: Enrichment of genes with higher expression levels 24 hours after infection with prgH− mutant compared to the response to infection with 14028 s wild type. GO Term analysis and clustering was done with the help of DAVID Bioinformatic Resources 6.7 at the National Institute of Allergy and Infectious Diseases (NIAID).
Complete lists of genes differentially expressed after infection with bacteria. Fourteen-day old Arabidopsis Col-0 seedlings were placed in MS/2 medium over night and then medium was inoculated with Salmonella Typhimurium strain 14028 s wild-type, E. coli DH5α, Pseudomonas syringae DC3000 for 2 hours or with 14028 s Salmonella wild type or its prgH− isogenic mutant for 24 hours. Transcriptome analysis was carried out using CATMA chips as described in . Two biological replicates and a dye swap repetition were used as a base for statistical analysis. Genes with the same patter in all hybridization events and with a p value≤0.05 were taken into account.
AmiGO enrichment analysis of genes up regulated exclusively during infection with prgH− mutant (http://www.arabidopsis.org/tools/bulk/go/index.jsp).
List of genes with the highest difference in expression levels between infections with prgH− mutant and wild-type Salmonella. Table represents log ratios between the mock treatments and the treatment with either prgH− mutant or wild-type Salmonella, and the difference in those two ratios (log prgH-log 14028 s ratio colon) calculated on the base of CATMA microarray analysis. A difference of 1 means that the expression level upon prgH− infection is 2 times higher than upon infection with 14028 s. Value of 1 = log2 1 difference (2× higher expression) between prgH− and 14028 s treatments.
The authors would like to thank Prof. David Holden for providing the Salmonella prgH− and ssaV− mutants. Zaid Pirzada, Alessandro Carreri and Stephanie Pateyron for their technical support.
Conceived and designed the experiments: AS IVP TN HH. Performed the experiments: AS IVP EB SP TN PM. Analyzed the data: AS IVP AVG SP TN. Contributed reagents/materials/analysis tools: MB. Wrote the paper: AS AVG IVP PV HH.
- 1. Fisher IS, Threlfall EJ (2005) The Enter-net and Salm-gene databases of foodborne bacterial pathogens that cause human infections in Europe and beyond: an international collaboration in surveillance and the development of intervention strategies. Epidemiol Infect 133: 1–7.IS FisherEJ Threlfall2005The Enter-net and Salm-gene databases of foodborne bacterial pathogens that cause human infections in Europe and beyond: an international collaboration in surveillance and the development of intervention strategies.Epidemiol Infect13317
- 2. Patel JC, Rossanese OW, Galan JE (2005) The functional interface between Salmonella and its host cell: opportunities for therapeutic intervention. Trends Pharmacol Sci 26: 564–570.JC PatelOW RossaneseJE Galan2005The functional interface between Salmonella and its host cell: opportunities for therapeutic intervention.Trends Pharmacol Sci26564570
- 3. Collazo CM, Galan JE (1997) The invasion-associated type-III protein secretion system in Salmonella. Gene 192: 51–59.CM CollazoJE Galan1997The invasion-associated type-III protein secretion system in Salmonella.Gene1925159
- 4. Hensel M (2000) Salmonella pathogenicity island 2. Mol Microbiol 36: 1015–1023.M. Hensel2000Salmonella pathogenicity island 2.Mol Microbiol3610151023
- 5. Kuhle V, Hensel M (2004) Cellular microbiology of intracellular Salmonella enterica: functions of the type III secretion system encoded by Salmonella pathogenicity island 2. Cell Mol Life Sci 61: 2812–2826.V. KuhleM. Hensel2004Cellular microbiology of intracellular Salmonella enterica: functions of the type III secretion system encoded by Salmonella pathogenicity island 2.Cell Mol Life Sci6128122826
- 6. Waterman SR, Holden DW (2003) Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cell Microbiol 5: 501–511.SR WatermanDW Holden2003Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system.Cell Microbiol5501511
- 7. McGhie EJ, Brawn LC, Hume PJ, Humphreys D, Koronakis V (2009) Salmonella takes control: effector-driven manipulation of the host. Curr Opin Microbiol 12: 117–124.EJ McGhieLC BrawnPJ HumeD. HumphreysV. Koronakis2009Salmonella takes control: effector-driven manipulation of the host.Curr Opin Microbiol12117124
