1. Ecology
  2. Plant Biology
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Blumenols as shoot markers of root symbiosis with arbuscular mycorrhizal fungi

  1. Ming Wang
  2. Martin Schäfer
  3. Dapeng Li
  4. Rayko Halitschke
  5. Chuanfu Dong
  6. Erica McGale
  7. Christian Paetz
  8. Yuanyuan Song
  9. Suhua Li
  10. Junfu Dong
  11. Sven Heiling
  12. Karin Groten
  13. Philipp Franken
  14. Michael Bitterlich
  15. Maria J Harrison
  16. Uta Paszkowski
  17. Ian T Baldwin  Is a corresponding author
  1. Max Planck Institute for Chemical Ecology, Germany
  2. University of Chinese Academy of Sciences, China
  3. Leibniz-Institute of Vegetable and Ornamental Crops, Germany
  4. Humboldt Universität zu Berlin, Germany
  5. Boyce Thompson Institute for Plant Research, United States
  6. University of Cambridge, United Kingdom
Research Article
Cite this article as: eLife 2018;7:e37093 doi: 10.7554/eLife.37093
7 figures, 5 tables and 2 additional files

Figures

Blumenol core structures and exemplary modifications.

(A) Structure of blumenol A, blumenol B and blumenol C. (B) Exemplary blumenol C derivatives. Glyc, glycoside.

https://doi.org/10.7554/eLife.37093.003
Figure 2 with 1 supplement
Combined targeted and untargeted metabolomics identified blumenol derivatives as AMF-indicative in-planta metabolic fingerprints in the roots and leaves of Nicotiana attenuata plants.

(A) Experimental set-up. EV and irCCaMK plants were co-cultured and inoculated with or without R. irregularis. Six weeks after inoculation (wpi), root samples were harvested for metabolite profiling. (B) Covariance network visualizing m/z features from UHPLC-qTOF-MS untargeted analysis (n = 8). Known compounds, including nicotine, phenylalanine and various phenolics, and unknown metabolites (Unk.) are enclosed in dashed ellipses. (C) Normalized Z-scores of m/z features were clustered using STEM Clustering; 5 of 8 significant clusters are shown in different colors and mapped onto the covariance network. The intensity variation (mean +SE) of 2 selected features (Compounds 1 and 2) are shown in bar plots (n.d., not detected). (D) Representative chromatograms of Compounds 1 and 2 in roots and leaves of plants with and without AMF inoculation, as analyzed by targeted UHPLC-triple quadrupole-MS metabolomics.

https://doi.org/10.7554/eLife.37093.004
Figure 2—figure supplement 1
Abundance of root blumenol derivatives correlates positively with root AMF colonization.

(A) Time lapse accumulations (3–7 weeks post inoculation, n ≥ 3 for each time point) of Compounds 1, 2, 3, 4 and 5 in roots of plants with (EV+, black lines with circles) and without (EV-, grey lines with triangle) AMF inoculation. The experiment was conducted with empty vector (EV) transformed plants. Data are means ±SE. (B) Abundance of Compounds 1, 2, 3, 4 and 5 relative to the transcript abundance of the R. irregularis specific housekeeping gene, Ri-tub (GenBank: EXX64097.1), as well as to the plant-derived marker genes RAM1, Vapyrin, STR1 and PT4 (Gene ID and transcripts abundance are listed in Data Set 1). The transcript abundance was quantified by q-PCR, relative to NaIF-5a (NCBI Reference Sequence: XP_019246749.1). The correlations among blumenol derivatives and the transcript abundances of marker genes were analyzed by linear regression (lm) models.

https://doi.org/10.7554/eLife.37093.005
Figure 2—figure supplement 1—source data 1

Source data for Source data for Figure 2—figure supplement 1.

https://doi.org/10.7554/eLife.37093.006
Figure 3 with 4 supplements
Compounds 1 and 2 are leaf markers of root AMF colonization in N. attenuata.

