Effect of plant systemic resistance elicited by biological and chemical inducers on the colonization of the lettuce and basil leaf apoplast by Salmonella enterica

Mitigation strategies to prevent microbial contamination of crops are lacking. We tested the hypothesis that induction of plant systemic resistance by biological (ISR) and chemical (SAR) elicitors reduces endophytic colonization of leaves by Salmonella enterica serovars Senftenberg and Typhimurium. S . Senftenberg had greater endophytic fitness than S . Typhimurium in basil and lettuce. The apoplastic population sizes of serovars Senftenberg and Typhimurium in basil and lettuce, respectively, were significantly reduced approximately 10- to 100-fold by root treatment with microbial inducers of systemic resistance compared with the H 2 O treatment. Rhodotorula glutinis effected the lowest population increase of S . Typhimurium in lettuce (120-fold) and S . Senftenberg in basil leaves (60-fold) compared with the H 2 O treatment over 10 days post-inoculation. Trichoderma harzianum and Pichia guilliermondii did not have any significant effect on S . Senftenberg in the basil apoplast. The chemical elicitors acidobenzolar-S-methyl and DL-β-amino-butyric acid inhibited S . Typhimurium multiplication in the lettuce apoplast 10- and 2-fold, respectively, compared with H 2 O-treated plants. All ISR and SAR inducers applied to lettuce roots in this study increased leaf expression of the defense gene PR1 , as did Salmonella apoplastic colonization in H 2 O-treated lettuce plants. Remarkably, both acidobenzolar-S-methyl- and R. glutinis -upregulation of PR1 was repressed by the presence of Salmonella in the leaves. However, enhanced PR1 expression was sustained longer and at greater levels upon elicitor treatment than by Salmonella induction alone. These results serve as proof of concept that priming of plant immunity may provide an intrinsic hurdle against the endophytic establishment of enteric pathogens in leafy vegetables. Importance Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast where they confront plant innate immunity. Plants may be triggered for induction of their defense signaling pathways by exposure to chemical or microbial elicitors. This priming for recognition of microbes by plant defense pathways has been used to inhibit plant pathogens and limit disease. Given that current mitigation strategies are insufficient in preventing microbial contamination of produce and associated outbreaks, we investigated the effect of plant induced resistance on S. enterica colonization of the lettuce and basil leaf apoplast in order to gain a proof of concept for the use of such an intrinsic approach to inhibit human pathogens in leafy vegetables.

Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem are required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

Silicon‐mediated manganese tolerance of cucumber: the apoplastic modulation

<p>An impressive body of Si research could be found in the literature despite the fact that, from a biochemical perspective, Si is a “monotonous” element largely uncharged and unreactive at physiological pH (forming mostly silicates and SiO<sub>2</sub> polymers). However, the detailed role of Si in plants remains unexploited, particularly the potential for its practical application. One of the main properties of Si intensively explored is the protection mechanism(s) against biotic and abiotic stresses, especially heavy metal stress. To investigate the effect of Si application on the Mn binding potential of the leaf apoplast, cucumber plants were grown in nutrient solutions with optimal (0.5 µM) or excessive (100 µM) Mn concentrations with or without Si supply to roots. Leaves were subjected to fractionated extraction of Mn revealing a relative distribution of Mn fractions in cucumber leaves: water-extractable (WE) Mn represents the soluble fraction in the cell walls; the protein-bound (PB) Mn fraction originates mostly from the symplast; while the cell wall-bound (CWB) Mn fraction represents Mn which is fixed to the wall structure. After the high Mn supply (100 µM), the concentration of WE Mn was 10-fold higher compared to control, while the relative proportion of the WE Mn fraction decreased from 56% in control to 23% in high Mn treatment. Si application did not affect WE and PB Mn fractions in the control treatment but significantly decreased these fractions in the high Mn treatment. On the other hand, the CWB Mn significantly increased in the leaves of Si-fed plants. Data obtained by fractionated Mn extraction are consistent with the relative proportion of free and bound Mn, estimated from the recorded electron paramagnetic resonance (EPR) signals of Mn<sup>2+</sup>. The EPR spectrum of a high spin Mn<sup>2+</sup> showed the characteristic six hyperfine lines whose intensity correlated with Mn treatments and, consequently, leaf concentrations of Mn. The results presented here demonstrated that Si supply increased the Mn binding properties of leaf cell walls in cucumber plants with simultaneously decreasing of the free apoplastic Mn<sup>2+</sup>, indicating the protective role of Si in smothering harmful (inter)actions of free Mn<sup>2+</sup> within plant tissue. Taken together, the leaf apoplast plays the central role in modulation of Mn toxicity and Si enhanced Mn tolerance in cucumber.</p><p>This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Contract No. 451-03-68/2020-14/200053).</p>

Leaf apoplastic alkalisation promotes transcription of the ABA synthesising enzyme Vp14 and stomatal closure in Zea mays L

The chloride component of NaCl-salinity causes the leaf apoplast to transiently alkalinize. This transition in pH reduces stomatal aperture. However, whether this apoplastic pH (pHapo) transient initiates stomatal closure by interacting with other chloride-stress-induced responses or whether the pH transient alone initiates stomatal closure is unknown. To clarify the problem, the transient alkalinization of the leaf apoplast was mimicked in intact maize (Zea mays L.) by infiltrating near-neutral pH buffers into the leaf apoplast. Effects of the pHapo transient could thus be investigated independently from other chloride-stress-derived effects. Microscopy-based ratiometric live pHapo-imaging was used to monitor pHapo in planta. LC-MS/MS and real-time-qRT-PCR leaf analyses showed that the artificially induced pHapo transient led to an increase in the concentrations of the stomata-regulating plant hormone abscisic acid (ABA) and in transcripts of the key ABA-synthesizing gene ZmVp14 in the leaf. Since stomatal aperture and stomatal conductance decreased according to pHapo, we conclude that the pHapo transient alone initiates stomatal closure. Therefore, the functionality does not depend on interactions with other compounds induced by chloride-stress. Overall, our data indicate that the pH of the leaf apoplast links chloride-salinity with the control of stomatal aperture via effects exerted on the transcription of ABA.

