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Redundancy among phospholipase D isoforms in resistance triggered by recognition of the Pseudomonas syringae effector AvrRpm1 in Arabidopsis thaliana.

Johansson ON, Fahlberg P, Karimi E, Nilsson AK, Ellerström M, Andersson MX - Front Plant Sci (2014)

Bottom Line: Recognition of microbial effectors or their activity by plant resistance (R)-proteins triggers a second line of defense resulting in effector triggered immunity (ETI).However, repression of PLD dependent PA production by n-butanol strongly inhibited the HR following Pseudomonas syringae effector recognition.Thus, the effect of mutations of PLDs could become detectable, and the corresponding genes can be proposed to be involved in the HR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Environmental Sciences, University of Gothenburg Gothenburg, Sweden.

ABSTRACT
Plants possess a highly sophisticated system for defense against microorganisms. So called MAMP (microbe-associated molecular patterns) triggered immunity (MTI) prevents the majority of non-adapted pathogens from causing disease. Adapted plant pathogens use secreted effector proteins to interfere with such signaling. Recognition of microbial effectors or their activity by plant resistance (R)-proteins triggers a second line of defense resulting in effector triggered immunity (ETI). The latter usually comprises the hypersensitive response (HR) which includes programmed cell death at the site of infection. Phospholipase D (PLD) mediated production of phosphatidic acid (PA) has been linked to both MTI and ETI in plants. Inhibition of PLD activity has been shown to attenuate MTI as well as ETI. In this study, we systematically tested single and double knockouts in all 12 genes encoding PLDs in Arabidopsis thaliana for effects on ETI and MTI. No single PLD could be linked to ETI triggered by recognition of effectors secreted by the bacterium Pseudomonas syringae. However, repression of PLD dependent PA production by n-butanol strongly inhibited the HR following Pseudomonas syringae effector recognition. In addition some pld mutants were more sensitive to n-butanol than wild type. Thus, the effect of mutations of PLDs could become detectable, and the corresponding genes can be proposed to be involved in the HR. Only knockout of PLDδ caused a loss of MTI-induced cell wall based defense against the non-host powdery mildew Erysiphe pisi. This is thus in stark contrast to the involvement of a multitude of PLD isoforms in the HR triggered by AvrRpm1 recognition.

No MeSH data available.


Related in: MedlinePlus

Phospholipase D (PLD) dependence of hypersensitive response (HR) induction in Arabidopsis following recognition of AvrRpm1 expressed by Pseudomonas syringae pv. tomato (Pst). Leaf discs were prepared from wild type, Col-0, Arabidopsis, infiltrated with Pst DC3000:AvrRpm1 suspended in 0.2, 0.4 (A), 0.6 or 0.8 % (B) tert- or n-butanol solutions and incubated in deionized water with the same alcohol at the same concentration for 6 h. The conductivity of the bathing solution was measured at the indicated time points. Average of six replicates and SD is shown. Lower case letters represent statistically significant different groups (one way ANOVA, p < 0.05) for the 6 h time point. The experiment was performed twice with similar results.
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Figure 1: Phospholipase D (PLD) dependence of hypersensitive response (HR) induction in Arabidopsis following recognition of AvrRpm1 expressed by Pseudomonas syringae pv. tomato (Pst). Leaf discs were prepared from wild type, Col-0, Arabidopsis, infiltrated with Pst DC3000:AvrRpm1 suspended in 0.2, 0.4 (A), 0.6 or 0.8 % (B) tert- or n-butanol solutions and incubated in deionized water with the same alcohol at the same concentration for 6 h. The conductivity of the bathing solution was measured at the indicated time points. Average of six replicates and SD is shown. Lower case letters represent statistically significant different groups (one way ANOVA, p < 0.05) for the 6 h time point. The experiment was performed twice with similar results.

Mentions: As PLDs are clearly involved in both PTI and ETI, we decided to test the involvement of individual Arabidopsis PLD genes on defense responses triggered by recognition of a bacterial effector. The tomato pathovar of Pseudomonas syringae DC3000 is normally highly virulent on wild type Arabidopsis. However, if the pathogen carries the AvrRpm1 effector gene, the AvrRpm1 protein is recognized by the Arabidopsis R-protein RPM1 (Grant et al., 1995). This recognition triggers induction of HR and programmed cell death in the plant. The latter can be measured as loss of electrolytes from leaf tissue into an aqueous solution (Hibberd, 1987; Mackey et al., 2002). To verify the involvement of PLD in HR triggered by AvrRpm1 recognition in Arabidopsis, leaf tissue infiltrated with Pst DC3000:AvrRpm1 was incubated in solution with different concentrations of n- or tert-butanol and the rate of cell death determined by measuring the electric conductance of the bathing solution (Figure 1). The primary alcohol n-butanol is known to inhibit PLD dependent formation of PA, as the alcohol is preferred over water to form an artificial phosphatidylalcohol by transphosphatidylation (Ella et al., 1997). Tert-butanol, on the other hand, is unable to do this. A concentration of 0.6% (v/v) n-butanol caused a decrease in the HR induced by AvrRpm1 recognition by about 40%, whereas 0.8% (v/v) of n-butanol caused an almost complete loss in cell death. Tert-butanol had only a slight effect on the HR as measured by electrolyte leakage, the effect of tert-butanol was apparent only at the two highest concentrations used.


