N-hydroxy-pipecolic acid is a mobile metabolite that induces systemic disease resistance in Arabidopsis

Article abstract of DOI:10.1073/pnas.1805291115

Systemic acquired resistance (SAR) is a global response in plants induced at the site of infection that leads to long-lasting and broad-spectrum disease resistance at distal, uninfected tissues. Despite the importance of this priming mechanism, the identity and complexity of defense signals that are required to initiate SAR signaling is not well understood. In this paper, we describe a metabolite, N-hydroxy-pipecolic acid (N-OH-Pip) and provide evidence that this mobile molecule plays a role in initiating SAR signal transduction in Arabidopsis thaliana. We demonstrate that FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), a key regulator of SAR-associated defense priming, can synthesize N-OH-Pip from pipecolic acid in planta, and exogenously applied N-OH-Pip moves systemically in Arabidopsis and can rescue the SAR-deficiency of fmo1 mutants. We also demonstrate that N-OH-Pip treatment causes systemic changes in the expression of pathogenesis-related genes and metabolic pathways throughout the plant and enhances resistance to a bacterial pathogen. This work provides insight into the chemical nature of a signal for SAR and also suggests that the N-OH-Pip pathway is a promising target for metabolic engineering to enhance disease resistance.

Full text of DOI:10.1073/pnas.1805291115

N-hydroxy-pipecolic acid is a mobile metabolite that
induces systemic disease resistance in Arabidopsis
a,2
a,1
b,1
b
a
b
c
Yun-Chu Chen , Eric C. Holmes , Jakub Rajniak , Jung-Gun Kim , Sandy Tang , Curt R. Fischer , Mary Beth Mudgett
,
b,2
and Elizabeth S. Sattely
a
b
Department of Biology, Stanford University, Stanford, CA 94305-5020; Department of Chemical Engineering, Stanford University, Stanford,
c
CA 94305-5020; and Chemistry, Engineering & Medicine for Human Health, Stanford University, Stanford, CA 94305-5020
Edited by Brian J. Staskawicz, University of California, Berkeley, CA, and approved April 16, 2018 (received for review March 28, 2018)
Systemic acquired resistance (SAR) is a global response in plants
induced at the site of infection that leads to long-lasting and
broad-spectrum disease resistance at distal, uninfected tissues.
Despite the importance of this priming mechanism, the identity
and complexity of defense signals that are required to initiate SAR
signaling is not well understood. In this paper, we describe a me-
tabolite, N-hydroxy-pipecolic acid (N-OH-Pip) and provide evidence
that this mobile molecule plays a role in initiating SAR signal trans-
duction in Arabidopsis thaliana. We demonstrate that FLAVIN-
DEPENDENT MONOOXYGENASE 1 (FMO1), a key regulator of
SAR-associated defense priming, can synthesize N-OH-Pip from
pipecolic acid in planta, and exogenously applied N-OH-Pip moves
systemically in Arabidopsis and can rescue the SAR-deficiency of
fmo1 mutants. We also demonstrate that N-OH-Pip treatment
causes systemic changes in the expression of pathogenesis-
related genes and metabolic pathways throughout the plant and
enhances resistance to a bacterial pathogen. This work provides
insight into the chemical nature of a signal for SAR and also sug-
gests that the N-OH-Pip pathway is a promising target for meta-
bolic engineering to enhance disease resistance.
(G3P) (11), dehydroabietinal (DA) (12), and pipecolic acid (Pip)
(13)] are associated with long-distance communication and signal
amplification during SAR (8, 14, 15). However, the onset of SAR
signaling also requires the uncharacterized enzyme FLAVIN-
DEPENDENT MONOOXYGENASE 1 (FMO1) (16–18).
Remarkably, treatment of fmo1 mutants with AzA, DA, or Pip
does not elicit systemic resistance, suggesting that a metabolite
produced by FMO1 plays a key role in the establishment of SAR
signaling (10, 12, 13). The biochemical function of FMO1 has
remained unknown.