- 8. Holden N, Pritchard L, Toth I (2009) Colonization outwith the colon: plants as an alternative environmental reservoir for human pathogenic enterobacteria. FEMS Microbiol Rev 33: 689–703.N. HoldenL. PritchardI. Toth2009Colonization outwith the colon: plants as an alternative environmental reservoir for human pathogenic enterobacteria.FEMS Microbiol Rev33689703
- 9. Rangel JM, Sparling PH, Crowe C, Griffin PM, Swerdlow DL (2005) Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002. Emerg Infect Dis 11: 603–609.JM RangelPH SparlingC. CrowePM GriffinDL Swerdlow2005Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002.Emerg Infect Dis11603609
- 10. Barak JD, Gorski L, Naraghi-Arani P, Charkowski AO (2005) Salmonella enterica virulence genes are required for bacterial attachment to plant tissue. Appl Environ Microbiol 71: 5685–5691.JD BarakL. GorskiP. Naraghi-AraniAO Charkowski2005Salmonella enterica virulence genes are required for bacterial attachment to plant tissue.Appl Environ Microbiol7156855691
- 11. Barak JD, Kramer LC, Hao LY (2011) Colonization of tomato plants by Salmonella enterica is cultivar dependent, and type 1 trichomes are preferred colonization sites. Appl Environ Microbiol 77: 498–504.JD BarakLC KramerLY Hao2011Colonization of tomato plants by Salmonella enterica is cultivar dependent, and type 1 trichomes are preferred colonization sites.Appl Environ Microbiol77498504
- 12. Berger CN, Brown DJ, Shaw RK, Minuzzi F, Feys B, et al. (2011) Salmonella enterica strains belonging to O serogroup 1,3,19 induce chlorosis and wilting of Arabidopsis thaliana leaves. Environ Microbiol. CN BergerDJ BrownRK ShawF. MinuzziB. Feys2011Salmonella enterica strains belonging to O serogroup 1,3,19 induce chlorosis and wilting of Arabidopsis thaliana leaves.Environ Microbiol
- 13. Dong Y, Iniguez AL, Ahmer BM, Triplett EW (2003) Kinetics and strain specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of Medicago sativa and Medicago truncatula. Appl Environ Microbiol 69: 1783–1790.Y. DongAL IniguezBM AhmerEW Triplett2003Kinetics and strain specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of Medicago sativa and Medicago truncatula.Appl Environ Microbiol6917831790
- 14. Golberg D, Kroupitski Y, Belausov E, Pinto R, Sela S (2011) Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs. Int J Food Microbiol 145: 250–257.D. GolbergY. KroupitskiE. BelausovR. PintoS. Sela2011Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs.Int J Food Microbiol145250257
- 15. Iniguez AL, Dong Y, Carter HD, Ahmer BM, Stone JM, et al. (2005) Regulation of enteric endophytic bacterial colonization by plant defenses. Mol Plant Microbe Interact 18: 169–178.AL IniguezY. DongHD CarterBM AhmerJM Stone2005Regulation of enteric endophytic bacterial colonization by plant defenses.Mol Plant Microbe Interact18169178
- 16. Kroupitski Y, Golberg D, Belausov E, Pinto R, Swartzberg D, et al. (2009) Internalization of Salmonella enterica in leaves is induced by light and involves chemotaxis and penetration through open stomata. Appl Environ Microbiol 75: 6076–6086.Y. KroupitskiD. GolbergE. BelausovR. PintoD. Swartzberg2009Internalization of Salmonella enterica in leaves is induced by light and involves chemotaxis and penetration through open stomata.Appl Environ Microbiol7560766086
- 17. Saggers EJ, Waspe CR, Parker ML, Waldron KW, Brocklehurst TF (2008) Salmonella must be viable in order to attach to the surface of prepared vegetable tissues. J Appl Microbiol 105: 1239–1245.EJ SaggersCR WaspeML ParkerKW WaldronTF Brocklehurst2008Salmonella must be viable in order to attach to the surface of prepared vegetable tissues.J Appl Microbiol10512391245
- 18. Schikora M, Schikora A, Kogel K-H, Koch W, Cremers D (2010) Probabilistic classification of disease symptoms caused by Salmonella on Arabidopsis plants. M. SchikoraA. SchikoraK-H KogelW. KochD. Cremers2010Probabilistic classification of disease symptoms caused by Salmonella on Arabidopsis plants.5th IEEE ISIF Workshop on Sensor Data Fusion: Trends, Solutions, Applications (SDF). Leipzig, Germany. 5th IEEE ISIF Workshop on Sensor Data Fusion: Trends, Solutions, Applications (SDF). Leipzig, Germany.
