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Functional analysis of Hyaloperonospora arabidopsidis RXLR effectors.

Pel MJ, Wintermans PC, Cabral A, Robroek BJ, Seidl MF, Bautor J, Parker JE, Van den Ackerveken G, Pieterse CM - PLoS ONE (2014)

Bottom Line: Multifactorial analysis of results collated from all experiments revealed that, except for RXLR20, all RXLR effector proteins tested affected plant immunity.For RXLR9 this was confirmed using a P. syringae ΔCEL-mediated effector delivery system.Together, the results show that many H. arabidopsidis RXLR effectors have small effects on the plant immune response, suggesting that suppression of host immunity by this biotrophic pathogen is likely to be caused by the combined actions of effectors.

View Article: PubMed Central - PubMed

Affiliation: Plant-Microbe Interactions, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands; Centre for BioSystems Genomics, Wageningen, The Netherlands.

ABSTRACT
The biotrophic plant pathogen Hyaloperonospora arabidopsidis produces a set of putative effector proteins that contain the conserved RXLR motif. For most of these RXLR proteins the role during infection is unknown. Thirteen RXLR proteins from H. arabidopsidis strain Waco9 were analyzed for sequence similarities and tested for a role in virulence. The thirteen RXLR proteins displayed conserved N-termini and this N-terminal conservation was also found in the 134 predicted RXLR genes from the genome of H. arabidopsidis strain Emoy2. To investigate the effects of single RXLR effector proteins on plant defense responses, thirteen H. arabidopsidis Waco9 RXLR genes were expressed in Arabidopsis thaliana. Subsequently, these plants were screened for altered susceptibility to the oomycetes H. arabidopsidis and Phytophthora capsici, and the bacterial pathogen Pseudomonas syringae. Additionally, the effect of the RXLR proteins on flg22-triggered basal immune responses was assessed. Multifactorial analysis of results collated from all experiments revealed that, except for RXLR20, all RXLR effector proteins tested affected plant immunity. For RXLR9 this was confirmed using a P. syringae ΔCEL-mediated effector delivery system. Together, the results show that many H. arabidopsidis RXLR effectors have small effects on the plant immune response, suggesting that suppression of host immunity by this biotrophic pathogen is likely to be caused by the combined actions of effectors.

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Effect of EDV-mediated delivery of Waco9 RXLR9 on P. syringae ΔCEL-induced callose deposition.Five-week-old Col-0 plants were infiltrated with P. syringae ΔCEL carrying YFP, RXLR9 or ATR13. After 12 h, leaves were harvested and stained with analine blue for the detection of callose deposition. Pictures were taken (A) and the number of callose spots was quantified (B). The combined results of three independent experiments are shown. Results represent mean ± SEM (n = 17–23) and asterisks indicate significant differences (ANOVA and Fisher’s LSD corrected for type I errors; p<0.05).
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pone-0110624-g008: Effect of EDV-mediated delivery of Waco9 RXLR9 on P. syringae ΔCEL-induced callose deposition.Five-week-old Col-0 plants were infiltrated with P. syringae ΔCEL carrying YFP, RXLR9 or ATR13. After 12 h, leaves were harvested and stained with analine blue for the detection of callose deposition. Pictures were taken (A) and the number of callose spots was quantified (B). The combined results of three independent experiments are shown. Results represent mean ± SEM (n = 17–23) and asterisks indicate significant differences (ANOVA and Fisher’s LSD corrected for type I errors; p<0.05).

Mentions: To further test the above observed RXLR trends independently of constitutive in planta expression, we used the bacterial effector detector vector (EDV) system. This system has been used successfully to deliver the H. arabidopsidis RXLR effector ATR13 into A. thaliana leaf cells [59]. It is based on the fusion of a candidate effector protein to the N-terminus of the type III secreted bacterial effector, AvrRPs4, allowing delivery of the effector into A. thaliana leaf cells by bacteria such as P. syringae pv. tomato DC3000 mutant ΔCEL, which strongly triggers MTI [54], [65]. Immune suppressive effects of the delivered putative effector can be tested by quantifying its effect on P. syringae ΔCEL-triggered callose deposition [39]. Waco9 RXLR29 has previously been shown to suppress pathogen-induced callose deposition [39]. Here, a different RXLR protein, RXLR9, was selected for analysis. RXLR9 was cloned in the EDV effector delivery system and expressed in the P. syringae ΔCEL mutant strain and delivered to A. thaliana by pressure infiltrating P. syringae ΔCEL (RXLR9) into leaves of Col-0 plants. As a negative control the same EDV system was used to deliver the YFP protein into plant cells, and EDV-ATR13 was used as a positive control [39], [59]. At 12 h after pathogen infiltration, the immune-suppressive effect of RXLR9 was evaluated by quantifying callose deposition in the infiltrated leaf tissue. Infiltration of Col-0 leaves with P. syringae ΔCEL (YFP) resulted in strong callose deposition at the site of tissue infiltration (Figure 8), confirming previous findings that P. syringae ΔCEL triggers a strong MTI response [39], [54], [65]. Also, infiltration of Col-0 with bacteria delivering ATR13 reduced the number of callose deposition sites, confirming the immune suppressive effect of ATR13 [59]. Infiltration with P. syringae ΔCEL delivering RXLR9 led to a reduction in callose production that was similar to that of ATR13. We therefore concluded that RXLR9 is able to suppress MTI.


