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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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N-terminal sequence conservation of H. arabidopsidis RXLR proteins.(A) The amino acid sequences of the 18 RLXR proteins of H. arabidopsidis isolate Waco9 identified by Cabral et al. [38] were grouped using BLASTp on the N-terminal 60 amino acids of these proteins. Color schemes show for each amino acid the similarity within the 11 amino acid region surrounding the specific amino acid. (B) N-terminal 60-amino acid regions of 130 Emoy2 RXLR proteins [18] were checked for similarity using BLASTp, leading to the identification of 23 groups containing at least two Emoy2 RXLRs. An alignment of the members of one of these identified groups is shown as example. RXL4, RXL5, RXL15, RXL26, RXL58, RXL76 are aligned and this group includes the homologs of RXLR13 (RXL76) and RXLR23 (RXL4) of H. arabidopsidis Waco9. Alignments are depicted as in (A).
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pone-0110624-g001: N-terminal sequence conservation of H. arabidopsidis RXLR proteins.(A) The amino acid sequences of the 18 RLXR proteins of H. arabidopsidis isolate Waco9 identified by Cabral et al. [38] were grouped using BLASTp on the N-terminal 60 amino acids of these proteins. Color schemes show for each amino acid the similarity within the 11 amino acid region surrounding the specific amino acid. (B) N-terminal 60-amino acid regions of 130 Emoy2 RXLR proteins [18] were checked for similarity using BLASTp, leading to the identification of 23 groups containing at least two Emoy2 RXLRs. An alignment of the members of one of these identified groups is shown as example. RXL4, RXL5, RXL15, RXL26, RXL58, RXL76 are aligned and this group includes the homologs of RXLR13 (RXL76) and RXLR23 (RXL4) of H. arabidopsidis Waco9. Alignments are depicted as in (A).

Mentions: RXLR proteins consist of an N-terminal signal peptide followed by an RXLR domain and a C-terminal effector domain. Based on their amino acid sequences, the 18 RXLR proteins identified by Cabral et al.[39] could be divided in four groups of two or three RXLR proteins each, and eight RXLR proteins that showed no similarity to the other RXLRs. Strikingly, within each of the four groups the N-terminus is highly conserved while the C-terminus is very divergent (Figure 1A). To investigate whether amino acid sequence conservation in the N-terminus of RXLR proteins is a common phenomenon, we aligned all 134 identified RXLR sequences in the genome of the sequenced H. arabidopsidis isolate Emoy2 [18]. Based on the first 60 amino acids of each RXLR protein, around 60 percent of the RXLRs can be placed in a group with at least one other RXLR protein and in most cases the similarity between proteins within one group is limited to the N-terminus (data not shown). In Figure 1B the amino acid sequence conservation pattern of the six H. arabidopsis RXLRs of isolate Emoy2 [18] that group with RXLR13 and RXLR23 from Waco9 is shown. The N-termini show an amino acid sequence similarity of 60%–80%, while the sequence similarity in the C-terminal parts is relatively low. Thus, conservation of the N-terminal seems to be common for RXLR proteins in H. arabidopsidis.


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)

N-terminal sequence conservation of H. arabidopsidis RXLR proteins.(A) The amino acid sequences of the 18 RLXR proteins of H. arabidopsidis isolate Waco9 identified by Cabral et al. [38] were grouped using BLASTp on the N-terminal 60 amino acids of these proteins. Color schemes show for each amino acid the similarity within the 11 amino acid region surrounding the specific amino acid. (B) N-terminal 60-amino acid regions of 130 Emoy2 RXLR proteins [18] were checked for similarity using BLASTp, leading to the identification of 23 groups containing at least two Emoy2 RXLRs. An alignment of the members of one of these identified groups is shown as example. RXL4, RXL5, RXL15, RXL26, RXL58, RXL76 are aligned and this group includes the homologs of RXLR13 (RXL76) and RXLR23 (RXL4) of H. arabidopsidis Waco9. Alignments are depicted as in (A).
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pone-0110624-g001: N-terminal sequence conservation of H. arabidopsidis RXLR proteins.(A) The amino acid sequences of the 18 RLXR proteins of H. arabidopsidis isolate Waco9 identified by Cabral et al. [38] were grouped using BLASTp on the N-terminal 60 amino acids of these proteins. Color schemes show for each amino acid the similarity within the 11 amino acid region surrounding the specific amino acid. (B) N-terminal 60-amino acid regions of 130 Emoy2 RXLR proteins [18] were checked for similarity using BLASTp, leading to the identification of 23 groups containing at least two Emoy2 RXLRs. An alignment of the members of one of these identified groups is shown as example. RXL4, RXL5, RXL15, RXL26, RXL58, RXL76 are aligned and this group includes the homologs of RXLR13 (RXL76) and RXLR23 (RXL4) of H. arabidopsidis Waco9. Alignments are depicted as in (A).
Mentions: RXLR proteins consist of an N-terminal signal peptide followed by an RXLR domain and a C-terminal effector domain. Based on their amino acid sequences, the 18 RXLR proteins identified by Cabral et al.[39] could be divided in four groups of two or three RXLR proteins each, and eight RXLR proteins that showed no similarity to the other RXLRs. Strikingly, within each of the four groups the N-terminus is highly conserved while the C-terminus is very divergent (Figure 1A). To investigate whether amino acid sequence conservation in the N-terminus of RXLR proteins is a common phenomenon, we aligned all 134 identified RXLR sequences in the genome of the sequenced H. arabidopsidis isolate Emoy2 [18]. Based on the first 60 amino acids of each RXLR protein, around 60 percent of the RXLRs can be placed in a group with at least one other RXLR protein and in most cases the similarity between proteins within one group is limited to the N-terminus (data not shown). In Figure 1B the amino acid sequence conservation pattern of the six H. arabidopsis RXLRs of isolate Emoy2 [18] that group with RXLR13 and RXLR23 from Waco9 is shown. The N-termini show an amino acid sequence similarity of 60%–80%, while the sequence similarity in the C-terminal parts is relatively low. Thus, conservation of the N-terminal seems to be common for RXLR proteins in H. arabidopsidis.

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