01 Plant innate immunity against human bacterial pathogens
3 Maeli Melotto1 *, Shweta Panchal2, Debanjana Roy2
4 department of Plant Sciences, University of California, Davis, CA, USA
5 2Department of Biology, University of Texas, Arlington, TX, USA
8 Correspondence:
9 Dr. Maeli Melotto
10 University of California, Davis
11 Department of Plant Sciences
12 One Shields Avenue
13 Davis, CA 95616
14 melotto@ucdavis.edu
16 Number of words: 5,217
17 Number of figures: 2
Certain human bacterial pathogens such as the enterohemorrhagic Escherichia coli and Salmonella enterica are not proven to be plant pathogens yet. Nonetheless, under certain conditions they can survive on, penetrate into, and colonize internal plant tissues causing serious food borne disease outbreaks. In this review, we highlight current understanding on the molecular mechanisms of plant responses against human bacterial pathogens and discuss salient common and contrasting themes of plant interactions with phytopathogens or human pathogens.
Keywords: leafy vegetables, fresh produce, Salmonella enterica, Escherichia coli O157:H7, plant defense
INTRODUCTION
Bagged greens in the market are often labelled "pre-washed", "triple-washed" or "ready-to-eat", and look shiny and clean. But are they really "clean" of harmful microbes? We cannot be so sure. Food safety has been threatened by contamination with human pathogens including bacteria, viruses, and parasites. Between 2000 and 2008, norovirus and Salmonella spp. contributed to 58% and 11% of forborne illnesses, respectively in the United States (Scallan et al., 2011). In those same years, non-typhoidal Salmonella alone was ranked as the topmost bacterial pathogen contributing to hospitalizations (35%) and deaths (28%) (Scallan et al., 2011). In 2007, 235 outbreaks were associated with a single food commodity; out of which 17% was associated with poultry, 16% with beef, and 14% with leafy vegetables that also accounted for the most episodes of illnesses (CDC, 2010).
Apart from the direct effects on human health, enormous economic losses are incurred by contaminated food products recalls. The eight-day recall of spinach in 2006 cost $350 million to the US economy (Hussain and Dawson, 2013). It should be realized that this is not the loss of one individual, but several growers, workers, and distributors. This is a common scenario for any multistate foodborne outbreak. Additionally, the skepticism of the general public towards consumption of a particular food product can lead to deficiencies of an important food source from the diet. Less demand would in turn lead to losses for the food industry. Economic analysis shows that money spent on prevention of foodborne outbreak by producers is much less than the cost incurred after the outbreak (Ribera et al., 2012).
Contamination of plants can occur at any step of food chain while the food travels from farm to table. Both pre-harvest and post-harvest steps are prone to contamination. Contaminated irrigation water, farm workers with limited means of proper sanitation, and fecal contamination in the farm by animals can expose plants to human pathogens before harvest of the edible parts (Lynch et al., 2009; Barak and Schroeder, 2012). After harvest, contamination can occur during unclean modes of transportation, processing, and bagging (Lynch et al., 2009). Mechanical damage during transport can dramatically increase the population of human pathogens surviving on the surface of edible plants (Aruscavage et al., 2008). Control measures to decrease pathogen load on plant surfaces have been defined by the Food Safety Modernization Act (US Food and Drug Administration) and Hazard Analysis and Critical Control Point system (HACCP). Using chlorine for post-harvest crop handling has been approved by US Department of Agriculture
64 (USDA) under the National Organic Program. However, some studies indicated that internalized
65 human pathogens escape sanitization (Seo and Frank, 1999; Saldana et al., 2011). Thus,
66 understanding the biology of human pathogen-plant interactions is now crucial to prevent
67 pathogen colonization of and survival in/on plants, and to incorporate additional, complementing
68 measures to control food borne outbreaks.
70 We reasoned that as plants are recognized vectors for human pathogens, enhancing the plant
71 immune system against them creates a unique opportunity to disrupt the pathogen cycle. In this
72 cross-kingdom interaction, the physiology of both partners contribute to the outcome of the
73 interactions (i.e., colonization of plants or not). Bacterial factors important for interaction with
74 plants have been discussed in recent, comprehensive reviews (Tyler and Triplett, 2008; Teplitski
75 et al., 2009; Berger et al., 2010; Barak and Schroeder, 2012; Brandl et al., 2013). Plant factors
76 contributing to bacterial contamination (or lack of) is much less studied and discussed. In this
77 review, we highlight current knowledge on plants as vectors for human pathogens, the molecular
78 mechanisms of plant responses to human bacterial pathogens, and discuss common themes of
79 plant defenses induced by phytopathogens and human pathogens. We have focused on human
80 bacterial pathogens that are not recognized plant pathogens such as Salmonella enterica and
81 Escherichia coli (Barak and Schroeder, 2012; Meng et al., 2013), but yet are major threats to
82 food safety and human health.
84 PLANT SURFACE: THE FIRST BARRIER FOR BACTERIAL INVADERS
86 The leaf environment has long been considered to be a hostile environment for bacteria. The leaf
87 surface is exposed to rapidly fluctuating temperature and relative humidity, UV radiation,
88 fluctuating availability of moisture in the form of rain or dew, lack of nutrients, and
89 hydrophobicity (Lindow and Brandl, 2003). Such extreme fluctuations, for example within a
90 single day, are certainly not experienced by pathogens in animal and human gut. Thus, it is
91 tempting to speculate that animal pathogens may not even be able to survive and grow in an
92 environment as dynamic as the leaf surface. However, the high incidence of human pathogens
93 such as S. enterica and E. coli O157:H7 on fresh produce, sprouts, vegetables, leading to
94 foodborne illness outbreaks indicate a certain level of human pathogen fitness in/on the leaf.
96 The plant surface presents a barrier to bacterial invaders by the presence of wax, cuticle, cell
97 wall, trichomes, and stomata. All except stomata, present a passive defense system to prevent
98 internalization of bacteria. Nonetheless, several bacteria are able to survive on and penetrate
99 within the plant interior. The surface of just one leaf is a very large habitat for any bacteria. The
100 architecture of the leaf by itself is not uniform and provides areas of different environmental
101 conditions. There are bulges and troughs formed by veins, leaf hair or trichomes, stomata, and
102 hydathodes that form microsites for bacterial survival with increased water and nutrient
103 availability, as well as temperature and UV radiation protection (Leveau and Lindow, 2001;
104 Miller et al., 2001; Brandl and Amudson, 2008; Kroupitski et al., 2009; Barak et al., 2011).
105 Indeed, distinct microcolonies or aggregates of S. enterica were found on cilantro leaf surfaces in
106 the vein region (Brandl and Mandrell, 2002) In addition, preference to the abaxial side of lettuce
107 leaf by S. enterica may be is an important strategy for UV avoidance (Kroupitski et al., 2011).
108 Conversion of cells to viable but non-culturable (VNBC) state in E. coli O157:H7 on lettuce
109 leaves may also be a strategy to escape harsh environmental conditions (Dinu and Bach, 2011).
110 Hence, localization to favorable microsites, avoidance of harsh environments, and survival by
111 aggregation or conversion to non-culturable state may allow these human pathogens to survive
112 and at times multiply to great extent on the leaf surface.
114 As stomata are abundant natural pores in the plant epidermis which serve as entrance points
115 for bacteria to colonize the leaf interior (intercellular space, xylem, and phloem), several studies
116 addressed the question as to whether human bacterial pathogens could internalize leaves through
117 stomata. Populations of E. coli O157:H7 and S. enterica SL1344 in the Arabidopsis leaf apoplast
118 can be as large as four logs per cm2 of leaf after surface-inoculation under 60% relative humidity
119 (Roy et al., 2013) suggesting that these bacteria can and access the apoplast of intact leaves.