- 19. Shirron N, Yaron S (2011) Active suppression of early immune response in tobacco by the human pathogen Salmonella Typhimurium. PLoS One 6: e18855.N. ShirronS. Yaron2011Active suppression of early immune response in tobacco by the human pathogen Salmonella Typhimurium.PLoS One6e18855
- 20. Prithiviraj B, Bais HP, Jha AK, Vivanco JM (2005) Staphylococcus aureus pathogenicity on Arabidopsis thaliana is mediated either by a direct effect of salicylic acid on the pathogen or by SA-dependent, NPR1-independent host responses. Plant J 42: 417–432.B. PrithivirajHP BaisAK JhaJM Vivanco2005Staphylococcus aureus pathogenicity on Arabidopsis thaliana is mediated either by a direct effect of salicylic acid on the pathogen or by SA-dependent, NPR1-independent host responses.Plant J42417432
- 21. Milillo SR, Badamo JM, Boor KJ, Wiedmann M (2008) Growth and persistence of Listeria monocytogenes isolates on the plant model Arabidopsis thaliana. Food Microbiol 25: 698–704.SR MililloJM BadamoKJ BoorM. Wiedmann2008Growth and persistence of Listeria monocytogenes isolates on the plant model Arabidopsis thaliana.Food Microbiol25698704
- 22. Takeuchi K, Matute CM, Hassan AN, Frank JF (2000) Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens to lettuce leaves. J Food Prot 63: 1433–1437.K. TakeuchiCM MatuteAN HassanJF Frank2000Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens to lettuce leaves.J Food Prot6314331437
- 23. Lang MM, Harris LJ, Beuchat LR (2004) Survival and recovery of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on lettuce and parsley as affected by method of inoculation, time between inoculation and analysis, and treatment with chlorinated water. J Food Prot 67: 1092–1103.MM LangLJ HarrisLR Beuchat2004Survival and recovery of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on lettuce and parsley as affected by method of inoculation, time between inoculation and analysis, and treatment with chlorinated water.J Food Prot6710921103
- 24. Barak JD, Gorski L, Liang AS, Narm KE (2009) Previously uncharacterized Salmonella enterica genes required for swarming play a role in seedling colonization. Microbiology 155: 3701–3709.JD BarakL. GorskiAS LiangKE Narm2009Previously uncharacterized Salmonella enterica genes required for swarming play a role in seedling colonization.Microbiology15537013709
- 25. Schikora A, Carreri A, Charpentier E, Hirt H (2008) The dark side of the salad: Salmonella Typhimurium overcomes the innate immune response of Arabidopsis thaliana and shows an endopathogenic lifestyle. PLoS One 3: e2279.A. SchikoraA. CarreriE. CharpentierH. Hirt2008The dark side of the salad: Salmonella Typhimurium overcomes the innate immune response of Arabidopsis thaliana and shows an endopathogenic lifestyle.PLoS One3e2279
- 26. Xu X, Chen C, Fan B, Chen Z (2006) Physical and Functional Interactions between Pathogen-Induced Arabidopsis WRKY18, WRKY40, and WRKY60 Transcription Factors. The Plant Cell Online 18: 1310–1326.X. XuC. ChenB. FanZ. Chen2006Physical and Functional Interactions between Pathogen-Induced Arabidopsis WRKY18, WRKY40, and WRKY60 Transcription Factors.The Plant Cell Online1813101326
- 27. Lara-Tejero M, Galan JE (2009) Salmonella enterica serovar Typhimurium pathogenicity island 1-encoded type III secretion system translocases mediate intimate attachment to nonphagocytic cells. Infect Immun 77: 2635–2642.M. Lara-TejeroJE Galan2009Salmonella enterica serovar Typhimurium pathogenicity island 1-encoded type III secretion system translocases mediate intimate attachment to nonphagocytic cells.Infect Immun7726352642
- 28. Behlau I, Miller SI (1993) A PhoP-repressed gene promotes Salmonella Typhimurium invasion of epithelial cells. J Bacteriol 175: 4475–4484.I. BehlauSI Miller1993A PhoP-repressed gene promotes Salmonella Typhimurium invasion of epithelial cells.J Bacteriol17544754484
