Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast

Jacob, Cristián; Panchal, Shweta; Melotto, Maeli

doi:10.21769/bioprotoc.2167

Cited by 21 publications

(20 citation statements)

References 7 publications

(8 reference statements)

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“…After incubation, leaves were rinsed twice in SDW for 1 min and then blotted on a paper towel. Then, the bacterial population was enumerated by serial-dilution plating as described by Jacob et al (2017). Three leaves from each genotype were used for each treatment and the experiment was repeated four times (n = 12) with independent batches of plants.…”

Section: Bacterial Attachment Assaymentioning

confidence: 99%

“…Surface-inoculation was conducted to evaluate the bacterial internalization rate (IR) and subsequent bacterial survival in the leaf intercellular space of lettuce genotypes. The protocol was adapted from those previously described for the pathosystem Arabidopsis thaliana-Pseudomonas syringae (Katagiri et al, 2002;Jacob et al, 2017). Lettuce plants (2.5-to 3-week-old) grown in peat moss pellets were used.…”

Section: Leaf Surface Inoculationmentioning

confidence: 99%

“…Four 0.2 cm 2 discs, punched with a cork-borer, were placed in a 1.7 mL centrifuge tube and ground in 100 µL of SDW. The bacterial population was enumerated by serial-dilution plating as described by Jacob et al (2017). To quantify the bacterial IR and subsequent apoplastic persistence, bacterial enumeration on day 0 was conducted in non-surface sterilized leaves; for the rest of the sampling points, leaves were gently washed in 2% (v/v) sodium hypochlorite for 1 min, 70% (v/v) ethanol for 1 min, rinsed in SDW for 1 min, and blotted onto a paper towel.…”

Section: Leaf Surface Inoculationmentioning

confidence: 99%

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Human Pathogen Colonization of Lettuce Dependent Upon Plant Genotype and Defense Response Activation

Jacob

Melotto

2020

Front. Plant Sci.

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Fresh produce contaminated with human pathogens may result in foodborne disease outbreaks that cause a significant number of illnesses, hospitalizations, and death episodes affecting both public health and the agribusiness every year. The ability of these pathogens to survive throughout the food production chain is remarkable. Using a genetic approach, we observed that leaf colonization by Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium 14028s) and Escherichia coli O157:H7 was significantly affected by genetic diversity of lettuce (Lactuca sativa L. and L. serriola L.). In particular, there was a significant variation among 11 lettuce genotypes in bacterial attachment, internalization, and apoplastic persistence after surface-and syringeinoculation methods. We observed a significant correlation of the bacterial leaf internalization rate with stomatal pore traits (width and area). Moreover, bacterial apoplastic populations significantly decreased in 9 out of 11 lettuce genotypes after 10 days of surface inoculation. However, after syringe infiltration, populations of E. coli O157: H7 and S. Typhimurium 14028s showed positive, neutral, or negative net growth in a 10day experimental period among seedlings of different lettuce types. The relative ability of the bacteria to persist in the apoplast of lettuce genotypes after syringe inoculation was minimally altered when assessed during a longer period (20 days) using 3.5-to 4-weekold plants. Interestingly, contrasting bacterial persistence in the lettuce genotypes Red Tide and Lollo Rossa was positively correlated with significant differences in the level of reactive oxygen species burst and callose deposition against S. Typhimurium 14028s and E. coli O157:H7 which are related to plant defense responses. Overall, we characterized the genetic diversity in the interaction between lettuce genotypes and enterobacteria S. Typhimurium 14028s and E. coli O157:H7 and discovered that this genetic diversity is linked to variations in plant immune responses towards these bacteria. These results provide opportunities to capitalize on plant genetics to reduce pathogen contamination of leaves.

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Section: Bacterial Attachment Assaymentioning

confidence: 99%

Section: Leaf Surface Inoculationmentioning

confidence: 99%

Section: Leaf Surface Inoculationmentioning

confidence: 99%

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Human Pathogen Colonization of Lettuce Dependent Upon Plant Genotype and Defense Response Activation

Jacob

Melotto

2020

Front. Plant Sci.