(A) Time lapse accumulations of Compounds 1 and 2 in leaves of EV plants with (EV+, red) or without (EV-, black) AMF-inoculation and of irCCaMK plants with AMF-inoculation (irCCaMK+, orange, covered by black) (means ± SE, n ≥ 5). (B) Leaf abundances of Compounds 1 and 2 (five wpi) of plants inoculated with different inoculum concentrations (means + SE, n ≥ 4); different letters indicate significant differences (p<0.05, one-way ANOVA followed by Fisher’s LSD). (C) Compounds 1 and 2 in leaf samples of EV and irCCaMK plants inoculated with (+) or without (-) AMF inoculum isolated from the plant’s native habitat (six wpi); different letters indicate significant differences (p<0.05, one-way ANOVA followed by Tukey’s HSD, n = 10). (D) Field experiment (Great Basin Desert, Utah, USA): Compounds 1 and 2 in leaf samples of EV (n = 20) and irCCaMK (n = 19) plants sampled eight weeks after planting. (Student’s t-test: ***p<0.001). (E) Representative images of WGA-488 stained roots of plants shown in B) (bar = 100 μm). (F) Leaf Compounds 1 and 2 relative to the percentage of root colonization by hyphae, arbuscules, vesicles and total root length colonization of the same plants (linear regression model). (G) Compounds 1 and 2 in 17 different tissues of plants with (+AMF, n = 3, red bars) or without (-AMF, n = 1, black bars) AMF-inoculation harvested at six wpi.

https://doi.org/10.7554/eLife.37093.008
Figure 3—figure supplement 1
AMF-induced accumulation of blumenol derivatives in roots and leaves of N. attenuata.

(A) Representative chromatograms of targeted tandem MS-based analyses of Compounds 3, 4 and 5 in roots (bottom panel) and leaves (top panel) of N. attenuata plants after inoculation with R. irregularis (+R. irregularis, red line, 6wpi) and in untreated control plants (Control, black line). Experiments were conducted with wild type (WT) plants. The respective precursor-to-product ion transitions are indicated at the top. (B), (D) Representative chromatograms of a high resolution MS-based analysis of Compounds 1, 3, 4 and 5 (B), as well as Compound 2 (D) in roots (bottom panel) and leaves (top panel) of N. attenuata plants after inoculation with R. irregularis. Extracted ion chromatograms (EIC) are labeled by colors and settings listed at the top. (C), (E) Comparison of fragmentation patterns of Compounds 1 (C) and 2 (E) in both tissues by high resolution tandem MS.

https://doi.org/10.7554/eLife.37093.009
Figure 3—figure supplement 2
Time course analysis of the root colonization by AMF and the corresponding accumulation of Compounds 1 and 2 in roots and leaves of N. attenuata.

(A) Root colonization in EV plants at different time points after inoculation with R. irregularis (2/3/4/5 wpi). H: hyphae; A: arbuscules; V: vesicles; Total: total root length colonization (means +SE; n = 8). (B, C) Abundances of Compounds 1 and 2 in roots (B) and leaves (C) of plants at different time points after inoculation with R. irregularis (2/3/4/5 wpi; means +SE; n ≥ 5). Different letters indicate significant differences (p<0.05, one-way ANOVA followed by Tukey’s HSD). n.d., not detected.

https://doi.org/10.7554/eLife.37093.010
Figure 3—figure supplement 2—source data 1

Source data for Source data for Figure 3—figure supplement 2.

https://doi.org/10.7554/eLife.37093.011
Figure 3—figure supplement 3
Root AMF colonization and abundance of Compound 1 in a second independently transformed irCCaMK line.

(A) Root colonization analysis in EV and irCCaMK (A-09-1208-6) plants. H: hyphae; A: arbuscules; V: vesicles; Total: total root length colonization (means +SE; EV, n = 9; irCCaMK; n = 7). (B) Transcript abundance of AMF marker genes in roots of EV and irCCaMK plants inoculated with R. irregularis (six wpi; means +SE; n = 6). (C) Compound 1 levels in roots and leaves of EV and irCCaMK plants inoculated with R. irregularis (six wpi; means +SE; n = 8).

https://doi.org/10.7554/eLife.37093.012
Figure 3—figure supplement 3—source data 1

Source data for Source data for Figure 3—figure supplement 3.

https://doi.org/10.7554/eLife.37093.013
Figure 3—figure supplement 4
Signals from Compound 1 are partially disturbed in field samples, but not for Compound 2.

