Metabolite profiling reveals a role for intercellular dihydrocamalexic acid in the response of mature Arabidopsis thaliana to Pseudomonas syringae

Article abstract of DOI:10.1016/j.phytochem.2021.112747

The leaf intercellular space is a site of plant-microbe interactions where pathogenic bacteria such as Pseudomonas syringae grow. In Arabidopsis thaliana, the biosynthesis of tryptophan-derived indolic metabolites is induced by P. syringae infection. Using high-resolution mass spectrometry-based profiling and biosynthetic mutants, we investigated the role of indolic compounds and other small molecules in the response of mature Arabidopsis to P. syringae. We observed dihydrocamalexic acid (DHCA), the precursor to the defense-related compound camalexin, accumulating in intercellular washing fluids (IWFs) without further conversion to camalexin. The indolic biosynthesis mutant cyp71a12/cyp71a13 was more susceptible to P. syringae compared to mature wild-type plants displaying age-related resistance (ARR). DHCA and structural analogs inhibit P. syringae growth (MIC ~ 500 μg/mL), but not at concentrations found in IWFs, and DHCA did not inhibit biofilm formation in vitro. However, infiltration of exogenous DHCA enhanced resistance in mature cyp71a12/cyp71a13. These results provide evidence that DHCA derived from CYP71A12 and CYP71A13 activity accumulates in the intercellular space and contributes to the resistance of mature Arabidopsis to P. syringae without directly inhibiting bacterial growth.

Full text of DOI:10.1016/j.phytochem.2021.112747

Phytochemistry 187 (2021) 112747
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Metabolite profiling reveals a role for intercellular dihydrocamalexic acid
in the response of mature Arabidopsis thaliana to Pseudomonas syringae
,b,c,
Christine J. Kempthornea
*, Alexander J. Nielsenb, Daniel C. Wilsonb, James McNultyb,
Robin K. Cameronb, David K. Liscombea
,c,1
a Vineland Research and Innovation Centre, 4890 Victoria Ave North Box 4000, Vineland Station, Ontario, L0R 2E0, Canada
b McMaster University, 1280 Main St W, Hamilton, Ontario, L8S 4L8, Canada
c Brock University, 1812 Sir Isaac Brock Way, St Catharines, Ontario, L2S 3A1, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords:
The leaf intercellular space is a site of plant-microbe interactions where pathogenic bacteria such as Pseudomonas
syringae grow. In Arabidopsis thaliana, the biosynthesis of tryptophan-derived indolic metabolites is induced by
P. syringae infection. Using high-resolution mass spectrometry-based profiling and biosynthetic mutants, we
investigated the role of indolic compounds and other small molecules in the response of mature Arabidopsis to
P. syringae. We observed dihydrocamalexic acid (DHCA), the precursor to the defense-related compound
camalexin, accumulating in intercellular washing fluids (IWFs) without further conversion to camalexin. The
indolic biosynthesis mutant cyp71a12/cyp71a13 was more susceptible to P. syringae compared to mature wild-
type plants displaying age-related resistance (ARR). DHCA and structural analogs inhibit P. syringae growth
Arabidopsis thaliana (L.) heynh
Brassicaceae
Age-related resistance
Intercellular space
Metabolomics
Antimicrobial
Indolic
Cytochrome P450
Dihydrocamalexic acid
(MIC ~ 500 μg/mL), but not at concentrations found in IWFs, and DHCA did not inhibit biofilm formation in
vitro. However, infiltration of exogenous DHCA enhanced resistance in mature cyp71a12/cyp71a13. These results
provide evidence that DHCA derived from CYP71A12 and CYP71A13 activity accumulates in the intercellular
space and contributes to the resistance of mature Arabidopsis to P. syringae without directly inhibiting bacterial
growth.
1. Introduction
response to pathogen exposure (Darvill and Albersheim, 1984; Pedras
et al., 2011) and localization of specialised metabolites to the intercel-
The leaf intercellular space comprises the air-filled area between
plant cells (including the cell wall) and fluids that accumulate along the
plant cell wall (O’Leary et al., 2014). This dynamic environment is the
site of beneficial and pathogenic plant-bacteria interactions; within
hours of pathogen detection, conditions in the intercellular space change
in an attempt to prevent bacterial colonization (Delaunois et al., 2014;
Naseem et al., 2017; O’Leary et al., 2016). Intercellular components can
be studied by infiltrating the leaf intercellular space with water, sol-
vents, or buffers, followed by collecting the intercellular washing fluids
through centrifugation. Intercellular washing fluids (IWFs) contain
plant and bacterial metabolites and components from the plant cell wall
(O’Leary et al, 2014, 2016; Soylu et al., 2005). Specialised metabolites
with antimicrobial activity (also known as phytoalexins) accumulate in
lular space may act to limit the pathogenicity of bacteria, but the
identity of these compounds and their mode(s) of action are not well
understood (Agrawal et al., 2010; Forcat et al., 2010; O’Leary et al,
2014, 2016; Soylu et al., 2005; Wilson et al., 2017).
Age is a relevant factor in the outcome of plant-pathogen and plant-
herbivore interactions, as many plants become more resistant to path-
`
ogens and pests as they mature (Develey-Riviere and Galiana, 2007;
Whalen, 2005). In the model species Arabidopsis thaliana, resistance to
Pseudomonas syringae, Hyaloperonospora arabidopsis (downy mildew),
and the herbivore Trichoplusia ni (cabbage looper larvae) increases with
age (Carviel et al., 2014; Kus et al., 2002; Rusterucci et al., 2005; Tucker
and Avila-Sakar, 2010). In several cases, age-related resistance (ARR)
has been associated with changes in specialised metabolite
* Corresponding author. Vineland Research and Innovation Centre, 4890 Victoria Ave North Box 4000, Vineland Station, Ontario, L0R 2E0, Canada.
E-mail addresses: christine.kempthorne@vinelandresearch.com (C.J. Kempthorne), david.liscombe@vinelandresearch.com (D.K. Liscombe).
1
This work was completed at the Vineland Research and Innovation Centre, 4890 Victoria Ave North Box 4000, Vineland Station, Ontario, L0R 2E0, Canada; the
Centre for Microbial Chemical Biology, 1200 Main St W MDCL-2330, Hamilton, Ontario, L8S 4K1, Canada; and the Departments of Biology and Chemistry and
Chemical Biology, McMaster University, 1280 Main St W, Hamilton, Ontario, L8S 4L8, Canada.
https://doi.org/10.1016/j.phytochem.2021.112747
Received 8 December 2020; Received in revised form 14 March 2021; Accepted 18 March 2021
Available online 3 April 2021
0031-9422/© 2021 Elsevier Ltd. All rights reserved.

C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
accumulation. For example, Mansfeld et al. (2017) used non-targeted
metabolomics to show that terpenoids and flavonoids in mature fruit
peels are a major contributor to cucumber fruit resistance against the
oomycete Phytophthora capsici. Similarly, Datura species produce tro-
pane alkaloids as they age, which are thought to contribute to devel-
polysaccharides, extracellular DNA, and proteins that provide a pro-
tective environment against antimicrobials (Costerton et al., 1999;
Donlan and Costerton, 2002). The formation of biofilm involves bacteria
sensing and responding to environmental cues, and bacteria in biofilms
have different metabolic and gene expression profiles than
free-swimming planktonic bacteria, which can contribute to antibiotic
˜
opmental resistance against herbivores (Karinho-Betancourt et al.,
´
2015). In A. thaliana, intercellular washing fluids from mature
ARR-responding plants exhibit antimicrobial activity against P. syringae
pathovar tomato (Pst) in vitro, and intercellular accumulation of salicylic
acid (SA) plays a role in inhibition of growth and biofilm formation of Pst
(Cameron and Zaton, 2004; Kus et al., 2002; Wilson et al., 2017). Bio-
films are composed of bacterial cell aggregates embedded in a matrix of
resistance or susceptibility (de Kievit et al., 1999; Masak et al., 2014).
Inhibition of virulence factors involved in biofilm formation but not
growth may be a successful strategy for plants since it reduces selection
for antibiotic-resistant populations (Lee et al., 2012; Lewis and Ausubel,
2006). Biofilm-like aggregation has been reported for P. syringae pv.
