Metabolic response induced by parasitic plant-fungus interactions hinder amino sugar and nucleotide sugar metabolism in the host

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1 Supplementary information Metabolic response induced by parasitic plant-fungus interactions hinder amino sugar and nucleotide sugar metabolism in the host Dong-Kyu Lee, Soohyun Ahn, Hae Yoon Cho, Hye Young Yun, Jeong Hill Park, Johan Lim, Jeongmi Lee, Sung Won Kwon

2 Supplementary Figure S1. Leaf samples of Crataegus pinnatifida (control, a; parasitized, b), Chaenomeles sinensis (control, c; parasitized, d) and Pyrus pyrifolia (control, e; parasitized, f). a b c d e f

3 Supplementary Figure S2. Four aeciospores and a single peridial cell of Gymnosporangium asiaticum on Pyrus pyrifolia var. culta (Makino) Nakai, specimen HY2926 ( 400).

4 Supplementary Figure S3. PCA score scatter plots of Crataegus pinnatifida (n=20), Chaenomeles sinensis (n=20) and Pyrus pyrifolia (n=20) with control (green) and parasitized (red) groups. a b c PC2 (19.1%) PC2 (25.0%) PC2 (17.6%) PC1 (47.4%) PC1 (45.9%) PC1 (54.5%)

5 Supplementary Figure S4. PCA score scatter plot of control (green) and parasitized (red), including whole samples of Rosaceae species (n=10 for each group, total n=60) Crataegus pinnatifida Chaenomeles sinensis Pyrus pyrifolia 5 PC2 (25.0%) PC1 (45.9%)

6 Supplementary Figure S5. Heat-map representation of unsupervised hierachical clustering analysis (HCA) class Ribose Inositol Sorbose Mannose Oleic acid Succinic acid Glucose Phosphoric acid Fructose Sorbitol Melibiose Citramalic acid Glyceric acid Xylose Xylitol Ribitol Mannitol Erythritol Glycerol Gluconic acid Malic acid Citric acid Maltose class Con Para CP_C3 CP_C1 CP_C10 CP_C7 CP_C5 CP_C6 CP_C4 CP_C2 CP_C8 CP_C9 CS_C6 CS_C1 CS_C4 CS_C2 CS_C10 CS_C8 CS_C9 CS_C7 CS_C5 CS_C3 PP_C3 PP_C2 PP_C9 PP_C10 PP_C6 PP_C8 PP_C7 PP_C5 PP_C1 PP_C4 CS_P7 CS_P5 CS_P8 CS_P6 CS_P3 CS_P2 CS_P4 CS_P1 CS_P9 CP_P5 CS_P10 CP_P10 CP_P7 CP_P6 CP_P9 CP_P8 CP_P3 CP_P4 CP_P2 CP_P1 PP_P5 PP_P4 PP_P3 PP_P2 PP_P7 PP_P6 PP_P8 PP_P9 PP_P1 PP_P10

7 Supplementary Figure S6. Substrates of ANM pathway from other metabolic pathways ANM pathway substrates Fructose and mannose metabolism Fructose Mannose Fucose Pentose and glucuronate interconversions Glucuronate Galacturonate Glycolysis/ Gluconeogenesis Glucose metabolism Red: not found in leaves

8 Supplementary Figure S7. Mapping of metabolic alterations on GM. Metabolites (p-value <0.05 as asterisk) between control (green) and parasitism (red) were colored based on PPMCC value from -1 (negative correlation, red) to +1 (positive correlation, blue) against fructose (a), glucose (b) and mannose (c). All values in bar graphs were mean + s.d., 30 replicates for each group. a b c

