As previously reported by Hauck et al. , Pst DC3000 did not induce strong callose deposition on its Arabidopsis plant host , although callose deposit frequency was significantly higher than in the water control . However, the Pst DC3000 type-three secretion system mutant induced 2.5 times more callose deposits than the wild type Pst DC3000 in Arabidopsis leaves . Altogether, our findings suggest that STm 14028s can induce a weak defense in lettuce leaves, similar to that of Pst DC3000 in Arabidopsis leaves. A major function of the SPI genomic region is to assemble the TTSS apparatus and encode effector proteins that could potentially suppress plant defenses. However, we observed that, unlike in the Arabidopsis-Pst DC3000 pathosystem where the TTSS is involved in suppressing plant immune response such as callose , the SPI-1 and SPI-2 regions of STm 14028s are not involved in this process in the lettuce system.Growth rates of STm 14028s, Mut3, and Mut9 in AWF and LSLB were determined during the log-phase of bacterial growth.As expected, there was minimal bacterial growth in water , indicating that residual nutrients in the inoculum were not transferred to LSLB or AWF to enhance growth. In an attempt to correlate the ability of the bacterium to survive within the apoplast with the ability to utilize apoplastic nutrients for growth, we included in this analysis Mut3 that contains a deletion of SPI-1 and adjacent genes and shows apoplastic persistence similar to the wild type STm 14028s .
When grown on LSLB,drainage pot both Mut3 and Mut9 had statistically significant lower growth rates than STm 14028s. Mut3, Mut9, and STm 14028s had growth rates of 2.78, 2.18, and 3.53 generations/hour, respectively . When grown in lettuce AWF, Mut3 and STm 14028s had similar growth rates, while the Mut9 growth rate was significantly lower . This finding suggests that the STm’s ability to persist in the apoplast may be linked to nutrient acquisition or the overall bacterial fitness in this niche that is dependent on yet-to-be determined gene and operon deleted in Mut9.The importance of food borne illness caused by contamination of produce by Salmonella spp. and the prevalence of contamination associated with leafy greens led us to investigate the molecular mechanisms allowing Salmonella spp. to use this alternate host for survival. As apoplastic populations of human pathogenic bacteria in lettuce are a potential risk for food borne illnesses due to persistence from production to consumption, we directed our focus on the bacterial internalization into leaves through stomata and endophytic survival. S. enterica internalization of leaves can occur through the stomatal pore . We were able to identify ten regions in the STm 14028s genome that may directly or indirectly contribute to the bacterium’s ability to open the stomatal pore facilitating its entry into the apoplast. Although it is not obvious which genes in those regions are specifically responsible for the observed phenotype on the leaf surface, the major metabolic functions of these regions are associated with sensing the environment, bacterium chemotaxis and movement, membrane transporters, and biosynthesis of surface appendices . Previously, these functions have been found to be associated with epiphytic fitness of bacterial phytopathogens . Furthermore, Kroupitski et al. observed that STm SL1344 aggregates near open stomata and uses chemotaxis and motility for internalization through lettuce stomata. Additionally, darkness prevents STm SL1344’s ability to re-open the stomatal pore and internalization into the leaves possibly due to the lack of chemo attractant leaching through closed stomata .
These findings suggest that close proximity to stomata may be required for Salmonella to induce opening of the pore. Therefore, STm invasion of the apoplast may be a consequence of a combined behavior of the bacterium on the phylloplane that can be modulated by plant-derived cues and,with this study, we have defined key genomic regions involved in this complex process. Not all the genomic regions required for initiation of the leaf colonization are essential for continuing bacterial survival as an endophyte . For instance, genes deleted from Mut3 and Mut6 [encoding unspecified membrane proteins, the PhoP/Q two-component system, SopE2 , phage genes, a transcriptional repressor , and some unspecified transporters] do not contribute to endophytic survival. Thus, these regions missing in Mut3/6 are potential targets for disrupting leaf surface colonization, but not endophytic persistence. This observation is not entirely surprising as the phylloplane and the apoplast environments are unique and they pose different challenges for bacterial survival in these niches. STm seems to have metabolic plasticity for adaptation to varying conditions in the leaf. For instance, STm SL1344 can shift its metabolism to utilize nutrients available in decaying lettuce and cilantro leaves and STm 14028s uses distinct metabolism strategies to colonize tomatoes and animal infection . We also observed that seven regions of the STm 14028s genome have opposite effects on the different phases of colonization. Mut1/2/4/5/7/8/10 seem to lack the ability to promote penetration into the leaf , but they show better fitness than that of the wild type strain in the apoplast . One hypothesis is that the increased bacterial population titers are due to lack of energy expenditure for maintaining large genomic segments that are not essential for survival as an endophyte, so that the excess energy can be spent on survival. However, this indirect effect of the deletion may not be valid for Mut4/10, where only small genomic regions are missing . Alternatively, these regions might encode for proteins that negatively affect bacterial survival in leaves.