Actinidiae and pv. Phaseolicola in the intercellular space of kiwifruit and
Fig. 1. Biosynthesis pathway of tryptophan-derived specialised metabolism in Arabidopsis thaliana (simplified). Dashed arrows indicate potential non-
enzymatic reactions. Multiple arrows indicate multiple reaction steps simplified for presentation. IAOx: indole-3-acetaldoxime, I3M: indole-3-
methylglucosinolate, IAN: indole-3-acetonitrile, ICHO: indole-3-carbaldehyde, ICOOH: indole-3-carboxylic acid, ICN: indole-3-carbonyl nitrile, 4-OH-ICN: 4-
hydroxyindole-3-carbonyl nitrile, NSP: nitrile-specifier protein, FOX1: flavin-dependent oxidoreductase, AAO1: Arabidopsis aldehyde oxidase I, GGP: gamma-
glutamyl peptidase, GGT: gamma-glutamyl transpeptidase DHCA: dihydrocamalexic acid. Modified from Rajniak et al. (2015); Müller et al. (2019).
2

C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
beans respectively (Ghods et al., 2015; Manoharan et al., 2015) and for
Pst in the intercellular space of A. thaliana leaves using GFP-expressing
bacteria (Wilson et al., 2017). In Arabidopsis, accumulation of trypto-
phan (trp)-derived indolic metabolites has been observed in response to
P. syringae (Forcat et al., 2010; Glazebrook and Ausubel, 1994; Rajniak
et al., 2015; Zhou et al., 1999).
Pastorczyk et al., 2020; Zhou et al., 1999). To examine the role of
CYP71A13 in the response of mature A. thaliana to Pst we initially
screened the cyp71a13 TDNA insertion mutant for ARR defects. The
cyp71a13 mutant had an inconsistent ARR response; it supported
wild-type levels of bacteria at 3 weeks old (Table S1.1), but at 7 weeks
old had a variable response to Pst, supporting wild-type levels of bacteria
in six of seven experiments (Table S1.2). The amino acid sequence of
CYP71A12 is 89% identical to CYP71A13 and can partially compensate
for the loss of CYP71A13 as demonstrated using in vitro and heterologous
pathway reconstitution and metabolite profiling of biosynthetic mutants
Biosynthesis of indolic metabolites in Arabidopsis leaves begins with
CYP79B2 and CYP79B3 producing indole-3-acetaldoxime (IAOx), fol-
lowed by CYP71A12-and CYP71A13-mediated conversion of IAOx to
two reactive indole-3-acetonitrile (IAN) intermediates, alpha-hydroxy
IAN and dehydro-IAN (Nafisi et al., 2007; Zhao, 2002, Fig. 1).
CYP71A12 and CYP71A13 form a metabolic branchpoint in the
biosynthesis of three main classes of defense-related indole molecules:
indole-3-carboxylic acid (ICOOH) derivatives, indole carbonylnitrile
¨
(Bottcher et al., 2009; Klein et al., 2013; Morten E. Møldrup et al.,
2013b; Müller et al., 2015; Nafisi et al., 2007; Pastorczyk et al., 2020),
which may explain the variable response to Pst of mature cyp71a13. The
partial redundancy of CYP71A12 and CYP71A13 and the tandem loca-
tion of these genes made it difficult to study the role of this pathway in
resistance until Müller et al. (2015) developed a cyp71a12/cyp71a13
double mutant through gene editing. The mature cyp71a12/cyp71a13
double mutant is consistently more susceptible to Pst, supporting 2- to
8-fold higher bacterial levels compared to mature Col-0 (Fig. 2,
Table S1.2).
¨
(ICN) derivatives, and camalexin (Bottcher et al., 2009; Klein et al.,
2013; Møldrup et al., 2013a; Müller et al., 2019, 2015; Nafisi et al.,
2007; Rajniak et al., 2015, Fig. 1). In the case of camalexin biosynthesis,
alpha-hydroxy IAN is converted to dehydro-IAN mainly through
CYP71A13 activity with minor contribution from CYP71A12 activity
(Klein et al., 2013; Rajniak et al., 2015). Dehydro-IAN is conjugated to
glutathione (GSH(IAN)) and catabolized to a cysteine conjugated form
(Cys (IAN)) through γ-glutamyl peptidase and/or γ-glutamyl trans-
peptidase activity (Geu-Flores et al., 2011; Møldrup et al., 2013b; Su
et al., 2011). Cys (IAN) cyclizes non-enzymatically to form dihy-
drocamalexic acid (DHCA) and is also the substrate for the multifunc-
tional enzyme CYP71B15, which catalyzes the formation of both DHCA
CYP71B15 acts downstream of CYP71A12 and CYP71A13 in the
biosynthesis of camalexin and the cyp71b15 mutant displayed a wild-
type ARR response to Pst inoculation by pressure infiltration into the
intercellular space regardless of age (Fig. 2, Table S1.1, S1.2). Previous
studies using pressure infiltration for inoculation have also reported that
CYP71B15 is not required for resistance to P. syringae (Glazebrook and
Ausubel, 1994). Rajniak et al. (2015) showed that cyp71b15 (4–5 weeks
post-germination) was more susceptible to Pst than Col-0 when using a
spray-inoculation technique meant to represent the natural infection
process more accurately, whereby bacteria on the leaf surface can only
enter the intercellular space via stomatal openings. Since we were
searching for potential intercellular antimicrobial compounds, we
pressure infiltrated bacteria directly into the intercellular space of leaves
to bypass the potential confounding effects of disruption of stomatal
signaling mechanisms that have been shown to occur in some mutants
for biosynthetic genes (Baccelli et al., 2014; Melotto et al., 2006). The
¨
and camalexin through two sequential reaction steps (Bottcher et al.,
2009; Schuhegger et al., 2006; Zhou et al., 1999, Fig. 1).
Alpha-hydroxy-IAN is converted to ICN via FOX1 activity or to
indole-3-carbaldehyde and indole-3-carboxylic acid via CYP71B6 and
¨
AAO1 activity (Bottcher et al., 2009; Rajniak et al., 2015). CYP71A12
activity, but not CYP71A13, is responsible for ICN biosynthesis, and
ICOOH biosynthesis in response to pathogens (Rajniak et al., 2015;
Pastorczyk et al., 2020). The expression of CYP71A13 was previously
reported as upregulated in mature wild type (Col-0) A. thaliana in
response to Pst at 12 h post-inoculation (Carviel et al., 2014). The flux of
CYP71A12 and CYP71A13 intermediates is affected by the type of
stressor plants are exposed to for induction of the pathway;
CYP71A12-mediated ICOOH biosynthesis in response to abiotic
stressors like UV exposure can be partially compensated for by
CYP71A13 (Müller et al., 2015), but in response to fungal pathogens
such as Plectosphaerella cucumerina CYP71A12 is required for accumu-
lation of ICOOH derivatives independent of CYP71A13 activity (Pas-
torczyk et al., 2020).
In this work, we sought to investigate the potential role of intercel-
lular indolic compounds involved in mature A. thaliana at 7 weeks post-
germination (vegetative phase). We screened the resistance phenotypes
of cyp71a13 and cyp71a12/cyp71a13 mutants to Pst and used a non-
targeted, high-resolution mass spectrometry-based approach to
analyze IWFs collected from resistant and susceptible genotypes to
identify indolic derivatives and other small molecules that become
localized to the intercellular space of leaves in response to Pst. The
identification of DHCA without further conversion to camalexin in IWFs
from mature Pst-infiltrated leaves led us to examine this compound’s
role in A. thaliana’s bacterial defense through growth inhibition, biofilm
inhibition, and in planta resistance assays.
2. Results and discussion
Fig. 2. The cyp71a12/cyp71a13 mutant supports higher levels of Pseu-
domonas syringae at 7 weeks post-germination compared to Col-0 and
cyp71b15. P. syringae pv. Tomato levels were quantified 3 days after inocula-
tion by pressure infiltration of fully expanded rosette leaves of (A) 3- or (B) 7-
week-old plants. Values represent the mean ± standard deviation of three
sample replicates (n = 3) consisting of 8 plants each. Different letters indicate
statistically significant differences (one-way ANOVA, Tukey’s honestly signifi-
cant difference [HSD], P < 0.05).