9 Supplementary Figure S8. Correlation analysis (based on PPMCC) results among whole metabolic changes in Crataegus pinnatifida (a, n=20), Chaenomeles sinensis (b, n=20) and Pyrus pyrifolia (c, n=20). Negative correlations (maximum -1, red) and positive correlations (maximum +1, blue) were displayed on heatmaps with clustering. a D-Citramalic acid Succinic acid Glyceric acid Shikimic acid Ribonic acid Glucose Fructose Sorbitol Oleic acid Stearic acid Melibiose Idonic acid Ribose Xylonic acid Glycerol Phosphoric acid Inositol Sucrose Mannose Sorbose Malic acid Maltose Citric acid Xylitol Xylose Mannitol Gluconic acid Ribitol Erythritol b Melibiose Mannose Ribose Stearic acid Xylitol Citric acid Inositol Glucose Ribonic acid Gluconic acid Mannitol Erythritol Xylonic acid Sorbose Sucrose Maltose Xylose Malic acid Succinic acid Shikimic acid Fructose D-Citramalic acid Glyceric acid Oleic acid Ribitol Idonic acid Glycerol Phosphoric acid Sorbitol Erythritol Ribitol Gluconic acid Mannitol Xylose Xylitol Citric acid Maltose Malic acid Sorbose Mannose Sucrose Inositol Phosphoric acid Glycerol Xylonic acid Ribose Idonic acid Melibiose Stearic acid Oleic acid Sorbitol Fructose Glucose Ribonic acid Shikimic acid Glyceric acid Succinic acid D-Citramalic acid Sorbitol Phosphoric acid Glycerol Idonic acid Ribitol Oleic acid Glyceric acid D-Citramalic acid Fructose Shikimic acid Succinic acid Malic acid Xylose Maltose Sucrose Sorbose Xylonic acid Erythritol Mannitol Gluconic acid Ribonic acid Glucose Inositol Citric acid Xylitol Stearic acid Ribose Mannose Melibiose c Shikimic acid Sorbitol Gluconic acid Sorbose Glyceric acid Ribose D-Citramalic acid Fructose Idonic acid Sucrose Oleic acid Xylonic acid Melibiose Glucose Mannose Succinic acid Malic acid Citric acid Stearic acid Glycerol Ribonic acid Xylitol Inositol Maltose Phosphoric acid Erythritol Xylose Mannitol Ribitol -1 (Neg corr) +1 (Pos corr) Ribitol Mannitol Xylose Erythritol Phosphoric acid Maltose Inositol Xylitol Ribonic acid Glycerol Stearic acid Citric acid Malic acid Succinic acid Mannose Glucose Melibiose Xylonic acid Oleic acid Sucrose Idonic acid Fructose D-Citramalic acid Ribose Glyceric acid Sorbose Gluconic acid Sorbitol Shikimic acid

10 Supplementary Figure S9. Interaction effect of group variable and accumulated sugar alcohols on the ANM supplies. Forty-five scatter plots (n = 15 for each species) with min-max normalized values of ANM supplies (fructose, arabinose and galactose on the y-axis) and accumulated sugar alcohols (ribitol, arabitol, erythritol, mannitol and xylitol on the x-axis). The interaction effect directions of the models were measured by the slope differences of two regression lines upon each group variable (control, green; parasitism, red). The significant models were denoted as *p < 0.05 and **p < Fructose CS PS CP CS PS CP CP PS CS Ribitol Erythritol Mannitol Xylitol ** * * ** * * X axis: normalized value of independent metabolite Y axis: normalized value of dependent metabolite * ** ** * * ** * ** ** * * **

11 Supplementary Figure S10. Mass spectra of derivatized metabolites. The list of metabolites were as follows: phosphoric acid (a), glycerol (b), succinic acid (c), glyceric acid (d), fumaric acid (e), serine (f), threonine (g), citramalic acid (h), malic acid (i), erythritol (j), aspartic acid (k), erythronic acid (l), 3-hydroxy-3-methylpentanedioic acid (m), xylonic acid (n), xylose (o), arabinose (p), ribose (q), xylitol (r), arabitol (s), ribitol (t), arabinofuranose (u), ribonic acid (v), idonic acid (w), citric acid (x), sorbose (y), mannose (z), fructose (aa), galactose (ab), mannitol (ac), sorbitol (ad), gluconic acid (ae), inositol (af), glucose (ah), linoleic acid (ah), oleic acid (ai), stearic acid (aj), sucrose (ak), maltose (al) and melibiose (am) (a) (b) (x10,000) (c) (d) (e) (f)