Several forward genetic screens searching for SAR-deficient
mutants identified multiple alleles of fmo1, ald1 (AGD2-LIKE
DEFENSE RESPONSE PROTEIN 1), and sard4 (SAR-
DEFICIENT 4), highlighting the importance of metabolites pro-
duced by these enzymes (16, 17, 19, 20). ALD1 and SARD4 are
involved in the biosynthesis of Pip (13, 21, 22). Irrigation of wild-
type Arabidopsis plants with Pip induces SAR (13), which suggested
that Pip might be a mobile SAR metabolite. Návarová et al. (13)
reported, however, that Pip could not trigger SAR in fmo1 mu-
tants. In addition, fmo1 plants accumulate high levels of Pip during
a late stage of infection compared with wild-type plants (13). These
findings feature FMO1 as a key missing link in the mechanism of
Pip-associated SAR.
systemic acquired resistance signaling plant natural products
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Arabidopsis thaliana N-hydroxy-pipecolic acid
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lants have developed a complex and dynamic innate immune
system that relies on sensing and signaling using small mol-
P
Significance
ecules for defense against pathogens (1, 2). At a primary site of
infection, plants respond to common molecular features of mi-
crobes (e.g., bacterial flagellin or fungal chitin, collectively known
as microbial-associated molecular patterns or MAMPs) and
pathogen-derived proteins, termed effectors, that enter the plant
cell (3). Immune responses to both classes of molecules (effector-
triggered or pattern-triggered; refs. 3 and 4) activate signal trans-
duction networks through the action of hormones such as salicylic
acid (SA), ethylene, and jasmonic acid, which cause changes in
defense gene expression and production of antimicrobial metabo-
lites at the site of infection (5). Interactions between these hor-
mones function synergistically and antagonistically to tailor
a specific immune response to different pathogens (2, 6). While
local defense is critical for limiting pathogen growth, plants also
possess the ability to prime and amplify immune responses at distal
sites. This global response is termed systemic acquired resistance
Plants lack circulating immune cells and instead rely on small
molecule chemistry for local and long-distance defense sig-
naling. Following pathogen attack, plants activate innate im-
mune pathways at the site of infection to limit pathogen
growth. Plants also possess the ability to prime similar immune
responses in uninfected tissues to prevent the spread of
pathogens or protect against new infections. Despite the im-
portance of systemic immunity, the mechanism for signaling is
not clear. In this study, we show that N-hydroxy-pipecolic acid
metabolites are mobile defense signals produced at the site of
bacterial infection and establish and amplify defense in un-
infected, distal tissues. Our study illuminates the chemical na-
ture of a mobile bioactive metabolite that confers pathogen
resistance throughout the plant.
(
SAR) (2, 7). SAR is critical for preventing the spread of pathogens
Author contributions: Y.-C.C., E.C.H., J.R., C.R.F., M.B.M., and E.S.S. designed research;
Y.-C.C., E.C.H., J.R., J.-G.K., S.T., and C.R.F. performed research; Y.-C.C., E.C.H., J.R.,
J.-G.K., C.R.F., S.T., M.B.M., and E.S.S. analyzed data; and Y.-C.C., E.C.H., M.B.M., and
E.S.S. wrote the paper.
and protecting against new infections (1, 2). Although much is
understood about how the immune response is activated locally,
the chemical nature of the plant-derived molecules that mediate
long-distance communication between the site of infection (pri-
mary) and distal uninfected (secondary, systemic) sites remain
elusive.
Genetic and mechanistic studies of the model plant Arabidopsis
thaliana have led to the identification of key components of the
SAR pathway, providing critical insight into the mechanisms
controlling long-lasting and broad-spectrum disease resistance (7).
Several plant-derived small molecules [e.g., SA (8), methyl salic-
ylate (MeSA) (9), azelaic acid (AzA) (10), glycerol-3-phosphate
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.
1
Y.-C.C. and E.C.H. contributed equally to this work.
2
To whom correspondence may be addressed. Email: mudgett@stanford.edu or sattely@
stanford.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1805291115/-/DCSupplemental.
