SUPPORTING INFORMATION – FULL LEGENDS
Figure S1. Hyphal growth of B. cinerea over 24 h on Col-0, etr1-1, eto2 and ctr1-1. Mean hyphal
growth of two 5 μL droplets of 1 x 105 spores mL-1 in Potato Dextrose Broth (PDB) on Col-0,
etr1-1, eto2 and ctr1-1 maintained at 24 °C with an 8 h photoperiod (80 μmol m-2 s-1 and 50-80
% relative humidity) were made at 2 h intervals over 24 h. Six replicates of each genotype x
time combination were made and the mean is plotted with their respective standard error. The
mean over 24 h (over all the time points) for each treatment is also shown.
Figure S2. Infection structures produced during the interaction of B. cinerea with Col-0, etr1-1,
eto2 and ctr1-1. Using the aniline blue staining method, a random field of view was selected for
each genotype x time (24, 36, 48 and 72 hours post infection) combination for 20 individual
samples and the occurrence of (i) % appressoria-like structures; (ii) % of thin, elongated
hyphae; (iii) % of no hyphal differentiation and (iv) % host autofluorescence were recorded.
This investigation was repeated 3 times and the mean was displayed with the standard error. *
Significantly different to Col-0 at that particular time point, P < 0.01.
Figure S3. Examples of non polar and polar spectra. Examples of spectra following Direct
Injection Electrospray Ionisation-Mass Spectrometry (DI-MS) of (i) polar and (ii) non-polar Col-0
plant extracts in negative mode, acquired over the m/z range 100-1400 Th
Figure S4. Data analysis pathway. (A) Initial data display and interpretation was based on
multivariate approaches; Multivariate Analysis of Variance (MANOVA) was used to describe the
significance of different factors in the experimental series as described in Johnson et al., (2007).
Non-supervised Principal Component Analysis (PCA) identified major sources of variation in the
dataset which could be associated with experiment classes. Discriminant Functional Analysis
(DFA) is based on derived Principal Components (PCs) and is supervised approach where a priori
differences between experimental classes are maximised.
(B) Due to the complex class
structure involving time, treatment and genotype, deducing important m/z differentiating
between given experimental classes. PC-DFA models were derived (1) where for each time
point all infected samples for a genotype were compared to equivalent Potato Dextrose Broth
(PDB) controls. This would suggest differences which were due to infection rather than use of
PDB. (2) Where an individual genotype is examined over all time points to target genotypic
changes over time. (3) Where all genotypes were examined at a single time point (as shown in
Fig 3B and 3C). This would reveal genotypic specific changes to infection. In each case, m/z
that appeared more positive than 1 standard deviation (STD) from the mean m/z loading, or
more negative than -1 STD, were accepted as the most important discriminatory loading
variables for that particular model. In a parallel approach. In a parallel approach, DI-MS spectra
for each replicate were averaged and mean uninfected subtracted from mean infected spectra
for each genotype and time point. The major m/z changes were ranked whilst analysis of
variance (ANOVA) provided a method for analysing individual m/z that significantly different
from uninfected controls and from other genotypes.
Those that appeared as having
significantly different m/z were ‘denoted as ‘*’. Those that were both discriminatory and
significantly different m/z at all three steps were denoted as ‘d*’.
Figure S5. Polar m/z contributing towards the discrimination between Col-0 and the three
mutants etr1-1, eto2 and ctr1-1, upon infection with B. cinerea. DI-MS polar discriminatory m/z
were displayed using a heat map and hierarchical cluster analysis. The m/z levels were derived
from subtraction of mean spectra for each B. cinerea infected mutant at each time point with
corresponding levels for infected Col-0 at the same time point. Indicated are (A) a clade of m/z
increased regulation upon infection with B. cinerea compared with Col-0, independent of
genotype; (B) a clade of m/z decreased in etr1-1 compared with Col-0 upon B. cinerea infection
over all three time points and increased in modulation in both ctr1-1 and eto2; (C) no consistent
response over the three time points after infection with B. cinerea in any of the three
genotypes; (D) small blocks of m/z showing increased regulation in the genotypes etr1-1 and
eto2 and decreased regulation in ctr1-1, compared with Col-0 upon infection with B. cinerea; (E)
a clade of m/z showing decreased regulation upon infection with B. cinerea in both eto2 and
ctr1-1, but increased modulation in etr1-1 compared with Col-0.