2.1. Susceptibility of indolic biosynthesis mutants to P. syringae
CYP71A12, CYP71A13, and CYP71B15 transcripts are upregulated in
response to a broad range of biotic and abiotic stressors, including heavy
metal salts, reactive oxygen species, UV irradiation, mechanical
wounding, fungal interaction, and P. syringae (Frerigmann et al., 2015;
Glawischnig et al., 2004; Müller et al., 2019; Nafisi et al., 2006;
3

C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
cyp71a12/cyp71a13 and cyp71b15 mutants both produce trace amounts
abiotic stressors (UV exposure and silver nitrate). ICOOH derivatives are
also constitutively produced but accumulate at elevated levels after
pathogen challenge in Arabidopsis (Bednarek et al., 2005; Hagemeier
et al., 2001; Iven et al., 2012; Pastorczyk et al., 2020). ICOOH is ester-
ified to the cell wall in response to P. syringae and has been hypothesized
to prevent localized bacterial aggregation or act as an antimicrobial
compound by intercalating into the bacterial membrane (Forcat et al.,
¨
of camalexin (Bottcher et al., 2009; Müller et al., 2015; Zhou et al.,
1999) yet differed in their resistance phenotypes, which indicated
camalexin is not contributing to Pst resistance in mature A. thaliana as an
antimicrobial and suggested a possible role for other trp-derived me-
tabolites produced as a result of CYP71A13 and/or CYP71A12 activity.
2.1.1. Non-targeted metabolite profiling of intercellular washing fluids in
response to P. syringae
2010;
Soylu
et
al.,
2005).
Indole-3-carboxylic
acid
beta-D-glucopyranosyl ester accumulated at 400–600 pmol in leaves of
6-week-old Arabidopsis in response to Pst at 48 h post-inoculation and
was hypothesized to function as a transport intermediate for ICOOH cell
wall conjugation (Hagemeier et al., 2001). Recent work by Pastorczyk
et al. (2020) confirmed that CYP71A12, but not CYP71A13, is required
for accumulation of two glycosylated ICOOH products (6-O-glucoside of
6-hydroxy-indole-3-carboxylic acid and indole-3-carboxylic acid
glucose ester) in the leaves of 4-week-old plants in response to the fungal
pathogen Plectosphaerella cucumerina.
As mentioned, CYP71A12 and CYP71A13 form a metabolic branch
point in the production of precursors that serve as a substrate for en-
zymes in trp-derived specialised metabolism, so it was unknown which,
if any, downstream compounds were present in the intercellular space in
response to Pst. Therefore, we used non-targeted, high-resolution mass
spectrometry-based metabolomics to examine the accumulation of all
detectable semi-polar metabolites in mature plants’ intercellular space
after Pst inoculation in Col-0, cyp71b15 (wild-type resistance despite
perturbation in trp-derived metabolite pathway downstream of
CYP71A12 and CYP71A13), and cyp71a12/cyp71a13 (increased sus-
ceptibility to Pst when mature compared to Col-0). We focused on
mature plants because of the increased susceptibility observed in mature
cyp71a12/cyp71a13 in ARR assays and because 7-week-old rosette
leaves were well suited for the infiltration-centrifugation technique
(O’Leary et al., 2014) used to collect required volumes of intercellular
washing fluids (IWFs) for metabolite profiling from mock- and Pst-ino-
culated plants. Minimal cytoplasmic contamination in IWFs was
confirmed by assessing chlorophyll levels spectrophotometrically (Baker
et al., 2012).
It is also possible that the peak corresponding to an ICOOH gluco-
pyranosyl ester is a degradation product of compounds derived from
indole carbonylnitriles (ICNs). ICN compounds are unstable and rapidly
degrade to ICOOH in aqueous solution (Rajniak et al., 2015). In the
context of our work, ICN degradation to ICOOH could occur between the
time bacteria (in aqueous solution) are first pressure infiltrated into the
leaf intercellular space and when IWFs are collected by centrifugation
24 h later, although given the role of CYP71A12 in ICN biosynthesis
(Fig. 1), we would expect a mass feature corresponding to glycosylated
ICOOH to be absent from cyp71a12/cyp71a13 if it was an ICN degra-
dation product.
To identify mass spectral features that may be important for Pst
resistance in mature plants, we first performed a pairwise comparison
via volcano plot between mock- and Pst-inoculated wild type IWFs
(Fig. S1). Principal component analysis (PCA) of all features showed that
the first two components account for 93.7% of the total variance in IWFs
run in positive ESI mode (Fig. S2A) and 90.4% of the total variance in
negative ESI mode (Fig. S2B). Mock- and Pst-inoculated samples formed
distinct clusters in both ESI modes, although the clusters are more
separated in negative ESI mode. In total, 26 features were found to be
significantly different between IWFs from wild type mock- and Pst-
inoculated leaves (Fig. S1; Table S2.1, 2.2, 2.3). Next, we compared the
accumulation of these 26 features between susceptible and resistant
genotypes by including IWFs collected from cyp71b15 (wild-type Pst
resistance) and cyp71a12/cyp71a13 (increased susceptibility to Pst)
using one-way ANOVA (Fig. S3). We pursued mass features that could be
functional in resistance to Pst, rather than features solely reflecting
perturbation of trp-derived specialised metabolism in cyp71a12/
cyp71a13. These compounds should have different accumulation pat-
terns in the resistant genotypes (Col-0 and cyp71b15) compared to sus-
ceptible ones (cyp71a12/cyp71a13). Two features were significantly
different between resistant (Col-0, cyp71b15) and susceptible
(cyp71a12/cyp71a13) genotypes (Fig. S3-E, Q): one at 6.96 min with a
nominal mass of m/z 322 corresponding to a glycosylated indole-3-
carboxylic acid (ICOOH), and the other at 7.32 min with a nominal
mass of m/z 247 corresponding to the camalexin precursor, dihydrox-
ycamalexic acid (DHCA).
Probing the contribution of ICOOH pathways in response to bacteria
in the intercellular space requires consideration of differences in
constitutive and induced ICOOH pathways, the complexity of down-
stream derivatives (e.g., glycosylated and amino acid conjugates, indole-
ring hydroxylations), and the fact that not all the genes involved in these
¨
pathways have been fully elucidated (Bottcher et al., 2014; Müller et al.,
2019; Pastorczyk et al., 2020; Tan et al., 2004). Further work examining
the resistance phenotype of cyp71a12 mutants in combination with
other genes with minor contribution to pathogen induced ICOOH
biosynthesis (i.e., cyp71b6, aao1) to Pst may reveal more about the role
of ICOOH compounds in disease resistance (Müller et al., 2019; Pas-
torczyk et al., 2020). Given the previously mentioned studies supporting
a role for ICOOH derivatives in pathogen resistance, we chose to pursue
investigation of the mass feature at 7.32 min that resembled DHCA.
2.1.2. Dihydrocamalexic acid accumulates in the intercellular space in
response to P.syringae
The mass spectral feature that eluted at 7.32 min with a nominal
mass of m/z 247 was of interest since it was only detected in IWFs
collected from mature resistant genotypes inoculated with Pst and was
not detected in mock-inoculated wild type plants and cyp71a12/
cyp71a13 regardless of treatment. This suggested that this compound
could be involved in defense against Pst and that it is not likely to be of
bacterial origin (Fig. S3-E). Based on the retention time and fragmen-
tation pattern of two independently synthesized standards, this feature
was confirmed to be the camalexin precursor, (S)-dihydrocamalexic acid
(DHCA; m/z 247.0541 [C12H10N2O2S + H]), with fragmentation pro-
ducing peaks at m/z 201.0520 ([C11H8N2S + H], loss of carboxylic acid),
and m/z 143.0604 ([C9H7N2+H], thiazole ring-opening or fragmenta-
The feature that eluted at 6.96 min with a nominal mass of m/z 322
was putatively identified as an indole-3-carboxylic acid beta-D-
glucopyranosyl ester with a mass error of ꢀ 1.9 ppm (m/z 322.0921
[C15H17NO7–H]). Fragmentation produced peaks at m/z 160.0392 cor-
responding to an indole-3-carboxylic acid aglycone ([C9H7NO2–H]) and
m/z 116.0503 corresponding to an indole ring ([C8H7N–H]) (Fig. S4),
which is consistent with fragmentation behavior for this compound
¨
tion) (Bottcher et al., 2009; Zandalinas et al., 2012, Fig. 3). At 24 hpi
with Pst, DHCA accumulated to ~0.3 μg/mL and ~0.24 μg/mL in IWFs
collected from mature wild-type and cyp71b15 plants, respectively
(Fig. 4). Mock-inoculated wild-type plants did not produce detectable
levels of intercellular DHCA, and mock-inoculated cyp71b15 accumu-
¨
noted by Bottcher et al. (2014, 2009). This feature was elevated in IWFs
from resistant plants (Col-0 and cyp71b15) compared to IWFs from
susceptible cyp71a12/cyp71a13 plants (Fig. S3-Q). Müller et al. (2015)
showed that 6-week-old cyp71a12/cyp71a13 produces reduced levels of
glycosylated ICOOH compared to wild-type Arabidopsis in response to
lated DHCA (~0.09 μg/mL), likely due to a block in camalexin biosyn-
thesis whereby a cysteine-conjugated indole-3-acetonitrile (Cys (IAN))
precursor accumulates, and non-enzymatically cyclizes to produce
¨
DHCA (Bottcher et al., 2009). DHCA is not readily degraded to other
4

C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
Fig. 3. Identification of dihydrocamalexic acid (DHCA) in intercellular washing fluids. Extracted ion chromatograms and mass spectra for DHCA (m/z
247.0541, [C12H10N2O2S + H] in intercellular washing fluids from Pseudomonas syringae-inoculated leaves compared to a synthetic standard. (A) Extracted ion
chromatograms and (B) MSMS (25 eV). n. d. Indicates compound not detected. Samples were run in positive electrospray ionization mode.