12 0 204 (g) (h) (i) (j) (k) (l)

13 0 (m) (n) (o) (p) (q) (r)

14 (s) (t) (u) (v) (w) (x)

15 0 2 (y) (z) (aa) (ab) (ac) (ad)

16 0 (ae) (af) (ag) (x10,000) (ah) (ai) (aj)

17 0 117 (ak) (al) (am) (x10,000)

18 Supplementary Table S1. List of identified metabolites and the differences between control and parasitized leaves. Retention time (min) Compound name Fragment ion (derivatized compound) a p-value NIST: putatively identified using NIST database (match score over 80/100) STD: identified using standard compound Fold change Identification NIST STD 8.04 Phosphoric acid, 3TMS 298(100), (60), 313(18), 45(11) Glycerol, 3TMS (100), (78), 205(43), 117(32), 103(31) Succinic acid, 2TMS (100), (52), 75(19), 148(16), 246(10) 3.0ⅹ Glyceric acid, 3TMS (100), (61), 189(31), 292(23), 102(17) 6.5ⅹ Fumaric acid, 2TMS 245(100), (57), (41), 143(23) NM 9.84 Serine, 3TMS (100), 204(95), (53), 100(27), (20) NM Threonine, 3TMS (100), 117(48), 218(41), 57(22), (17) NM Citramalic acid, 3TMS (100), (53), 247(41), 75(23), 115(15) 2.3ⅹ Malic acid, 2TMS (100), (53), 233(18) Erythritol, 4TMS (100), (57), 216(51), 103(34), 205(28) 1.3ⅹ Aspartic acid, 3TMS 232(100), (98), 100(32), (22), 218(16) NM 13. Erythronic acid, 4TMS (100), (51), 292(24), 117(14), 103(11) NM H-3-MP acid, 3TMS b (100), (47), 247(31), 115(18), 231(18) NM Xylonic acid, 3TMS (100), (29), (27), 129(18), 102(11) Xylose, 4TMS, 1 MO (100), 103(77), (49), 307(32), (28) , 4TMS (100), 103(88), (43), 307(29), (25) 3.0ⅹ Ribose, 4TMS, 1 MO (100), 103(80), (44), 307(28), (24) Xylitol, 5TMS (100), (64), 103(42), (40), 205(21) , 5TMS (100), (63), 103(44), (41), 205(22) 3.9ⅹ Ribitol, 5TMS (100), (57), (43), 103(40), 205(27) 2.5ⅹ Arabinofuranose, 4TMS (100), (92), (31), 232(10) NM Ribonic acid, 5TMS (100), 292(45), (36), 103(30), (29) Idonic acid, 4TMS (100), (36), (28), 103(24), 436(20) 8.2ⅹ Citric acid, 4TMS (100), 2(71), (59), 347(17), 375(14) Sorbose, 5TMS (100), 103(76), (52), 307(32), (24) Mannose, 5TMS, 1 MO (100), 319(48), 205(41), (39), 160(26) Fructose, 5TMS, 1 MO (100), 103(), (53), 307(33), (24) 9.0ⅹ , 5TMS, 1 MO (100), 319(49), 205(40), (39), (21) ⅹ Mannitol, 6TMS (100), 319(56), (43), 205(39), (35) 1.7ⅹ Sorbitol, 6TMS (100), 319(53), (42), 205(36), 103(29) 3.6ⅹ Gluconic acid, 6TMS (100), (36), 333(28), 292(25), 103(15) myo-inositol, 6TMS (100), (60), 305(58), (44), 318(31) Glucose, 5TMS, 1 MO (100), 319(63), 205(38), (36), 103(21) Linoleic acid, 1TMS 75(100), (97), 67(77), 81(70), 55(54) NM Oleic acid, 1TMS (100), 117(94), 75(90), 129(64), 55(56) 1.54ⅹ Stearic acid, 1TMS 117(100), (75), 75(56), 341(55), 132(54) Sucrose, 8TMS (100), 361(88), 216(37), (25), 103(22) Maltose, 8TMS (100), 361(86), 204(59), (34), (31) Melibiose, 8TMS 204(100), (66), (29), 205(28), 361(27)

