Method S1. Reverse phase HPLC for determination of sinapoylmalate content

in leaf material.

Sinapate esters were extracted from 100 mg fresh leaf material of three-week-old

plants in 750 µl 50 % (v/v) methanol at 65°C for 1.5 h. After centrifugation at

13’000 rpm for 10 min supernatants were filtered (Miniosart RC 4 syringe filter,

Sartorius Stedim Biotech GmbH) and analyzed by reverse phase HPLC on a

Nucleosil C18 column (EC 150/4,6 Nucleosil 100-5 C18; Macherey-Nagel) using

a gradient from 2 % acetonitrile in 1.5 % acetic acid to 40 % acetonitrile in 1.5 %

acetic acid at a flow rate of 1 ml min-1. Only a single fluorescence peak at a

retention time of 24.5 min was detected at 335 nm excitation and 460 nm emission

wavelengths (FP 920 Intelligent Fluorescence Detector; Jasco). Since the area of

the latter peak was significantly and reproducibly reduced in ref3-3, brt1-1 and

pen2 BRT1dsRNAi lines compared to extracts of the wild type or pen2 controls

while it was completely absent in fah1-2 mutants, the compound very likely was

sinapoylmalate. Since commercial sinapoylmalate standard compounds are not

available, sinapoylmalate content could not be quantified absolutely; instead, it is

presented as peak area relative to the pen2 control.

Figure S1. Callose-encased haustorium of P. pachyrhizi in a mesophyll cell of

Arabidopsis pen2 pad4 sag101. P. pachyrhizi -infected leaves were stained with trypan

blue 48 h after inoculation , destained in in chloral hydrate and subsequently stained with

aniline blue. Micrographs were taken under UV epifluorescence. Scale bar corresponds to

20 µm.

Figure S2. Frequencies of haustoria formation in infected Arabidopsis

genotypes only slightly increase after 2 dpi. Leaves were harvested two,

three and four days after inoculation with P. pachyrhizi and the frequency of

haustoria development was determined microscopically after trypan blue

staining. Per genotype and point of time at least 300 penetration events on

three different plants were analyzed.

Pen

etra

tion

even

ts w

ith h

aust

oriu

m (

%)

2 d

pi

2 dp

i

4 dp

i

2 dp

i

3 dp

i

4 dp

i

0

10

20

30

40

50

60

pen2 pen2pad4sag101

pen2brt1

4 dp

i

A

CL16dsRNAi (Col-0)

wt (Col-0) brt1

brt1 BRT1:BRT1-GFP

0

20

40

60

80

100

120

pen2 w

t

pen2

CL1

6 ds

RNAi

brt1

ref3

fah1

B

Sina

poyl

mal

ate

abun

danc

e re

lativ

e to

pen

2 (%

)

Figure S3. Silencing of BRT1 by dsRNAi results in hyperfluorescent trichomes under UV-

light (A) and reduction of sinapoylmalate levels in leaves (B). Photos of leaves of 4-week-old

plants were taken under UV-light (CAMAG, Berlin, Germany) at 366 nm. Note that

hyperfluorescent trichomes are observed only for the brt1 mutant and for CL16dsRNAi

transgenic lines but neither for the wild type or brt1BRT1:BRT1-GFP complementation plants

(A). For HPLC analysis (B) equal amounts (100mg) of leaf material of four plants of a given

genotype were pooled and subjected to methanol extraction. Only a single fluorescence peak at a

retention time of 24.5 min was detected by HPLC (excitation wavelength: 335 nm; emission

wavelength: 460 nm) which likely corresponds to sinapoylmalate since its abundance is reduced

in brt1 and the ref3 mutant and completely absent in fah1. Relative sinapoylmalate abundance is

shown as peak area relative to pen2. Mean values and SD of three independent leaf extracts are

shown.