¨
products (Bottcher et al., 2009), so in the absence of CYP71B15 in a
localized area like the intercellular space, it could accumulate without
being converted to camalexin. Bednarek et al. (2005) observed both
DHCA and camalexin as root exudates from 4-week-old Arabidopsis
thaliana grown in liquid culture after infection with the oomycete
Pythium sylvacticum. Camalexin was not detected in the intercellular
space, regardless of treatment or genotype (Table S3).
to produce these intermediates (Geu-Flores et al., 2011; Klein et al.,
2013; Møldrup et al., 2013a, 2013b). In cytosolic camalexin biosyn-
thesis, γ-glutamyl peptidase 1 (GGP1) and 3 (GGP3) are the enzymes
involved in the catabolism of GSH(IAN) to Cys (IAN), and double mu-
tants for these genes have reduced camalexin production in response to
chemical inducers (Geu-Flores et al., 2011). The γ-glutamyl trans-
peptidases GGT1 and GGT2 are not likely to be involved in cytosolic
camalexin production as GGT1 is not upregulated in response to
chemical inducers and GGT2 is not expressed in leaves (Destro et al.,
2011; Martin et al., 2007). However, given that GGT1 and GGT2 are
localized to the apoplast, they could be involved in the production of
DHCA in the intercellular space in response to Pst (Martin et al., 2007;
Ohkama-Ohtsu et al., 2007). In that case, GGT1 and GGT2 involvement
in DHCA biosynthesis would require transport of a glutathione-IAN
conjugate such as γ-GSH-, Gly-Cys-, or Cys-Glu-(IAN) to the intercel-
lular space.
DHCA derivatives (DHCA methyl ester [C13H12N2O2S], glutathione-
conjugated DHCA [C17H18N4O4S], and glycosylated DHCA
[C17H17N3O5S]) that accumulate in response to silver nitrate treatment
¨
(Bottcher et al., 2009) were not detected in IWFs from mock- or Pst-i-
noculated leaves, but this does not rule out the possibility that DHCA
metabolites are transported to the intercellular space where the activity
of an as-yet-unidentified apoplastic process could then form DHCA.
DHCA may be directly transported to the intercellular space, but the
release of DHCA from CYP71B15 would have to occur during the
sequential conversion of Cys (IAN) to DHCA and then camalexin
(Schuhegger et al., 2006). Recent research by Mucha and colleagues
(Mucha et al., 2019) suggest that metabolons consisting of CYP71A12,
CYP71A13, and CYP71B15 provide efficient channeling of bioactive
indolic intermediates to form camalexin; it is therefore unlikely that
appreciable levels of DHCA and other intermediates are released from
these enzyme complexes during camalexin biosynthesis.
Mucha et al. (2019) revealed a possible mechanism for how bioactive
intermediates can be released during camalexin biosynthesis. They
showed that glutathione-S-transferase (GST) U4 physically interacts
with CYP71A13 and negatively regulates camalexin biosynthesis in
response to pathogens. The regulatory mechanism is unknown, but the
authors discussed the possibility that GSTs interact with an
ER-associated camalexin biosynthetic metabolon to transport in-
termediates to other subcellular compartments. In the context of our
work, intercellular DHCA may be accumulating as a result of transport of
IAN conjugates from the camalexin biosynthetic metabolon, followed by
cytosolic and/or intercellular GGP/GGT activity and non-enzymatic
cyclization resulting in accumulation of DHCA without further conver-
sion to camalexin by CYP71B15. This model for DHCA accumulation in
Glutathione-conjugated IAN (GSH(IAN)), Cys (IAN), or in-
termediates formed during catabolism of GSH(IAN) to Cys (IAN) may be
transported to the intercellular space where they could be converted
enzymatically or non-enzymatically to DHCA. This part of the camalexin
pathway is difficult to elucidate due to functional redundancy in enzyme
families and in vitro substrate promiscuity of the enzymes hypothesized
5

C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
Zaton, 2004; Wilson et al., 2017; Fig. S7). Since current intercellular
profiling techniques require full hydration of the leaf for downstream
profiling, IWF measurements represent the amount of compound
washed out of the intercellular space and do not account for the possi-
bility of localized concentration gradients (Gentzel et al., 2019; Wilson
et al., 2017). For example, plant metabolites may accumulate at sites of
bacterial aggregation or in the cell wall at high concentrations.
2.1.4. Dihydrocamalexic acid does not inhibit P. syringae biofilm formation
Indole-3-acetonitrile (~100
μg/mL) and indole-3-acetaldehyde
(~100 μg/mL) have been shown to inhibit biofilm formation of the
bacterial pathogens E. coli O157:H7 and Pseudomonas aeruginosa,
potentially by interfering with intercellular signaling and affecting the
expression of genes required for biofilm formation without affecting
bacterial growth (Lee et al., 2012). To test the activity of DHCA against
Pst biofilm formation, a spectrophotometric 96-well plate biofilm assay
was used (see Materials and Methods; O’Toole, 2011). To ensure that
these tests would reflect reduced biofilm formation rather than inhibi-
tion of growth, DHCA concentrations were chosen to avoid the reduction
of bacterial growth (0.3–18
it has bioactivity against P. syringae biofilms, inhibiting their formation
in vitro at concentrations as low as 2 M (~270 ng/mL). As previously
observed (Wilson et al., 2017), biofilm formation was reduced in the
μg/mL). SA was used as a positive control as
μ
presence of 18 μg/mL SA at 48 and 72 h (Fig. 6A). However, DHCA did
Fig. 4. Quantification of dihydrocamalexic acid (DHCA) (m/z 247.0541,
[C12H10N2O2S þ H]) in intercellular washing fluids (IWFs). DHCA levels
measured in IWFs from Col-0, cyp71a12/cyp71a13, and cyp71b15 (all 7-weeks
post-germination) 24 h after inoculation with P. syringae (Pst) or 10 mM MgCl2
(mock-inoculation) measured by UPLC-MS electrospray ionization in positive
mode (ESI+). Values represent the mean ± standard deviation of three sample
replicates (n = 3). Different letters indicate statistically significant differences
(one-way ANOVA, Tukey’s honestly significant difference [HSD], P < 0.05).
not significantly affect biofilm formation at any concentrations or time
points tested (Fig. 6B). These results show that DHCA does not inhibit
biofilm formation of Pst in vitro.
2.1.5. Structure-activity relationship of DHCA chirality and indole moiety
To examine how structural components of DHCA may affect its
antimicrobial activity against planktonic (free individual cells not part
of an aggregate) Pst, structure-activity studies were completed using
camalexin and synthetic enantiomers of phenylalanine and tyrosine-
derived analogs of DHCA (Fig. 5). Camalexin differs structurally in
that it lacks chirality provided by the carboxylic acid functional group
on carbon 4 of the thiazole ring of DHCA. The antimicrobial activity of
camalexin was similar to that reported for DHCA; camalexin has a MIC
Standard curves were prepared using synthetic DHCA (2 pg–6 μg on column).
the intercellular space (Fig. S5) is speculative at this time and mass
features corresponding to IAN conjugates were not detected in IWFs at
24 hpi with Pst regardless of genotype or treatment (Table S3). Further
work analyzing IWFs and resistance phenotypes of double, triple, or
quadruple GGP and GGT mutants, GST4U knockouts and
over-expressors, as well as screening for candidate genes encoding
glutathione-conjugate ABC transporters (DeRidder and Goldsbrough,
2006; Lu et al., 1997; Tommasini et al., 1998), may reveal the genes and
mechanisms involved in DHCA accumulation in the intercellular space
in response to bacterial induction of the camalexin pathway.
of ~500
μg/mL when incubated with P. syringae pv. Maculicola in
camalexin-spiked IWFs (Rogers et al., 1996), and a MIC of ~250 μg/mL
when incubated with Pst in HIM medium (Fig. 5B).
