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Auto-acetylation on K289 is not essential for HopZ1a-mediated plant defense suppression.

Rufián JS, Lucía A, Macho AP, Orozco-Navarrete B, Arroyo-Mateos M, Bejarano ER, Beuzón CR, Ruiz-Albert J - Front Microbiol (2015)

Bottom Line: We have previously shown that HopZ1a suppresses both local [effector-triggered immunity (ETI)] and systemic immunity [systemic acquired resistance (SAR)] triggered by the heterologous effector AvrRpt2.HopZ1a acetyltransferase activity has been reported to require the auto-acetylation of the effector on a specific lysine (K289) residue.Our results indicate that, while the HopZ1a(K289R) mutant is impaired to some degree in its virulence and avirulence activities, is by no means phenotypically equivalent to the catalytically inactive HopZ1a(C216A), since it is still able to trigger a defense response that induces detectable macroscopic HR and effectively protects Arabidopsis from infection, reducing growth of P. syringae within the plant.

View Article: PubMed Central - PubMed

Affiliation: Departamento Biología Celular, Genética y Fisiología, Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" - Universidad de Málaga - Consejo Superior de Investigaciones Científicas Málaga, Spain.

ABSTRACT
The Pseudomonas syringae type III-secreted effector HopZ1a is a member of the HopZ/YopJ superfamily of effectors that triggers immunity in Arabidopsis. We have previously shown that HopZ1a suppresses both local [effector-triggered immunity (ETI)] and systemic immunity [systemic acquired resistance (SAR)] triggered by the heterologous effector AvrRpt2. HopZ1a has been shown to possess acetyltransferase activity, and this activity is essential to trigger immunity in Arabidopsis. HopZ1a acetyltransferase activity has been reported to require the auto-acetylation of the effector on a specific lysine (K289) residue. In this paper we analyze the relevance of autoacetylation of lysine residue 289 in HopZ1a ability to suppress plant defenses, and on the light of the results obtained, we also revise its relevance for HopZ1a avirulence activity. Our results indicate that, while the HopZ1a(K289R) mutant is impaired to some degree in its virulence and avirulence activities, is by no means phenotypically equivalent to the catalytically inactive HopZ1a(C216A), since it is still able to trigger a defense response that induces detectable macroscopic HR and effectively protects Arabidopsis from infection, reducing growth of P. syringae within the plant. We also present evidence that the HopZ1a(K289R) mutant still displays virulence activities, partially suppressing both ETI and SAR.

No MeSH data available.


Related in: MedlinePlus

HopZ1aK289R partially suppresses AvrRpt2-triggered immunity. (A) Western blot showing PR1 accumulation in Col-0 leaves inoculated with 5 × 105 cfu/ml of DC3000 expressing AvrRpt2 (pAME8) alone or co-expressing AvrRpt2 with HopZ1a (pAME33), HopZ1aC216A (pAME34), or HopZ1aK289R (pJRU10). Ten micrograms of total protein were loaded per sample, and Coomassie staining is shown as loading control. The signal intensity for each band was quantified using Fiji distribution of ImageJ software and is shown below the blot. The experiment was repeated twice with similar results. (B) Canceled-out indices (COIs) measuring growth within a mixed infection of DC3000 co-expressing AvrRpt2 and any of the three HopZ1a variants: wild-type HopZ1a (pAME33), HopZ1aC216A (pAME34) or HopZ1aK289R (pJRU10), in relation to growth of DC3000 expressing only the corresponding HopZ1a: wild-type HopZ1a (pAME30Gm), HopZ1aC216A (pAME27Gm), or HopZ1aK289R (pMAM1Gm). COIs are calculated as the output ratio between the strain expressing both effectors and the strain expressing just one, divided by their input ratio. Each COI value represents the means of two independent experiments with three biological replicates each. Error bars represent the standard error. Mean values marked with the same letter are not significantly different from each other as established by Student’s t-test (P < 0.05).
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Figure 2: HopZ1aK289R partially suppresses AvrRpt2-triggered immunity. (A) Western blot showing PR1 accumulation in Col-0 leaves inoculated with 5 × 105 cfu/ml of DC3000 expressing AvrRpt2 (pAME8) alone or co-expressing AvrRpt2 with HopZ1a (pAME33), HopZ1aC216A (pAME34), or HopZ1aK289R (pJRU10). Ten micrograms of total protein were loaded per sample, and Coomassie staining is shown as loading control. The signal intensity for each band was quantified using Fiji distribution of ImageJ software and is shown below the blot. The experiment was repeated twice with similar results. (B) Canceled-out indices (COIs) measuring growth within a mixed infection of DC3000 co-expressing AvrRpt2 and any of the three HopZ1a variants: wild-type HopZ1a (pAME33), HopZ1aC216A (pAME34) or HopZ1aK289R (pJRU10), in relation to growth of DC3000 expressing only the corresponding HopZ1a: wild-type HopZ1a (pAME30Gm), HopZ1aC216A (pAME27Gm), or HopZ1aK289R (pMAM1Gm). COIs are calculated as the output ratio between the strain expressing both effectors and the strain expressing just one, divided by their input ratio. Each COI value represents the means of two independent experiments with three biological replicates each. Error bars represent the standard error. Mean values marked with the same letter are not significantly different from each other as established by Student’s t-test (P < 0.05).