120 Several microscopy studies indicated association of pathogens on or near guard cells. For
121 instance, S. enterica serovar Typhimurium SL1344 was shown to internalize arugula and iceberg
122 lettuce through stomata and bacterial cells were located in the sub-stomatal space (Golberg et al.,
123 2011). However, no internalization of SL1344 was observed into parsley where most cells were
124 found on the leaf surface even though stomata were partially open (Golberg et al., 2011). Cells of
125 S. enterica serovar Typhimurium MAE110 (Gu et al., 2011), enteroaggregative E. coli (Berger et
126 al., 2009a), and E. coli O157:H7 (Saldana et al., 2011) were found to be associated with stomata
127 in tomato, arugula leaves, and baby spinach leaves, respectively. In the stem E. coli O157:H7
128 and Salmonella serovar Typhimurium were found to be associated with the hypocotyl and the
129 stem tissues including epidermis, cortex, vascular bundles, and pith when seedlings were
130 germinated from contaminated seeds (Deering et al., 2011a; Deering et al., 2011b).
132 The plant rhizosphere is also a complex habitat for microorganisms with different life styles
133 including plant beneficial symbionts and human pathogens. Nutritionally rich root exudate has
134 been documented to attract S. enterica to lettuce roots (Klerks et al., 2007a). Although bacteria
135 cannot directly penetrate through root cells, sites at the lateral root emergence and root cracks
136 provide ports of entry for S. enterica and E. coli O157:H7 into root tissues (Cooley et al., 2003;
137 Dong et al., 2003; Klerks et al., 2007b; Tyler and Triplett, 2008), and in some instances between
138 the epidermal cells (Klerks et al., 2007b). High colonization of S. enterica has been observed in
139 the root-shoot transition area (Klerks et al., 2007b). Once internalized both bacterial pathogens
140 have been found in the intercellular space of the root outer cortex of Medicago truncatula
141 (Jayaraman et al., 2014). Salmonella enterica was found in the parenchyma, endodermis,
142 pericycle, and vascular system of lettuce roots (Klerks et al., 2007b) and in the inner root cortex
143 of barley (Kutter et al., 2006). A detailed study on the localization of E. coli O157:H7 in live root
144 tissue demonstrated that this bacterium can colonize the plant cell wall, apoplast, and cytoplasm
145 (Wright et al., 2013). Intracellular localization of E. coli O157:H7 seems to be a rare event as
146 most of the microscopy-based studies show bacterial cells in the intercellular space only.
147 Bacterial translocation from roots to the phyllosphere may be by migration on the plant surface
148 in a flagellum-dependent manner (Cooley et al., 2003) or presumably through the vasculature
149 (Itoh et al., 1998; Solomon et al., 2002). The mechanism for internal movement of enteric
150 bacterial cells from the root cortex to the root vasculature through the endodermis and casparian
151 strips and movement from the roots to the phyllosphere through the vascular system is yet to be
152 demonstrated.
154 Several outbreaks of S. enterica have also been associated with fruits, especially tomatoes.
155 Salmonella enterica is unlikely to survive on surface of intact fruits (Wei et al., 1995) raising the
156 question: what are the routes for human pathogenic bacteria penetration into fruits? It has been
157 suggested that S. enterica can move from inoculated leaves (Barak et al., 2011), stems, and
158 flowers (Guo et al., 2001) to tomato fruits. However, the rate of internal contamination of fruits
159 was low (1.8%) when leaves were surface-infected with S. enterica (Gu et al., 2011). The
160 phloem has been suggested as the route of movement of bacteria to non-inoculated parts of the
161 plant as bacterial cells were detected in this tissue by microscopy (Gu et al., 2011). Figure 1
162 depicts the observed phyllosphere and rhizosphere niches colonized by bacteria in/on intact
163 plants and probable sources of contamination.
165 PERCEPTION OF HUMAN PATHOGENS BY THE PLANT IMMUNE SYSTEM
167 Plants possess a complex innate immune system to ward off microbial invaders (Jones and
168 Dangl, 2006). Plants are able to mount a generalized step-one response that is triggered by
169 modified/degraded plant products or conserved pathogen molecules. These molecules are known
170 as damage or pathogen associated molecular patterns (DAMP/PAMP). In many cases, conserved
171 PAMPs are components of cell walls and surface structures such as flagellin,
172 lipopolysaccharides, and chitin (Zeng et al., 2010). Examples of intracellular PAMPs exist such
173 as the elongation factor EF-Tu (Kunze et al., 2004). PAMPs are recognized by a diverse set of
174 plant extracellular receptors called pattern-recognition receptors (PRRs) that pass intracellular
175 signals launching an army of defense molecules to stop the invasion of the pathogens. This
176 branch of the immune system known as pathogen-triggered immunity (PTI) is the first line of
177 active defense against infection.
179 Human pathogen on plants (HPOP) is an emerging field that only recently has caught the
180 attention of plant biologists and phytopathologists. A few studies have been reported in the last
181 5-10 years, which focused on the most well studied PAMPs, flagellin and lipopolysaccharide
182 (LPS), in the interaction of human pathogens with plants. Table 1 lists the plants, bacterial
183 strains, and method details for such studies.
185 Flagellin perception
187 Flagellin, the structural component of flagellum in bacteria, is involved in bacterial attachment
188 and motility on the plant (Cooley et al., 2003), is recognized by plant through the FLS2 receptor
189 (Garcia et al., 2013), and induces plant defenses (Garcia et al., 2013; Meng et al., 2013). Similar
190 to the well-studied PTI elicitor flg22 (Felix et al., 1999), the flg22 epitope of S. enterica serovar
191 Typhimurium 14028 is also an effective PAMP and elicitor of downstream immune responses in
192 Arabidopsis (Garcia et al., 2013), tobacco, and tomato plants (Meng et al., 2013). Flagellum-
193 deficient mutants of S. enterica serovar Typhimurium 14028 are better colonizers of wheat,
194 alfalfa, and Arabidopsis roots as compared to the wild type bacterium (Iniguez et al., 2005)
195 further suggesting that the Salmonella flagellum induces plant defenses that may restrict bacterial
196 colonization of several plant organs. However, the Salmonella flg22 peptide is not the only
197 PAMP for elicitation of plant immune response as fls2 mutant of Arabidopsis still shows a low
198 level of PTI activation in response to this PAMP (Garcia et al., 2013).
200 Purified flagellin or derived epitopes of E. coli O157:H7 has not been used to induce plant
201 defenses. However, flagellum-deficient mutant of this strain does not activate the SA-dependent
202 BGL2 gene promoter as much as the wild type strain and shows larger population in Arabidopsis
203 than the wild type strain (Seo and Matthews, 2012) further suggesting that surface structures in
204 the bacterial cell are perceived by plants.
206 The differences in responses observed could be attributed to the presence of other microbial
207 signatures eliciting plant defense. Variations in plant response to S. enterica flagellin could be
208 owed to host-strain specificity as well. Although flagellin sequences from S. enterica strains and
209 other bacteria are highly conserved, even a minor change of five amino acids in the flg22 epitope
210 leads to reduced activation of PTI in Arabidopsis, tobacco, and tomato plants (Garcia et al.,
211 2013). Adding to the specificity, it has also been shown that Brassicaceae and Solanocecae plants
212 recognize specific flagellin (Robatzek et al., 2007; Clarke et al., 2013). Hence, evolving
213 variations in flagellin sequences could be a strategy employed by the pathogens to avoid plant
214 recognition, which in turn leads to the development of pathogen-specific immune responses in
215 the plant.
217 Flagella also play an important role in bacterial behavior on the plant. Several studies have
218 pointed out to the usefulness of flagella for attachment to leaf surfaces and movement on plant
219 surfaces (Berger et al., 2009a; Berger et al., 2009b; Saldana et al., 2011; Shaw et al., 2011;
220 Xicohtencatl-Cortes et al., 2009).
222 LPS perception
224 Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacterial pathogens
225 of animals and plants. In the animal host, LPS is a well-characterized PAMP that is recognized
226 by host Toll-like receptor 4 (de Jong et al., 2012). In plants however, receptors for LPS have not
227 been discovered yet. Nonetheless, current evidence suggests that human pathogen-derived LPS
228 can be perceived by plants resulting in PTI activation. For instance, on the leaf surface, purified
229 LPS from Pseudomonas aeruginosa, S. Minnesota R595, and E. coli O55:B5 induces strong
230 stomatal closure in Arabidopsis (Melotto et al., 2006). Purified LPS from Salmonella triggers of
231 ROS production and extracellular alkalinization in tobacco cell suspension (Shirron and Yaron,
232 2011) but not on tomato leaves (Meng et al., 2013) suggesting that LPS recognition may be
233 either dependent on experimental conditions or variable among plant species.