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We and others have found untargeted metabolite analysis of
Arabidopsis genetic mutants to be a powerful approach for the
identification of small molecules associated with fitness pheno-
types (either previously characterized or suggested by tran-
scriptome analysis) (23). Examples include the identification and
characterization of cytochromes P450 involved in phytoalexin
production (24) and iron acquisition (25). Given that FMO1 is
one of the genes most responsive to biotic stress (as indicated by
analysis of previously reported Arabidopsis microarray data
summarized in SI Appendix, Fig. S9) and that genetic data sug-
gest molecules generated by FMO1 are required for initiating
SAR (16–18), we sought to apply an untargeted metabolomics
approach to determine the products of FMO1 and their function.
Here, we report the discovery of glycosylated N-hydroxypipecolic
acid (N-OGlc-Pip) in Arabidopsis. We provide evidence that the
aglycone, N-OH-Pip, is the direct product of FMO1 and has a
central role in SAR signal transduction (26). We also demon-
strate that exogenously applied N-OH-Pip can rescue the SAR-
deficient response of fmo1 mutants, cause systemic changes in
expression of pathogenesis-related genes and metabolic path-
ways throughout the plant (and distal to the site of application),
and enhance resistance to the bacterial pathogen, Pseudomonas
syringae. In addition, we provide biochemical evidence that Ara-
bidopsis FMO1 catalyzes N-hydroxylation of the nonproteinogenic
amino acid Pip to N-OH-Pip using transient expression assays in
Nicotiana benthamiana. Taken together, our data indicate that N-
OH-Pip is a key signaling molecule that is required to initiate SAR
in Arabidopsis.
added to the seedling media 24 h before metabolite analysis to
elicit expression of enzymes associated with defense and SAR
(including FMO1). This analysis revealed a major mass signal
that is present in WT plants in response to Pst treatment but not
2
Mock treatment (10 mM MgCl ) and was absent from all fmo1
plants (Fig. 1A and SI Appendix, Fig. S1 A and B). We propose
this signal corresponds to the O-glycosylated form of the me-
tabolite N-OH-Pip, based on comparison of the MS/MS
spectrum to an authentic synthetic standard of the N-OH-Pip
aglycone (SI Appendix, Figs. S2 and S3). Although N-OH-Pip
has not previously been observed as a naturally occurring me-
tabolite in plants or other organisms, pipecolic acid has been
observed in Arabidopsis and tomato (13, 27) and was shown to be
associated with the SAR response (13). Therefore, we hypoth-
esized that FMO1 catalyzes the N-hydroxylation of Pip to pro-
duce N-OH-Pip. Glycosylated N-OH-Pip (N-OGlc-Pip) detect-
able in Arabidopsis plant extracts is likely the product of
unknown UDP glycosyltransferases that further processes
N-OH-Pip (Fig. 1C). We did not detect free N-OH-Pip in WT
seedlings elicited with Pst by LC-MS analysis (SI Appendix, Fig.
S1B). These data suggest that the N-OH-Pip aglycone does not
accumulate in cells and/or is an unstable metabolite.
Measurement of N-OH-Pip Derivatives in Adult Plants. We also
measured the level of N-OGlc-Pip in adult WT and fmo1 plants
(
both in lower and upper leaves), after treatment of lower leaves
with 10 mM MgCl (Mock) or Pst expressing the type III se-
2
cretion (T3S) effector gene avrRpt2 (Pst avrRpt2). Pst avrRpt2 is
an avirulent strain that induces effector-triggered immunity
Results
Untargeted Metabolomics of Arabidopsis fmo1 Seedlings. To dis-
cover metabolites whose production is dependent on FMO1, we
used liquid chromatography-mass spectrometry (LC-MS)-based
untargeted metabolomics to compare the composition of meth-
anolic extracts from two sets of 12-d-old seedlings grown hy-
droponically: A. thaliana Col-0 WT and a T-DNA insertion
mutant of FMO1, fmo1-1 (16), herein referred to as fmo1. The
bacterial pathogen P. syringae pathovar tomato DC3000 (Pst) was
(
(
ETI) and SAR signaling in resistant RPS2 Arabidopsis plants
28–30). N-OGlc-Pip was detected in the lower and upper leaves
of WT only after infection of lower leaves with Pst avrRpt2, but
not in fmo1 plants (Fig. 1B). As in seedlings, we were not able to
detect free N-OH-Pip by LC-MS. Collectively, these data in-
dicate that FMO1 is required for the production of N-OGlc-Pip
in Arabidopsis.