To examine how the indole ring contributes to growth inhibition
against Pst, analogs of DHCA were synthesized with the indole ring
replaced with a phenyl (Compounds 1 and 2 in Fig. 5) or a phenol
(Compounds 3 and 4 in Fig. 5) group. Stereochemistry had a marked
effect on antimicrobial activity for the phenyl ring analogs as analog 1
lacked growth inhibitory activity against Pst at any of the concentrations
tested (Fig. 5C). Analog 2 was more effective at inhibiting the growth of
2.1.3. Bioactivity of dihydrocamalexic acid on P. syringae growth
To examine the ability of DHCA to inhibit the growth of Pst, bacteria
were incubated with concentrations of 0.05–1000 μg/mL DHCA and
Pst compared to DHCA, with a similar MIC as camalexin (~250 μg/mL;
bacterial growth was measured spectrophotometrically (OD600). Hrp-
inducing minimal (HIM) medium was used for all in vitro assays in this
work to mimic the conditions of the intercellular space and induce
bacterial virulence gene expression in the bacteria (Huynh et al., 1989;
Kim et al., 2009; Rahme et al., 1992). To test for Pst’s ability to use
DHCA as a nutrient source, assays with DHCA as the sole source of
carbon or nitrogen and sulfur were performed using concentrations that
Fig. 5D); however, it was less bactericidal than DHCA and camalexin, as
1 mg/mL of analog 2 was not lethal to Pst (Fig. S8). Replacement of the
indolic ring with a phenol moiety causes loss of antimicrobial activity
regardless of stereochemistry as Pst growth was unaffected by incuba-
tion with analogs 3 and 4 (Fig. 5E and F).
DHCA may have a similar mode of action as camalexin for Pst growth
inhibition but is not as effective due to the carboxylic acid moiety.
Camalexin rapidly disrupts membrane integrity in P. syringae, and due to
its lipophilic nature, it does not dissociate from dying cells such that any
surviving bacterial cells continue to grow (Rogers et al., 1996). The
hydroxyl substitution on the ring of compounds 3 and 4 may render the
molecule less lipophilic, disrupting the association of camalexin and
possibly DHCA with bacterial membranes, whereas the more lipophilic
phenyl ring of compounds 1 and 2 still allows for membrane association.
However, compound 1 did not inhibit Pst growth, while compound 2
did, indicating that chirality may affect the antibacterial activity of
DHCA. Indeed, chirality can result in vastly different outcomes on the
target organism, including adverse toxic effects or a loss of bioactivity
showed little to no bacterial growth inhibition (1–32 μg/mL). Pst sur-
vived, but no growth was observed, indicating that Pst did not use DHCA
as a nutrient source (Fig. S6).
DHCA displayed inhibitory activity against Pst at concentrations
above 96 μg/mL but did not inhibit growth at the range of concentra-
tions observed in IWFs (0.3
μ
g/mL to 2.4
μ
g/mL). DHCA had a minimum
inhibitory concentration (MIC) of ~500
μ
g/mL and a minimum bacte-
ricidal concentration (MBC) of ~1 mg/mL (Fig. 5A). In comparison, the
MIC and MBC of salicylic acid, an intercellular compound with antimi-
crobial activity that contributes Pst observed in mature plants, is
approximately 250 μg/mL and 500 μg/mL, respectively (Cameron and
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C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
Fig. 5. Growth of Pseudomonas syringae (Pst) in the presence of dihydrocamalexic acid (DHCA), camalexin, or DHCA analogs in vitro. Dose-dependent effect
of (S)-dihydrocamalexic acid (A), camalexin (B), (1) (R)-2-(phenyl)-4,5-dihydrothiazole-4-carboxylic acid (C), (2) (S)-2-(phenyl)-4,5-dihydrothiazole-4-carboxylic
acid (D), (3) (R)-2-(4-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid (E), and (4) (S)-2-(4-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid (F) on the
growth of Pst in Hrp-inducing minimal medium as measured by turbidity (OD600) after incubation for 68 h at room temperature (approximately 25 ◦C). Each data
point is the mean ± SD of three wells per concentration from a 96-well non-tissue-culture-treated plate.
Fig. 6. Effect of dihydrocamalexic acid (DHCA) and salicylic acid (SA) on biofilm formation of Pseudomonas syringae (Pst) in vitro. Dose-dependent effect of
(A) SA and (B) DHCA on Pst biofilm formation in Hrp-inducing minimal medium as measured by crystal violet staining of surface-adherent cells and de-staining with
acetic acid (OD570) after stationary incubation for 24, 32, 48, or 60 h. Each data point is the mean ± SD of five wells per concentration from a 96-well non-tissue-
culture-treated plate. Different letters indicate statistically significant differences (one-way ANOVA, Tukey’s honestly significant difference [HSD], P < 0.05). Ns
indicates not significant. Bars (from left to right) within each timepoint are: 18
μg/mL, 4.5 μg/mL, 1.2 μg/mL, 0.3 μg/mL, and 0 μg/mL. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)
(Hutt and O’Grady, 1996; Smith, 2009). Comparing the antimicrobial
activity of (S)-DHCA and (R)-DHCA could clarify whether chirality is a
contributing factor to bacterial growth inhibition. To our knowledge,
this is the first study examining antimicrobial activity of DHCA. Addi-
tional investigation is required to understand the bio-transformations
and potential target interactions between DHCA and bacteria.
2.2. Dihydrocamalexic acid treatment restores bacterial resistance in
cyp71a12/cyp71a13
The absence of DHCA in IWFs from cyp71a12/cyp71a13 does not
necessarily indicate a role for DHCA in resistance to Pst. Rather, our
results may be due to the blockage of DHCA production in cyp71a12/
cyp71a13. In vitro assays demonstrated that DHCA inhibited Pst growth
at concentrations higher than those quantified in IWF samples, and
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C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
DHCA did not inhibit biofilm formation of Pst. However, in vitro anti-
microbial studies do not capture the complex metabolic and physio-
logical changes occurring in plants responding to pathogens (Bednarek
and Osbourn, 2009). If DHCA is important for the response of mature
plants to Pst, then reintroducing the compound should restore resistance
intercellular washing fluids. Both compounds accumulated in response
to Pst and to different extents in mature resistant and susceptible ge-
notypes. We confirmed that differential accumulation had a function in
plant defense since cyp71a12/cyp71a13 was more susceptible to Pst and
defective for intercellular DHCA accumulation. Also, exogenous infil-
tration of DHCA into the intercellular space at 24 hpi with Pst increased
the resistance response of mature cyp71a12/cyp71a13 b y reducing
bacterial levels in leaves. Since DHCA does not exhibit antibiofilm ac-
tivity in vitro and is weakly antimicrobial against Pst growth in vitro at
concentrations quantified in IWFs, our results suggest that the mecha-
nism of action for DHCA bioactivity against Pst is not likely through
direct antimicrobial activity. Our results also suggest that DHCA, IAN-
conjugates, or other precursors are transported to the intercellular
space in response to bacterial invasion in A. thaliana. These results serve
as a reminder that the localization of compounds is an important
consideration in plant defense investigations. Overall, this work in-
dicates a role for CYP71A13 and CYP71A12 in the mature A. thaliana
response to P. syringae through the accumulation of intercellular indolic
compounds.
to cyp71a12/a13 plants. To test this, exogenous DHCA (0.07
0.25 g/mL) was pressure-infiltrated into the intercellular space of
rosette leaves 24 h post-inoculation with Pst.
Infiltration of DHCA at a concentration similar to that quantified in
μg/mL or
μ
wild type IWFs (0.25 μg/mL; 1.02 μM) 24 h after mock or Pst inoculation
restored wild-type levels of resistance in mature cyp71a12/cyp71a13,
causing a 10-fold reduction in bacterial levels compared to mock-
inoculated plants (Fig. 7). Infiltration of a lower level of DHCA (0.07
μ
g/mL; 0.28 μM) resulted in a modest increased resistance in cyp71a12/
cyp71a13, causing a 1.6-fold reduction in bacterial levels compared to
mock-inoculated plants (Fig. 7). Wild-type resistance to Pst in mature
plants was not significantly affected by infiltration of either concentra-
tion of DHCA and did not exhibit an enhanced resistance response.