Figure S6. Displaying targeted biosynthetic pathways based on tentatively identified m/z. (Ai) a
key devised to display the modulation and discrimination of individual m/z in etr1-1, ctr1-1 and
eto2 (compared with Col-0) at 12, 24 and 48 hours post infection (hpi) with B. cinerea (Aii)
example of Ai. The m/z significantly increased in etr1-1 at 12 and 24 hpi compared with Col-0,
but was not discriminatory.
Within ctr1-1 at 24 hpi, this m/z decreased in modulation
compared with Col-0 and was discriminatory. At 48 hpi it only appeared to be discriminatory.
In eto2, this m/z appeared to be discriminatory and significantly modulated compared with Col0 over all time points after B. cinerea infection. (B) A display of tentatively identified
discriminatory m/z and those significantly modulated within etr1-1, ctr1-1 and eto2 compared
with Col-0 at 12, 24 and 48 hpi with B. cinerea, within the biosynthesis of the core
hydroxycinnamates and sinapate esters, using negative mode DI-MS polar data Indicated on
the pathway are mutants (as described in Table 1). (C) A display of tentatively identified
discriminatory m/z and those significantly modulated within etr1-1, ctr1-1 and eto2 compared
with Col-0 at 12, 24 and 48 hpi with B. cinerea, within the reduction of the hydrocinnamic acid
CoA thioester intermediates to produce the p-coumaryl-‘H unit’, Indicated on the pathway are
mutants (as described in Table 1).
Figure S7. Confirmation of key metabolites within discriminatory pathways using tandem-MS.
MS-MS spectra of (Ai) phenylalanine standard; (Aii) phenylalanine in an etr1-1 sample 48 hours
post inoculation (hpi) with B. cinerea; (Bi) ferulate standard; (Bii) ferulate in a Col-0 sample 48
hpi; (Ci) sinapate standard; (Cii) sinapate in an eto2 sample 48 hpi; (Di) sinapyl alcohol standard
and (Dii) sinapyl alcohol in a Col-0 sample 48 hpi.
Figure S8. Control Spatial imaging targeting the hydroxyl groups in phenolic and aliphatic
structures in lignin (3400-3300 cm-1; Boeriu et al., 2004). Light (i) and (ii) corresponding Fourier
Transform Infra Red (FT-IR) images (A) Col-0; (B) etr1-1; (C) eto2; (D) ref3-2; (E) pap1-D and (F)
fah1-2 at 24 hour post infection (hpi). The arrows denote areas of interest, where (4) shows
the PDB droplet. Bar = 50 μm. The colour bar map represent the intensity of the IR signal
absorbed, white-pink-red showing high and blue showing low.
Figure S9. Variation in symptoms in wild type and mutant Arabidopsis genotypes following
challenge with different B. cinerea strains. Hierarchical clustering analysis of the symptom
development of Col-0, etr1-1 and fah1 (described in Table 1), caused by 5 μL droplets of 1 x
105 spores mL-1 from B. cinerea strains (described in Table 2) in Potato Dextrose Broth. Red
indicates less virulent “resistant” while green denotes more virulent “susceptibility”. The
colour code indicates the host from which each strain was isolated. Two main clades are
indicated; (A) contains B. cinerea strains which elicit different symptoms to iMi169558 (iMi) on
Arabidopsis genotypes whilst with (B) symptoms are broadly equivalent to those seen with
iMi169558.
Figure S10. Multivariate analyses of symptom variation in wild type and mutant Arabidopsis
genotypes following challenge with different B. cinerea strains. Symptom development of Col-0,
etr1-1 and fah1 (described in Table 1), caused by 5 μL droplets of 1 x 105 spores mL-1 from B.
cinerea strains (described in Table 2) in Potato Dextrose Broth were scored for resistance or
susceptibility (as described in Table S1). The colour and symbol codes indicate the host from
which each strain was isolated. (A) Principal component analysis (PCA) indicated failed to
detect similarities in symptoms elicited from the host based on original host, except for strains
originating from tomato (the grouping being highlighted by a pink stripe). (B) Discriminant
Function Analysis (DFA) based on 3 Principal Components (PCs) explaining 99.5% of the
variation indicated that the symptoms elicited by B. cinerea strains originally isolated from
grape were the most variable. (C) Examination of the PC1 loadings (derived from (A)) suggested
that the symptoms elicited by the different strains from Col-0 were the major scores of
variation.