Functional analysis of Hyaloperonospora arabidopsidis RXLR effectors.

Pel MJ, Wintermans PC, Cabral A, Robroek BJ, Seidl MF, Bautor J, Parker JE, Van den Ackerveken G, Pieterse CM - PLoS ONE (2014)

Effect of EDV-mediated delivery of Waco9 RXLR9 on P. syringae ΔCEL-induced callose deposition.Five-week-old Col-0 plants were infiltrated with P. syringae ΔCEL carrying YFP, RXLR9 or ATR13. After 12 h, leaves were harvested and stained with analine blue for the detection of callose deposition. Pictures were taken (A) and the number of callose spots was quantified (B). The combined results of three independent experiments are shown. Results represent mean ± SEM (n = 17–23) and asterisks indicate significant differences (ANOVA and Fisher’s LSD corrected for type I errors; p<0.05).
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Related In: Results  -  Collection

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pone-0110624-g008: Effect of EDV-mediated delivery of Waco9 RXLR9 on P. syringae ΔCEL-induced callose deposition.Five-week-old Col-0 plants were infiltrated with P. syringae ΔCEL carrying YFP, RXLR9 or ATR13. After 12 h, leaves were harvested and stained with analine blue for the detection of callose deposition. Pictures were taken (A) and the number of callose spots was quantified (B). The combined results of three independent experiments are shown. Results represent mean ± SEM (n = 17–23) and asterisks indicate significant differences (ANOVA and Fisher’s LSD corrected for type I errors; p<0.05).
Mentions: To further test the above observed RXLR trends independently of constitutive in planta expression, we used the bacterial effector detector vector (EDV) system. This system has been used successfully to deliver the H. arabidopsidis RXLR effector ATR13 into A. thaliana leaf cells [59]. It is based on the fusion of a candidate effector protein to the N-terminus of the type III secreted bacterial effector, AvrRPs4, allowing delivery of the effector into A. thaliana leaf cells by bacteria such as P. syringae pv. tomato DC3000 mutant ΔCEL, which strongly triggers MTI [54], [65]. Immune suppressive effects of the delivered putative effector can be tested by quantifying its effect on P. syringae ΔCEL-triggered callose deposition [39]. Waco9 RXLR29 has previously been shown to suppress pathogen-induced callose deposition [39]. Here, a different RXLR protein, RXLR9, was selected for analysis. RXLR9 was cloned in the EDV effector delivery system and expressed in the P. syringae ΔCEL mutant strain and delivered to A. thaliana by pressure infiltrating P. syringae ΔCEL (RXLR9) into leaves of Col-0 plants. As a negative control the same EDV system was used to deliver the YFP protein into plant cells, and EDV-ATR13 was used as a positive control [39], [59]. At 12 h after pathogen infiltration, the immune-suppressive effect of RXLR9 was evaluated by quantifying callose deposition in the infiltrated leaf tissue. Infiltration of Col-0 leaves with P. syringae ΔCEL (YFP) resulted in strong callose deposition at the site of tissue infiltration (Figure 8), confirming previous findings that P. syringae ΔCEL triggers a strong MTI response [39], [54], [65]. Also, infiltration of Col-0 with bacteria delivering ATR13 reduced the number of callose deposition sites, confirming the immune suppressive effect of ATR13 [59]. Infiltration with P. syringae ΔCEL delivering RXLR9 led to a reduction in callose production that was similar to that of ATR13. We therefore concluded that RXLR9 is able to suppress MTI.

Bottom Line: Multifactorial analysis of results collated from all experiments revealed that, except for RXLR20, all RXLR effector proteins tested affected plant immunity.For RXLR9 this was confirmed using a P. syringae ΔCEL-mediated effector delivery system.Together, the results show that many H. arabidopsidis RXLR effectors have small effects on the plant immune response, suggesting that suppression of host immunity by this biotrophic pathogen is likely to be caused by the combined actions of effectors.

View Article: PubMed Central - PubMed

Affiliation: Plant-Microbe Interactions, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands; Centre for BioSystems Genomics, Wageningen, The Netherlands.

ABSTRACT
The biotrophic plant pathogen Hyaloperonospora arabidopsidis produces a set of putative effector proteins that contain the conserved RXLR motif. For most of these RXLR proteins the role during infection is unknown. Thirteen RXLR proteins from H. arabidopsidis strain Waco9 were analyzed for sequence similarities and tested for a role in virulence. The thirteen RXLR proteins displayed conserved N-termini and this N-terminal conservation was also found in the 134 predicted RXLR genes from the genome of H. arabidopsidis strain Emoy2. To investigate the effects of single RXLR effector proteins on plant defense responses, thirteen H. arabidopsidis Waco9 RXLR genes were expressed in Arabidopsis thaliana. Subsequently, these plants were screened for altered susceptibility to the oomycetes H. arabidopsidis and Phytophthora capsici, and the bacterial pathogen Pseudomonas syringae. Additionally, the effect of the RXLR proteins on flg22-triggered basal immune responses was assessed. Multifactorial analysis of results collated from all experiments revealed that, except for RXLR20, all RXLR effector proteins tested affected plant immunity. For RXLR9 this was confirmed using a P. syringae ΔCEL-mediated effector delivery system. Together, the results show that many H. arabidopsidis RXLR effectors have small effects on the plant immune response, suggesting that suppression of host immunity by this biotrophic pathogen is likely to be caused by the combined actions of effectors.

Show MeSH
Related in: MedlinePlus