Article Figures & Data
Figures
- Figure 1.
Comparative analysis of transcript accumulation in M. truncatula roots after treatment with A. euteiches zoospores for 2 h. A, Venn diagram showing the numbers of differentially expressed genes. Total RNA isolated from the roots of 1-week-old seedlings (blue) and 6-week-old plants (green) nontreated or treated with A. euteiches zoospores was subjected to transcript profiling using the Affymetrix M. truncatula GeneChip array. Numbers of significantly upregulated (red) and downregulated (green) genes in A. euteiches-treated roots in comparison to nontreated roots are shown (P ≤ 0.01, n = 3). B. Enriched GO molecular function terms (P < 0.05) associated with the core sets of differentially regulated genes in the treatment of seedlings and adult plants are shown with –log10 transformed P-values indicated on x axes (full data in Supplemental Dataset S1). The GO function “terpene synthase activity” is outlined in red. FMN, Flavin mono nucleotide.
- Figure 2.
Induction of MtTPS10 expression in M. truncatula roots by treatment with A. euteiches zoospores. Roots of 1-week-old seedlings (A and C) and 6-week-old plants (B) were treated with A. euteiches zoospores (Ae) or mock-treated with swamp-water (mock). MtTPS10 transcript levels were determined by RT-qPCR at 2 hpi (A and B) or at time points as indicated (C). Data are shown as the mean ± se. His-3-like was used as a reference gene and statistical analysis was done using Student’s t test (n ≥ 3 independent samples); *P < 0.05. n.s., not significant.
- Figure 3.
Expression of MtTPS10 is specifically induced by infection with oomycetes and is restricted to roots. (A) MtTPS10 transcript accumulation in 1-week-old Medicago A17 seedlings inoculated with A. euteiches zoospores (Ae), A. euteiches zoospores boiled for 10 min (dead Ae), and P. palmivora zoospores (Pp) for 2 h. Controls were treated with water (mock). B, MtTPS10 transcript accumulation was studied in 1-week-old M. truncatula A17 seedlings inoculated with spores of R. irregularis (Ri), spores of R. irregularis boiled for 10 min (dead Ri), and spores of C. trifolii (Ci), and in 1-week-old seedling roots treated with 100 mm NaCl or 50 µm MeJA or wounded by squeezing. All samples were analyzed 2 h after treatment. Control plants were either mock-treated, treated with Tween20, or left unwounded. C, Two-week-old M. truncatula A17 plants were infected with A. euteiches for 4 weeks and MtTPS10 transcript accumulation was analyzed in shoots and roots by RT-qPCR. Note that MtTPS10 transcripts were barely detected in leaves of either noninfected or infected plants. Data in A to C are shown as the mean ± se. His-3 like was used as the reference gene and statistical analysis was performed using ANOVA and Tukey’s honest significant difference (HSD) test (n = 3); lowercase letters a and b indicate significantly different values (P < 0.05). D to F, MtTPS10 is induced in all root cells after infection by A. euteiches. A 2-kb region of the MtTPS10 promoter fused to the GUS-encoding gene was transformed in M. truncatula roots via A. rhizogenes-mediated root transformation. Transformed roots were then treated with A. euteiches zoospores for 2 hpi and 24 hpi; controls were not treated. After treatment, roots were stained with X-Gluc and embedded in PEG for sectioning. Pictures were taken from roots (D), root tips (E), and cross sections of 10 µm thickness (F). Bars represent 100 µm (D and E) and 50 µm (F).
- Figure 4.
MtTPS10 is a cytosolic protein and produces a blend of sesquiterpenes. A, N. benthamiana protoplasts were transformed with the MtTPS10 encoding sequence fused to mCherry. Fluorescence images were taken using confocal laser scanning microscopy. Note the localization of MtTPS10 (green) within the cytosol surrounding the chloroplasts visible by chlorophyll autofluorescence (red). Scale bars = 10 µm. B, Products of MtTPS10 identified after heterologous expression in S. cerevisiae are shown. MtTPS10 was expressed together with FPPS and HMGCR (tHMGCR) and the resulting products were analyzed using liquid-injection GC-MS (black line). Yeast cells expressing FPPS and HMGCR alone served as the control (pink line). C, Products of MtTPS10 identified after heterologous expression in S. cerevisiae and N. benthamiana leaves are shown. MtTPS10 was expressed together with FPPS and HMGCR. Yeast cultures and infiltrated leaves were analyzed using SPME-coupled GC-MS. Note that in B and C, several sesquiterpenes and sesquiterpene alcohols occurred upon expression of MtTPS10 and could be annotated by comparison of MS spectra based on library suggestions: (1) longipenene, (2) ylangene, (3) longicyclene, (5) farnesene, (6) α-himachalene (9) alloaromadendrene, (10) β-himachalene, (11) bisabolene, (12) himachalol, (13) α-bisabolol, and (14) shyobunol (D) The main product (peak 12) was purified, subjected to NMR analysis and identified as himachalol (Supplemental Fig. S2; Supplemental Table S1).