1
1
1
1
1
1
1
06
05
04
03
02
01
00
106
105
104
103
102
101
100
A
B
WT fmo1 WT fmo1
L
U
L
U
L
U
L
U
MgCl2
Pst avrRpt2
MgCl2
Pst avrRpt2
MgCl2
Pst
Col-0 WT
Col-0 fmo1
C
O
OH
N
O
OH
O
OH
OH
O
OH
ALD1
At2g13810)
SARD4
(At5g52810)
FMO1
O
(
NH (At1g19250)
N
N
Glc
Lys
Pip
N-OH-Pip
N-OGlc-Pip
Fig. 1. Untargeted metabolomics of Arabidopsis seedlings elicited with Pst implicates FMO1 in the production of pipecolic acid derivatives. (A) Ion abun-
dances for N-OGlc-Pip (gray bars) detected in extracts isolated from Arabidopsis Col-0 WT and fmo1 seedlings grown hydroponically and elicited with P.
syringae pv. tomato DC3000 (Pst). Levels represent the mean ± STD of six biological replicates. Levels reported as zero indicate no detection of metabolites.
6
(
B) Ion abundances for N-OGlc-Pip (gray bars) in lower (L) and upper (U) leaves of adult Arabidopsis Col-0 plants 48 hpi with a 5 × 10 cfu/mL suspension of Pst
expressing the T3S effector avrRpt2 (Pst avrRpt2). Levels represent the mean ± STD of six biological replicates. Levels reported as zero indicate no detection of
metabolites. (C) Proposed biosynthetic activity for Arabidopsis FMO1. The Arabidopsis enzymes ALD1 and SARD4 convert lysine to pipecolic acid (Pip) (22).
FMO1 is proposed to hydroxylate Pip to N-OH-Pip. Unknown enzymes are proposed to glycosylate N-OH-Pip to produce N-OGlc-Pip, the molecule identified in
the untargeted metabolite analysis.
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Biochemical Activity of Arabidopsis FMO1. Arabidopsis FMO1
expressed and purified as a 6×-His fusion protein (FMO1-
tative FAD and NADP domains for FMO1 catalytic activity (18).
N-OGlc-Pip was not detected in N. benthamiana leaf extracts
when Arabidopsis FMO1 was expressed, suggesting that N. ben-
thamiana either does not have the necessary glycosyl transferases
or that they are not expressed under our experimental conditions.
6×-His) from Escherichia coli lacked the cofactor FAD and was
not catalytically active in any of our in vitro biochemical assays.
However, we were able to detect FMO1-dependent activity when
Arabidopsis FMO1 was transiently expressed in N. benthamiana
only after feeding pipecolic acid and then analyzing products
present in leaf methanolic extracts (Fig. 2 and SI Appendix, Fig.
S1 C–E). As shown in Fig. 2, FMO1-expressing leaves accumu-
lated free N-OH-Pip. They also contained a second metabolite
with an m/z of 100 and a putative structure of N-hydroxy
piperideine (Fig. 2 and SI Appendix, Fig. S4), which we propose
is the result of oxidative decarboxylation of N-OH-Pip. We noted
that the mass signal m/z 100 can be produced in samples of
synthetic N-OH-Pip after heating in buffer. It is also present in
Col-0 seedlings after Pst treatment and absent from fmo1 seed-
lings (SI Appendix, Fig. S1B), suggesting that the m/z 100 me-
tabolite is likely FMO1-derived. In addition, N-OH-Pip levels in
N. benthamiana extracts decreased between 28 and 48 h after
infiltration (hpi) while the abundance of m/z 100 increased (SI
Appendix, Fig. S1C), further suggesting that N-OH-Pip is un-
stable in planta and may convert to m/z 100 over time. N. ben-
thamiana leaves expressing two FMO1 active-site mutants,
FMO1 G17A/G19A or FMO1 G215A, did not produce N-OH-
Pip or m/z 100 at 28 hpi (Fig. 2 and SI Appendix, Fig. S1 D and
E). The abundance of the mutant proteins was less than WT
FMO1 at this time point (SI Appendix, Fig. S1F). At 48 hpi,
protein abundance was similar (SI Appendix, Fig. S1F); however,
N-OH-Pip was not detected in extracts expressing FMO1 G17A/
G19A or FMO1 G215A (SI Appendix, Fig. S1E). Taken together,
these data demonstrate that FMO1 can catalyze the hydroxylation
of Pip in planta (Fig. 2) and support the requirement of the pu-
N-OH-Pip Treatment of Arabidopsis Leaves Is Sufficient to Induce SAR.