Leaf samples were harvested from glasshouse-(top panel) and field-grown, Utah, 2016 (bottom panel) plants for analysis. Representative chromatograms of two samples of each genotype, EV (red) and irCCaMK (black), are shown. Grey area indicates the peak integration window used for the quantification of Compound 1.

https://doi.org/10.7554/eLife.37093.014
Transcript abundance of classical arbuscular mycorrhizal symbiosis-marker genes do not respond in leaves of mycorrhizal and control N. attenuata plants.

The transcript abundance (relative to NaIF-5a) of classical root marker genes was analyzed in leaves of N. attenuata plants in the presence (+R. irregularis, black bars) and absence (control, white bars) of root colonization with R. irregularis. The marker genes include the R. irregularis specific housekeeping gene, Ri-tub, as well as the plant-derived marker genes CCaMK, Vapyrin, PT4, STR1 and RAM1. Leaf samples were harvested six wpi and analyzed by qPCR. Data represent means +SE (n ≥ 3), n.d., not detected.

https://doi.org/10.7554/eLife.37093.016
Different biotic and abiotic stresses do not elicit accumulations of Compounds 1 and 2 in leaves.

(A–D) Representative leaves of N. attenuata plants subjected to different stresses (right leaf), as well as the untreated controls (left leaf): (A) Manduca sexta feeding for 10 days; (B) Botrytis cinerea infection for five days. (C) Infection for two weeks with Agrobacterium tumefaciens carrying the Tobacco Rattle Virus; (D) Dehydration for three days. For each treatment, four biological replicates were used. (E) Accumulation of Compounds 1 and 2 in treated samples from (A–D). n.d., not detected.

https://doi.org/10.7554/eLife.37093.018
Figure 6 with 2 supplements
AMF-indicative Compounds 1 and 2 in shoots of mycorrhizal plants originate from the roots.

(A) Hierarchical clustering analysis of transcript abundance from RNA-seq of methylerythritol 4-phosphate (MEP) and (apo)carotenoid biosynthetic genes (for details see Figure 6—figure supplement 1A). (B) Compounds 1, 2 (AMF-specific) and 6 (not AMF-specific) in AMF-inoculated i-irPDS and EV plants. On each plant, a single stem leaf (leaf 0) was elicited with 100 μM DEX-containing paste for three weeks; treated and adjacent, untreated control leaves (leaf −1 and leaf +1) were harvested. Representative leaves are shown (bleaching indicates PDS silencing); (means +SE, n ≥ 6). The same leaf positions in i-irPDS and EV plants were compared by Student’s t-tests. (C) Contents of Compounds 1, 2 and 6 in the roots and shoots of seedlings whose roots were dipped for 1 d into an aqueous solution with (treatment) or without (control) AMF-indicative blumenols. (D) Model of blumenol distribution in plants with (right panel) and without (left panel) AMF colonization. The model illustrates constitutive blumenols (e.g., Compound 6 in N. attenuata) and AMF-indicative ones (e.g., Compounds 1 and 2 in N. attenuata) and their inferred transport.

https://doi.org/10.7554/eLife.37093.019
Figure 6—figure supplement 1
Foliar levels of Compounds 1 and 2 are derived from roots.