235 Genetic evidence suggests that the high activity of SA-dependent BGL2 gene promoter in
236 Arabidopsis is dependent on the presence of LPS in E. coli O157:H7 as higher activity of this
237 promoter was observed in the wild type bacterial as compared to its LPS mutant (Seo and
238 Matthews, 2012). However, LPS-dependent responses seem not to be sufficient to restrict
239 bacterial survival on plants as the population titer of E. coli O157:H7 LPS mutant or wild type in
240 plant is essentially the same (Seo and Matthews, 2012). Additionally, live S. Typhimurium cells
241 do not induce ROS in epidermal tissue of tobacco (Shirron and Yaron, 2011) suggesting that, at
242 least Salmonella, can suppress LPS-induced ROS and extracellular alkalinization.
244 Similar to flagellin, the O-antigen moiety of LPS is not only important for plant perception of
245 bacterial cells, but also for bacterial attachment, fitness, and survival on plants (Barak et al.,
246 2007; Berger et al., 2011; Marvasi et al., 2013).
248 Functional output of bacterium perception
250 One of the earliest PTI responses in plants is stomatal closure that greatly decreases the rate of
251 pathogen entry into plant's internal tissues. This response requires molecular components of PTI
252 including such as flagellin and LPS perception and hormone perception and signaling (Melotto et
253 al., 2006; Melotto et al., 2008; Zeng and He, 2010; Sawinski et al., 2013). Stomatal immunity is
254 also triggered by the presence of human pathogens S. enterica serovar Typhimurium SL1344 and
255 E. coli O157:H7 (Melotto et al., 2006; Kroupitski et al., 2009; Roy et al., 2013), albeit at various
256 levels. For instance, E. coli O157:H7 induces a strong stomatal immunity and Salmonella
257 SL1344 elicits only a transient stomatal closure in both Arabidopsis (Melotto et al., 2006; Roy et
258 al., 2013) and lettuce (Kroupitski et al., 2009; Roy et al., 2013) suggesting that the bacterial
259 strain SL1344 can either induce weaker or subvert stomata-based defense. Active suppression of
260 stomatal closure by SL1344 may be unlikely because it cannot re-open dark-closed stomata (Roy
261 et al., 2013). However, it is possible that signaling pathways underlying bacterium-triggered and
262 dark-induced stomatal closure are not entirely overlapping and SL1344 acts on immunity-
263 specific signaling to subvert stomatal closure.
265 PLANT INTRACELLULAR RESPONSE TO HUMAN PATHOGENS
267 Recognition of PAMPs by PRRs leads to several hallmark cellular defense responses that are
268 categorized based on the timing of response. Zipfel and Robatzek (2010) have discussed that
269 early responses occur within seconds to minutes of recognition including ion fluxes, extracellular
270 alkalinization, and oxidative burst. Intermediate responses occur within minutes to hours
271 including stomatal closure, ethylene production, mitogen-activated protein kinase (MAPK)
272 signaling, and transcriptional reprogramming. Late responses occur from hours to days and
273 involve callose deposition, salicylic acid accumulation, and defense gene transcription.
275 These hallmark plant cellular defenses have also been tested for both E. coli and S. enterica
276 (Figure 2). In particular, S. enterica infection results in the induction of MPK3/MPK6 kinase
277 activity and plant defense-associated genes PDF1.2, PR1, and PR2 in Arabidopsis leaves
278 (Schikora et al., 2008) as well as PR1, PR4, and PR5 in lettuce (Klerks et al., 2007b). MPK6
279 activation in Arabidopsis is independent of FLS2 (Schikora et al., 2008), indicating that flagellin
280 is not the only active PAMP of Salmonella and plant response to other PAMPs may converge at
281 MAPK signaling. Direct comparison of the PR1 gene expression in Arabidopsis indicated that
282 both E. coli O157:H7 and Salmonella SL1344 are able to induce this defense marker gene,
283 however at difference levels (Roy et al., 2013). The PR1 gene induction is low in SL1344-
284 infected plants indicating that immune responses are either weaker or are suppressed by
285 Salmonella.
287 A few studies (Table 1) have addressed the role of plant hormones in response to endophytic
288 colonization of human bacterial pathogens:
290 Ethylene signaling. The ethylene-insensitive mutant of Arabidopsis, ein2, supports higher
291 Salmonella 14028 inside whole seedlings as compared to the wild type Col-0 plants (Schikora et
292 al., 2008). Furthermore, addition of a specific inhibitor of ethylene mediated signaling, 1293 methylcyclopropene (1-MCP), to the growth medium resulted in increased S. enterica 14028
294 endophytic colonization of Medicago truncatula, but not M. sativum, roots and hypocotyls
295 (Iniguez et al., 2005) suggesting that the role of endogenous ethylene signaling maybe be
296 specific to each plant-bacterium interaction. However, ethylene signaling may play a contrasting
297 role during fruit contamination. Tomato mutants (rin and nor) with defects in ethylene synthesis,
298 perception, and signal transduction show significantly reduced Salmonella proliferation within
299 their fruits as compared to the wild type control (Marvasi et al., 2014).
301 Jasmonic acid. Similar to the ein2 mutant, the coronatine-insensitive mutant of Arabidopsis,
302 coi1-16, also supports high Salmonella 14028 inside whole seedlings (Schikora et al., 2008).
303 Along with the induction of the jasmonate-responsive gene PDF1.2 addressed in the same study
304 and mentioned above, it seems that jasmonate signaling is also an important component to
305 restrict Salmonella infection in, at least, Arabidopsis. These results are surprising as coi1 mutants
306 are well known to have increased resistant to various bacterial pathogen of plants, such as P.
307 syringae, but not to fungal or viral pathogens (Feys et al., 1994; Kloek et al., 2001).
309 Salicylic acid. Two genetic lines of Arabidopsis has been extensively used to determine the role
310 of salicylic acid (SA) in plant defenses against phytopathogens, the transgenic nahG plant that
311 cannot accumulate SA (Friedrich et al., 1995) and the null mutant npr1 that is disrupted in both
312 SA-dependent and -independent defense responses (Ton et al., 2002). Both of these plant lines
313 support higher populations of Salmonella 14028 inside their roots (Iniguez et al., 2005) and
314 seedlings (Schikora et al., 2008) as compared to the wild type plant. NPR1-dependent signaling
315 is important reduce the population of the curli-negative strain of E. coli O157:H7 43895 but not
316 for the curli-positive strain 86-24 in Arabidopsis leaves (Seo and Matthews, 2012). Although
317 only a few strains of Salmonella and E. coli have been used, there is an emerging patterns
318 suggesting that SA itself and activation of SA-signaling can potentially restrict HPOP.
320 In attempts to understand the overall cellular transcriptional response to human bacterial
321 pathogens, global transcriptomic analyses have been used. Thilmony et al. (2006) showed that E.
322 coli O157:H7 regulates PTI-associated genes in Arabidopsis leaves, albeit in a flagellin-
323 independent manner. A similar transcriptomic analysis with medium-grown Arabidopsis
324 seedlings 2h after inoculation with S. enterica serovar Typhimurium 14028, E. coli K-12, and P.
325 syringae pv. tomato DC3000 showed a strong overlap among genes responsive to each bacterial
326 infection suggesting a common mechanism of plant basal response towards bacteria (Schikora et
327 al., 2011). Gene expression analysis of Medicago truncatula seedlings root-inoculated with only
328 two bacterial cells per plant indicated that 83 gene probes (30-40% of each data set) were
329 commonly regulated in response to S. enterica and E. coli O157:H7 (Jayaraman et al., 2014). All
330 together, these studies indicate that each human pathogenic bacterium can modulate specific
331 plant genes beyond a basal defense response; however the mechanisms for plant-bacterium
332 specificity are largely unknown.