To test if N-OH-Pip treatment alone is sufficient to induce SAR
and rescue the SAR deficiency of fmo1 mutants, we infiltrated
three lower leaves of Arabidopsis WT and fmo1 plants with
1
1
0 mM MgCl (Mock) or 10 mM MgCl containing 1 mM Pip or
2
2
mM N-OH-Pip. We used 1 mM of each metabolite because it
was previously established that watering plants with 1 mM Pip
elicits SAR in the leaves of WT but not fmo1 mutants (13). After
chemical incubation for 24 h, one upper leaf of each plant was
5
inoculated with a 1 × 10 cfu/mL suspension of P. syringae
pathovar maculicola strain ES4326 (Psm ES4326), a virulent
bacterium (Fig. 3A). At 3 d post inoculation (dpi), bacterial
growth was quantified (Fig. 3B), and leaf symptoms were pho-
tographed (Fig. 3C). Both WT and fmo1 plants treated with
N-OH-Pip contained significantly less Psm ES4326 in infected
upper leaves compared with plants treated with Mock or Pip
(
Fig. 3B). N-OH-Pip treatment also reduced symptom develop-
ment (i.e., leaf yellowing and tissue collapse) (Fig. 3C). These
results suggest that treatment of leaves with N-OH-Pip, but not
Pip, is sufficient to induce SAR and this does not require FMO1.
N-OH-Pip Treatment of Arabidopsis Leaves Is Sufficient To Induce
Metabolite Production and Gene Transcription Associated with SAR.
Next, we examined the ability of N-OH-Pip to systemically in-
duce the production of SAR-associated metabolites and mRNAs
in Arabidopsis leaves. Three lower leaves of WT and fmo1 plants
2 2
were infiltrated with 10 mM MgCl (Mock) or 10 mM MgCl
containing 1 mM Pip or 1 mM N-OH-Pip. After 48 h, the three
treated lower leaves and three untreated upper leaves were
pooled independently, and then analyzed by LC-MS and quan-
titative real-time PCR (qRT-PCR) for measurement of metab-
olites and mRNAs, respectively.
O
OH
NH
O
OH
OH
OH
FMO1
N
N
For metabolite profiling, we quantified Pip, N-OH-Pip, N-
OGlc-Pip, m/z 100, and two canonical defense metabolites, the
phytoalexin camalexin and SA-Glucoside (SA-Glc) (Fig. 4 A and
B). After infiltration of the lower leaves of both WT and fmo1
plants with N-OH-Pip, we observed accumulation of camalexin
and SA-Glc in both lower and upper leaves (Fig. 4B). Our
findings are consistent with reports showing that FMO1 is re-
quired to stimulate camalexin and SA-Glc synthesis and accu-
mulation in distal, uninfected leaves during SAR (31).
Although we did not detect free N-OH-Pip, we observed ac-
cumulation of N-OGlc-Pip and m/z 100 in both lower and upper
leaves of all plants (Fig. 4B), suggesting rapid metabolism to
these derivatives. These data show that local treatment of puri-
fied N-OH-Pip alone, even in the absence of infection, can in-
duce systemic changes in defense metabolite production across
the plant and this does not require FMO1. In contrast, the in-
filtration of lower leaves of WT or fmo1 plants with Pip did not
induce changes in N-OH-Pip metabolites, camalexin, or SA-Glc.
Given that fmo1 plants are unable to produce N-OH-Pip, the
detection of N-OH-Pip metabolites (N-OGlc-Pip and m/z 100) in
the upper leaves after N-OH-Pip treatment of lower leaves (Fig.
4B) provides evidence that one or more of these metabolites are
mobile. Taken together, these data indicate that N-OH-Pip or
rapidly formed derivatives move systemically through the plant
to induce metabolic changes during SAR.