Figure S11. Transcriptional regulation of key genes involved in monolignol and ferulate
biosynthesis. Monolignol and ferulae biosynthetic pathways derived from Plant Metabolic
Network (http://plantcyc.org/).
Genes encoding key biosynthetic enzymes are indicated.
Figures in parenthesis represent metabolite masses. Inset: Table showing log2 ratio of gene
expression in infected compared to uninfected leaf tissue for key biosynthetic genes. In one
experiment whole leaves were harvested 24 h after inoculation, and in the second, leaf disks
were cut 0-6 mm and 6-12 mm from the edge of the lesion after 24 h. NS – no significant
change in expression. Coloured boxes represent the direction of change in expression of the
indicated genes on the pathway diagram.
Table S1. Devised scoring method of ‘susceptible’ and ‘resistant’ macroscopic symptom
development in Arabidopsis in response to B. cinerea. A weighted scoring method was devised
for the responses of Col-0, etr1-1, eto2 and ctr1-1 upon inoculation with B. cinerea up to 96
hours post inoculation (hpi), under standard conditions, where the susceptible symptoms were
conferred a range of negative scores and the resistant symptoms were given positive scores.
An arbitrary weighted score could be produced arithmetically from the lesion scores of
replicates (A) ‘susceptible response’ the negative arbitrary scores for each defined lesion type
where the more negative values equated to more progressive and diseased symptoms; (B)
‘resistant response’ the positive arbitrary scores for each defined response type, where the
more positive values were associated with penetration failure and reduced disease.
Table S2. Multivariate analysis of variance (MANOVA) of DI-MS of extracts from Botrytis cinerea
infected Arabidopsis leave using 8 PCs accounting for >99 % total explained variance, derived
from the DI-MS data. PCA was employed to reduce data dimensionality and to visualise trends
within the datasets, but it provided no measure of significance., MANOVA was applied to the
first 8 PCs accounting for >99 % total explained variance to permit the statistical evaluation of
main treatments and, more importantly, their interactions within the experimental design. The
power of MANOVA was demonstrated with a three-factor investigation. The first factor was
replicate experiment, which had two levels; the second factor was genotype, which consisted of
Col-0, etr1-1, eto2 and ctr1-l samples, taken 12 h after treatment with either B. cinerea or
controls sprayed with PDB, which constituted the third factor. For each replicate experiment,
six samples were collected for each genotype x treatment and analysed in the negative mode DI-MS over the m/z range 100-1400 Th. The variability of the individual plants represented the
residual or error line in the MANOVA and was used to assess the three main effects and their
interactions df1 was the numerator degrees of freedom, df2 was the denominator df, F was
the value of the F statistic and P was the probability.
Table S3. Selecting biosynthetic pathway showing discrimination and significantly modulated
m/z. The percentage of discriminatory and significantly (P< 0.05) modulated m/z (d*) for the
chosen biosynthetic pathways was used to target importance metabolite changes. In the
example pathways given; (white block) in all of the mutants showed less that <25 % of the
mutants contained discriminatory metabolites when compared to Col-0; (grey block) in at least
one mutant, the pathway >25 <50 % of the metabolites were discriminatory when compared
with Col-0; (black block) in a least one mutant, the biosynthetic pathway >50 % of the
metabolites were d* compared with Col-0 AND in addition to some opposite changes in
accumulation seen by the mutants and with Col-0.
Table S4. Summary of confirmed key metabolites within discriminatory pathways. The high
resolution Linear Triple Quadrupole (LTQ)-Orbitrap hybrid system was used to unambiguously
confirm key metabolites within discriminatory pathways by determination of the accurate
mass. The precursor ions represented by these m/z were also subjected to MS-MS and the
product ions matched those of standards.