334 CAN HUMAN PATHOGENIC BACTERIA INDUCE ETI IN PLANT CELLS?
336 Successful virulent pathogens of plants are able to defeat this army plant defense by employing
337 its own set of artillery (such as the type three secretion system effectors and phytotoxins) and
338 cause disease in the host plant (Melotto and Kunkel, 2013; Xin and He, 2013). In incompatible
339 interactions (i.e., low bacterial colonization and no disease on leaves), the host plant already has
340 pre-evolved molecules (R proteins) that recognize these effectors and cause a specific defense
341 response to this pathogen. This specific response is called effector-triggered immunity (ETI).
342 Because the type 3-secretion system (T3SS) is important for the virulence of both animal and
343 plant pathogenic bacteria on their natural hosts as evidenced by the use of bacterial mutants, it is
344 reasonable to expect that T3SS would be important for HPOP as well. However, animal and
345 plant cell surfaces are structurally different; the plant cells wall seems to be impenetrable by the
346 secretion needle of the extracellular animal pathogens (Salmonella and E. coli) as discussed by
347 He et al. (2004) raising the question of how these effectors can reach the plant cytoplasm and
348 interfere with plant defenses. To date, there is no evidence for the ability of human pathogens to
349 inject T3SS effectors inside plant cells. It is possible that the T3SS is still active on the plant cell
350 surface and the effectors are secreted into the plant apoplast. If that is the case, however, plant
351 membrane receptors would be necessary to recognize the effectors and trigger plant cellular
352 responses. Nevertheless, it has been observed that the T3SS mutant of E. coli O157:H7, escN,
353 has reduced ability to attach to and colonize baby spinach leaves similar to the fliC mutant
354 (Saldana et al., 2011). Furthermore, apoplastic population of T3SS structural mutants of S.
355 enterica serovar Typhimurium 14028 (invA, prgH, ssaV, and ssaJ) is smaller than that of the
356 wild type bacterium in Arabidopsis leaves (Schikora et al., 2011) and plant defense-associated
357 genes are up-regulated for longer time by the prgH mutant than wild type Salmonella in
358 Arabidopsis seedlings (Garcia et al., 2013). Contrary to these findings, Iniguez et al. (2005)
359 reported that two Salmonella 14028 T3SS-SPI1, the structural mutant spaS and the effector
360 mutant sipB, hypercolonize roots and hypocotyls of M. sativum and fail to induce SA-dependent
361 PR1 promoter in Arabidopsis leaves. More studies need to be conducted to conclude whether
362 T3SS of Salmonella acts as "recognizable" surface structure similar to flagellum and/or as a
363 conduit to deliver effectors in plant tissues and trigger ETI. It is worth mentioning that T3SS and
364 effectors of the phytopathogen P. syringae pv. syringae have functions on ETI as well as
365 bacterial fitness on plant surface (Lee et al., 2012) and the filamentous T3SS protein EspA is
366 required for E. coli O157:H7 attachment to arugula leaves (Shaw et al., 2008).
368 The invA structural mutant, that is defective in all T3SS-1 system-associated phenotypes,
369 induces high ROS and extracellular alkalinizing in tobacco BY-2 cell suspension and
370 hypersensitive reaction (HR) in tobacco leaves as compared to the wild type strain (Shirron and
371 Yaron, 2011) suggesting that T3SS is important for this suppression of immunity. However,
372 Shirron and Yaron (2011) also reported that plant response to the regulatory mutant phoP that
373 modulates the expression of many effector proteins and membrane components (Dalebroux and
374 Miller, 2014), is no different to that of the wild type bacterium. These findings raised the
375 question whether the phenotypes observed are due to the T3SS structure itself or due to the
376 translocated effectors. A recent report shows that transient expression of the type three effector
377 of Salmonella 14028 SseF in tobacco plants elicits HR, and this response is dependent on the
378 SGT1 protein (Ustun et al., 2012). This study suggests that SseF can induce resistant-like
379 response in plants and requires resistance (R) protein signaling components. Ustun et al. (2012)
380 and Shirron and Yaron (2011) also showed that Salmonella 14028, which is able to deliver the
381 SseF effector, cannot induce HR or any disease-like symptoms in tobacco leaves. Thus, it
382 remains to be determined what would be the biological relevance of ETI in the Salmonella and
383 other human pathogenic bacteria in their interaction with plants in nature.
386 GENOTYPIC VARIABILITY IN PLANT SALMONELLA AND E. COLI INTERACTIONS
388 Although S. enterica and E. coli O157:H7 have not been traditionally known to be closely
389 associated with plants and modulate plant's physiology, the evidence tells us otherwise. An
390 arms-race evolution in both the human pathogen and the plant is therefore, expected. A few
391 studies (methodology details described in Table 1) have addressed whether genetic variability
392 among plant species or within the same plant species (i.e., cultivars, varieties, and ecotypes) can
393 be correlated with differential bacterial behavior and/or colonization of plants. Barak et al.
394 (2011) described that different tomato cultivars can harbor different levels of S. enterica
395 population after inoculation via water (sprinkler imitation) indicating plant factors may control
396 the ability of bacterial to colonize the phyllosphere. However, they also found that the cultivar
397 with the smallest S. enterica population also had the lowest number of speck lesions when
398 infected with the tomato pathogen Pst DC3000 (Barak et al., 2011), suggesting that strong basal
399 defense in this cultivar may account for low bacterial colonization. On a comparative study of S.
400 enterica contamination of several crop species, Barak et al. (2008) reported that seedlings from
401 Brassicaceae family have higher contamination than carrot, tomato, and lettuce when grown on
402 contaminated soil. Seedling contamination correlated with the Salmonella population in the
403 phyllosphere of all crop species, except tomato.
405 Golberg et al. (2011) reported variations in internalization of Salmonella SL1344 in different
406 leafy vegetables and fresh herbs using confocal microscopy. Internalization incidence (% of
407 microscopic fields containing bacterial cells) was high in iceberg lettuce and arugula, moderate
408 in romaine lettuce, red lettuce, basil, and low in parsley and tomato. Attraction to stomata was
409 seen in iceberg lettuce and basil, not in arugula, parsley, and tomato. Brandl and Amudson
410 (2008) reported that the age of romaine lettuce leaves is correlated with population size of E. coli
411 O157:H7 and S. enterica Thompson on leaves. Young leaves (inner) harbor greater number of
412 cells than middle aged leaves. These authors also observed that exudates on the surface of
413 younger leaves have higher nitrogen content than that of older leaves, which may contribute to
414 determining the bacterial population size on the leaf. Thus, it is tempting to speculate that the
415 genetic variability existent among plant genotypes regarding the chemical composition of their
416 organ exudates may be a determinant for human pathogen behavior (such as chemotaxis and
417 tropism towards stomata and roots) and ability to colonize plants.
419 Finally, Mitra et al. (2009) studied the effect of different methods of inoculation on
420 internalization and survival of E. coli O157:H7 in three cultivars of spinach. Among the organs
421 studied, the spinach phylloplane and the stem provided the most and least suitable niche for this
422 bacterium colonization, respectively. Although the leaf surface was the best "territory" for E.
423 coli, the leaf morphologies of each cultivar affected the ability of this bacterium to survive.
425 Collectively, all these studies point out that the plant genotype, age, leaf morphology,
426 chemical composition of exudates, and the primarily infected organ affect the outcome of
427 bacterial colonization of plants and the process may not be a generalized phenomenon,
428 consequently shaping specific human pathogen and plant interactions.
CONCLUDING REMARKS
The fundamental understanding of plant association with human bacterial pathogens that do not cause visual or macroscopic symptom in the plant, but yet are major food contaminants, are in its infancy. Both plant and bacterial factors are critical for these cross-kingdom interactions and emerging evidence suggests an overlap between plant molecular responses to human pathogens and phytopathogens. The future challenge will be to determine how these interactions differ. As this field of research is relatively new, we see differences in conclusions from different laboratories regarding multiplication versus decline in bacterial populations overtime and disease-like symptoms versus HR on inoculated plants. These differences are mainly associated with differences in methods of inoculation, bacterial strains, inoculum concentration, plant age, and plant cultivation methods (e.g., growth on medium, soil, or hydroponic solutions). Standard procedures for model systems, consensus, and collaborations must be developed among food scientists, microbiologists, plant pathologists, and molecular biologists to elucidate the specificity of each plant-bacterium interaction and avoid discrepancies in making general conclusions. A major point to be resolved is whether the observed plant defenses against Salmonella and its PAMPs are due to low recognition and/or active suppression. If Salmonella suppression of the plant immunity is a cause of weak defense responses, the major question becomes what is the responsible factor? This line of research might lead to a whole new paradigm that otherwise could not be revealed by only studying plant associations with its own natural pathogens.
