RESUMEN
Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of Salmonella enterica serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety.
RESUMEN
Bacterium-triggered stomatal closure is a functional output of plant immunity, also known as stomatal defense. This is an early response mediated by the recognition of pathogen-associated molecular patterns (PAMPs) by the plant's pathogen recognition receptors (PRRs). Several approaches to analyzing stomatal movement in response to bacteria have been described, but difficulties in fine-tuning the experimental procedures still exist. Here we provide a detailed method for assessing stomatal defense via high-quality microscopic imaging and explain trouble-shooting steps to obtaining robust data. Although this procedure requires minimal manipulation of the leaf sample, it is crucial to control all environmental conditions and extrinsic variables that could interfere with stomatal movement. The method described here is also suitable for in vivo characterization of stomatal response in new pathosystems and can be used in conjunction with other profiling assays to gain a detailed understanding of early PAMP-triggered immunity (PTI).