Scholarly article on topic 'Plant innate immunity against human bacterial pathogens'

Plant innate immunity against human bacterial pathogens Academic research paper on "Biological sciences"

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Front. Microbiol.
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Academic research paper on topic "Plant innate immunity against human bacterial pathogens"

1 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


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


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.


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.


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.


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.


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.


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.


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.


Work in our laboratory is supported by funding from the US National Institute of Allergy and Infectious Disease (R01AI068718).


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688 689

690 Table 1. Experimental conditions used in the studies reporting plant response to pathogenic Salmonella and E. coli.


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


Flagellin and LPS perception

S. enterica kflhDC,

serovar Newport flaN

(RM1655); E.

coli O157:H7












Sickle; M.



A. thaliana



S. enterica serovar Typhimurium 14028s

S. enterica



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


Seedling inoculation

S yringe


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


7.5 log cfu/ml No

Microscopy Yes

SL1344, 14028s


*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 inoculation

1 x 108 cfu/ml No

2 x 108 cfu/ml No

лАС Лт


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



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


1 x 108 cfu/ml Yes

Microscopy No Microscopy No

Microscopy, Yes plating


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







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



Yes for some


Microscopy, No 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





Grape, San


Root seedling root dose response Yes for Microscopy, Yes

some plating


Genotypic variability

Seedling Soil 1 x 104 cfu/ml No Plating Yes

phyllo sphere inoculation

Nyarous, Yellow Pear

Brandl and


Mitra et al., 2009



Barak et al., 2011


Golberg et al., 2011

Lettuce, arugula, parsley, tomato, basil

Parris Island


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




Leaf, stem, root

Seedling phyllosphere and leaf


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

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