Combined with our antimicrobial studies, these results suggest that
DHCA affects Pst pathogenicity via mechanisms other than direct anti-
microbial activity (e.g., by affecting enzyme activity, receptor binding,
affecting plant defense or bacterial virulence signaling) or requires other
compounds (e.g., ICOOH-beta-D-glucopyranosyl ester) for additive/
synergistic interactions to be fully effective – mechanisms plants use to
avoid the development of bacterial resistance (Bednarek and Osbourn,
2009; Khameneh et al., 2019; Lewis and Ausubel, 2006; Silva et al.,
2016). In vitro studies have demonstrated that camalexin inhibits
glycolysis, TCA pathway gene expression and reduces mycelial growth
in A. brassicicola (Pedras et al., 2014). Camalexin has also been shown to
inhibit fungal detoxification enzymes involved in the metabolism of the
plant defense compounds brassinin and cyclobrassinin in isolates from
canola and brown mustard (Pedras and Abdoli, 2017). Likewise, DHCA
may interfere with Pst virulence genes or proteins through binding in-
teractions or affecting changes in plant or bacterial gene expression. If
this is the case, it would explain why the concentration of DHCA in IWFs
from resistant plants does not directly inhibit bacterial growth in vitro
and why mature cyp71b15 does not display enhanced resistance to Pst
despite an ~6.5-fold increase in intercellular DHCA compared to mature
wild type plants.
4. Experimental
4.1. General experimental procedures
4.1.1. Plant growth
Arabidopsis thaliana (L.) Heynh (Brassicaceae) seeds were surface
sterilized (70% ethanol followed by a solution of 30% bleach and 0.1%
Tween 20 in sterile water), rinsed with ddH2O, and suspended in 0.1%
phytoblend. Seeds stratified for 2–3 days at 4 ◦C before plating on
Murashige and Skoog (MS) basal salt medium (Murashige and Skoog,
1962) and germinated in under continuous light (22 ◦C, light intensity
~90
μ
mol/m2/s, ~45% humidity). At 6–7 days, seedlings were trans-
planted to soil (Sunshine Mix No. 1 – JUIC Ltd.) prepared with 2 L of 1
g/L 20-20-20 fertilizer per tray. To maintain high humidity, seedlings
were covered with a plastic transparent dome for 48 h in growth
chambers and grown at 22 ◦C–24 ◦C at ~80% relative humidity under a
9-h photoperiod (average light intensity ~120
μ
mol/m2/s). Plants were
fertilized with 1 g/L of 20–20-20 fertilizer at 3 weeks post-germination
(wpg) and 5 wpg. At 3- and 7-weeks post-germination (wpg), plants
were used in disease resistance assays (described below). All mature
plants were 7 wpg and in vegetative phase (no bolts or flowers present).
3. Conclusion
Using a metabolomics approach with two biosynthetic mutants in the
A. thaliana trp-derived specialised metabolism pathway, two mass
spectral features (corresponding to a glycosylated indole-3-carboxylic
acid derivative and dihydrocamalexic acid) were identified in
4.1.2. Plant lines
All mutant lines are Arabidopsis thaliana (L.) Heynh (Brassicaceae) in
the Columbia (Col-0) accession background. The cyp71a12
(GABI_127H03) mutant line was obtained from The Arabidopsis
Fig. 7. Exogenous infiltration of dihy-
drocamalexic acid (DHCA) and bacterial
quantification of Pseudomonas syringae in
rosette leaves of 7-week old Col-0 and
cyp71a12/cyp71a13. DHCA (0.07
μg/mL
or 0.25
μg/mL) was applied via pressure
infiltration to 7-week-old plants at 24 h post-
inoculation with P. syringae or mock solution
(0.06% DMSO in 10 mM MgCl2). Bacterial
levels were quantified at
3 days post-
inoculation with Pst. Values represent the
mean ± standard deviation of three sample
replicates (n = 3) consisting of 8 plants each.
Different letters indicate statistically signifi-
cant differences (ANOVA, Tukey’s honestly
significant difference [HSD], P < 0.05).
8

C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
Information Resource (TAIR). The cyp71b15 (pad3-1) line was obtained
from J. Glazebrook (University of Minnesota, St. Paul, MN, USA). The
cyp71a12/a13-1 line was provided by E. Glawischnig (TUM Campus
Straubing for Biotechnology and Sustainability, Technical University of
Munich, Germany). Individual plants homozygous for the cyp71a13-1 T-
DNA insertion were confirmed using PCR as described in Nafisi et al.
(2007). Gene-specific primers designed to identify wild-type CYP71A13:
5′-GTAAGAGAAGACGAGGTAAATGC-3’ (forward) and 5′-CTTCTGAT-
CAGTTCCGTCATCG-3’ (reverse). The primer sequence used to identify
the T-DNA left border in cyp71a13-1 was 5′-GGAACAA-
CACTCAACCCTATCTCG-3’ (LBe). The TALENs-mediated 5 base pair
deletion in CYP71A12 was confirmed via sequencing using primers 5′-
TGACATTCCGCAATCTGAAAACC-3’ (forward) and 5′- GGGA-
GAAGGATTTGTCCAGGG-3’ (reverse).
and turbidity (700 nm) as outlined in Baker et al. (2012) and IWFs were
flash frozen in liquid nitrogen, stored at ꢀ 80 ◦C, and transported to the
Vineland Research and Innovation Centre under dry ice for analysis.
Each of three replicates contained leaves from 13 individual plants (13
fully expanded rosette leaves).
4.1.6. Accession numbers
Sequence data for genes from this article can be found in the Gen-
Bank/EMBL data libraries under the following Arabidopsis Genome
Initiative identifiers: CYP71A12 (At2g30750), CYP71A13 (At2g30770),
CYP71B15 (At3g26830).
4.2. Chemicals
Unless otherwise noted, all chemicals were purchased from Sigma-
Aldrich (>95% purity). An initial stock of dihydrocamalexic acid for
use as an UPLC-MS standard for DHCA identification was generously
provided by E. Glawischnig (TUM Campus Straubing for Biotechnology
and Sustainability, Technical University of Munich, Germany). Indole-3-
carbonitrile and D-cysteine were purchased from Toronto Research
Chemicals. UPLC-MS grade acetonitrile, water, and formic acid were
purchased from Fisher Scientific.
4.1.3. Disease resistance assays
Virulent P. syringae pv. Tomato (Pst) DC3000 (pVSP61) (Whalen
et al., 1991) cultures were prepared in King’s B (KB) medium supple-
mented with 50
μg/mL kanamycin (plasmid resistance) and grown
overnight with agitation at 200 rpm for ~16 h at 22–25 ◦C to mid-log
phase (OD600 = 0.2 to 0.6). Cultures were re-suspended and diluted to
106 colony forming units (cfu) per mL of 10 mM MgCl2. Medium-sized
rosette leaves (fully expanded) were inoculated by pressure infiltration
of dilute cultures into the abaxial (underside) surface of three leaves per
plant using a needle-less 1 mL syringe. Control mock-inoculations con-
sisted of 10 mM MgCl2. Leaf disks were removed from leaves of 3- or
7-week-old plants at 3 days post-inoculation (dpi) using a cork-borer (6
mm diameter) for a total of three replicates of eight leaf disks each (one
leaf disk per leaf per plant) sampled from 24 plants per treatment. Pst
was isolated from tissue by submerging leaf disks in sterile 0.1% Silwet
L-77 in 10 mM MgCl2 at 200 rpm for 1 h. In planta Pst was then quan-
tified by plating a dilution series on KB medium with kanamycin (50
4.3. Metabolite profiling
4.3.1. Preparation of intercellular washing fluids
100 μl aliquots of IWF were diluted in 60 μl of acetonitrile (5%) in
water acidified with 0.1% formic acid. Samples were sonicated in ice
water for 5 min, vortexed, sonicated for another 5 min, then centrifuged
at 20,000×g for 2 min. The supernatant was filtered with 0.2 μm GHP
filters (Acrodisc) and transferred to LC-certified vials (Waters) with 250
μ
l glass inserts (Chromatographic Specialties Inc). Quality control pools
μ
g/mL) and rifampicin (100
μg/mL).
consisting of equal amounts of individual extracted IWFs were prepared
in LC-certified vials (Waters).
