Figure 1.
Hpa development and scheme for aligning Illumina sequence reads.
(A) Trypan blue staining in three-week-old Arabidopsis Col-0 plants at 1, 3 and 5 dpi with Hpa Emoy2 and Waco9. Black arrows indicate the parts in which HR cell death was observed. (B) Work-flow scheme to separate Illumina sequencing reads from Arabidopsis and Hpa.
Table 1.
Summary of transcriptome data in Arabidopsis inoculated with Hpa.
Table 2.
The number of genes detected in this study.
Figure 2.
Expression pattern of Hpa predicted effectors and potential cis-regulatory elements in Hpa.
(A) Expression pattern of predicted effectors expressed in at least one of three infections (1, 3, and 5 dpi) with Hpa Waco9. Expression levels were represented as TPM (tags per million) of total reads mapped to Hpa genome. Red lines indicate predicted effectors induced more than two fold at 3 dpi compared to the expression level in conidiospores (cs). Single and double asterisks indicate expression pattern of HaRxL76 and HaRxL62, respectively. A right line chart is magnification of left one. (B) Average expression pattern of genes in the indicated groups during the infection with Hpa Waco9. The induction levels compared to the level in cs were indicated by value of log2. (C to K) Distribution of motifs in coexpressed genes of Hpa and P. infestans. Nucleotide conservation of (C) the INR-FPR motif in “induced effectors”, (F) Motif I in “induced effectors” and (I) Motif II in “non-induced effectors” is displayed as sequence logos, based on hits within 200 nt (INR-FPR) and 500 nt (Motif I and II) upstream of the start codon. Bar charts indicate the percent of promoters within each group that contain (D, E) the INR-FPR motif within 200 nt and (G, H) Motif I and (J, K) Motif II within 500 nt upstream of the start codon. The analysis was done in promoters from (D, G, J) Hpa and (E, H, K) P. infestans. Asterisks indicate statistically significant over-representation of the motifs compared to population in “all genes” (p<1e-4), which is shown in Table S4.
Figure 3.
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.
Figure 4.
Arabidopsis genes differentially expressed after inoculation with Hpa Emoy2 and Waco9.
(A) The number of Arabidopsis genes significantly upregulated or downregulated at 1, 3 and 5 dpi with Hpa Emoy2 and Waco9. (B) Assessment of overlap of genes significantly upregulated at 1 dpi with Hpa Emoy2 and at 3 and 5 dpi with Hpa Waco9, and classification into Group I (yellow), II (blue), III (purple) and IV (red). (C) Expression pattern of genes categorized into Group I, II, III and IV. The relative expression (in log2 ratios) is colored red for induction and green for repression as illustrated in the fold change color bars. (D) Percentage of genes with significantly enriched gene ontology (GO) terms in Group I (yellow), II (blue), III (purple) and IV (red), compared to the background (grey). Y-axis: percentage of genes that fall within each given GO annotation class.
Figure 5.
Hpa suppresses PR1 expression induced by SA in infected cells.
(A) Expression pattern of 871 BTH-inducible genes reported by Wang et al. (2006) [48] after inoculation with Hpa Emoy2 and Waco9. The relative expression (in log2 ratios) is colored red for induction and green for repression as illustrated in the fold change color bars. (B) Expression of PR1 in Arabidopsis at 1, 3, and 5 dpi with Hpa Emoy2 and Waco9. The expression level was determined by qRT-PCR using specific primers for PR1 and indicated as relative fold induction compared to water-treated samples (mock). Expression of EF-1α was used to normalize the expression value in each sample. Data are means ± SDs from three biological replicates. (C) GUS staining in three-week-old Arabidopsis leaves containing PR1 promoter fused GUS (PR1::GUS) at 1 and 2 dpi with Hpa Emoy2 and at 6 dpi with Hpa Waco9. Lower images are magnified upper images. Black and red asterisks indicate Hpa-haustoriated and non-haustoriated mesophyll cells, respectively. cs, conidiospore. Scale bars = 40 µm. (D) GUS staining in Hpa-infected PR1::GUS lines 8 hours after treatment with SA (200 µM). The leaves at 4 dpi with Hpa Waco9 or spraying water (mock) were infiltrated with SA or water (mock). Red arrows indicate Hpa-haustoriated cells. Scale bars = 100 µm.
Figure 6.
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).