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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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HaRxL62 reduces responsiveness to SA.(A) Expression level of PR1 8 hours after treatment with SA (100 µM) in ten-day-old Col-0 plants (WT), npr1 mutants and transgenic lines expressing the indicated Hpa predicted effectors. The expression level was determined by qRT-PCR using specific primers for PR1 and indicated as relative fold induction compared to the expression level in WT after SA treatment. Expression of EF-1α was used to normalize the expression value in each sample. Data are means from three biological replicates showing quantiles. Data analysis was carried using one-way ANOVA followed by Tukey's HSD (honestly significant difference). Genotypes showing significant differences (p<0.01) are marked with different alphabets (B) Hpa growth on three-week-old Col-0 plants (WT), npr1 mutants and two independent transgenic lines expressing HaRxL62 (HaRxL62-1 and HaRxL62-2) and HaRxLL464 (HaRxLL464-1 and HaRxLL464-2) pretreated with SA (10 µM) or water (mock). The plants 24 hours after spray treatment with SA or water were inoculated with Hpa Waco9. Conidiospores were harvested and counted at 6 dpi. Different letters indicate significantly different values at p<0.05 (one-way ANOVA, Tukey's HSD).
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ppat-1004443-g006: HaRxL62 reduces responsiveness to SA.(A) Expression level of PR1 8 hours after treatment with SA (100 µM) in ten-day-old Col-0 plants (WT), npr1 mutants and transgenic lines expressing the indicated Hpa predicted effectors. The expression level was determined by qRT-PCR using specific primers for PR1 and indicated as relative fold induction compared to the expression level in WT after SA treatment. Expression of EF-1α was used to normalize the expression value in each sample. Data are means from three biological replicates showing quantiles. Data analysis was carried using one-way ANOVA followed by Tukey's HSD (honestly significant difference). Genotypes showing significant differences (p<0.01) are marked with different alphabets (B) Hpa growth on three-week-old Col-0 plants (WT), npr1 mutants and two independent transgenic lines expressing HaRxL62 (HaRxL62-1 and HaRxL62-2) and HaRxLL464 (HaRxLL464-1 and HaRxLL464-2) pretreated with SA (10 µM) or water (mock). The plants 24 hours after spray treatment with SA or water were inoculated with Hpa Waco9. Conidiospores were harvested and counted at 6 dpi. Different letters indicate significantly different values at p<0.05 (one-way ANOVA, Tukey's HSD).

Mentions: Histochemical GUS analysis in Hpa-infected PR1::GUS lines showed that Hpa suppresses SA-inducible PR1 expression specifically in the haustoriated cells into which RxLR effectors are delivered (Figure 5D). To identify Hpa effectors which participate in the suppression, the level of PR1 expression after treatment with SA was checked in transgenic lines expressing Hpa predicted effectors and the SA-insensitive npr1 mutants [49], as a positive control. Nine Hpa effector-expressing lines showed more susceptibility to Hpa compared to wild type (WT) Col-0 plants [8], [10] (Figure S4 and Table S9). HaRxL62-expressing lines showed a five-fold reduction in expression level of PR1 compared to WT after SA treatment, whereas no significant reduction was observed in eight other Hpa effector-expressing lines, including HaRxLL464-expressing lines (Figure 6A). To evaluate the effect of HaRxL62 on Hpa growth after treatment with SA, WT plants, npr1 mutants and HaRxL62- and HaRxLL464-expressing lines were treated with SA or water as a mock treatment and, 24 hours later, inoculated with Hpa Waco9 (Figure 6B). Although water-treated WT plants were susceptible to Hpa Waco9, no Hpa growth was observed in SA-treated WT plants. As expected, SA did not trigger resistance to Hpa in npr1 mutants. In HaRxLL464-expressing plants treated with SA, essentially no Hpa spores were observed as observed for WT plants, whereas there were countable Hpa spores in HaRxL62-expressing plants treated with SA (Figure 6B), consistent with reduction in expression level of PR1 after treatment with SA (Figure 6A). As shown in Figure 2A, HaRxL62 was the second-highest expressed Hpa effector at 3 dpi. These results suggest that HaRxL62, a highly-expressed effector during infection, reduces responsiveness to SA.