(A) Transcript abundance of MEP and apocarotenoid pathway biosynthetic genes (based on homologies to tomato, Arabidopsis and tobacco). Plant materials from the same experimental set-up as in Figure 2A were used for sequencing. Data are means +SE (n = 3) generated by RNA-seq and the abundance of each transcript is expressed in TPM (Transcripts per kilobase of exon model per million mapped reads). Transcripts were analyzed in roots (left panel, orange background) and leaf tissues (right panel, green background) of EV and irCCaMK plants with (EV+ and irCCaMK+ respectively) and without (EV- and irCCaMK- respectively) inoculations with R. irregularis. Gene abbreviations; CRTISO: carotenoid isomerase; GGPPs: geranylgeranyl diphosphate synthase; PSY: phytoene synthase; PDS: phytoene desaturase; ZDS: ζ-carotene de-saturase; Z-ISO: ζ- carotene isomerase; CCD: carotenoid cleavage dioxygenase; MAX1: cytochrome P450-type monooxygenase CYP711A1; DXS: 1-deoxy-D-xylulose 5-phosphate synthase; DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase; MCT: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK: 4-(cytidine 5′-diphospho)−2-C-methyl-D-erythritol kinase; MDS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS: 4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; D27: carotenoid isomerase. (B) Representative chromatograms from a targeted tandem MS-based analysis of Compounds 1, 2 and 6 in stem sap fluid of N. attenuata plants after R. irregularis inoculation (+AMF, red line, 6wpi) and of untreated control plants (Control, black line). The respective precursor-to-product ion transitions are indicated at top. (C) Accumulations of Compounds 1, 2 and 6 in non AMF-inoculated plants after local silencing of the carotenoid biosynthesis in the DEX-treated leaf. The experiment was performed with plants harboring a transformation construct mediating the chemically-inducible silencing of the phytoene desaturase (i-irPDS), as well as with empty vector (EV) plants. On each plant, a single stem leaf (leaf 0) was treated with a 100 μM dexamethasone (DEX) containing lanolin paste for 3 weeks. The adjacent, untreated leaves (leaf −1 and leaf +1) were harvested as controls. Representative leaves are shown (bleaching indicates functional PDS silencing). Data are means +SE (n ≥ 6). For statistical analysis, the samples from the same leaf positions in i-irPDS and EV plants were compared by Student’s t test. (D) Contents of Compounds 1, 2 and 6 in the roots (red bars) and shoots (blue bars) of seedlings whose roots were dipped into an aqueous solution with or without addition of the respective blumenols. Seedlings were incubated for 1d before analysis. Data are means +SE (shoot, n = 3; root, n = 1). The data originate from the same experiment presented in Figure 6C.

https://doi.org/10.7554/eLife.37093.020
Figure 6—figure supplement 2
Compound 6 is constitutively produced in shoots of N. attenuata and not indicative of AMF associations.

(A) Representative chromatograms from a targeted tandem MS-based analysis of Compound 6 in leaves of N. attenuata (bottom panel) and as comparison, a blumenol A-9-O-glucoside (roseoside) standard (top panel). The precursor-to-product ion transitions are indicated. (B) Time lapse accumulations of Compound 6 in roots of EV plants with (EV+, green line) or without (EV-, black line) AMF-inoculation. Data represent means ±SE (n ≥ 3). (C) Time lapse accumulations of Compound 6 in leaves of EV plants with (EV+, red line) or without (EV-, black line) AMF-inoculation and of irCCaMK plants with AMF-inoculation (irCCaMK+, orange line). Data represent means ±SE (n ≥ 5). (D) Comparison of the abundances of Compound 6 in leaves of plants inoculated with different inoculum concentrations, samples were harvested at 5 weeks-post-inoculation (wpi). Data are means +SE (n ≥ 4). Different letters indicate significant differences (p<0.05, one-way ANOVA followed by Fisher’s LSD). (E) Field experiment (Great Basin Desert, Utah, USA): leaf samples of EV (n = 20) and irCCaMK (n = 19) plants were sampled 8 weeks after planting and amounts of Compound 6 were analyzed. For statistical analysis, Student’s t test was applied. (F) Abundance of Compound 6 relative to the transcript abundance of the R. irregularis specific housekeeping gene, Ri-tub (GenBank: EXX64097.1), as well as to the plant derived marker genes RAM1, Vapyrin, STR1 and PT4.The transcript abundance was quantified by q-PCR, relative to NaIF-5a (NCBI Reference Sequence: XP_019246749.1). The correlation between Compound 6 and transcript abundance of marker genes was analyzed by linear regression (lm) models. (G) Distribution of Compound 6 in different plant tissues, as indicated, of plants with (+AMF, n = 3, red bars) or without (-AMF, n = 1, black bars) AMF-inoculation. Samples were harvested at six wpi.

https://doi.org/10.7554/eLife.37093.022
Figure 7 with 2 supplements
AMF-indicative changes in blumenols in aerial plant parts are valuable research tools providing accurate assessments of functional AMF associations in high-throughput screenings of multiple plant and AMF species.

