A phenylpropanoid diglyceride associates with the leaf rust resistance Lr34res gene in wheat

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Supplemental Figures

A phenylpropanoid diglyceride associates with the leaf rust resistance

Lr34res gene in wheat

Nandhakishore Rajagopalan

1 _{, Yuping Lu}

1 _{, Ian W. Burton}

2 _{, Fanny}

Monteil-Rivera

3 _{, Annamaria Halasz}

4 _{, Elsa Reimer}

5 _{, Rebecca Tweidt}

6 _{, Anita}

Brûlé-Babel

7 _{, Hadley R. Kutcher}

6 _{, Frank M. You}

8 _{, Sylvie Cloutier}

8 _{, Miroslava}

Cuperlovic-Culf

9 _{, Colin W. Hiebert}

5 _{, Brent D. McCallum}

5 _{, Michele C.}

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0 20 40 60 80 100 120 Intens. [mAU] 200 220 240 260 280 300 320 340 360 380 Wavelength[nm] 315 nm Time [min] 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 0 100 200 300 Intens. [mAU] 20.8

Supplemental Figure S1. Characterization of the P2 metabolite isolated for

Tc+Lr34res flag leaves during LC-MS/MS validation. A) HPLC/UV (310 nm)

chromatogram of P2 sample. B) UV spectrum of the major peak at 20.8 min.

0 20 40 60 80 100 120 Intens. [mAU] 200 220 240 260 280 300 320 340 360 380 Wavelength[nm] 315 nm Time [min] 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 0 100 200 300 Intens. [mAU] 20.8

A

B

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Supplemental Figure S2. 1D NMR data arising from the P2 (CFG) metabolite

sample. A) 1D proton NMR spectrum. B) 1D carbon NMR Spectrum

C -9 C -13 C -19 C -4 C -5 C -7 C -15 C -17, 21 C -1 C -16 C -2 C -18, 20 C -3 C -8 C -14 C -6 C -11 C -12 C -10 C -22

A

B

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H22 – C5

H10 – C9 H12 – C13 H17,21 – C19

Supplemental Figure S3 A & B. 60 ms HMBC spectrum used in confirmation of the

final structure of the P2 (CFG) metabolite A) Annotated 60 ms HMBC spectrum

showing primarily 3-bond correlations B) Structure of the P2 9CFG) metabolite

showing key HMBC correlations.

H7 – C9 H15 – C13 H8 – C1 H14 – C16 H8 – C9 H14 – C13 H18,20 – C19 H10 – C12 H12 – C10 H10,12 – C11 H11 – C10,12 1 2 3 4 5 6 7 8 9 10 11 13 12 14 15 16 17 ₁₈ 19 20 22 21

A

B

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H3 – C5 H3 – C4 H2 – C7 H2 – C4 H6 – C7 H6 – C4 H17,21 – C15 H17 – C21 H21 – C17 H15 – C17,21 H6 – C2 H2 – C6 H7 – C2 H7 – C6 H3 – C1 H18,20 – C16 H18 – C20 H20 – C18

Supplemental Figure S3 C. 60 ms HMBC spectrum used in confirmation of the

final structure of the P2 (CFG) metabolite. Expansion of aromatic and vinyl region

of HMBC.

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Df Sum Sq Mean Sq F value Pr(>F) Lines_tested 1 4.16 4.16 5.560 0.022499 * Infection_condition 1 33.23 33.23 44.372 2.43e-08 *** DPI 3 7.22 2.41 3.216 0.030934 * Lines_tested:Infection_condition 1 1.16 1.16 1.550 0.219138 Lines_Tested:DPI 3 0.95 0.32 0.422 0.737917 Infection_condition:DPI 3 17.62 5.87 7.842 0.000233 *** Lines_tested:Infection_condition:DPI 3 1.51 0.50 0.674 0.572391

Supplemental Figure S4: Summary of multifactorial ANOVA analysis of the accumulation

of CFG in Tc versus Tc+Lr34res (RL6058)/ Tc+Lr22a / Tc+Lr67res lines infected and

**uninfected with leaf rust over time. * P < 0.05; P < 0.01; * < 0.001.**

Df Sum Sq Mean Sq F value Pr(>F)

Lines_tested 1 0.683 0.683 1.715 0.19654 Infection_condition 1 13.253 13.253 33.292 5.62e-07 *** DPI 3 1.198 0.399 1.003 0.39959 Lines_tested:Infection_condition 1 1.095 1.095 2.752 0.10366 Lines_tested:DPI 3 0.550 0.183 0.461 0.71096 Infection_condition:DPI 3 6.519 2.173 5.459 0.00261 ** Lines_tested:Infection_condition:DPI 3 0.526 0.175 0.441 0.72488

Df Sum Sq Mean Sq F value Pr(>F)

Lines_tested 1 0.004 0.004 0.007 0.933220 Infection_condition 1 15.092 15.092 27.911 3.05e-06 *** DPI 3 9.694 3.231 5.976 0.001515 ** Lines_tested:Infection_condition 1 0.644 0.644 1.190 0.280712 Lines_tested:DPI 3 2.565 0.855 1.581 0.206186 Infection_condition:DPI 3 10.839 3.613 6.682 0.000734 *** Lines_tested:Infection_condition:DPI 3 0.713 0.238 0.440 0.725558

A phenylpropanoid diglyceride associates with the leaf rust resistance Lr34res gene in wheat

Supplemental Figures