- 29. Hensel M, Shea JE, Raupach B, Monack D, Falkow S, et al. (1997) Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2. Mol Microbiol 24: 155–167.M. HenselJE SheaB. RaupachD. MonackS. Falkow1997Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2.Mol Microbiol24155167
- 30. Kubori T, Sukhan A, Aizawa SI, Galan JE (2000) Molecular characterization and assembly of the needle complex of the Salmonella Typhimurium type III protein secretion system. Proc Natl Acad Sci U S A 97: 10225–10230.T. KuboriA. SukhanSI AizawaJE Galan2000Molecular characterization and assembly of the needle complex of the Salmonella Typhimurium type III protein secretion system.Proc Natl Acad Sci U S A971022510230
- 31. Marlovits TC, Kubori T, Lara-Tejero M, Thomas D, Unger VM, et al. (2006) Assembly of the inner rod determines needle length in the type III secretion injectisome. Nature 441: 637–640.TC MarlovitsT. KuboriM. Lara-TejeroD. ThomasVM Unger2006Assembly of the inner rod determines needle length in the type III secretion injectisome.Nature441637640
- 32. Galan JE, Ginocchio C, Costeas P (1992) Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J Bacteriol 174: 4338–4349.JE GalanC. GinocchioP. Costeas1992Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family.J Bacteriol17443384349
- 33. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640–6645.KA DatsenkoBL Wanner2000One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc Natl Acad Sci U S A9766406645
- 34. Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, et al. (2003) DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4: P3.G. Dennis JrBT ShermanDA HosackJ. YangW. Gao2003DAVID: Database for Annotation, Visualization, and Integrated Discovery.Genome Biol4P3
- 35. Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44–57.W. Huang daBT ShermanRA Lempicki2009Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat Protoc44457
- 36. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, et al. (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415: 977–983.T. AsaiG. TenaJ. PlotnikovaMR WillmannWL Chiu2002MAP kinase signalling cascade in Arabidopsis innate immunity.Nature415977983
- 37. Lu D-P, Christopher D (2008) Endoplasmic reticulum stress activates the expression of a sub-group of protein disulfide isomerase genes and AtbZIP60 modulates the response in Arabidopsis thaliana. Molecular Genetics and Genomics 280: 199–210.D-P LuD. Christopher2008Endoplasmic reticulum stress activates the expression of a sub-group of protein disulfide isomerase genes and AtbZIP60 modulates the response in Arabidopsis thaliana.Molecular Genetics and Genomics280199210
- 38. Murray SL, Ingle RA, Petersen LN, Denby KJ (2007) Basal resistance against Pseudomonas syringae in Arabidopsis involves WRKY53 and a Protein with homology to a Nematode Resistance Protein. Molecular Plant-Microbe Interactions 20: 1431–1438.SL MurrayRA IngleLN PetersenKJ Denby2007Basal resistance against Pseudomonas syringae in Arabidopsis involves WRKY53 and a Protein with homology to a Nematode Resistance Protein.Molecular Plant-Microbe Interactions2014311438
- 39. Carbon S, Ireland A, Mungall CJ, Shu S, Marshall B, et al. (2009) AmiGO: online access to ontology and annotation data. Bioinformatics 25: 288–289.S. CarbonA. IrelandCJ MungallS. ShuB. Marshall2009AmiGO: online access to ontology and annotation data.Bioinformatics25288289
- 40. Thimm O, Blasing O, Gibon Y, Nagel A, Meyer S, et al. (2004) MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J 37: 914–939.O. ThimmO. BlasingY. GibonA. NagelS. Meyer2004MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes.Plant J37914939
- 41. Prithiviraj B, Weir T, Bais HP, Schweizer HP, Vivanco JM (2005) Plant models for animal pathogenesis. Cell Microbiol 7: 315–324.B. PrithivirajT. WeirHP BaisHP SchweizerJM Vivanco2005Plant models for animal pathogenesis.Cell Microbiol7315324