ACKNOWLEDGMENTS
Work in our laboratory is supported by funding from the US National Institute of Allergy and Infectious Disease (R01AI068718).
REFERENCES
Aruscavage, D., Miller, S.A., Lewis Ivey, M.L., Lee, K., and LeJeune, J.T. (2008). Survival and dissemination of Escherichia coli O157:H7 on physically and biologically damaged lettuce plants. J. Food. Prot. 71, 2384-2388.
Barak, J.D., Jahn, C.E., Gibson, D.L., and Charkowski, A.O. (2007). The role of cellulose and O-antigen capsule in the colonization of plants by Salmonella enterica. Mol. Plant-Microbe Interact. 20, 1083-1091.
Barak, J.D., Liang, A., and Narm, K-E. (2008). Differential attachment and subsequent contamination of agricultural crops by Salmonella enterica. Appl. Environ. Microbiol. 74, 5568-5570.
Barak, J.D., Kramer, L.C., and Hao, L. (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.
Barak, J.D and Schroeder, B.K. (2012). Interrelationships of Food Safety and Plant Pathology: The Life Cycle of Human Pathogens on Plants. Annu. Rev. Phytopathol. 50, 241-266.
476 Berger, C.N., Shaw, R.K., Ruiz-Perez, F., Nataro, J.P., Henderson, I.R., Pallen, M.J., and
477 Frankel, G. (2009a). Interaction of enteroaggregative Escherichia coli with salad leaves.
478 Environ. Microbiol. Rep. 1, 234-239.
479 Berger, C.N., Shaw, R.K., Brown, D.J., Mather, H., Clare, S., Dougan, G., Pallen, M.J., and
480 Frankel, G. (2009b). Interaction of Salmonella enterica with basil and other salad leaves.
481 ISME J. 3, 261-265.
482 Berger, C.N., Sodha, S.V., Shaw, R.K., Griffin, P. M., Pink, D., Hand, P., and Frankel, G.
483 (2010). Fresh fruit and vegetable as vehicles for the transmission of human pathogens.
484 Environ. Microbiol. 12, 2385-2397.
485 Berger, C.N., Brown, D. J., Shaw, R.K., Minuzzi, F., Feys, B., and Frankel, G. (2011).
486 Salmonella enterica strains belonging to O serogroup 1,3,9 induce chlorosis and wilting of
487 Arabidopsis thaliana leaves. Environ. Microbiol. 13, 1299-1308.
488 Brandl, M.T., Cox, C.E., and Teplitski, M. (2013). Salmonella interactions with plants and their
489 associated microbiota. Phytopathology 103, 316-325.
490 Brandl, M.T., and Amundson, R. (2008). Leaf age as a risk factor in contamination of lettuce
491 with Escherichia coli O157:H7 and Salmonella enterica. Appl. Environ. Microbiol. 74, 2298492 2306.
493 Brandl, M.T., and Mandrell, R.E. (2002). Fitness of Salmonella enterica serovar Thompson in
494 the cilantro phyllosphere. Appl. Environ. Microbiol. 68, 3614-3621.
495 CDC 2010. Surveillance for foodborne disease outbreaks: United States, 2007. Morb. Mortal.
496 Wkly. Rep. 59, 973-1008.
497 Clarke, C.R., Chinchilla, D., Hind, S.R., Taguchi, F., Miki, R., Ichinose, Y., Martin, G.B.,
498 Leman, S., Felix, G., and Vinatzer, B.A. (2013). Allelic variation in two distinct
499 Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses
500 but not bacterial motility. New Phytol. 200, 847-680.
501 Cooley, M B., Miller, W. G., and Mandrell, R.E. (2003). Colonization of Arabidopsis thaliana
502 with Salmonella enterica and enterohemorrhagic Escherichia coli O157:H7 and competition
503 by Enterobacter asburiae. Appl. Environ. Microbiol. 69, 4915-4926.
504 Dalebroux, Z.D., and Miller, S.I. (2014). Salmonellae PhoPQ regulation of the outer membrane
505 to resist innate immunity. Curr. Opin. Microbiol. 17, 106-113.
506 de Jong, H.K., Parry, C.M., van der Poll, T., and Wiersinga, W.J. (2012). Host-pathogen
507 interaction in invasive Salmonellosis. PLoS Pathog. 8(10), e1002933. doi:
508 10.1371/journal.ppat.1002933.
509 Deering, A.J., Pruitt, R.E., Mauer. L.J., and Reuhs. B.L. (2011a). Identification of the cellular
510 location of internalized Escherichia coli O157:H7 in mung bean, Vigna radiata, using
511 immunocytochemical techniques. J. Food Prot. 74, 1224-1230.
512 Deering, A.J., Pruitt. R.E., Mauer. L.J., and Reuhs. B.L. (2011b). Examination of the
513 internalization of Salmonella serovar Typhimurium in peanut, Arachis hypogaea, using
514 immunocytochemical techniques. Food Res. Int. 45, 1037-1043.
515 Dinu, L. D., and Bach, S. (2011). Induction of viable but nonculturable Escherichia coli
516 O157:H7 in the phyllosphere of lettuce: a food safety risk factor. Appl. Environ. Microbiol.
517 77, 8295-8302.
518 Dong, Y., Iniguez, A. L., Ahmer, B. M., and Triplett, E. W. (2003). Kinetics and strain
519 specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of
520 Medicago sativa and Medicago truncatula. Appl. Environ. Microbiol. 69, 1783-1790.
521 Felix, G., Duran, J.D., Volko, S., and Boller, T. (1999). Plants have a sensitive perception system
522 for the most conserved domain of bacterial flagellin. Plant J. 18, 265-276.
523 Feys, B.J.F., Benedetti, C.E., Penfold, C.N., and Turner, J.G. (1994). Arabidopsis mutants
524 selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl
525 jasmonate, and resistant to a bacterial pathogen. Plant Cell. 6, 751-759.
526 Friedrich, L., Vernooij, B., Gaffney, T., Morse, A., and Ryals, J. (1995). Characterization of
527 tobacco plants expressing a bacterial salicylate hydroxylase gene. Plant Mol. Biol. 29, 959528 968.
529 Garcia, A. V., Charrier, A., Schikora, A., Bigeard, J., Pateyron, S., de Tauzia-Moreau, M., L.,
530 Evrard, A., Mithofer, A., Martin-Magniette, M.L., Virlogeux-Payant, I., and Hirt, H. (2013).
531 Salmonella enterica flagellin is recognized via FLS2 and activates PAMP-triggered 2
532 immunity in Arabidopsis thaliana. Mol. Plant. E-pub Nov. 6, 2013. doi:10.1093/mp/sst145.
533 Golberg, D., Kroupitski, Y., Belausov, E., Pinto, R., and Sela, S. (2011). Salmonella
534 Typhimurium internalization is variable in leafy vegetables and fresh herbs. Int. J. Food
535 Microbiol. 145, 250-257.
536 Gu, G., Hu, J., Cevallos-Cevallos, J.M., Richardson, S.M., Bartz, J.A., and van Bruggen, A.H. C.
537 (2011). Internal colonization of Salmonella enterica serovar Typhimurium in tomato plants.
538 PLoS ONE 6, e27340. doi: 10.1371/journal.pone.0027340.
539 Guo, X., Chen, J., Brackett, R.E., and Beuchat, L.R. (2001). Survival of Salmonellae on and in
540 tomato plants from the time of inoculation at flowering and early stages of fruit development
541 through fruit ripening. Appl. Environ. Microbiol. 67, 4760-4764.
