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Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid.

Asai S, Rallapalli G, Piquerez SJ, Caillaud MC, Furzer OJ, Ishaque N, Wirthmueller L, Fabro G, Shirasu K, Jones JD - PLoS Pathog. (2014)

Bottom Line: By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1.By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells.Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA.

View Article: PubMed Central - PubMed

Affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich, United Kingdom; Center for Sustainable Resource Science, RIKEN, Tsurumi, Yokohama, Kanagawa, Japan.

ABSTRACT
Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

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Hpa Waco9 overcomes recognition by RPP4, but not RPP5.(A) The number of predicted effectors expressed in Hpa Emoy2 and/or Waco9. (B) Expression of ATR1 and ATR5 in Hpa Emoy2 and Waco9 conidiospores (cs) and the infections in Arabidopsis Col-0. The expression level was determined by qRT-PCR using specific primers for ATR1 and ATR5. Expression of Hpa actin was used to normalize the expression value in each sample. Data are means ± SDs from three biological replicates. (C) Illumina sequencing reads coverage in genomic region including ATR1. Region indicated in red is of ATR1. (D) Resistance (R) and susceptibility (S) to Hpa Emoy2 and Waco9 in seven-day-old 3860, RPP1-Nd-transformed 3860 (3860:RPP1Nd), CW84 and RPP5-Ler_transformed CW84 (CW84:RPP5Ler) plants. The plants inoculated with Hpa Emoy2 and Waco9 were photographed at 6 dpi.
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ppat-1004443-g003: Hpa Waco9 overcomes recognition by RPP4, but not RPP5.(A) The number of predicted effectors expressed in Hpa Emoy2 and/or Waco9. (B) Expression of ATR1 and ATR5 in Hpa Emoy2 and Waco9 conidiospores (cs) and the infections in Arabidopsis Col-0. The expression level was determined by qRT-PCR using specific primers for ATR1 and ATR5. Expression of Hpa actin was used to normalize the expression value in each sample. Data are means ± SDs from three biological replicates. (C) Illumina sequencing reads coverage in genomic region including ATR1. Region indicated in red is of ATR1. (D) Resistance (R) and susceptibility (S) to Hpa Emoy2 and Waco9 in seven-day-old 3860, RPP1-Nd-transformed 3860 (3860:RPP1Nd), CW84 and RPP5-Ler_transformed CW84 (CW84:RPP5Ler) plants. The plants inoculated with Hpa Emoy2 and Waco9 were photographed at 6 dpi.

Mentions: Transcriptome analysis revealed that 355 and 366 predicted effectors were expressed in conidiospores and/or infections with Hpa Emoy2 and Waco9, respectively (Table 2). Of these, 339 predicted effectors were expressed in both Hpa Emoy2 and Waco9, whereas 16 and 27 predicted effectors were expressed in only Hpa Emoy2 and Waco9, respectively (Figure 3A and Table S5). ATR5, an effector recognized by RPP5 [40], was found among the 339 predicted effectors expressed in both Hpa Emoy2 and Waco9 (Figure 3B and Table S5). The Waco9 allele of ATR5 is identical to the Emoy2 allele. Surprisingly, while ATR1 was expressed in Hpa Emoy2, no tag corresponding to ATR1 in Hpa Waco9 was detected (Figure 3B and Table S5). We resequenced Hpa Waco9 genome using an Illumina Genome Analyzer II, and found that the genomic region that includes ATR1 is deleted in Waco9 (Figure 3C). These results suggest that Hpa Waco9 can infect plants containing functional RPP1, but not plants containing functional RPP5. To evaluate this possibility, several Arabidopsis accessions were inoculated with Hpa Emoy2 and Waco9. ATR1 from Hpa Emoy2 is recognized by RPP1-Nd from Arabidopsis Nd-1 accession and RPP1-WsA and RPP1-WsB from Arabidopsis Ws-2 accession (the accession previously reported as Ws-0 in our laboratory is in fact Ws-2) [35]. As expected, Arabidopsis Nd-1 and Ws-2 are resistant to Hpa Emoy2, but susceptible to Hpa Waco9 (Figure S2). We also checked the phenotype on an Arabidopsis RIL 3860 (3860), a recombinant inbred line from a cross between Col-5 and Nd-1 that lacks RPP1-Nd, and a transgenic 3860 line containing the functional RPP1-Nd gene (3860:RPP1Nd) [35]. Like Arabidopsis Nd-1 and Ws-2, 3860:RPP1Nd is resistant to Hpa Emoy2, but susceptible to Hpa Waco9, whereas Arabidopsis 3860 is susceptible to both Hpa Emoy2 and Waco9 (Figure 3D). On the other hand, no Hpa sporulation was observed on Arabidopsis Ler-0 accession containing functional RPP5, RPP5-Ler, inoculated with Hpa Emoy2 and Waco9 (Figure S2). To confirm if Hpa Emoy2 and Waco9 are recognized by RPP5-Ler, Arabidopsis CW84, a broadly Hpa-susceptible recombinant inbred line generated from a cross between Col-0 and Ws-2 [47], and CW84 transformants containing RPP5-Ler (CW84:RPP5Ler) [40] were inoculated with Hpa Emoy2 and Waco9. Like Arabidopsis Ler-0, CW84:RPP5Ler is resistant to both Hpa Emoy2 and Waco9, whereas Arabidopsis CW84 is susceptible to both Hpa isolates (Figure 3D). These results indicate that Hpa Waco9 overcomes recognition by RPP1, but not RPP5, through the deletion of ATR1 from its genome.


Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid.