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“…Inoculated plants were immediately incubated under the following conditions: 25°C, 80 ± 10% relative humidity, 12 h of photoperiod (100 μmol.m − 2 .sec − 1 ) for the duration of the experiment. Bacterial population in the plant apoplast was determined as previously described [70,76]. Data points represent the mean (n = 12) ± SE of three leaves (each with two technical replicates) of one plant per data point in two independent experiments.…”

Section: Bacterial Pathogenesis Assaysmentioning

confidence: 99%

Novel molecular components involved in callose-mediated Arabidopsis defense against Salmonella enterica and Escherichia coli O157:H7

2020

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Background: Food contamination with Salmonella enterica and enterohemorrhagic Escherichia coli is among the leading causes of foodborne illnesses worldwide and crop plants are associated with > 50% of the disease outbreaks. However, the mechanisms underlying the interaction of these human pathogens with plants remain elusive. In this study, we have explored plant resistance mechanisms against these enterobacteria and the plant pathogen Pseudomonas syringae pv. tomato (Pst) DC3118, as an opportunity to improve food safety. Results: We found that S. enterica serovar Typhimurium (STm) transcriptionally modulates stress responses in Arabidopsis leaves, including induction of two hallmark processes of plant defense: ROS burst and cell wall modifications. Analyses of plants with a mutation in the potentially STm-induced gene EXO70H4 revealed that its encoded protein is required for stomatal defense against STm and E. coli O157:H7, but not against Pst DC3118. In the apoplast however, EXO70H4 is required for defense against STm and Pst DC3118, but not against E. coli O157: H7. Moreover, EXO70H4 is required for callose deposition, but had no function in ROS burst, triggered by all three bacteria. The salicylic acid (SA) signaling and biosynthesis proteins NPR1 and ICS1, respectively, were involved in stomatal and apoplastic defense, as well as callose deposition, against human and plant pathogens. Conclusions: The results show that EXO70H4 is involved in stomatal and apoplastic defenses in Arabidopsis and suggest that EXO70H4-mediated defense play a distinct role in guard cells and leaf mesophyll cells in a bacteriadependent manner. Nonetheless, EXO70H4 contributes to callose deposition in response to both human and plant pathogens. NPR1 and ICS1, two proteins involved in the SA signaling pathway, are important to inhibit leaf internalization and apoplastic persistence of enterobacteria and proliferation of phytopathogens. These findings highlight the existence of unique and shared plant genetic components to fight off diverse bacterial pathogens providing specific targets for the prevention of foodborne diseases.

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“…Pathogenesis assay was conducted using Pst DC3000, as described previously (Jacob et al, 2017;Katagiri et al, 2002). Briefly, plants were dip inoculated with 1 3 10 8 CFU/mL or vacuum infiltrated with 1 3 10 6 CFU/ mL of bacterial suspension containing 0.03% or 0.004% of Silwet L-77 (Lehle Seeds), respectively.…”

Section: Pathogenesis Assaymentioning

confidence: 99%

The common bean COK‐4 and the Arabidopsis FER kinase domain share similar functions in plant growth and defence

Azevedo

Gonçalves‐Vidigal

Oblessuc

et al. 2018

Molecular Plant Pathology

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Receptor-like kinases are membrane proteins that can be shared by diverse signalling pathways. Among them, the Arabidopsis thaliana FERONIA (FER) plays a role in the balance between distinct signals to control growth and defence. We have found that COK-4, a putative kinase encoded in the common bean anthracnose resistance locus Co-4, which is transcriptionally regulated during the immune response, is highly similar to the kinase domain of FER. To assess whether COK-4 is a functional orthologue of FER, we expressed COK-4 in the wild-type Col-0 and the fer-5 mutant of Arabidopsis and evaluated FER-associated traits. We observed that fer-5 plants show an enhanced apoplastic and stomatal defence against Pseudomonas syringae. In addition, the fer-5 mutant shows reduced biomass, smaller guard cell size, greater number of stomata per leaf area, fewer leaves, faster transition to reproductive stage and lower seed weight per plant than the wild-type Col-0. Except for the stomatal complex length and number of stomata, COK-4 expression in fer-5 lines partially or completely rescued both defence and developmental defects of fer-5 to the wild-type level. Notably, COK-4 may have an additive effect to FER, as the expression of COK-4 in Col-0 resulted in enhanced defence and growth phenotypes in comparison with wild-type Col-0 plants. Altogether, these findings indicate that the common bean COK-4 shares at least some of the multiple functions of the Arabidopsis FER kinase domain, acting in both the induction of plant growth and regulation of plant defence.

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Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast

Cited by 21 publications

References 7 publications

Human Pathogen Colonization of Lettuce Dependent Upon Plant Genotype and Defense Response Activation

Human Pathogen Colonization of Lettuce Dependent Upon Plant Genotype and Defense Response Activation

Novel molecular components involved in callose-mediated Arabidopsis defense against Salmonella enterica and Escherichia coli O157:H7

The common bean COK‐4 and the Arabidopsis FER kinase domain share similar functions in plant growth and defence

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