Mucilaginous Secretions in the Xylem and Leaf Apoplast of the Swamp Palm Mauritia flexuosa L.f. (Arecaceae)

Mauritia flexuosa palms inhabit wetland environments in the dry, seasonal Brazilian savanna (Cerrado) and produce mucilaginous secretions in the stem and petiole that have a medicinal value. The present study sought to characterize the chemical natures of those secretions and to describe the anatomical structures involved in their synthesis. Chemical analyzes of the secretions, anatomical, histochemical analyses, and electron microscopy studies were performed on the roots, stipes, petioles, and leaf blades. Stipe and petiole secretions are similar, and rich in cell wall polysaccharides and pectic compounds such as rhamnose, arabinose, xylose, mannose, galactose, and glucose, which are hydrophilic largely due to their hydroxyl and carboxylate groups. Mucilaginous secretions accumulate in the lumens of vessel elements and sclerenchyma fibers of the root, stipe, petiole, and foliar veins; their synthesis involves cell wall loosening and the activities of dictyosomes. The outer faces of the cell walls of the parenchyma tissue in the mesophyll expand to form pockets that rupture and release pectocellulose substances into the intercellular spaces. The presence of mucilage in the xylem, extending from the roots to the leaf veins and continuous with the leaf apoplast, and sub-stomatal chambers suggest a strategy for plant water economy.

Common and unique Arabidopsis proteins involved in stomatal susceptibility to Salmonella enterica and Pseudomonas syringae

ABSTRACT Salmonella enterica is one of the most common pathogens associated with produce outbreaks worldwide; nonetheless, the mechanisms uncovering their interaction with plants are elusive. Previous reports demonstrate that S. enterica ser. Typhimurium (STm), similar to the phytopathogen Pseudomonas syringae pv. tomato (Pst) DC3000, triggers a transient stomatal closure suggesting its ability to overcome this plant defense and colonize the leaf apoplast. In order to discover new molecular players that function in the stomatal reopening by STm and Pst DC3000, we performed an Arabidopsis mutant screening using thermal imaging. Further stomatal bioassay confirmed that the mutant plants exo70h4-3, sce1-3, bbe8, stp1, and lsu2 have smaller stomatal aperture widths than the wild type Col-0 in response to STm 14028s. The mutants bbe8, stp1 and lsu2 have impaired stomatal movement in response to Pst DC3000. These findings indicate that EXO70H4 and SCE1 are involved in bacterial-specific responses, while BBE8, STP1, and LSU2 may be required for stomatal response to a broad range of bacteria. The identification of new molecular components of the guard cell movement induced by bacteria will enable a better understanding of the initial stages of plant colonization and facilitate targeted prevention of leaf contamination with harmful pathogens.

Pseudomonas syringaeIncreases Water Availability in Leaf Microenvironments via Production of Hygroscopic Syringafactin

ABSTRACTThe epiphytic bacteriumPseudomonas syringaestrain B728a produces the biosurfactant syringafactin, which is hygroscopic. The water-absorbing potential of syringafactin is high. Syringafactin attracts 250% of its weight in water at high relative humidities but is less hygroscopic at lower relative humidities. This finding suggests that the benefit of syringafactin to the producing cells is strongly context dependent. The contribution of syringafactin to the water availability around cells on different matrices was assessed by examining the water stress exhibited by biosensor strains expressinggfpvia the water-stress-activatedproUpromoter. Wild-type cells exhibited significantly less green fluorescent protein (GFP) fluorescence than a syringafactin-deficient strain on dry filters in atmospheres of high water saturation, as well as on leaf surfaces, indicating greater water availability. When infiltrated into the leaf apoplast, wild-type cells also subsequently exhibited less GFP fluorescence than the syringafactin-deficient strain. These results suggest that the apoplast is a dry but humid environment and that, just as on dry but humid leaf surfaces, syringafactin increases liquid water availability and reduces the water stress experienced byP. syringae.IMPORTANCEMany microorganisms, including the plant pathogenPseudomonas syringae, produce amphiphilic compounds known as biosurfactants. While biosurfactants are known to disperse hydrophobic compounds and to reduce water tension, they have other properties that can benefit the cells that produce them. Leaf-colonizing bacteria experience frequent water stress, since liquid water is present only transiently on or in leaf sites that they colonize. The demonstration that syringafactin, a biosurfactant produced byP. syringae, is sufficiently hygroscopic to increase water availability to cells, thus relieving water stress, reveals thatP. syringaecan modify its local habitat both on leaf surfaces and in the leaf apoplast. Such habitat modification may be a common role for biosurfactants produced by other bacterial species that colonize habitats (such as soil) that are not always water saturated.