(A) Root colonization analysis in two N. attenuata accessions (UT/AZ). H: hyphae; A: arbuscules; V: vesicles; Total: total root length colonization (n = 4; Student’s t-test, *p<0.05, **p<0.01, ***p<0.001). (B) Representative images of trypan blue stained roots (six wpi; bar = 100 μm). (C) Compound 2 in roots and leaves of UT and AZ plants with and without AMF-inoculation (means +SE, n = 8). (D) Heatmap of the normalized abundance of foliar Compound 2 in plants of a UT-AZ RIL population (728 plants) planted across a 7,200 m2 field plot. (E) QTL mapping analysis of the data from D. The QTL on linkage group three contains NaNOPE1, an AMF-associated gene, in addition to others. LOD, logarithm of the odds ratio. (F) Blumenol contents of different crop and model plants with and without AMF inoculation (S. lycopersicum (n = 6), T. aestivum (n = 10), H. vulgare (n = 5): eight wpi; M. truncatula (n = 3): seven wpi; S. tuberosum (n = 5): six wpi; B. distachyon (n = 4): five wpi). Different plant and AMF species were used as indicated (means +SE; n.d., not detected).

https://doi.org/10.7554/eLife.37093.025
Figure 7—figure supplement 1
Phenotypes of UT and AZ accessions and field plot planting design.

(A) Representative N. attenuata plants of the UT and AZ accessions in the rosette stages of growth (12 days after potting). (B) Transcripts of marker genes in roots responding to AMF colonization in UT and AZ after six wpi inoculated with R. irregularis were quantified by qPCR in the same samples as in Figure 4A–C (n = 8); Student’s t test *p<0.05, **p<0.01, ***p<0.001. (C) Field plot of 728 sampled individual plants in Utah, USA, 2017.

https://doi.org/10.7554/eLife.37093.026
Figure 7—figure supplement 2
AMF-indicative changes in blumenols in aerial plant part are valuable research tools providing accurate assessments of functional AMF associations of multiple plant and AMF species (continued from Figure 7F).

Blumenol contents of different crop plants with and without AMF inoculation (T. aestivum: eight wpi, n = 5; H. vulgare: eight wpi, n = 10; S. lycopersicum with F. mossae: 6 wpi, n = 5; S. lycopersicum with R. irregularis: 11 wpi, n = 6; B. distachyon: five wpi, n = 4; M. truncatula: seven wpi, n = 3). Different plant and AMF species were used, as indicated; means +SE, n.d., not detected.

https://doi.org/10.7554/eLife.37093.028
Figure 7—figure supplement 2—source data 1

Source Code File for QTL analysis.