Mentions: PR1 accumulated to similar levels in leaves inoculated with DC3000 expressing AvrRpt2 or DC3000 co-expressing AvrRpt2 and HopZ1aC216A, while PR1 accumulation was clearly reduced in leaves inoculated with DC3000 co-expressing AvrRpt2 and HopZ1a (Figure 2A). In leaves inoculated with DC3000 co-expressing AvrRpt2 and the HopZ1aK289R mutant protein, we could not detect differences in PR1 accumulation in comparison to leaves inoculated with either DC3000 expressing AvrRpt2 alone or with HopZ1aC216A (Figure 2A)


Auto-acetylation on K289 is not essential for HopZ1a-mediated plant defense suppression.

Rufián JS, Lucía A, Macho AP, Orozco-Navarrete B, Arroyo-Mateos M, Bejarano ER, Beuzón CR, Ruiz-Albert J - Front Microbiol (2015)

HopZ1aK289R partially suppresses AvrRpt2-triggered immunity. (A) Western blot showing PR1 accumulation in Col-0 leaves inoculated with 5 × 105 cfu/ml of DC3000 expressing AvrRpt2 (pAME8) alone or co-expressing AvrRpt2 with HopZ1a (pAME33), HopZ1aC216A (pAME34), or HopZ1aK289R (pJRU10). Ten micrograms of total protein were loaded per sample, and Coomassie staining is shown as loading control. The signal intensity for each band was quantified using Fiji distribution of ImageJ software and is shown below the blot. The experiment was repeated twice with similar results. (B) Canceled-out indices (COIs) measuring growth within a mixed infection of DC3000 co-expressing AvrRpt2 and any of the three HopZ1a variants: wild-type HopZ1a (pAME33), HopZ1aC216A (pAME34) or HopZ1aK289R (pJRU10), in relation to growth of DC3000 expressing only the corresponding HopZ1a: wild-type HopZ1a (pAME30Gm), HopZ1aC216A (pAME27Gm), or HopZ1aK289R (pMAM1Gm). COIs are calculated as the output ratio between the strain expressing both effectors and the strain expressing just one, divided by their input ratio. Each COI value represents the means of two independent experiments with three biological replicates each. Error bars represent the standard error. Mean values marked with the same letter are not significantly different from each other as established by Student’s t-test (P < 0.05).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4495678&req=5

