Active Suppression of Early Immune Response in Tobacco by the Human Pathogen Salmonella Typhimurium

The persistence of enteric pathogens on plants has been studied extensively, mainly due to the potential hazard of human pathogens such as Salmonella enterica being able to invade and survive in/on plants. Factors involved in the interactions between enteric bacteria and plants have been identified and consequently it was hypothesized that plants may be vectors or alternative hosts for enteric pathogens. To survive, endophytic bacteria have to escape the plant immune systems, which function at different levels through the plant-bacteria interactions. To understand how S. enterica survives endophyticaly we conducted a detailed analysis on its ability to elicit or evade the plant immune response. The models of this study were Nicotiana tabacum plants and cells suspension exposed to S. enterica serovar Typhimurium. The plant immune response was analyzed by looking at tissue damage and by testing oxidative burst and pH changes. It was found that S. Typhimurium did not promote disease symptoms in the contaminated plants. Live S. Typhimurium did not trigger the production of an oxidative burst and pH changes by the plant cells, while heat killed or chloramphenicol treated S. Typhimurium and purified LPS of Salmonella were significant elicitors, indicating that S. Typhimurium actively suppress the plant response. By looking at the plant response to mutants defective in virulence factors we showed that the suppression depends on secreted factors. Deletion of invA reduced the ability of S. Typhimurium to suppress oxidative burst and pH changes, indicating that a functional SPI1 TTSS is required for the suppression. This study demonstrates that plant colonization by S. Typhimurium is indeed an active process. S. Typhimurium utilizes adaptive strategies of altering innate plant perception systems to improve its fitness in the plant habitat. All together these results suggest a complex mechanism for perception of S. Typhimurium by plants

Laser scanning confocal images showing Z stacks projections imaging of infected <i>N. tabacum</i> plants showing oxidative burst in parenchyma cells.

<p>Plant tissue was loaded with H₂DCF-DA, washed, and infected with <i>S.</i> Typhimurium (A, C) and <i>P. syringae</i> (B, D). Epidermal tissues of the plant were examined by laser scanning confocal microscopy. The pseudocolor key was included and was applied to glow scale images (C and D). The laser setting, microscope filters and all other image parameters were identical in both images.</p

Elicitation of response in <i>N. tabacum</i> mature leaves.

<p>Leaf panels were infiltrated with approximately 7 log CFU ml⁻¹ of (A) <i>S.</i> Typhimurium, (B) <i>P. syringae</i> pv. tomato, (C) <i>P. fluorescens</i>, (D) <i>S.</i> Typhimurium <i>ΔinvA</i> mutant; and (E) <i>S.</i> Typhimurium <i>ΔrfaH</i> mutant. Leaf tissue collapse was evident within 24 h in plants infiltrated with <i>P. syringae</i> pv. tomato (B), chlorotic primary lesions were induced in resistant leaves by <i>P. fluorescens</i> 48 h following plants infiltratation (C). No visible reaction to infiltration of <i>S.</i> Typhimurium wt and <i>ΔrfaH</i> mutant was observed (A and E). However, necrosis was evident 4 days following infiltration with <i>Salmonella ΔinvA</i> mutant (D).</p

Laser scanning confocal imaging of the elicited- oxidative burst in stomata cells of <i>N. tabacum</i>.

<p>Epidermal tissue was loaded with H₂DCF-DA, washed, and examined by LSCM. <i>S.</i> Typhimurium (A) and <i>P. syringae</i> (B) were added during the time course of image acquisition. The laser setting, microscope filters and all other image parameters were identical in both images.</p

Extracellular pH increase of the culture medium of tobacco BY-2 cells after elicitation.

<p>Pyranine (3 µg mL⁻¹) was added to the supernatant of the cells and aliquots of these suspensions were transferred to the wells of 96-well plates, to which bacteria (approximately 8 log CFU g cells⁻¹) were subsequently added. Resulting fluorescence from the cell suspension cultures was plotted against the pyranine fluorescence/pH curve. <i>S</i>. Typhimurium (WT and null mutants Δ<i>invA</i> and Δ<i>rfaH</i>); <i>P. syringae</i>; pure <i>Salmonella</i>'s LPS (LPS) and chloramphnicol treated <i>Salmonella</i> (CM <i>Salmonella</i>). Control cells were treated with the non-quenching medium alone (“control”). Values are pH means of 8 results, and SD values are shown. Means not followed by the same capital letter (A or B) are significantly different (<i>P</i><0.05).</p

Cytokinins Induce Transcriptional Reprograming and Improve Arabidopsis Plant Performance under Drought and Salt Stress Conditions

In nature, annual plants respond to abiotic stresses by activating a specific genetic program leading to early flowering and accelerated senescence. Although, in nature, this phenomenon supports survival under unfavorable environmental conditions, it may have negative agro-economic impacts on crop productivity. Overcoming this genetic programing by cytokinins (CK) has recently been shown in transgenic plants that overproduce CK. These transgenic plants displayed a significant increase in plant productivity under drought stress conditions. We investigated the role of CK in reverting the transcriptional program that is activated under abiotic stress conditions and allowing sustainable plant growth. We employed 2 complementary approaches: Ectopic overexpression of CK, and applying exogenous CK to detached Arabidopsis leaves. Transgenic Arabidopsis plants transformed with the isopentyltransferase (IPT) gene under the regulation of the senescence associated receptor kinase (SARK) promoter displayed a significant drought resistance. A transcriptomic analysis using RNA sequencing was performed to explore the response mechanisms under elevated CK levels during salinity stress. This analysis showed that under such stress, CK triggered transcriptional reprograming that resulted in attenuated stress-dependent inhibition of vegetative growth and delayed premature plant senescence. Our data suggest that elevated CK levels led to stress tolerance by retaining the expression of genes associated with plant growth and metabolism whose expression typically decreases under stress conditions. In conclusion, we hypothesize that CK allows sustainable plant growth under unfavorable environmental conditions by activating gene expression related to growth processes and by preventing the expression of genes related to the activation of premature senescence