https://doi.org/10.7554/eLife.37093.029

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (N. attenuata)A-09-1212-1Groten et al. (2015), DOI: 10.1111/pce.12561Stably silenced in CCaMK via RNAi
Genetic reagent (N. attenuata)A-09-1208-6Groten et al. (2015), DOI: 10.1111/pce.12561Stably silenced in CCaMK via RNAi
Genetic reagent (N. attenuata)A-11-92−4 ×
A-11-325-4
Schäfer et al. (2013), DOI: 10.1111/tpj.12301Chemically-inducible silenced in PDS via RNAi
Genetic reagent (N. attenuata)A-04-266-3Bubner et al. (2006),
DOI: 10.1007/s00299-005-0111-4
Empty vector control
Biological sample (N. attenuata)AZ-UT RILZhou et al. (2017), DOI: 10.1016/j.cub.2017.03.017Biparental QTL mapping population
Table 1
1H and 13C NMR data for compounds 1–4 and 7.
https://doi.org/10.7554/eLife.37093.031
No.Compound 1Compound 2Compound 3Compound 4Compound 7
Pos.δHmult.,
J [Hz]
δCδHmult.,
J [Hz]
δCδHmult.,
J [Hz]
δCδHmult.,
J [Hz]
δCδHmult.,
J [Hz]
δC
1--202.3--203.1--202.2--202.3--200.9
26.06dd,
1.8/1.8
121.36.40s128.56.06s br121.35.81s br125.25.82s126.4
3--172.4--172.6--172.2--169.8--171.5
41.92dd,
5.2/5.2
47.82.64m46.31.92dd,
5.2/5.2
48.01.97dd,
5.0/5.0
52.4--79.2
5--37.2--36.9--37.2--37.2--42.9
62.59
2.02
d, 17.5
d, 17.5
48.52.03
2.60
d, 17.4
d, 17.4
48.02.59
2.02
d, 17.6
d, 17.6
47.72.49
1.98
d, 17.3
d, 17.3
48.02.13
2.16
d, 18.0
d, 18.0
50.8
71.66
1.82
m m26.81.62
1.88
m m27.61.66
1.82
m m27.11.69
1.80
m m26.51.83
2.07
m m34.6
81.63m37.11.60m36.01.63m37.21.68
1.61
m m37.41.49
1.78
m m32.7
93.82dd, 6.2/
11.7
77.23.80m77.73.83dd, 6.3/
11.7
77.73.83dd, 6.3/
11.6
77.73.80ddd, 6.3/
11.8/11.8
78.2
101.24d, 6.221.61.21d, 6.121.91.24d, 6.321.91.25d, 6.321.91.24d, 6.321.9
114.32
4.16
dd,
17.8/1.8
dd,
17.8/1.8
64.9--160.14.33
4.16
m/m64.92.05d, 1.224.92.04d, 1.021.7
121.02s28.41.01s28.41.02s28.61.02s28.71.01s24.3
131.12s27.51.12s27.51.11s27.51.10s27.41.09s23.7
1'4.31d, 7.9103.84.30d, 7.9104.14.32d, 7.9103.84.32d, 7.8104.04.31d, 7.9104.0
2'3.15dd,
7.9/9.0
75.03.13dd,
7.9/8.9
75.13.15dd, 7.9/
9.0
75.13.16dd,
7.8/9.0
75.23.14dd,
7.9/8.9
75.1
3'3.33dd,
9.0/9.0
77.93.35dd,
8.9/8.9
78.03.33m77.83.34dd,
9.0/9.0
78.03.33dd,
8.9/8.9
77.8
4'3.27dd,
9.0/9.0
71.43.27dd,
8.9/8.9
71.53.33m71.43.33dd,
9.0/9.0
71.53.27dd,
8.9/8.9
71.4
5'3.25m77.63.25m77.63.45m77.03.44m76.93.24m77.7
6'3.85
3.65
dd,
11.8/2.2
dd,
11.8/5.5
62.53.84
3.66
dd,
2.0/12.3
dd,
5.0/12.3
62.64.11
3.78
dd,
11.7/2.0
dd,
11.7/5.7
69.64.11
3.79
dd,
11.6/1.6
dd,
11.6/5.9
69.73.85
3.65
d, 11.7
dd,
4.5/11.7
62.3
1''4.40d, 7.9104.64.40d, 7.8104.8
2''3.21dd,
7.9/9.0
74.93.21dd,
7.8/9.0
75.0
3''3.34dd,
9.0/9.0
77.93.34dd, 9.0/9.077.9
4''3.28dd,
9.0/9.0
71.43.28dd,
9.0/9.0
71.5
5''3.26m77.93.26ddd,
9.0/5.4/1.8
78.0
6''3.87
3.66
dd,
11.9/2.0
dd,
11.9/5.2
62.53.86
3.66
dd,
11.6/1.8
dd,
11.6/5.4
62.7
  1. s, singlet; s br, broad singlet; d, doublet; dd, doublet of doublet; m, multiplet

Table 2
MRM-settings used for targeted blumenol analysis.
https://doi.org/10.7554/eLife.37093.032
Nr.Compound nameRTQ1 [m/z]*, †Q3 [m/z] ‡, § (CE [V])
111-hydroxyblumenol C-Glc¶, **2.82+389.22227.16 (-2.5), 209.15 (-7.5), 191.14 (-12.5), 163.10 (-15), 149.10 (-17.5)
211-carboxyblumenol C-Glcf¶, **3.22+403.22241.16 (-2.5), 223.15 (-7.5), 177.10 (-15), 195.14 (-12.5)
+241.16 #223.15 (-5), 177.10 (-15), 195.14 (-10)
311-hydroxyblumenol C-Glc-Glc ¶, **2.5+551.27389.22 (-2.5), 227.16 (-7.5), 209.15 (-10), 191.14 (-15), 149.10 (-20)
4Blumenol C – Glc-Glc ¶, **3.47+535.27373.22 (-2.5), 211.00 (-10), 193.10 (-17.5), 135.00 (-22.5), 109.00 (-22.5)
5Blumenol C - Glc ¶, ††4.18+373.22211.20 (-6), 193.16 (-9), 175.10 (-15), 135.12 (-16), 109.10 (-20)
6Blumenol A - Glc¶, ††2.51- 385.20153.10 (14)
+387.20225.15 (-5), 207.14 (-8), 149.10 (-18), 135.12 (-16), 123.08 (-23)
7Blumenol B - Glc¶, **2.5+389.22227.16 (-5), 209.15 (-7.5), 191.14 (-12.5), 153.10 (-17.5), 149.10 (-17.5)
8Blumenol C – Glc-GlcU¶, ‡‡3.25+549.27373.22 (-2.5), 211.00 (-10), 193.10 (-17.5), 135.00 (-22.5), 109.00 (-22.5)
and 3.38
911-hydroxylumenol C – Glc-Rha‡‡2.8+535.27389.22 (-2.5), 227.16 (-7.5), 209.15 (-10), 191.14 (-15), 149.10 (-20)
10Blumenol C – Glc-Rha¶, ‡‡4.1+519.27373.22 (-2.5), 211.00 (-10), 193.10 (-17.5), 135.00 (-22.5), 109.00 (-22.5)
11Hydroxyblumenol C-Hex-Pen‡‡2.5+521.27389.22 (-2.5), 227.16 (-7.5), 209.15 (-10), 191.14 (-15), 149.10 (-20)
D6-ABA††4.5- 269.17159.00 (10)
  1. RT: retention time