542 He, S.Y., Nomura, K, and Whittam, T.S. (2004). Type III protein secretion mechanism in
543 mammalian and plant pathogens. Biochim. Biophys. Acta. 1694, 181-206.
544 Hussain, M.A., and Dawson, C.O. (2013). Economic impact of food safety outbreaks on food
545 businesses. Foods. 2, 585-589.
546 Iniguez, A.L., Dong, Y., Carter, H.D., Ahmer, B.M.M., Stone, J.M., and Triplett, E.W. (2005).
547 Regulation of enteric endophytic bacterial colonization by plant defenses. Mol. Plant-
548 Microbe Interact. 18,169-178.
549 Itoh, Y., Sugita-Konishi, Y., Kasuga, F., Iwaki, M., Hara-Kudo, Y., Saito, N., Noguchi, Y.,
550 Konuma, H., and Kumagai, S. (1998). Enterohemorrhagic Escherichia coli O157:H7 present
551 in radish sprouts. Appl. Environ. Microbiol. 64, 1532-1535.
552 Jayaraman, D., Valdes-Lopez, O., Kaspar, C.W., Ane, J-M (2014). Response of Medicago
553 truncatula Seedlings to Colonization by Salmonella enterica and Escherichia coli O157:H7.
554 PLoS ONE 9(2), e87970. doi:10.1371/journal.pone.0087970.
555 Jones, J.D.G. and Dangl, J.L. (2006). The plant immune system. Nature 444, 323-329.
556 Klerks, M.M., Franz, E., vanGent-Pelzer, M., Zijlstra, C., and van Brüggen, A.H.C. (2007a).
557 Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-
558 microbe factors influencing the colonization efficiency. ISME J. 1, 620-631.
559 Klerks, M.M., vanGent-Pelzer, M., Franz, E., Zijlstra, C., and van Bruggen, A.H.C. (2007b).
560 Physiological and molecular responses of Lactuca sativa to colonization by Salmonella
561 enterica serovar Dublin. Appl. Environ. Microbiol. 73, 4905-4914.
562 Kloek, A.P., Verbsky, M.L., Sharma, S.B., Schoelz, J.E., Vogel, J., Klessig, D.F., and Kunkel,
563 B.N. (2001). Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana
564 coronatine-insensitive (coil) mutation occurs through two distinct mechanisms. Plant J 26,
565 509-522.
566 Kroupitski, Y., Golberg, D., Belausov, E., Pinto, R., Swartzberg, D., Granot, D. and Sela, S.
567 (2009). Internalization of Salmonella enterica in leaves is induced by light and involves
568 chemotaxis and penetration through open stomata. Appl. Environ. Microbiol. 75, 6076-6086.
569 Kroupitski, Y., Pinto, R., Belausov, E., and Sela, S. (2011). Distribution of Salmonella
570 typhimurium in romaine lettuce leaves. Food Microbiol. 28, 990-997.
571 Kunze, G., Zipfel, C., Robatzek, S., Niehaus, K., Boller, T., and Felix, G. (2004). The N
572 terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants.
573 Plant Cell 16, 3496-3507.
574 Kutter, S., Hartmann, A., and Schmid, M. (2006). Colonization of barley (Hordeum vulgare)
575 with Salmonella enterica and Listeria spp. FEMSMicrobiol Ecol. 56, 262-271.
576 Lee, J., Teitzel, G.M., Munkvold, K., del Pozo, O., Martin, G.B., Michelmore, R.W., and
577 Greenberg, J.T. (2012). Type III secretion and effectors shape the survival and growth
578 pattern on Pseudomonas syringae on leaf surfaces. Plant Physiol. 158, 1803-1818.
579 Leveau, J.H., and Lindow, S.E. (2001). Appetite of an epiphyte: quantitative monitoring of
580 bacterial sugar consumption in the phyllosphere. Proc. Natl. Acad. Sci. USA. 98, 3446-3453.
581 Lindow, S. E., and Brandl, M.T. (2003). Microbiology of the phyllosphere. Appl. Environ.
582 Microbiol. 69, 1875-1883.
583 Lynch, M.F., Tauxe, R.V., and Hedberg, C.W. (2009). The growing burden of foodborne
584 outbreaks due to contaminated fresh produce: risks and opportunities. Epidemiol. Infect. 137,
585 307-315.
586 Marvasi, M., Cox, C.E., Xu, Y., Noel, J.T., Giovannoni, J.J., and Teplitski, M. (2013).
587 Differential regulation of Salmonella typhimurium genes involved in O-antigen capsule
588 production and their role in persistence within tomato fruit. Mol. Plant-Microbe Interact. 26,
589 793-800.
590 Marvasi, M., Noel, J.T., George, A.S., Farias, M.A., Jenkins, K.T., Hochmuth, G., Xu, Y.,
591 Giovanonni, J.J., Teplitski, M. (2014). Ethylene signaling affects susceptibility of tomatoes
592 to Salmonella. Microbial Biotechnology (accepted).
593 Melotto, M. and Kunkel, B.N. (2013). "Virulence strategies of plant pathogenic bacteria," in
594 The Prokaryotes - Prokaryotic Physiology and Biochemistry, 4th edition, eds. E. Rosenberg,
E. Stackebrand, E. F. DeLong, F. Thompson, S. Lory (Springer-Verlag, Berlin Heidelberg), 61-82.
Melotto, M., Underwood, W., and He, S. Y. (2008). Role of stomata in plant innate immunity and foliar bacterial diseases. Ann. Rev. Phytopathol. 46, 101-122.
Melotto, M., Underwood, W., Koczan, J., Nomura, K., and He, S. Y. (2006). Plant stomata function in innate immunity against bacterial invasion. Cell 126, 969-980.
Meng, F., Altier, C. and Martin, G.B. (2013). Salmonella colonization activates the plant immune system and benefits from association with plant pathogenic bacteria. Environ. Microbiol. 9, 2418-2430.
Miller, W. G., Brandl, M.T., Quiñones, B., and Lindow, S.E. (2001). Biological sensor for sucrose availability: relative sensitivities of various reporter genes. Appl. Environ. Microbiol. 67, 1308-1317.
Mitra, R., Cuesta-Alonso, E., Wayadande, A., Talley, J., Gilliland, S., and Fletcher, J. (2009). Effect of route of introduction and host cultivar on the colonization, internalization, and movement of the human pathogen Escherichia coli O157:H7 in spinach. J. Food Prot. 72, 1521-1530.
Ribera, L.A., Palma, M.A., Paggi, M., Knutson, R., Masabni, J.G., and Anciso, J. (2012). Economic analysis of food safety compliance costs and foodborne illness outbreaks in the United States. Hort. Technology 22, 150-156.
Robatzek, S., Bittel, P., Chinchilla, D., Köchner, P., Felix, G., Shiu, S.-H., and Boller, T. (2007). Molecular identification and characterization of the tomato flagellin receptor LeFLS2, an orthologue of Arabidopsis FLS2 exhibiting characteristically different perception specificities. Plant Mol. Biol. 64, 539-547.
Roy, D., Panchal, S. and Melotto, M. (2013). Escherichia coli O157:H7 induces stronger plant immunity than Salmonella enterica Typhimurium SL1344. Phytopathology 103:326-332.
Saldaña, Z., Sánchez, E., Xicohtencatl-Cortes, J., Puente, J.L., and Girón, J.A. (2011). Surface structures involved in plant stomata and leaf colonization by shiga-toxigenic Escherichia coli O157:H7. Front. Microbiol. 2, 119. doi: 10.3389/fmicb.2011.00119.
Sawinski K., Mersmann S., Robatzek, S., and Böhmer, M. (2013). Guarding the green: pathways to stomatal immunity. Mol. Plant-Microbe Interact. 26, 626-632.
Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M.A., Roy, S.L., Jones, J.L., and Griffin, P.M. (2011). Foodborne illness acquired in the United States--major pathogens. Emerg. Infect. Dis. 17, 7-15.
Schikora, A., Carreri, A., Charpentier, E., and 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. doi: 10.1371/journal.pone.0002279.