Asai S, Rallapalli G, Piquerez SJ, Caillaud MC, Furzer OJ, Ishaque N, Wirthmueller L, Fabro G, Shirasu K, Jones JD - PLoS Pathog. (2014)

Hpa Waco9 overcomes recognition by RPP4, but not RPP5.(A) The number of predicted effectors expressed in Hpa Emoy2 and/or Waco9. (B) Expression of ATR1 and ATR5 in Hpa Emoy2 and Waco9 conidiospores (cs) and the infections in Arabidopsis Col-0. The expression level was determined by qRT-PCR using specific primers for ATR1 and ATR5. Expression of Hpa actin was used to normalize the expression value in each sample. Data are means ± SDs from three biological replicates. (C) Illumina sequencing reads coverage in genomic region including ATR1. Region indicated in red is of ATR1. (D) Resistance (R) and susceptibility (S) to Hpa Emoy2 and Waco9 in seven-day-old 3860, RPP1-Nd-transformed 3860 (3860:RPP1Nd), CW84 and RPP5-Ler_transformed CW84 (CW84:RPP5Ler) plants. The plants inoculated with Hpa Emoy2 and Waco9 were photographed at 6 dpi.
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Related In: Results  -  Collection

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ppat-1004443-g003: Hpa Waco9 overcomes recognition by RPP4, but not RPP5.(A) The number of predicted effectors expressed in Hpa Emoy2 and/or Waco9. (B) Expression of ATR1 and ATR5 in Hpa Emoy2 and Waco9 conidiospores (cs) and the infections in Arabidopsis Col-0. The expression level was determined by qRT-PCR using specific primers for ATR1 and ATR5. Expression of Hpa actin was used to normalize the expression value in each sample. Data are means ± SDs from three biological replicates. (C) Illumina sequencing reads coverage in genomic region including ATR1. Region indicated in red is of ATR1. (D) Resistance (R) and susceptibility (S) to Hpa Emoy2 and Waco9 in seven-day-old 3860, RPP1-Nd-transformed 3860 (3860:RPP1Nd), CW84 and RPP5-Ler_transformed CW84 (CW84:RPP5Ler) plants. The plants inoculated with Hpa Emoy2 and Waco9 were photographed at 6 dpi.
Mentions: Transcriptome analysis revealed that 355 and 366 predicted effectors were expressed in conidiospores and/or infections with Hpa Emoy2 and Waco9, respectively (Table 2). Of these, 339 predicted effectors were expressed in both Hpa Emoy2 and Waco9, whereas 16 and 27 predicted effectors were expressed in only Hpa Emoy2 and Waco9, respectively (Figure 3A and Table S5). ATR5, an effector recognized by RPP5 [40], was found among the 339 predicted effectors expressed in both Hpa Emoy2 and Waco9 (Figure 3B and Table S5). The Waco9 allele of ATR5 is identical to the Emoy2 allele. Surprisingly, while ATR1 was expressed in Hpa Emoy2, no tag corresponding to ATR1 in Hpa Waco9 was detected (Figure 3B and Table S5). We resequenced Hpa Waco9 genome using an Illumina Genome Analyzer II, and found that the genomic region that includes ATR1 is deleted in Waco9 (Figure 3C). These results suggest that Hpa Waco9 can infect plants containing functional RPP1, but not plants containing functional RPP5. To evaluate this possibility, several Arabidopsis accessions were inoculated with Hpa Emoy2 and Waco9. ATR1 from Hpa Emoy2 is recognized by RPP1-Nd from Arabidopsis Nd-1 accession and RPP1-WsA and RPP1-WsB from Arabidopsis Ws-2 accession (the accession previously reported as Ws-0 in our laboratory is in fact Ws-2) [35]. As expected, Arabidopsis Nd-1 and Ws-2 are resistant to Hpa Emoy2, but susceptible to Hpa Waco9 (Figure S2). We also checked the phenotype on an Arabidopsis RIL 3860 (3860), a recombinant inbred line from a cross between Col-5 and Nd-1 that lacks RPP1-Nd, and a transgenic 3860 line containing the functional RPP1-Nd gene (3860:RPP1Nd) [35]. Like Arabidopsis Nd-1 and Ws-2, 3860:RPP1Nd is resistant to Hpa Emoy2, but susceptible to Hpa Waco9, whereas Arabidopsis 3860 is susceptible to both Hpa Emoy2 and Waco9 (Figure 3D). On the other hand, no Hpa sporulation was observed on Arabidopsis Ler-0 accession containing functional RPP5, RPP5-Ler, inoculated with Hpa Emoy2 and Waco9 (Figure S2). To confirm if Hpa Emoy2 and Waco9 are recognized by RPP5-Ler, Arabidopsis CW84, a broadly Hpa-susceptible recombinant inbred line generated from a cross between Col-0 and Ws-2 [47], and CW84 transformants containing RPP5-Ler (CW84:RPP5Ler) [40] were inoculated with Hpa Emoy2 and Waco9. Like Arabidopsis Ler-0, CW84:RPP5Ler is resistant to both Hpa Emoy2 and Waco9, whereas Arabidopsis CW84 is susceptible to both Hpa isolates (Figure 3D). These results indicate that Hpa Waco9 overcomes recognition by RPP1, but not RPP5, through the deletion of ATR1 from its genome.

Bottom Line: By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1.By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells.Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA.

View Article: PubMed Central - PubMed

Affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich, United Kingdom; Center for Sustainable Resource Science, RIKEN, Tsurumi, Yokohama, Kanagawa, Japan.

ABSTRACT
Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

Show MeSH
Related in: MedlinePlus