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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

Additative effects of n-butanol and loss of single PLD genes on HR cell death following recognition of AvrRpm1. Leaf discs were prepared from the indicated lines, infiltrated with Pst DC3000:AvrRpm1 in 0.6% tert- or n-butanol as indicated and incubated in deionized water with the same alcohol. Col-0 treated with tert- and n-butanol is included in all experiments (A–E) together with the indicated subset of PLD knock out mutants. The loss of cellular electrolytes was measured as the conductance of the bathing solution 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 6: Additative effects of n-butanol and loss of single PLD genes on HR cell death following recognition of AvrRpm1. Leaf discs were prepared from the indicated lines, infiltrated with Pst DC3000:AvrRpm1 in 0.6% tert- or n-butanol as indicated and incubated in deionized water with the same alcohol. Col-0 treated with tert- and n-butanol is included in all experiments (A–E) together with the indicated subset of PLD knock out mutants. The loss of cellular electrolytes was measured as the conductance of the bathing solution 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: While none of the tested pld mutants displayed a clear reduction in effector induced HR, the involvement of PLDs in this defense reaction was apparent as treatment with n-butanol clearly affected the plants ability to mount HR and formation of PBut in connection with this was observed. We thus reasoned that the PLD activity in response to AvrRpm1 recognition is likely caused by the activation of several PLD isoforms and that the individual contributions might be so small that the single knock outs show no phenotype. Thus, if the overall activity of PLD is lowered by addition of n-butanol, it might be possible to detect the effect of loss of single PLD isoforms. To test this, wild type (Col-0) and all the pld mutants were infiltrated with 0.6% n-butanol together with Pst DC3000:AvrRpm1 (Figure 6). As a control, the wild type was also treated with 0.6% tert-butanol. As expected, 0.6% n-butanol caused a significant reduction in ion leakage following AvrRpm1 recognition compared to treatment with 0.6% tert-butanol in wild type. The single mutants pldα1, pldα2, pldβ1, pldβ2, pldδ, pldζ1, pldζ2, and pldε all displayed 10–20% statistically significant reductions in HR compared to wild type when treated with 0.6% n-butanol (Figures 6A–C). The mutants pldγ1 and pldγ2 also displayed a statistically significant reduction in ion leakage induced after AvrRpm1 recognition, this effect was however smaller than for the other mutants (Figure 6D). Finally, the double mutants pldβ1 pldβ2 and pldα1pldδ, as well as the triple mutant pldβ1 pldβ2 pldδ, displayed a similar conditional reduction of ion leakage as shown for the single mutants (Figure 6E).


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)

Additative effects of n-butanol and loss of single PLD genes on HR cell death following recognition of AvrRpm1. Leaf discs were prepared from the indicated lines, infiltrated with Pst DC3000:AvrRpm1 in 0.6% tert- or n-butanol as indicated and incubated in deionized water with the same alcohol. Col-0 treated with tert- and n-butanol is included in all experiments (A–E) together with the indicated subset of PLD knock out mutants. The loss of cellular electrolytes was measured as the conductance of the bathing solution 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 6: Additative effects of n-butanol and loss of single PLD genes on HR cell death following recognition of AvrRpm1. Leaf discs were prepared from the indicated lines, infiltrated with Pst DC3000:AvrRpm1 in 0.6% tert- or n-butanol as indicated and incubated in deionized water with the same alcohol. Col-0 treated with tert- and n-butanol is included in all experiments (A–E) together with the indicated subset of PLD knock out mutants. The loss of cellular electrolytes was measured as the conductance of the bathing solution 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: While none of the tested pld mutants displayed a clear reduction in effector induced HR, the involvement of PLDs in this defense reaction was apparent as treatment with n-butanol clearly affected the plants ability to mount HR and formation of PBut in connection with this was observed. We thus reasoned that the PLD activity in response to AvrRpm1 recognition is likely caused by the activation of several PLD isoforms and that the individual contributions might be so small that the single knock outs show no phenotype. Thus, if the overall activity of PLD is lowered by addition of n-butanol, it might be possible to detect the effect of loss of single PLD isoforms. To test this, wild type (Col-0) and all the pld mutants were infiltrated with 0.6% n-butanol together with Pst DC3000:AvrRpm1 (Figure 6). As a control, the wild type was also treated with 0.6% tert-butanol. As expected, 0.6% n-butanol caused a significant reduction in ion leakage following AvrRpm1 recognition compared to treatment with 0.6% tert-butanol in wild type. The single mutants pldα1, pldα2, pldβ1, pldβ2, pldδ, pldζ1, pldζ2, and pldε all displayed 10–20% statistically significant reductions in HR compared to wild type when treated with 0.6% n-butanol (Figures 6A–C). The mutants pldγ1 and pldγ2 also displayed a statistically significant reduction in ion leakage induced after AvrRpm1 recognition, this effect was however smaller than for the other mutants (Figure 6D). Finally, the double mutants pldβ1 pldβ2 and pldα1pldδ, as well as the triple mutant pldβ1 pldβ2 pldδ, displayed a similar conditional reduction of ion leakage as shown for the single mutants (Figure 6E).

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