4.1.4. DHCA rescue assays
4.3.2. UPLC/ESI-QTOF MSE and MS/MS
A. thaliana (7 wpg) rosette leaves (medium size, fully expanded)
were inoculated with Pst as outlined in section 4.1.3. At 24-h post-
Chromatographic separation was performed on an Acquity I-Class
UPLC (Waters, Manchester, UK) equipped with an Acquity UPLC BEH
inoculation with bacteria, DHCA (70 μg/mL (284 μM) or 250 ng/mL
(1.02 μM)) was pressure-infiltrated into leaves. Mock controls consisted
C18 1.7 μm column (Waters) using a binary solvent mixture consisting of
of 10 mM MgCl2 with an equivalent amount of DMSO (the solvent used
for DHCA) added. Once thoroughly dried (~1 h), plants were returned
to growth chambers. Bacterial growth was quantified as outlined in
section 4.1.3.
solvent A (5% acetonitrile in water with 0.1% formic acid) and solvent B
(acetonitrile with 0.1% formic acid), with 5 l (IWFs) sample injection at
μ
infusion flow rate of 0.3 mL/min. The gradient was set for solvent B at
0–40% for 0.0–22.5 min, 100% over 23.0–24.5 min, decreasing to 0%
over 25.0–26.0 min (Rogachev and Aharoni, 2011). Analytic data were
acquired on a Xevo G2-XS QTOF (Waters) with a capillary voltage of 3
kV (ESI+) or 2.5 kV (ESI-) and sample cone voltage of 40 eV. MSE data
were acquired in positive and negative electrospray ionization (ESI)
modes and in ESI + for dihydrocamalexic acid MS/MS. A survey scan
time of 0.25 s in continuum data format with an acquisition mass range
of 50–1200 Da was used for MSE with a desolvation temperature of
500 ◦C, cone gas flow of 50 L/h, and desolvation gas flow of 800 L/h. For
MSE, the low collision energy was 6 eV and the high ramp collision
energy was 15–35 eV. MS/MS data for DHCA was acquired over 5–10
min at a mass range of 50–1200 Da and set mass of 247.05 Da [M+H]
using a scan time of 0.25 s in continuum format at 25 eV collision en-
4.1.5. Intercellular washing fluid collection for metabolite profiling
Inoculations of mock- and Pst-solutions was completed as described
in Section 4.1.3. The infiltration-centrifugation method developed by
O’Leary et al. (2014) was used with some modifications for metabolite
profiling. Fully expanded medium rosette leaves from mature (7 wpg)
plants were detached at the petiole with a razor blade at 24 hpi with
mock solution or Pst. Leaves were weighed to 1 g fresh weight (FW) and
rinsed for 5 s in chilled, distilled water to remove surface contamination
and then placed in a 60 mL syringe filled with 30 mL of chilled, distilled
water. Using a gloved finger, negative pressure was created in the sy-
ringe by pulling the plunger out to the 60 mL mark and slowly releasing.
This was repeated approximately three times per sample until leaves
were thoroughly infiltrated (darkened transparent color throughout and
negative buoyancy). Excess surface liquid was removed by blotting and
the leaves were again weighed. Infiltrated leaves were stacked between
ergy. Leucine enkephelin (200 pg/μl in 50:50 acetonitrile/water with
0.1% formic acid) was used as a reference calibrant with LockSpray ion
source (Waters; infusion flow rate 10
measurement.
μl/min) for exact mass
layers of Parafilm and secured around a 1000 μl pipette tip placed in a
cut 20 mL syringe bottom with the tip placed in a 1.5 mL microfuge tube
4.3.3. Data acquisition
(petioles facing upward) and centrifuged for 6 min at 600×g in a
Extracts for each treatment and blanks were all injected in ran-
domized order. Quality control samples were initially injected 10 times
sequentially to equilibrate the column, then every 6 runs in both ESI+
and ESI- modes. Peak alignment, quality control assessment, peak
picking, pseudomolecular ion (adduct) composition, and normalization
swinging bucket rotor at 4 ◦C, resulting in 150–200
μl of IWF collected
per sample. After collection, leaves were visually examined to ensure
IWFs were removed from all leaves (no water-soaked patches). Cyto-
plasmic contamination was assayed by measuring chlorophyll (664 nm)
9

C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
to all mass features was performed in Progenesis QI (Non-Linear Dy-
namics). Suitability of data for downstream analysis was assessed by
ensuring tight clustering of quality control pooled samples in principal
component analysis considering all samples. Mass features were
manually processed in MassLynx 4.1 (Waters) to filter out background
and identify molecular ions of interest.
were dried over sodium sulphate and concentrated using rotary evapo-
ration. The crude residue was purified by filtering over a short silica
column (elution with 100% ethyl acetate), followed by solvent removal.
Yield = 156 mg of off-white solid (75% yield). m. p. = 110–111 ◦C
(recrystallized from EtOAc, lit. 114–115 ◦C).1 1H NMR (500 MHz,
CDCl3) δ 9.06 (s, 1H), 7.89–7.83 (m, 2H), 7.54–7.50 (m, 1H), 7.43 (t, J
= 7.6 Hz, 2H), 5.38 (t, J = 9.3 Hz, 1H), 3.76 (dd, J = 10.3, 7.9 Hz, 1H),
3.72 (dd, J = 10.2, 8.6 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 173.6,
173.4, 132.5, 132.0, 128.8, 128.8, 77.8, 35.1. For (R)-2-Phenyl-4,5-
dihydrothiazole-4-carboxylic acid (derived from L-cysteine) 1, [alpha]
D25 = +37.1 (c 0.36, MeOH). For (S)-2-phenyl-4,5-dihydrothiazole-4-
carboxylic acid 2 (derived from D-cysteine), [alpha]D21 = ꢀ 34.9 (c
0.37, MeOH). ESI HRMS expected for C10H10NO2S (M + H)+: 208.0427
found 208.0404.
4.3.4. Untargeted data analysis and compound identification
Putative metabolite identification was completed by: establishing
fragmentation patterns (fragmentation in mass spectra for low and high
energy (first and second function), published spectral data, adducts,
neutral loss); estimating elemental composition (Range C0-100, H0-100,
N0-5, O0-30, S0-3, unsaturation, 5 ppm ≥ m/z 300, 2 mDa ≤200); pu-
tative identification from publicly available and in-house customized
natural products databases and commercially available standards where
applicable. Mass error was calculated using the formula: (observed m/z –
theoretical m/z)/(theoretical m/z * 106). Theoretical m/z were calcu-
lated in Mass Lynx 3.1. Elemental composition was calculated using
ChemCalc Molecular formula based on monoisotopic mass (http
://www.cheminfo.org; Patiny and Borel, 2013). Features of interest
were selected by pairwise comparison (t-test) between wild-type (Col-0)
mock- and Pst-inoculated IWFs, using median fold change threshold
(>2) and t-test threshold (P < 0.05, two-tailed, equal variance) volcano
plots. The resulting features of interest were compared across all data-
sets (Col-0, cyp71a12/cyp71a13, and cyp71b15 mock- and Pst-inocu-
lated) using normalized abundance plots and ANOVA testing (P < 0.05,
Tukey’s HSD). Two synthesized standards of dihydrocamalexic acid
(2–5000 pg on column) and a commercially available standard of
camalexin (1–5000 pg on column) were quantified in positive-ion mode;
a commercially available standard of salicylic acid (100–5000 ng on
column) was quantified in negative-ion mode. An internal standard of
deuterated salicylic acid (-d6) was added to each sample for normali-
zation and relative quantification.
2-(4-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid (mono-
hydrate) 3 and 4.
4-cyanophenol (119 mg, 1.00 mmol, 1.0 eq.), cysteine (242 mg, 2.00
mmol, 2.0 eq.), and sodium bicarbonate (336 mg, 4.00 mmol, 4.0 eq.)
were added to 4 mL of degassed absolute ethanol in a pressure vial. The
vial was sealed under nitrogen and heated with stirring in a 100 ◦C oil
bath for 24 h. The reaction was removed from the oil bath and cooled to
room temperature and the ethanol was removed using rotary evapora-
tion. The residue was washed with 2 mL of ethyl acetate, suspended in 2
mL of water, cooled to 0 ◦C and carefully quenched by dropwise addition
of 2 M HCl until the mixture was pH 2 (indicator paper). The resulting
mixture was extracted with ethyl acetate (3 × 4 mL) and the combined
organic extracts were dried over sodium sulphate before concentration
using rotary evaporation. The resulting solid was washed with 0.5 mL of
water to remove trace salts and dried leaving the desired product. Yield
= 178 mg (80% yield) of a white powder. m. p. = 151–153 (decompo-
sition; powder recovered from MeOH; lit. 151–153 ◦C).1 1H NMR (500
MHz, DMSO) δ 12.92 (s, 1H), 10.15 (s, 1H), 7.63 (d, J = 8.7 Hz, 2H),
6.84 (d, J = 8.7 Hz, 2H), 5.21 (dd, J = 9.3, 8.1 Hz, 1H), 3.65 (dd, J =
11.1, 9.3 Hz, 1H), 3.55 (dd, J = 11.1, 8.1 Hz, 1H). 13C NMR (126 MHz,
DMSO) δ 172.1, 167.7, 160.7, 130.1, 123.4, 115.5, 78.1, 34.8. For (R)-2-
(4-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid 3 (derived
from L-cysteine), [alpha]D25 = ꢀ 12.4 (c 0.1, MeOH). For (S)-2-(4-
hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid 4 (derived from
D-cysteine), [alpha]D25 = +12.3 (c 0.1, MeOH). ESI HRMS expected for
4.4. Synthesis of dihydrocamalexic acid and structural analogs
All reactions were performed in sealed glass vials or round-bottom
flasks under nitrogen atmosphere. 1H and 13C spectra were recorded
on Bruker AV 500, 600 or 700 MHz spectrometers in CDCl3, DMSO‑d6, or
MeOD. Bulk solvent removal was performed by rotary evaporation
under reduced pressure. For reactions with solvent volumes under 3 mL,
the solvent was evaporated under a stream of nitrogen. Column chro-
matographic purification was performed using Silicycle silica gel
(40–63 μM, 230–400 mesh) with technical grade solvents. Yields are
reported for spectroscopically pure compounds, unless stated otherwise.