    CE: collision energy

  2. Glc: glucose

    GlcU: glucuronic acid

  3. Rha: rhamnose

    Hex: hexose

  4. Pen: pentose

    *Resolution: 0.7

  5. [M + H]+ or [M-H]- if not stated differently

    Resolution: 2

  6. § Quantifiers are depicted in bold

    # [M + H-Glc]+

  7. Verified by high resolution MS

    **Verified by NMR

  8. ††Optimized with commercial available standards

    ‡‡Transitions predicted based on structural similar compounds and literature information

Table 3
MRM-settings for the analysis of selected blumenols in N. attenuata.
https://doi.org/10.7554/eLife.37093.033
Nr.Compound nameRTQ1 [m/z]*, †Q3 [m/z]‡, § (CE [V])
111-hydroxyblumenol C-Glc¶, **2.82+389.22227.16 (-2.5), 209.15 (-7.5), 191.14 (-12.5), 163.10 (-15), 149.10 (-17.5)
211-carboxyblumenol C-Glc¶, **3.22+403.22241.16 (-2.5), 223.15 (-7.5), 177.10 (-15), 195.14 (-12.5)
+241.16#223.15 (-5), 177.10 (-15), 195.14 (-10)
6Blumenol A - Glc ¶, ††2.51- 385.20153.10 (14)
+387.20225.15 (-5), 207.14 (-8), 149.10 (-18), 135.12 (-16), 123.08 (-23)
D6-ABA††4.0- 269.17159.00 (10)
  1. RT: retention time

    CE: collision energy

  2. Glc: glucose

    Hex: hexose

  3. Pen: pentose

    *Resolution: 0.7

  4. [M + H]+ or [M-H]- if not stated differently

    Resolution: 2

  5. §Quantifiers are depicted in bold

    #[M + H-Glc]+

  6. Verified by high resolution MS

    **Verified by NMR

  7. ††Optimized with commercial available standards

Table 4
Sequences of primers used for qPCR-based analysis of AMF-colonization rates.
https://doi.org/10.7554/eLife.37093.034
GeneForward primerReversed primer
NaIF-5aGTCGGACGAAGAACACCATTCACATCACAGTTGTGGGAGG
NaRAM1ACGGGGTCTATCGCTCCTTGTGCACCAGTTGTAAGCCAC
NaVapyrinGGTCCCAAGTGATTGGTTCACGACCTTCAAAGTCAACTGAGTCAA
NaSTR1TCAGGCTTCCACCTTCAATATCTGACTCTCCGACGTTCTCCC
NaPT4GGGGCTCGTTTCAATGATTAAACACGATCCGCCAAACAT
NaCCaMKTTGGAGCTTTGTTCTGGTGGTATACTTGCCCCGTGTAGCG
NaNOPE1ACTTGATGCCATGTTTCAGAGCTCCAATTCGCGATAAGCTGGT
Ri-TUBTGTCCAACCGGTTTTAAAGTAAAGCACGTTTGGCGTACAT

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures.

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