- Figure 5.
The Tnt1 insertion line NF10408 is defective in MtTPS10 expression, fails to produce sesquiterpene volatiles, and shows enhanced susceptibility to infection with A. euteiches. A, One-week-old seedlings of BG and tps10 lines were treated with A. euteiches zoospores for 2 h. Total RNA from roots was subjected to RT-qPCR. Transcript levels of MtTPS10 determined using primers binding upstream of the Tnt1 insertion site (see Supplemental Fig. S5A) were significantly reduced in roots of tps10 mutant plants compared to BG plants. Data are shown as the mean ± se. Actin was used as the reference gene and statistical analysis was done using ANOVA followed by Tukey’s honest significant difference (HSD) test (n = 3 independent samples); lowercase letters a and b indicate significantly different values (P < 0.05). B, Volatiles produced by roots of BG and tps10 mutant plants with and without infection by A. euteiches. Volatile compounds induced during the first 12 h after treatment with zoospores were adsorbed on PDMS strips and analyzed by thermal desorption-GC-MS. The labeled peaks correspond to (1) longipenene, (2) ylangene, (3) longicyclene, (4) longifolene, (7) isoshyobunone, (8) 7-epi-cis-sesquisabinene hydrate, (9) alloaromadendrene, (10) β-himachalene, (15) nerolidol, (16) fokienol, (17) ethyl iso-allocholate, (18) cholest-8-en-3-β-ol acetate, (19) cyclononasiloxane, (20) thunbergol, (21) nerolidyl propionate, and (22) heptasiloxane. The compounds representing peaks 1 to 3 and 9 and 10 were also identified in extracts from yeast expressing MtTPS10. Note that the major compound identified in yeast (himachalol [peak 12 in Fig. 4B]) is not detectable among root volatiles, possibly because of M. truncatularoot modification. C and D, Biomass of shoots (C) and roots (D) of BG and tps10 plants noninfected or infected by A. euteiches, as shown in C. tps10 mutant plants were strongly affected by infection with A. euteiches as visible by the diminished biomass of shoots and roots, whereas plants of the BG line did not show significant alterations in growth. Data are shown as the mean ± se (n = 18 independent samples). Statistical analysis was done using Tukey’s honest significant difference (HSD) test; ***P < 0.001; *P < 0.05. E and F, Two-week-old seedlings of BG and tps10 mutant lines were infected with A. euteiches zoospores and harvested 4 weeks later. Total RNA from roots was subjected to RT-qPCR. Transcript levels of MtTPS10 (E) were significantly reduced in roots of tps10 mutant plants compared to BG plants, whereas 5.8S rRNA of A. euteiches was strongly enhanced in tps10 roots in comparison to BG roots (F). Data are shown as the mean ± se. Actin was used as the reference gene and statistical analysis was done using ANOVA followed by Tukey’s honest significant difference (HSD) test (n = 3 independent samples); *P < 0.05.
- Figure 6.
Expression of amiR targeting MtTPS10 (TPS10-amiR) reduced MtTPS10 expression and enhanced susceptibility to A. euteiches. M. truncatula cv R108 plants were transformed with an empty vector (EV) or a TPS10-amiR (tps) construct using A. rhizogenes-mediated root transformation and were subsequently infected with A. euteiches for 4 weeks. MtTPS10 transcript accumulation (A) and 5.8S rRNA of A. euteiches (B) were determined using RT-qPCR. Actin was used as the reference gene. Data from single transformed roots are shown. The red bars show the mean ± se for all four plants. Statistical analysis was performed using Tukey’s honest significant difference (HSD) test; *P < 0.05; **P < 0.01.
- Figure 7.
Products of MtTPS10 inhibit mycelial growth of A. euteiches. A, A. euteiches mycelial pieces were placed on plates and different amounts of the yeast extract containing MtTPS10 products (given in nmol himachalol per plate) were added at the edges of plates. Application of solvent only (pentane) or extracts from yeasts expressing the empty vector (EV) served as controls. After 1 week of mycelial growth, photographs were taken (Supplemental Fig. S4) and areas covered by mycelium were calculated using ImageJ. Data are shown as the mean ± se (n = 3 independent samples). Different letters indicate significant differences according to one-way ANOVA with Tukey’s honest significant difference (HSD) test, P < 0.05. B, Zoospores of A. euteiches were incubated for 1 h with different concentrations of yeast extract containing MtTPS10 products (himachalol plus some minor compounds) followed by plating on agar to allow germination. Pictures were taken 24 h later. Incubation with extracts from yeast expressing the empty-vector (EV) construct served as the control. Note the diminished hyphal growth at 250 µm himachalol and the complete inhibition of zoospore germination at 500 µm himachalol in the medium. Scale bar = 1 mm for all micrographs.
Additional Files
Supplemental Data
Supplemental Figures, Tables, and Data
Files in this Data Supplement:
- Supplemental Data - Supplemental Figures 1-7 and Supplemental Tables 1-4
- Supplemental Data - Supplemental Data