Coupling constants are recorded in Hz and chemical shifts are reported
in ppm downfield of TMS. HRMS (ESI+) was performed on a Waters
Micromass Q-ToF Ultima Global. The synthesis of DHCA and derivatives
is based on Maltsev et al. (2013).
C
10H8NO3S (M-H)-: 222.0230 found 222.0236.
(R)-2-(3-Indolyl)-4,5-dihydrothiazole-4-carboxylic acid (DHCA):
3-cyanoindole (36 mg, 0.25 mmol, 1.0 eq.), L-cysteine (60 mg, 0.50
mmol, 2.0 eq.) and sodium bicarbonate (84 mg, 1.00 mmol, 4.0 eq.)
were added to 1 mL of degassed ethanol in a microwave vial. The vial
was sealed under nitrogen and heated with stirring in a 100 ◦C oil bath
for 36 h. The reaction was removed from the oil bath and cooled to room
temperature and the ethanol was removed using rotary evaporation. The
residue was washed with 1 mL of ethyl acetate, suspended in 0.5 mL of
water, cooled to 0 ◦C and carefully quenched by dropwise addition of 2
M HCl until the mixture was pH 2 (indicator paper), resulting in the
precipitation of a small amount of tan solid. The solution was stored in a
refrigerator overnight and the solid was filtered and washed with 2 mL
of water, leaving the desired product. Yield = 26 mg of a tan powder
(42% yield). m. p. = 145–148 ◦C (decomposition; powder precipitated
2-Phenyl-4,5-dihydrothiazole-4-carboxylic acid 1 and 2.
Benzonitrile (103 mg, 1.00 mmol, 1.0 eq.), cysteine (181 mg, 1.50
mmol, 1.5 eq.) and sodium bicarbonate (126 mg, 1.50 mmol, 1.5 eq.)
were added to a degassed solution of methanol (2.6 mL) and water (1.7
mL) in round-bottom flask under nitrogen atmosphere. Then, 50 μL of a
1 M solution of NaOH was added to the reaction mixture and the flask
was sealed under nitrogen and allowed to stir overnight. MeOH was
removed by rotary evaporation and the reaction mixture was washed
◦
with 2 mL of diethyl ether before being cooled to 0 C and carefully
quenched by dropwise addition of 2 M HCl until the mixture was pH 2
(indicator paper) resulting in the solution becoming cloudy. The mixture
was then extracted with ethyl acetate (3 × 4 mL), the organic extracts
10

C.J. Kempthorne et al.
Phytochemistry 187 (2021) 112747
1
from H2O). H NMR (600 MHz, MeOD) δ 8.36 (s, 1H), 8.05–7.99 (m,
Dunnett’s MCT) statistical analyses were performed in Metaboanalyst
3.1 (www.metaboanalyst.ca; Xia and Wishart, 2016), R, and Prism 9.0
(GraphPad). One-way ANOVA was used to determine statistically sig-
nificant differences in bacterial levels in planta with normality and ho-
mogeneity of variance assessed by the Sharpio-Wilk test and Levene’s
test. Means with unequal variances were log-transformed before anal-
ysis. Tukey’s HSD post hoc test was used for comparisons (p < 0.05)
among mutants and wild-type plants. All non-metabolomic statistical
tests were completed in R and Prism 6.0 and 7.0 (GraphPad). Molecules
were drawn in ChemDraw (PerkinElmer).
1H), 7.58–7.53 (m, 1H), 7.37–7.30 (m, 2H), 5.29–5.24 (m, 1H),
4.05–3.93 (m, 2H). 13C NMR (151 MHz, MeOD) δ 177.1, 172.6, 138.8,
135.9, 126.0, 125.4, 124.4, 121.1, 114.2, 107.1, 69.2, 35.6. [alpha]D25
= ꢀ 62.9 (c 0.2, MeOH). ESI HRMS expected for C12H11N2O2S (M + H)+:
247.0536 found 247.0532.
4.5. Antimicrobial activity assessment
4.5.1. Planktonic bacterial growth assays
Minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) of tested indolic compounds and salicylic acid
were determined using the micro-dilution broth method (Wiegand et al.,
2008). Pst overnight cultures (OD600 = 0.2 to 0.6) were prepared for
growth assays as outlined in Wilson et al. (2017) in Hrp-inducing min-
imal (HIM) liquid medium (Huynh et al., 1989). Serial dilutions of
compounds were prepared in ethanol (SA, camalexin) or DMSO (SA,
DHCA, and DHCA analogs). A series of two-fold dilutions of DHCA, SA
camalexin, or DHCA analogs were prepared ranging from 0.05 to 1
mg/mL in HIM medium in sterile non-tissue-culture-treated 96-well
microtiter plates (Corning Life Sciences). Bacterial inoculum was
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We are especially grateful to Erich Glawischnig (TUM Campus
Straubing for Biotechnology and Sustainability, Technical University of
Munich, Germany) for providing the cyp71a12/a13-1 double mutant
seed and a synthesized standard of dihydrocamalexic acid to confirm our
metabolite identification. Thank you to Jane Glazebrook (University of
Minnesota, St. Paul, MN, USA) for providing cyp71b15 (pad3-1) mutant
seed, E. Weretilnyk (McMaster University, Dept. Of Biology) for
providing thermocycler access, and J. Wilson (McMaster University,
Dept. Of Biology) for providing access to the Biotek Synergy 2 plate
reader for biofilm assays. In vitro antibacterial assays were performed at
the McMaster Centre for Microbial Chemical Biology. This work has
been supported by Growing Forward 2 and Canada Foundation for
Innovation funding awarded to Vineland Research and Innovation
Centre, an Ontario Graduate Scholarship to C. Kempthorne, an NSERC
Discovery Grant to J. McNulty, NSERC Scholarships to A. Nielsen and D.
Wilson, and an NSERC Discovery and Research Infrastructure grant to R.
Cameron.
added to each well to obtain a final optical density value of OD600
=
0.05. Solvent (DMSO or ethanol) and sterile (media only) controls were
◦
included. Plates were incubated at room temperature (24–26 C) in a
plate reader with shaking (Tecan Sunrise). Optical density (OD600) was
measured every 15 min for 68 or 72 h. The MIC was assessed visually as
the lowest concentration of compound showing total bacterial growth
inhibition during the mid-exponential growth phase of controls (~36 h)
without killing bacteria. The MBC of each compound was determined as
the lowest concentration to kill bacteria, as defined by sub-cultures from
pooled wells exhibiting no bacterial growth on KB media plates without
antibiotics to prevent additive interactions between compounds creating
false MBC results (Balouiri et al., 2016). Each experiment was performed
at least twice, and representative data are presented.
4.5.2. Dihydrocamalexic acid bacterial metabolism assays
To test for Pst’s ability to utilize dihydrocamalexic acid (DHCA) as a
carbon source or as a nitrogen and sulfur source, bacteria were grown in
Hrp-inducing minimal medium with DHCA as outlined in section 4.6.1
with the following alterations. To test the ability of Pst to use DHCA as a
carbon source, bacteria were incubated with DHCA in HIM medium
without a carbon source (lacking fructose). To test for ability of Pst to use
DHCA as a nitrogen and/or sulfur source, bacteria were incubated with
DHCA in HIM medium without a nitrogen and sulfur source (lacking
ammonium sulphate).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.phytochem.2021.112747.
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