19 Supplementary Table S2. Multivariate (VIP value > 0) and univariate (p-value < 0.05) statistical parameters of 26 marker metabolites with whole data (n=60) implying importance for discriminating control and parasitized groups. Metabolites VIP value p-value Erythritol ⅹ ⅹ ⅹ10-10 Citric acid Maltose myo-inositol Mannitol ⅹ ⅹ10-8 Xylitol Malic acid Glucose Ribitol ⅹ10-7 Glycerol Gluconic acid Phosphoric acid Fructose ⅹ10-8 Mannose Ribose 3 Sorbitol 1 3.6ⅹ10-13 Sorbose Succinic acid <0 3.0ⅹ10-4 Glyceric acid <0 6.5ⅹ10-13 Citramalic acid <0 2.3ⅹ10-4 Oleic acid <0 1.54ⅹ10-6 Melibiose < Xylose <

20 Supplementary Table S3. Metabolic pathway enrichment results. Metabolic pathway Pentose and glucuronate interconversions Adjusted p-value Total metabolites included Matched Matched/Total metabolites (%) metabolism Fructose and mannose metabolism Starch and sucrose metabolism Glyoxylate and dicarboxylate metabolism Biosynthesis of plant hormones Citrate cycle (TCA cycle) Biosynthesis of alkaloids derived from histidine and purine Biosynthesis of phenylpropanoids Biosynthesis of alkaloids derived from terpenoid and polyketide Ascorbate and aldarate metabolism Biosynthesis of alkaloids derived from shikimate pathway Biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid Amino sugar and nucleotide sugar metabolism Biosynthesis of terpenoids and steroids

21 Supplementary Table S4. Enzymatic pathways about metabolic flows from AMN supplies (fructose, arabinose and galactose) to five sugar alcohols. Enzymes were designated as enzyme entry in KEGG pathway and modules. Enzymes and modules in PPP were colored as green. Fructose Ribitol Erythritol Mannitol Xylitol Ribitol EC: M00007 EC: EC: EC: Fructose Fructose-6P Ribose-5P Ribose Ribulose Ribitol EC: M00007 EC: EC: EC: Fructose Fructose-6P Ribose-5P Ribose Ribulose EC: Xylulose-5P EC: EC: EC: EC: EC: Xylulose EC: EC: EC: Fructose Fructose-6P Erythrose-4P Erythritol EC: or EC: EC: EC: Fructose Fructose-6P Mannitol-1P Mannitol EC: EC: EC: EC: EC: EC: Fructose Fructose-6P Xylulose-5P Xylulose Xylitol EC: EC: Ribulose Ribitol EC: Erythritol Mannitol Xylitol Leloir pathway Ribitol Erythritol Mannitol Xylitol EC: EC: EC: EC: EC: EC: Ribulose Ribose Ribose-5P Erythrose-4P Erythritol EC: EC: EC: M00007 EC: EC: Ribulose Ribose Ribose-5P Fructose-6P Mannitol-1P Mannitol EC: EC: EC: EC: EC: EC: EC: EC: EC: Ribulose Ribose Ribose-5P Xylulose-5P Xylulose Xylitol EC: EC: EC: EC: P Glucose-1P Glucose-6P Leloir pathway M00006 EC: EC: Glucose-6P Ribulose-5P Ribulose Ribitol Leloir pathway M00006 EC: EC: Glucose-6P Ribulose-5P Ribulose Leloir pathway EC: EC: EC: Glucose-6P Fructose-6P Erythrose-4P Erythritol Leloir pathway EC: EC: EC: Glucose-6P Fructose-6P Mannitol-1P Mannitol EC: Leloir pathway M00006 EC: EC: EC: EC: Glucose-6P Ribulose-5P Xylulose-5P Xylulose Xylitol