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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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Expression levels of H. arabidopsidis RXLR genes in A. thaliana.Semi-quantitative analysis of the expression levels of the RXLR transgenes from H. arabidopsidis Waco9 in A. thaliana accession Col-0. Expression levels were assessed in two independent transgenic lines using RXLR gene-specific primers. Depicted are ethidium bromide-stained agarose gels with PCR products after 20, 25 or 30 cycles of PCR amplification. The PCR product of the A. thaliana actin gene (At3g18780) was used as internal control (25 cycles of PCR amplification).
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pone-0110624-g002: Expression levels of H. arabidopsidis RXLR genes in A. thaliana.Semi-quantitative analysis of the expression levels of the RXLR transgenes from H. arabidopsidis Waco9 in A. thaliana accession Col-0. Expression levels were assessed in two independent transgenic lines using RXLR gene-specific primers. Depicted are ethidium bromide-stained agarose gels with PCR products after 20, 25 or 30 cycles of PCR amplification. The PCR product of the A. thaliana actin gene (At3g18780) was used as internal control (25 cycles of PCR amplification).

Mentions: The RXLR gene transcripts of H. arabidopsidis identified by Cabral et al.[39] are expressed during infection, suggesting a role in pathogen virulence. While for other oomycete pathogens transformation protocols have been established [12], [61], [62], it is currently still not possible to transform H. arabidopsidis. Hence, in order to investigate the function of H. arabidopsis effector proteins in the infection process, A. thaliana plants were transformed to constitutively express a single RXLR effector gene. Of the 18 RXLR effector genes identified by Cabral et al.[39], the coding region without the signal peptide of 13 was successfully cloned behind the constitutive 35S CaMV promoter and transformed into A. thaliana (Table 1). Independent lines of RXLR transgenes that were segregating for a single transgene and had different levels of expression were selected (Figure 2). To check if expression of the RXLR effector genes affected plant growth and development, the rosette diameters and leaf morphology were monitored during a growth period of five weeks. In none of the transgenic lines did expression of the RXLR gene lead to a noticeably altered plant phenotype (data not shown).


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)

Expression levels of H. arabidopsidis RXLR genes in A. thaliana.Semi-quantitative analysis of the expression levels of the RXLR transgenes from H. arabidopsidis Waco9 in A. thaliana accession Col-0. Expression levels were assessed in two independent transgenic lines using RXLR gene-specific primers. Depicted are ethidium bromide-stained agarose gels with PCR products after 20, 25 or 30 cycles of PCR amplification. The PCR product of the A. thaliana actin gene (At3g18780) was used as internal control (25 cycles of PCR amplification).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4222755&req=5

pone-0110624-g002: Expression levels of H. arabidopsidis RXLR genes in A. thaliana.Semi-quantitative analysis of the expression levels of the RXLR transgenes from H. arabidopsidis Waco9 in A. thaliana accession Col-0. Expression levels were assessed in two independent transgenic lines using RXLR gene-specific primers. Depicted are ethidium bromide-stained agarose gels with PCR products after 20, 25 or 30 cycles of PCR amplification. The PCR product of the A. thaliana actin gene (At3g18780) was used as internal control (25 cycles of PCR amplification).
Mentions: The RXLR gene transcripts of H. arabidopsidis identified by Cabral et al.[39] are expressed during infection, suggesting a role in pathogen virulence. While for other oomycete pathogens transformation protocols have been established [12], [61], [62], it is currently still not possible to transform H. arabidopsidis. Hence, in order to investigate the function of H. arabidopsis effector proteins in the infection process, A. thaliana plants were transformed to constitutively express a single RXLR effector gene. Of the 18 RXLR effector genes identified by Cabral et al.[39], the coding region without the signal peptide of 13 was successfully cloned behind the constitutive 35S CaMV promoter and transformed into A. thaliana (Table 1). Independent lines of RXLR transgenes that were segregating for a single transgene and had different levels of expression were selected (Figure 2). To check if expression of the RXLR effector genes affected plant growth and development, the rosette diameters and leaf morphology were monitored during a growth period of five weeks. In none of the transgenic lines did expression of the RXLR gene lead to a noticeably altered plant phenotype (data not shown).

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