Schikora, A., Virlogeux-Payant, I., Bueso, E., Garcia, A.V., Nilau, T., Charrier, A., Pelletier, S., Menanteau, P., Baccarini, M., Velge, P., and Hirt, H. (2011). Conservation of Salmonella infection mechanisms in plants and animals. PLoS ONE 6, e24112. doi: 10.1371/journal.pone.0024112.
636 Seo, K.H., and Frank, J.F. (1999). Attachment of Escherichia coli O157:H7 to lettuce leaf
637 surface and bacterial viability in response to chlorine treatment as demonstrated by using
638 confocal scanning laser microscopy. J. Food Prot. 62, 3-9.
639 Seo, S., and Matthews, K.R. (2012). Influence of the plant defense response to Escherichia coli
640 O157:H7 cell surface structures on survival of that enteric pathogen on plant surfaces. Appl.
641 Environ. Microbiol. 78, 5882-5889.
642 Shaw, R. K., Berger, C. N., Feys, B., Knutton, S., Pallen, M. J., and Frankel, G. (2008).
643 Enterohemorrhagic Escherichia coli exploits EspA filaments for attachment to salad leaves.
644 Appl. Environ. Microbiol. 74, 2908-2914.
645 Shaw, R.K., Berger, C.N., Pallen, M.J., Sjöling, A., and Frankel, G. (2011). Flagella mediate
646 attachment of enterotoxigenic Escherichia coli to fresh salad leaves. Environ. Microbiol.
647 Rep. 3, 112-117.
648 Shirron, N., and Yaron, S. (2011). Active suppression of early immune response in tobacco by
649 the human pathogen Salmonella Typhimurium. PLoS ONE 6, e 1885 5. doi:
650 10.1371/journal.pone.0018855.
651 Solomon, E.B., Yaron, S., and Matthews, K.R. (2002). Transmission of Escherichia coli
652 O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its
653 subsequent internalization. Appl. Environ. Microbiol. 68, 397-400.
654 Teplitski, M., Barak, J.D., and Schneider, K.R. (2009). Human enteric pathogens in produce: un-
655 answered ecological questions with direct implications for food safety. Curr. Opin.
656 Biotechnol. 20,166-171.
657 Thilmony, R., Underwood, W., and He, S. Y. (2006). Genome-wide transcriptional analysis of
658 the Arabidopsis thaliana interaction with the plant pathogen Pseudomonas syringae pv.
659 tomato DC3000 and the human pathogen Escherichia coli O157:H7. Plant J. 46, 34-53.
660 Ton, J., De Vos, M., Robben, C., Buchala, A., Metraux, J. P., Van Loon, L. C., and Pieterse, C.
661 M. (2002). Characterization of Arabidopsis enhanced disease susceptibility mutants that are
662 affected in systemically induced resistance. Plant J. 29, 11-21.
663 Tyler, H.L and Triplett, E.W. (2008). Plants as a habitat for beneficial and/or human pathogenic
664 bacteria. Annu. Rev. Phytopathol. 46, 53-73.
665 Üstün, S., Müller, P., Palmisano, R., Hensel, M., and Börnke, F. (2012). SseF, a type III effector
666 protein from the mammalian pathogen Salmonella enterica, requires resistance-gene-
667 mediated signaling to activate cell death in the model plant Nicotiana benthamiana. New
668 Phytol. 194, 1046-1060.
669 Wei, C. I., Huang, J. M., Lin, W. F., Tamplin, M. L., and Bartz, J. A. (1995). Growth and
670 survival of Salmonella Montevideo on tomatoes and disinfection with chlorinated water. J.
671 Food Prot. 8, 829-836.
672 Wright, K.M., Chapman, S., McGeachy, K., Humphrism, S., Campbell, E., Toth, I.K., and
673 Holden, N.J. (2013). The endophytic lifestyle of Escherichia coli O157:H7: Quantification
674 and internal localization in roots. Phytopathology 103:333-340.
675 Xicohtencatl-Cortes, J., Sánchez Chacón, E., Saldaña, Z., Freer, E., and Girón, J.A. (2009).
676 Interaction of Escherichia coli O157:H7 with leafy green produce. J. Food Prot. 72, 1531677 1537.
678 Xin, X.-F., and He, S.Y. (2013). Pseudomonas syringae pv. tomato DC3000: A model pathogen
679 for probing disease susceptibility and hormone signaling in plants. Annu. Rev. Phytopathol.
680 51, 473-498.
681 Zeng, W., and He, S.Y. (2010). A prominent role of the flagellin receptor FLAGELLIN-
682 SENSING2 in mediating stomatal response to Pseudomonas syringae pv. tomato DC3000 in
683 Arabidopsis. Plant Physiol. 153, 1188-1198.
684 Zeng, W., Melotto, M., and He, S.Y. (2010). Plant stomata: a checkpoint of host immunity and
685 pathogen virulence. Curr. Opin. Biotech. 21, 599-603.
686 Zipfel, C., and Robatzek, S. (2010). Pathogen-associated molecular pattern-triggered immunity:
687 Veni, vidi...? Plant Physiol. 154, 551-554.
688 689
690 Table 1. Experimental conditions used in the studies reporting plant response to pathogenic Salmonella and E. coli.
Reference
Plant genotype
Wild type bacterium
Mutant bacterium
Plant tissue used for detection
Inoculation Inoculum Surface
method concentration sterilization Methods
Intact plant tissue for infection
Cooley et al., 2003
A. thaliana Col-0
*Iniguez et al., 2005
A. thaliana, M.
truncatula, wheat, M. sativa
*Shirron and Yaron, 2011
Tobacco
Flagellin and LPS perception
S. enterica kflhDC,
serovar Newport flaN
(RM1655); E.
coli O157:H7
Odwalla
(RM1484J;
Enterobacter
asburiae
(RM3638)
Trenton;
truncatula
Jester,
Jermalong,
Gaerten
Mutant
Sickle; M.
sativa
CUS101;
A. thaliana
Col-0,
npr1-4,
S. enterica serovar Typhimurium 14028s
S. enterica
serovar
Typhimurium
AinvA, ArfaH, AphoP
Roots and shoots of seedling and adult plants, leaves, flowers, seeds, chaff
spaS, sipB, Root and fliC, fljB hypocotyl
Soil, seed, root
inoculation
Seedling inoculation
S yringe
infiltration;
1 x 104 or 1 x No 106 cfu/ml for root
inoculation; 1 x 108 cfu/ml for seed inoculation and 1 x 108 cfu/g for soil inoculation
dose response Yes
Microscopy, Yes plating
Plating
7.5 log cfu/ml No
Microscopy Yes
SL1344, 14028s
irrigation
*Seo and Matthews, 2012
*Garcia et al., 2013
Meng et al., 2013
A. thaliana Col-0, npr1-1
A. thaliana Col-0, fls2
E. coli O157:H7 ЛfliC, 43895, 86-24 ЛcsgD, Лм>аа1
Whole plant Dipping
Tobacco, tomato
S. enterica serovar Typhimurium 14028s, SL1344; S. enterica serovar Senftenberg 20070885
S. enterica serovar Typhimurium 14028s
prgH, fliC, fljB, hrcC
Seedling
Seedling inoculation
1 x 108 cfu/ml No
2 x 108 cfu/ml No
лАС Лт
ЛЫА, ЛЫЮ,
AsirA, Л&чтАВ,
Л^А, Лр^Н
Syringe infiltration
2 x 104 cfu/ml No
Plating Yes
Plating Yes
Plating Yes
Melotto et al. 2006
Kroupitski et al., 2009
*Roy et al., 2013
A. thaliana Col-0, fls2 E. coli O157:H7
Stomatal immunity
Lettuce
Iceberg
A. thaliana, A. thaliana lettuce Col-0 ostl-
2, Butter Lettuce
S. enterica fliGHI,
serovar cheY
Typhimurium SL1344
S. enterica serovar Typhimurium SL1344; E. coli
Epidermal 1 x 108 cfu/ml peels
Leaf pieces 1 x 108 cfu/ml No submersion
Dipping
1 x 108 cfu/ml Yes
Microscopy No Microscopy No
Microscopy, Yes plating
O157:H7
Plant intracellular response
Thilmony et al., 2006
Klerks et al., 2007b
Schikora et al., 2008
Saldana et al., 2011
Schikora et al., 2011
Ustun et al., 2012
A. thaliana Col-0 Lettuce Tamburo
A. thaliana Col-0
Baby spinach
A. thaliana Col-0
Tomato, tobacco, pepper
E. coli O157:H7 fliC
S. enterica serovars Dublin; E. coli JM109
S. enterica serovar Typhimurium 14028s
E. coli O157:H7
Tomato
maker;
tobacco
Domin.,
pepper
ECW-10R
S. enterica serovar Typhimurium 14028s
Transient expression of S. enterica SseF in tobacco
escN, tir, eae, espFu, espP, fliC, qseB, hcpA, ecpA, elfA, csgA, csgD, bscA
prgH, invA, ssaV, ssaF
Seedling phyllosphere
Seedling, leaf
Vacuum infiltration
1 x 108 cfu/ml
Manure 1x 107 cfu/g
contamination manure or 1 x Seedling inoculation
Seedling inoculation, leaf vacuum infiltration
Leaf pieces submersion
Syringe infiltration
Transient expression by syringe infiltration of vector organism
10' cfu/ml inoculum
3 x 108 cfu/ml Yes
1 x 107 cells
1.7 x 108 cfu/ml
2 x 108 cfu/ml
Plating
Plating
Yes for some
experiments
Microscopy, No plating
Plating
Plating
Jayaramanet M. truncatulc S. enterica.
