Mechanistic modeling of light-induced chemotactic infiltration of bacteria into leaf stomata

Light is one of the factors that can play a role in bacterial infiltration into leafy greens by keeping stomata open and providing photosynthetic products for microorganisms. We model chemotactic transport of bacteria within a leaf tissue in response to photosynthesis occurring within plant mesophyll. The model includes transport of carbon dioxide, oxygen, bicarbonate, sucrose/glucose, bacteria, and autoinducer-2 within the leaf tissue. Biological processes of carbon fixation in chloroplasts, and respiration in mitochondria of the plant cells, as well as motility, chemotaxis, nutrient consumption and communication in the bacterial community are considered. We show that presence of light is enough to boost bacterial chemotaxis through the stomatal opening and toward photosynthetic products within the leaf tissue. Bacterial chemotactic ability is a major player in infiltration, and plant stomatal defense in closing the stomata as a perception of microbe-associated molecular patterns is an effective way to inhibit the infiltration.

: a) A leaf cross section with b) an illustration of cell organs. c) A schematic of a chloroplast with various functions that lead to photosynthesis in plant cells. The chloroplast image inspired by Campbell et al. [1]. Note that only organs and functions that are of interest in the present study are shown. The signal transduction and actuator modules are not phosphorylated and repress the tumbling and elongate the biased motion.

LuxS Quorum sensing:
Increase in intracellular AI-2 concentration can induce biofilm formation, and increase motility, chemotaxis and cell adherence.

AI-2 influx:
Through LsrACD membrane proteins, mediated by LsrB periplasmic protein AI-2 efflux: Through TqsA membrane protein Glucose-6phosphate Glucose uptake: The phosphotransferase system (PTS) transfers glucose across cytoplasmic membrane, mediated by IIA and EI cytoplasmic proteins. The process uses a high energy phosphate group from phosphoenolpyruvate. Transport of species, i, (i.e., CO 2 and O 2 ) in the gas and water phases are governed by: where R i,w is an arbitrary source term (mol/m 3 · s). Assuming the equilibrium between gas and 78 water phases to be described by Henry's law [19]: and plugging into Eq. 4, the total concentration of each species in the REV is defined as: Using Eq. S4, Eq. S2 can be rewritten as: Finally, by adding Eq. S1 and Eq. S5 and applying Eq. S4, the combined transport equation in the 82 REV is obtained as: where D i,eff is effective diffusivity of species i in the porous media: Input data for the simulations are shown in ity (mol/m 3 · s) was calculated as: Here, α t is the reciprocal of the leaf thickness (1/m), which is used to get a volumetric value for The CO 2 compensation point without dark respiration (Pa), Γ * , is defined as the partial pressure 98 of CO 2 at which no net assimilations occur [47]. The temperature dependence of Γ * was reported Note that free water refers to the intercellular water, and bound water refers to intracellular water.
where θ = 0.97. The light limited rate of electron transport (mol/m 2 · s), J ll , is determined from 104 the amount of the available light (mol/m 2 · s), I, to be absorbed by the chlorophyll pigments that 105 can vary by the light wavelength [20]: where α P SII = 0.5 is the fraction of absorbed photons driving PS II electron transport, and 107 Φ P SII = 0.85 mol/mol is the maximum quantum efficiency of PS II in electron transport. The 108 distribution of light within the leaf tissue was calculated using Beer-Lambert's law: where a chl is the absorption coefficient of chlorophyll a which depends on the specific absorption 110 (m 2 /mol) [20,34], a * chl , and the density of chlorophyll a within the leaf tissue (mol/m 3 ), ρ chl : The profile of chlorophyll density within spinach leaves was obtained from Vogelmann and Evans [20] 112 who measured the chlorophyll fluorescence profiles within spinach leaves (Fig. Da).

113
The light saturated rate of electron transport (mol/m 2 · s) is defined as: where β is defined here as the relative photosynthetic capacity whose profile was obtained from TPU-limited: A value of 9.19×10 −6 mol/m 2 ·s was adopted for the TPU rate (in Eq. 18) (mol/m 2 · 118 s), T p [19]. So, the volumetric value of TPU rate is: at 37 • C and 220 rpm. This was followed by a second overnight incubation in fresh LB broth.

126
The bacterial culture were then harvested by two successive centrifugation steps (Sorrvall legend 127 RT+centrifuge, Thermo Scientific, USA) at 2700 g for 10 min to efficiently remove the LB broth.

128
The cell pellets were resuspended in sterile 0.85% NaCl (saline) solution and the concentration of  b. 100 µmol/m 2 · s, or were kept in the dark for 45 min. After illumination, samples (5 mm × 5 mm) 166 from three arbitrary locations of each leaf were cut and immediately used for microscopy. For 167 each experimental condition, data of stomatal aperture were gathered from more than 100 stomata.

168
The measurements were done using ImageJ software. The results of measurements of the stomatal 169 aperture when using various light wavelengths as well as dark condition, are shown in Fig. H.   b.

Bacteria: ampicillin-resistant E. coli K-12 MG1655
Figure J: Results of the colony growth of ampicillin-resistant E. coli K-12 MG1655 on LB-agar medium containing 100 µg/ml ampicillin. The inoculated leaves were exposed to white light, from adaxial side, with an intensity of 100 µmol/m 2 · s for 2 h. b.

Test 4
Bacteria: ampicillin-resistant E. coli K-12 MG1655 Figure K: Results of the colony growth of ampicillin-resistant E. coli K-12 MG1655 on LB-agar medium containing 100 µg/ml ampicillin. The inoculated leaves were exposed to blue light, from adaxial side, with an intensity of 100 µmol/m 2 · s for 2 h. b.

Bacteria: ampicillin-resistant E. coli K-12 MG1655
Figure L: Results of the colony growth of ampicillin-resistant E. coli K-12 MG1655 on LB-agar medium containing 100 µg/ml ampicillin. The inoculated leaves were exposed to green light, from adaxial side, with an intensity of 100 µmol/m 2 · s for 2 h.  Figure N: Results of the colony growth of kanamycin-resistant E. coli K-12 BW25113 (∆ CheZ) on LB-agar medium containing 30 µg/ml kanamycin. The inoculated leaves were exposed to white light, from adaxial side, with an intensity of 100 µmol/m 2 · s for 2 h.