This interesting observation is worth future investigation. Intriguingly, we found that genes deleted in Mut9 are important for re-opening the stomatal pore and successful endophytic survival. This deletion includes SPI-2 that functions in the production of the TTSS-2 apparatus, effectors, and a two component regulatory system of this island , which are important for the virulence of STm in animal systems . The contribution of the TTSS-2 apparatus and effectors to the bacterium’s ability to colonize the phyllosphere has been studied in several laboratories and it is largely dependent on the plant species analyzed . Nonetheless, so far there is no evidence for the ability of STm to inject TTSS effectors inside plant cells . Furthermore, the STm 14028s ssaV-structural mutant, that cannot form the TTSS-2 apparatus , survives in the lettuce cv. Romit 936 to the same extent as the wild type bacterium after surface inoculation . Our data also support the notion that the TTSS- 2 is not involved in STm ability to induce or subvert defenses, such as callose deposition in lettuce cv. Salinas . While studies in other plant systems have suggested that TTSS and encoded effectors may contribute to bacterial survival in the plant environment or in some cases are detrimental for bacterial colonization of plant tissues , it has become evident that the TTSS-2 within the SPI-2 region is not relevant in the STm 14028s-lettuce leaf interaction. It is important to note that SPI-2 is a genomic segment of roughly 40 kb with 42 open reading frames arranged into 17 operons . It is present in all pathogenic serovars and strains of S. enterica, but only partially present in species of a more distant common ancestor, such as S. bongori . Besides encoding structural and regulatory components of the TTSS-2 , SPI2 also carries genes coding for a tetrathionate reductase complex, a cysteine desulfurase enzyme complex, membrane transport proteins, murein transpeptidases, as well as genes with still uncharacterized functions . Thus, it is possible that genes and operons, other than the ones associated with TTSS-2,gallon pot may have a function in the bacterium colonization of the lettuce leaf. To date, it has not been demonstrated whether STm 14028s can access and utilize nutrients from the apoplast of intact lettuce leaves. Although nutrients in the apoplast might be limiting , it has been hypothesized that Salmonella may scavenge nutrients to persist in the plant environment and/or adjust its metabolism to synthesize compounds that are not readily available at the colonization site. For instance, a mutant screen analysis indicated that STm 14028s requires genes for biosynthesis of nucleotides, lipopolysaccharide, and amino acids during colonization of tomato fruits . Moreover, plants might secrete antimicrobial compounds into the apoplast as a plant defense mechanism, imposing a stressful condition to the microbial invader . Therefore, considering that subversion of plant defenses is not a function of the TTSS-2 in the apoplast of lettuce , it is possible that the Mut9 population reduces 20 fold over 21 days due to its inability to obtain nutrients from this niche and/or to cope with plant defenses. Although Mut9 shows reduced growth on lettuce leaf AWF , additional experimentation is required to distinguish between these two possibilities. It is tempting to speculate, however, that the tetrathionate reductase gene cluster within SPI-2 or the sulfur mobilization operon deleted in Mut9 might be involved in this process.
Particular to the ttr operon, TtrAB forms the enzyme complex, TtrC anchors the enzyme to the membrane, whereas TtrS and TtrR are the sensor kinase and DNA-binding response regulator, respectively . The reduction of tetrathionate by this membrane-localized enzyme is part of the Salmonella’s anaerobic respiration . Intriguingly, the use of tetrathionate as an electron acceptor during propanediol and ethanolamine utilization by the bacterium has been suggested to occur in macerated leaf tissue . A significant number of genes involved in the PDU ,EUT , and cobalamin pathways as well as the ttrC gene are upregulated in STm SL1344 when co-inoculated with the soft rot pathogen Dickeya dadantii onto cilantro and lettuce leaf cuts . Altogether, these findings suggest that these biochemical pathways may occur in both soft rot contaminated and healthy leaves. Considering that the encounter of the plant with a pathogenic bacterium triggers molecular action and reaction in both organisms overtime, it is not surprising that multiple regions of the STm 14028s genome may be required for lettuce leaf colonization. For instance, Goudeau et al. reported that 718 genes of the STm SL1344 genome were transcriptionally regulated upon exposure to degrading lettuce cell wall. In any case, further studies using single-gene mutants are still required to identify the specific genes and functions within each MGD mutant that are involved in the interaction between STm 14028s and lettuce cultivar Salinas. Butenolides are lactone-containing heterocyclic molecules with important biochemical and physiological roles in plant life. Although previously recognized as secondary metabolites, some types of butenolides were recently classified as plant hormones . Strigolactones are carotenoid-derived molecules bearing essential butenolide moieties that were originally described as chemical cues promoting seed germination of parasitic Striga species . It has since become evident that SLs are involved in controlling a wide range of plant developmental processes, including root architecture, establishment of mycorrhiza, stature and shoot branching, seedling growth, senescence, leaf morphology and cambial activity . SLs are synthesized via a sequential cleavage of alltrans-β-carotene by DWARF27 and the resulting 9-cis-β-carotene by MORE AXILLARY GROWTH3 and 4 . The SL precursor carlactone is then transported through the xylem and biologically active SLs are formed by MAX1 and its homologs and LATERAL BRANCHING OXIDOREDUCTASE . Cumulative evidence supports the idea that the DWARF14 α/β-fold hydrolase functions as a SL receptor and is required for the perception of the SL signal in Petunia , rice , Arabidopsis and pea . Upon binding, D14 proteins hydrolyze SL by action of its conserved Ser-His-Asp catalytic triad, followed by thermal destabilization of the proteins . As a consequence, the structural rearrangement of D14 proteins in the presence of SL enables the protein to physically interact with the F-box proteins MAX2 and SMAX1-LIKE family proteins SMXL 6, 7 and 8 to form a Skp-Cullin-Fbox ubiquitin ligase complex that polyubiquitinates SMXLs and targets them for degradation by the 26S proteasome. The subsequent signaling events are largely unknown, but tentatively the mechanism is similar to other systems employing targeted protein degradation . In Arabidopsis, two paralogs of AtD14 have been identified . One paralog, KARRIKIN INSENSITIVE2 was identified in a mutant in Ler background which showed insensitivity to karrikin , a butenolide-type germination stimulant from smoke water . Although both AtD14 and KAI2 signaling pathways converge upon MAX2 and might employ similar mechanisms to transduce the signal, the two proteins regulate separate physiological events.