Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct;7(10):e1002291.
doi: 10.1371/journal.ppat.1002291. Epub 2011 Oct 6.

A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000

Affiliations

A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000

Weiqing Zeng et al. PLoS Pathog. 2011 Oct.

Abstract

Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata), multiplication in the intercellular space (apoplast) of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR) is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst) DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord) mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA)-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA), and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA) biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC) protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to implicate an important role of stress-associated protein translation in stomatal guard cell signaling in response to microbe-associated molecular patterns and bacterial infection.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characterization of scord mutants.
(A) Pst DC3118 population at 3 dpi after dip-inoculation at 1×108 CFU/ml. (B) Stomata apertures (µm) from leaf peels incubated with Pst DC3118 (1×108 CFU/ml) for 1 h. (C) Pst DC3118 populations when inoculated by infiltration at 1×106 CFU/ml. (D) Pst DC3000 population at 3 dpi after dip-inoculation at 1×107 CFU/ml. Differences were detected by comparing each scord mutant with Col-7 for (A), (C) and (D) Statistical analyses for this and following figures are described in MATERIALS and METHODS.
Figure 2
Figure 2. Stomatal closure responses of scord mutants to SA.
Stomatal apertures (µm) were measured from leaf peels incubated with SA (100 µM) for 1 h.
Figure 3
Figure 3. Salicylic acid (SA) levels in leaves of Col-7 and scord mutants.
Numbers represent means and standard errors of values from 4 different plants. Similar results were obtained from two different experiments. (A) Plants without treatment. (B) Plants were infiltrated with Pst DC3000 at 1×108 CFU/ml. Leaves were collected at 12 hpi.
Figure 4
Figure 4. Responses of scord mutants to ABA.
(A) Stomatal apertures of Col-7 and scord1, -3, -5, -6, -7 and -8 in response to ABA (10 µM) in leaf peel assay. (B) Appearance of 5-week-old scord7 and Col-7 plants after being withheld water for 10 days. (C) Water loss of detached leaves. Four plants and 4 to 5 leaves from each plant were sampled for Col-7 or scord7. Values are means with standard errors displayed. (D) Germination rates of Col-7 and scord7 seeds on MS plates supplemented with ABA. Values are means with standard errors. (E) Stomata apertures of Col-7 and scord7 in response to dark. Leaf peels were incubated in buffer under light or in the dark for 3 h before stomatal aperture measurements.
Figure 5
Figure 5. Morphological phenotypes of scord mutants of 4 to 5 weeks old.
(A) Rosettes. (B) Stomata densities of leaf abaxial surfaces. (C) Scanning electron microscopy pictures of leaf abaxial surfaces of Col-7 and scord6 plants. Note that raised central ridges in stomata are missing in the scord6 mutant.
Figure 6
Figure 6. Characterization of SCORD5 (At1g64550).
(A) The intron/exon structure of At1g64550 and T-DNA insertion sites in scord5 (scord5-1) and 188G03 (scord5-2) mutants. Locations of primers used in this study are indicated. (B) and (D) PCR reactions using genomic DNA from Col-7 and scord5 (scord5-1) (B) or 188G03 (scord5-2) (D) as template. (C) and (E) RT-PCR reactions using total RNA from Col-7 and scord5 (scord5-1) (C) or 188G03 (scord5-2) (E) as template. The RT-PCR product of ACT1 (Arabidopsis actin 1; At2g37620) was used as a loading control. (F) Stomata apertures of Col-0, scord5-1, and scord5-2 leaf peels incubated with H2O or Pst DC3118 (1×108 CFU/ml) for 1 h. (G) Stomata apertures of Col-7, scord5-1 and scord5 mutant plants complemented with a 5.4-kb-genomic region of At1g64550, in response to Pst DC3118 (1×108 CFU/ml) for 1 h.
Figure 7
Figure 7. scord5 mutant plants have normal flg22-induced responses other than stomatal closure response.
(A) Stomata apertures of Col-7 and scord5-1 leaf peels treated with flg22 (10 µM). (B) H2O2-dependent luminescence of luminol induced by flg22 (100 nM) in Col-7 and scord5-1seedlings. (C) Callose deposition induced by flg22 (10 nM) in Col-7 and scord5-1 leaves. (D) and (E) Populations of Pst DC3000 at 2 dpi after inoculation with dipping (D; 1×108 CFU/ml) or infiltration (E; 1×106 CFU/ml) following spray with H2O or flg22 (3 µM) a day earlier. Differences were detected by comparing H2O and flg22 treatment. Experiments in (B) and (C) were repeated two and three times, respectively.
Figure 8
Figure 8. Stomatal closure response in the ila-3 mutant.
(A) Stomata apertures of Col-7, scord5 and ila-3 in response to Pst DC3118 (1×108 CFU/ml) at 1 hpi. (B) Four-week-old rosettes.

Similar articles

Cited by

References

    1. Whalen M, Innes R, Bent A, Staskawicz B. Identification of Pseudomonas syringae pathogens of Arabidopsis thaliana and a bacterial gene determining avirulence on both Arabidopsis and soybean. Plant Cell. 1991;3:49–59. - PMC - PubMed
    1. Preston GM. Pseudomonas syringae pv. tomato: the right pathogen, of the right plant, at the right time. Mol Plant Pathol. 2000;1:263–275. - PubMed
    1. Katagiri F, Thilmony R, He SY. The Arabidopsis thaliana-Pseudomonas syringae interaction. In: Somerville CR, Meyerowitz EM, editors. The Arabidopsis Book. Rockville: American Society of Plant Biologists; 2002. pp. 1–35. - PMC - PubMed
    1. Mitchell RE. Coronatine production by some phytopathogenic Pseudomonads. Physiol Plant Pathol. 1982;20:83–89.
    1. Ma SW, Morris VL, Cuppels DA. Characterization of a DNA region required for production of the phytotoxin coronatine by Pseudomonas syringae pv. tomato. Mol Plant Microbe Interact. 1991;4:69–77.

Publication types