al., 2014 serovars
Schwarzengrund, Enteritidis, Mbandaka, Havana, Cubana; E. coli 0157:H7 serovars Odwalla, EDL933, H2439, C7927, 96A 13466
Barak et al., 2008
Radish, Lettuce S. enterica
tomato, Balady serovars Baildon
broccoli, Aswan, 05x-02123,
turnip, carrot. Salinas 88, Cubana
lettuce, Little Gem, 98A9878,
cilantro, PI251246, Enteritidis 99A-
parsley, Pavane, 23, Havana
spinach, Valmaine, 98A4399,
radicchio, Iceburg, La Mbandaka
endive Brillante, 99A1670,
Paris Island, Newport
Parade, 96E01152C-TX,
Calmar; Poona 00 A3 563,
Tomato Schwarzengrund
Brandywine, 96E01152C
Paste,
Maker,
Soldacki,
Stupics,
Grape, San
Marzano,
Root seedling root dose response Yes for Microscopy, Yes
some plating
experiments
Genotypic variability
Seedling Soil 1 x 104 cfu/ml No Plating Yes
phyllo sphere inoculation
Nyarous, Yellow Pear
Brandl and
Amudson,
Mitra et al., 2009
Lettuce
Spinach
Barak et al., 2011
Tomato
Golberg et al., 2011
Lettuce, arugula, parsley, tomato, basil
Parris Island
Bordeaux,
H7996, Yellow Pear, Nyagous, LA2838A, LA3172, LA3556, LA0337, LA1049, MicroTom, Money Maker
Iceberg, Romaine, Red Ruby
E. coli O157:H7 H1827; S. enterica serovar Thompson RM1987
E. coli O157:H7
S. enterica serovars Baildon 99A 23, Cubana 98A 9878, Enteritidis 05x-02123, Havana 98A4399, Mbandaka 99A 1670, Newport 96 E01153c-TX, Poona 00A 3563, Schwarzenfrund 96 E01152c-TX
S. enterica
serovar
Typhimurium
SL1344
Leaf, stem, root
Seedling phyllosphere and leaf
inoculation
Leaf drop, leaf
infiltration, soil drench, stem puncture
inoculation; water
inoculation; leaf dipping
1 x 105 cfu/ml No
pieces/leaf submersion
1 x 106 cfu/ml Yes
1 x 108 cfu/ml No
Microscopy, Yes plating
Microscopy, Yes plating, BAX PCR assay
Microscopy, Yes plating, BAX PCR assay
1 x 108 cfu/ml No
Microscopy Leaf pieces and intact leaves
691 * indicates articles that have also reported plant intracellular responses to bacteria.
693 Figure 1. Schematic representation of human pathogen (HP) association with plants. (A)
694 Pathogens are introduced to soil through contaminated irrigation water, fertilizers, manure, and
695 pesticides (1). HPs are attracted to rhizosphere (2; Klerks et al., 2007a) and penetrate root tissues
696 at the sites of lateral root emergence, root cracks as well as root-shoot transition area (3; Cooley
697 et al., 2003; Dong et al., 2003; Klerks et al., 2007b; Tyler and Triplett 2008). HPs were found to
698 live on the leaf surface near veins (Brandl and Mandrell 2002), in the leaf apoplast (intercellular
699 space) (Barak et al., 2011; Brandl and Mandrell 2002; Dinu and Bach 2011; Kroupitski et al.,
700 2009; Gu et al., 2011; Niemira 2007; Roy et al., 2013; Solomon et al., 2003), and sometimes
701 with affinity for abaxial side of leaf (e.g., S. enterica; (Kroupitski et al., 2011) (4). Salmonella
702 enterica Typhimurium can enter tomato plants via leaves and move through vascular bundles
703 (petioles and stems) (5) into non-inoculated leaves (6) and fruits (8) (Gu et al., 2011). HPs are
704 also found to be associated with flower (7; (Cooley et al., 2003; Guo et al., 2001). Salmonella
705 could travel from infected leaves (4), stems (5), and flowers (7) to colonize the fruit interior (the
706 diagram represents a cross-section of a fruit) and fruit calyx (8) (Janes et al., 2005; Barak et al.,
707 2011; Guo et al., 2001). Escherichia coli O157:H7 has also been observed in the internal parts of
708 the apple and the seeds following contamination of the flower (8) (Burnett et al., 2000).
709 Movement on the plant surface has also been observed (9; Cooley et al., 2003). Epiphytically of
710 Salmonella and E. coli O157:H7 can aggregate near stomata and sub-stomatal space (10; Shaw
711 et al., 2008; Golberg et al., 2011; Gu at al., 2011; Berger at al., 2009; Saldana et al., 2011), reach
712 the sub-stomatal cavity and survive/colonize in the spongy mesophyll (Solomon et al., 2002a;
713 Wachtel et al., 2002; Warriner et al., 2003; Jablasone et al., 2005; Franz et al., 2007).
714 Salmonella cells were observed near trichomes (10; Barak et al., 2011; Gu et al., 2011). (B) Stem
715 cross-section showing bacteria located in different tissues (Ep = epidermis, C = cortex, V =
716 vascular tissue, Pi = pith) (Deering et al., 2011a; Deering et al., 2011b). (C) Root cross-section
717 showing bacteria on the root surface, internalizing between the epidermal cells, and colonizing
718 root outer and inner cortex, endodermis (En), pericycle (P) and vascular system (Kutter et al.,
719 2006; Klerks et al., 2007a; Klerks et al., 2007b; Jayaraman et al., 2014).
721 Figure 2. Plant cellular defense responses against human pathogens. (A) Upon reception of
722 PAMP (flagellin, LPS) through PRR (FLS2 and putatively others), Salmonella spp. trigger
723 downstream plant defense responses which include ROS production, MPK3/6, salicylic acid
724 (SA) signaling through NPR1, jasmonic acid (JA) and ethylene (ET) signaling, defense-
725 associated gene induction, and extracellular alkalinization. All these cellular events ultimately
726 lead to stomatal closure, antimicrobial activity, and plant defense. (B) Escherichia coli PAMPs
727 (curli, LPS, flagellin, EPS) are also perceived by PRRs (FLS2 and putatively others) present on
728 plant cell surface which triggers the induction of the SA-dependent BGL2 promoter activity and
729 PR1 gene expression. Only components that have been directly demonstrated experimentally are
730 included in the diagram. Plant defense responses in case of both these human pathogens are
731 strain specific as well as plant cultivar specific.
Figure 1.TIF
Copyright of Frontiers in Microbiology is the property of Frontiers Media S.A. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