Redundancy among phospholipase D isoforms in resistance triggered by recognition of the Pseudomonas syringae effector AvrRpm1 in Arabidopsis thaliana.

Johansson ON, Fahlberg P, Karimi E, Nilsson AK, Ellerström M, Andersson MX - Front Plant Sci (2014)

Phospholipase D (PLD) dependence of hypersensitive response (HR) induction in Arabidopsis following recognition of AvrRpm1 expressed by Pseudomonas syringae pv. tomato (Pst). Leaf discs were prepared from wild type, Col-0, Arabidopsis, infiltrated with Pst DC3000:AvrRpm1 suspended in 0.2, 0.4 (A), 0.6 or 0.8 % (B) tert- or n-butanol solutions and incubated in deionized water with the same alcohol at the same concentration for 6 h. The conductivity of the bathing solution was measured at the indicated time points. Average of six replicates and SD is shown. Lower case letters represent statistically significant different groups (one way ANOVA, p < 0.05) for the 6 h time point. The experiment was performed twice with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230166&req=5

Figure 1: Phospholipase D (PLD) dependence of hypersensitive response (HR) induction in Arabidopsis following recognition of AvrRpm1 expressed by Pseudomonas syringae pv. tomato (Pst). Leaf discs were prepared from wild type, Col-0, Arabidopsis, infiltrated with Pst DC3000:AvrRpm1 suspended in 0.2, 0.4 (A), 0.6 or 0.8 % (B) tert- or n-butanol solutions and incubated in deionized water with the same alcohol at the same concentration for 6 h. The conductivity of the bathing solution was measured at the indicated time points. Average of six replicates and SD is shown. Lower case letters represent statistically significant different groups (one way ANOVA, p < 0.05) for the 6 h time point. The experiment was performed twice with similar results.
Mentions: As PLDs are clearly involved in both PTI and ETI, we decided to test the involvement of individual Arabidopsis PLD genes on defense responses triggered by recognition of a bacterial effector. The tomato pathovar of Pseudomonas syringae DC3000 is normally highly virulent on wild type Arabidopsis. However, if the pathogen carries the AvrRpm1 effector gene, the AvrRpm1 protein is recognized by the Arabidopsis R-protein RPM1 (Grant et al., 1995). This recognition triggers induction of HR and programmed cell death in the plant. The latter can be measured as loss of electrolytes from leaf tissue into an aqueous solution (Hibberd, 1987; Mackey et al., 2002). To verify the involvement of PLD in HR triggered by AvrRpm1 recognition in Arabidopsis, leaf tissue infiltrated with Pst DC3000:AvrRpm1 was incubated in solution with different concentrations of n- or tert-butanol and the rate of cell death determined by measuring the electric conductance of the bathing solution (Figure 1). The primary alcohol n-butanol is known to inhibit PLD dependent formation of PA, as the alcohol is preferred over water to form an artificial phosphatidylalcohol by transphosphatidylation (Ella et al., 1997). Tert-butanol, on the other hand, is unable to do this. A concentration of 0.6% (v/v) n-butanol caused a decrease in the HR induced by AvrRpm1 recognition by about 40%, whereas 0.8% (v/v) of n-butanol caused an almost complete loss in cell death. Tert-butanol had only a slight effect on the HR as measured by electrolyte leakage, the effect of tert-butanol was apparent only at the two highest concentrations used.

Bottom Line: Recognition of microbial effectors or their activity by plant resistance (R)-proteins triggers a second line of defense resulting in effector triggered immunity (ETI).However, repression of PLD dependent PA production by n-butanol strongly inhibited the HR following Pseudomonas syringae effector recognition.Thus, the effect of mutations of PLDs could become detectable, and the corresponding genes can be proposed to be involved in the HR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Environmental Sciences, University of Gothenburg Gothenburg, Sweden.

ABSTRACT
Plants possess a highly sophisticated system for defense against microorganisms. So called MAMP (microbe-associated molecular patterns) triggered immunity (MTI) prevents the majority of non-adapted pathogens from causing disease. Adapted plant pathogens use secreted effector proteins to interfere with such signaling. Recognition of microbial effectors or their activity by plant resistance (R)-proteins triggers a second line of defense resulting in effector triggered immunity (ETI). The latter usually comprises the hypersensitive response (HR) which includes programmed cell death at the site of infection. Phospholipase D (PLD) mediated production of phosphatidic acid (PA) has been linked to both MTI and ETI in plants. Inhibition of PLD activity has been shown to attenuate MTI as well as ETI. In this study, we systematically tested single and double knockouts in all 12 genes encoding PLDs in Arabidopsis thaliana for effects on ETI and MTI. No single PLD could be linked to ETI triggered by recognition of effectors secreted by the bacterium Pseudomonas syringae. However, repression of PLD dependent PA production by n-butanol strongly inhibited the HR following Pseudomonas syringae effector recognition. In addition some pld mutants were more sensitive to n-butanol than wild type. Thus, the effect of mutations of PLDs could become detectable, and the corresponding genes can be proposed to be involved in the HR. Only knockout of PLDδ caused a loss of MTI-induced cell wall based defense against the non-host powdery mildew Erysiphe pisi. This is thus in stark contrast to the involvement of a multitude of PLD isoforms in the HR triggered by AvrRpm1 recognition.

No MeSH data available.


Related in: MedlinePlus