Figure 2: HopZ1aK289R partially suppresses AvrRpt2-triggered immunity. (A) Western blot showing PR1 accumulation in Col-0 leaves inoculated with 5 × 105 cfu/ml of DC3000 expressing AvrRpt2 (pAME8) alone or co-expressing AvrRpt2 with HopZ1a (pAME33), HopZ1aC216A (pAME34), or HopZ1aK289R (pJRU10). Ten micrograms of total protein were loaded per sample, and Coomassie staining is shown as loading control. The signal intensity for each band was quantified using Fiji distribution of ImageJ software and is shown below the blot. The experiment was repeated twice with similar results. (B) Canceled-out indices (COIs) measuring growth within a mixed infection of DC3000 co-expressing AvrRpt2 and any of the three HopZ1a variants: wild-type HopZ1a (pAME33), HopZ1aC216A (pAME34) or HopZ1aK289R (pJRU10), in relation to growth of DC3000 expressing only the corresponding HopZ1a: wild-type HopZ1a (pAME30Gm), HopZ1aC216A (pAME27Gm), or HopZ1aK289R (pMAM1Gm). COIs are calculated as the output ratio between the strain expressing both effectors and the strain expressing just one, divided by their input ratio. Each COI value represents the means of two independent experiments with three biological replicates each. Error bars represent the standard error. Mean values marked with the same letter are not significantly different from each other as established by Student’s t-test (P < 0.05).
Mentions: PR1 accumulated to similar levels in leaves inoculated with DC3000 expressing AvrRpt2 or DC3000 co-expressing AvrRpt2 and HopZ1aC216A, while PR1 accumulation was clearly reduced in leaves inoculated with DC3000 co-expressing AvrRpt2 and HopZ1a (Figure 2A). In leaves inoculated with DC3000 co-expressing AvrRpt2 and the HopZ1aK289R mutant protein, we could not detect differences in PR1 accumulation in comparison to leaves inoculated with either DC3000 expressing AvrRpt2 alone or with HopZ1aC216A (Figure 2A)

Bottom Line: We have previously shown that HopZ1a suppresses both local [effector-triggered immunity (ETI)] and systemic immunity [systemic acquired resistance (SAR)] triggered by the heterologous effector AvrRpt2.HopZ1a acetyltransferase activity has been reported to require the auto-acetylation of the effector on a specific lysine (K289) residue.Our results indicate that, while the HopZ1a(K289R) mutant is impaired to some degree in its virulence and avirulence activities, is by no means phenotypically equivalent to the catalytically inactive HopZ1a(C216A), since it is still able to trigger a defense response that induces detectable macroscopic HR and effectively protects Arabidopsis from infection, reducing growth of P. syringae within the plant.

View Article: PubMed Central - PubMed

Affiliation: Departamento Biología Celular, Genética y Fisiología, Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" - Universidad de Málaga - Consejo Superior de Investigaciones Científicas Málaga, Spain.

ABSTRACT
The Pseudomonas syringae type III-secreted effector HopZ1a is a member of the HopZ/YopJ superfamily of effectors that triggers immunity in Arabidopsis. We have previously shown that HopZ1a suppresses both local [effector-triggered immunity (ETI)] and systemic immunity [systemic acquired resistance (SAR)] triggered by the heterologous effector AvrRpt2. HopZ1a has been shown to possess acetyltransferase activity, and this activity is essential to trigger immunity in Arabidopsis. HopZ1a acetyltransferase activity has been reported to require the auto-acetylation of the effector on a specific lysine (K289) residue. In this paper we analyze the relevance of autoacetylation of lysine residue 289 in HopZ1a ability to suppress plant defenses, and on the light of the results obtained, we also revise its relevance for HopZ1a avirulence activity. Our results indicate that, while the HopZ1a(K289R) mutant is impaired to some degree in its virulence and avirulence activities, is by no means phenotypically equivalent to the catalytically inactive HopZ1a(C216A), since it is still able to trigger a defense response that induces detectable macroscopic HR and effectively protects Arabidopsis from infection, reducing growth of P. syringae within the plant. We also present evidence that the HopZ1a(K289R) mutant still displays virulence activities, partially suppressing both ETI and SAR.

No MeSH data available.


Related in: MedlinePlus