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)

HaRxL62 reduces responsiveness to SA.(A) Expression level of PR1 8 hours after treatment with SA (100 µM) in ten-day-old Col-0 plants (WT), npr1 mutants and transgenic lines expressing the indicated Hpa predicted effectors. The expression level was determined by qRT-PCR using specific primers for PR1 and indicated as relative fold induction compared to the expression level in WT after SA treatment. Expression of EF-1α was used to normalize the expression value in each sample. Data are means from three biological replicates showing quantiles. Data analysis was carried using one-way ANOVA followed by Tukey's HSD (honestly significant difference). Genotypes showing significant differences (p<0.01) are marked with different alphabets (B) Hpa growth on three-week-old Col-0 plants (WT), npr1 mutants and two independent transgenic lines expressing HaRxL62 (HaRxL62-1 and HaRxL62-2) and HaRxLL464 (HaRxLL464-1 and HaRxLL464-2) pretreated with SA (10 µM) or water (mock). The plants 24 hours after spray treatment with SA or water were inoculated with Hpa Waco9. Conidiospores were harvested and counted at 6 dpi. Different letters indicate significantly different values at p<0.05 (one-way ANOVA, Tukey's HSD).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4199768&req=5

ppat-1004443-g006: HaRxL62 reduces responsiveness to SA.(A) Expression level of PR1 8 hours after treatment with SA (100 µM) in ten-day-old Col-0 plants (WT), npr1 mutants and transgenic lines expressing the indicated Hpa predicted effectors. The expression level was determined by qRT-PCR using specific primers for PR1 and indicated as relative fold induction compared to the expression level in WT after SA treatment. Expression of EF-1α was used to normalize the expression value in each sample. Data are means from three biological replicates showing quantiles. Data analysis was carried using one-way ANOVA followed by Tukey's HSD (honestly significant difference). Genotypes showing significant differences (p<0.01) are marked with different alphabets (B) Hpa growth on three-week-old Col-0 plants (WT), npr1 mutants and two independent transgenic lines expressing HaRxL62 (HaRxL62-1 and HaRxL62-2) and HaRxLL464 (HaRxLL464-1 and HaRxLL464-2) pretreated with SA (10 µM) or water (mock). The plants 24 hours after spray treatment with SA or water were inoculated with Hpa Waco9. Conidiospores were harvested and counted at 6 dpi. Different letters indicate significantly different values at p<0.05 (one-way ANOVA, Tukey's HSD).
Mentions: Histochemical GUS analysis in Hpa-infected PR1::GUS lines showed that Hpa suppresses SA-inducible PR1 expression specifically in the haustoriated cells into which RxLR effectors are delivered (Figure 5D). To identify Hpa effectors which participate in the suppression, the level of PR1 expression after treatment with SA was checked in transgenic lines expressing Hpa predicted effectors and the SA-insensitive npr1 mutants [49], as a positive control. Nine Hpa effector-expressing lines showed more susceptibility to Hpa compared to wild type (WT) Col-0 plants [8], [10] (Figure S4 and Table S9). HaRxL62-expressing lines showed a five-fold reduction in expression level of PR1 compared to WT after SA treatment, whereas no significant reduction was observed in eight other Hpa effector-expressing lines, including HaRxLL464-expressing lines (Figure 6A). To evaluate the effect of HaRxL62 on Hpa growth after treatment with SA, WT plants, npr1 mutants and HaRxL62- and HaRxLL464-expressing lines were treated with SA or water as a mock treatment and, 24 hours later, inoculated with Hpa Waco9 (Figure 6B). Although water-treated WT plants were susceptible to Hpa Waco9, no Hpa growth was observed in SA-treated WT plants. As expected, SA did not trigger resistance to Hpa in npr1 mutants. In HaRxLL464-expressing plants treated with SA, essentially no Hpa spores were observed as observed for WT plants, whereas there were countable Hpa spores in HaRxL62-expressing plants treated with SA (Figure 6B), consistent with reduction in expression level of PR1 after treatment with SA (Figure 6A). As shown in Figure 2A, HaRxL62 was the second-highest expressed Hpa effector at 3 dpi. These results suggest that HaRxL62, a highly-expressed effector during infection, reduces responsiveness to SA.

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