- 42. Klerks MM, Franz E, van Gent-Pelzer M, Zijlstra C, van Bruggen AH (2007) Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-microbe factors influencing the colonization efficiency. ISME J 1: 620–631.MM KlerksE. FranzM. van Gent-PelzerC. ZijlstraAH van Bruggen2007Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-microbe factors influencing the colonization efficiency.ISME J1620631
- 43. Alfano JR, Collmer A (2004) Type III secretion system effector proteins: double agents in bacterial disease and plant defense. Annu Rev Phytopathol 42: 385–414.JR AlfanoA. Collmer2004Type III secretion system effector proteins: double agents in bacterial disease and plant defense.Annu Rev Phytopathol42385414
- 44. Boureau T, Routtu J, Roine E, Taira S, Romantschuk M (2002) Localization of hrpA-induced Pseudomonas syringae pv. tomato DC3000 in infected tomato leaves. Mol Plant Pathol 3: 451–460.T. BoureauJ. RouttuE. RoineS. TairaM. Romantschuk2002Localization of hrpA-induced Pseudomonas syringae pv. tomato DC3000 in infected tomato leaves.Mol Plant Pathol3451460
- 45. Haapalainen M, van Gestel K, Pirhonen M, Taira S (2009) Soluble plant cell signals induce the expression of the type III secretion system of Pseudomonas syringae and upregulate the production of pilus protein HrpA. Mol Plant Microbe Interact 22: 282–290.M. HaapalainenK. van GestelM. PirhonenS. Taira2009Soluble plant cell signals induce the expression of the type III secretion system of Pseudomonas syringae and upregulate the production of pilus protein HrpA.Mol Plant Microbe Interact22282290
- 46. Wang D, Weaver ND, Kesarwani M, Dong X (2005) Induction of protein secretory pathway is required for systemic acquired resistance. Science 308: 1036–1040.D. WangND WeaverM. KesarwaniX. Dong2005Induction of protein secretory pathway is required for systemic acquired resistance.Science30810361040
- 47. Li J, Zhao-Hui C, Batoux M, Nekrasov V, Roux M, et al. (2009) Specific ER quality control components required for biogenesis of the plant innate immune receptor EFR. Proc Natl Acad Sci U S A 106: 15973–15978.J. LiC. Zhao-HuiM. BatouxV. NekrasovM. Roux2009Specific ER quality control components required for biogenesis of the plant innate immune receptor EFR.Proc Natl Acad Sci U S A1061597315978
- 48. Craig A, Ewan R, Mesmar J, Gudipati V, Sadanandom A (2009) E3 ubiquitin ligases and plant innate immunity. J Exp Bot 60: 1123–1132.A. CraigR. EwanJ. MesmarV. GudipatiA. Sadanandom2009E3 ubiquitin ligases and plant innate immunity.J Exp Bot6011231132
- 49. Thilmony R, Underwood W, He SY (2006) Genome-wide transcriptional analysis of the Arabidopsis thaliana interaction with the plant pathogen Pseudomonas syringae pv. tomato DC3000 and the human pathogen Escherichia coli O157:H7. Plant J 46: 34–53.R. ThilmonyW. UnderwoodSY He2006Genome-wide transcriptional analysis of the Arabidopsis thaliana interaction with the plant pathogen Pseudomonas syringae pv. tomato DC3000 and the human pathogen Escherichia coli O157:H7.Plant J463453
- 50. Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, et al. (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16: 2089–2103.C. LurinC. AndresS. AubourgM. BellaouiF. Bitton2004Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis.Plant Cell1620892103
- 51. Sclep G, Allemeersch J, Liechti R, De Meyer B, Beynon J, et al. (2007) CATMA, a comprehensive genome-scale resource for silencing and transcript profiling of Arabidopsis genes. BMC Bioinformatics 8: 400.G. SclepJ. AllemeerschR. LiechtiB. De MeyerJ. Beynon2007CATMA, a comprehensive genome-scale resource for silencing and transcript profiling of Arabidopsis genes.BMC Bioinformatics8400
- 52. Crowe ML, Serizet C, Thareau V, Aubourg S, Rouze P, et al. (2003) CATMA: a complete Arabidopsis GST database. Nucleic Acids Res 31: 156–158.ML CroweC. SerizetV. ThareauS. AubourgP. Rouze2003CATMA: a complete Arabidopsis GST database.Nucleic Acids Res31156158