Pip (fed)
N-OH-Pip
m/z = 100.075
08
07
06
05
04
03
02
01
00
1
1
1
1
1
1
1
1
1
A
A
A
A
5
5
A
5A
MO1
21
MO1
21
MO1
21
F
F
F
AG19
G
AG19
G
AG19
G
1
1
1
7
17
17
O
G
G
G
MO
M
MO1
F
F
F
MO1
MO1
F
MO1
F
F
Fig. 2. Overexpression of Arabidopsis FMO1 in N. benthamiana converts Pip
to N-OH-Pip. Ion abundance of Pip (green bars), N-OH-Pip (blue bars), and an
unknown metabolite, m/z = 100.075 (orange bars) in leaves of N. ben-
thamiana expressing Arabidopsis Col-0 WT FMO1-6×-His or two mutants,
FMO1-G17A/G19A-6×-His (a FAD binding mutant) or FMO1-G215A-6×-His
a NADP binding mutant). One millimolar Pip was coinfiltrated with an A.
tumefaciens strain carrying FMO1-6×-His, FMO1-G17A/G19A-6×-His, or
FMO1-G215A-6×-His, and then leaves were harvested 28 hpi. N-OH-Pip and
the unknown metabolite were identified as FMO1-dependent signals using
an untargeted analysis. Levels represent the mean ± STD of three biological
replicates. Levels reported as zero indicate no detection of metabolites.
Pathway represents proposed reactions occurring in N. benthamiana.
+
(
For transcript profiling, we quantified the mRNA abundance
for known SAR marker genes, which were reported to be in-
duced in untreated upper leaves of WT plants but not fmo1
plants in response to bacterial infection (i.e., Psm ES4326) of
lower leaves (32). These included genes dependent on SA
Chen et al.
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Fig. 3. Infiltration of N-OH-Pip into Arabidopsis Col-
WT and fmo1 leaves inhibits growth of virulent
0
Psm ES4326 in distal leaves. (A) Photograph showing
a representative plant and leaves used for SAR ex-
periments. Typically, leaf numbers 7, 8, and 9 were
used for chemical treatments (treated lower leaf)
and leaf numbers 11, 12, or 13 was challenged with
bacteria (upper leaf). (B) Titer of Psm ES4326 in
challenged upper leaves of Arabidopsis WT and
fmo1 plants. Three lower leaves of Col-0 WT and
fmo1 were infiltrated with 10 mM MgCl
2
(Mock) or
1
0 mM MgCl containing 1 mM Pip or 1 mM N-OH-
2
Pip. After chemical incubation for 24 h, one upper
leaf of each WT and fmo1 plant was inoculated
5
with a 1 × 10 cfu/mL suspension of Psm ES4326. The
number of Psm ES4326 was quantified 3 d later to
measure resistance of leaves. Bars (black, WT; gray,
fmo1) represent the mean ± STD of four biological
replicates. Asterisks denote the significant differ-
ences between indicated samples using a one-tailed
t test (**P < 0.01). The experiment was repeated
twice with similar results. (C) Symptoms of Arabi-
dopsis WT and fmo1 upper leaves infected with Psm
ES4326 following the chemical treatment described
in B. Leaves were photographed 3 dpi. The SAR ex-
periment was repeated twice with similar results.
[
(
pathogenesis-related protein (PR) 1], partially dependent on SA
PR2 and PR5), or independent of SA (ALD1; AGD2-like de-
N-OH-Pip Irrigation Induces SAR in Arabidopsis WT and fmo1 Plants.
Previously, Návarová et al. (13) showed that irrigation with Pip
was sufficient to recover SAR activity in ald1 plants but not in
fmo1 plants. We tested if irrigation with N-OH-Pip could simi-
larly induce SAR in WT and fmo1 plants. SAR assays were
performed as described previously (13) with slight modification.
WT and fmo1 plants were drenched with water, 1 mM Pip, or
1 mM N-OH-Pip by root application. One day later, three lower
fense response protein 1; SAG13, senescence-associated gene 13;
and ICS1, isochorismate synthase 1). We also monitored ALD1,
SARD4 (SAR deficient 4 protein), and FMO1, three genes in the
proposed N-OH-Pip metabolic pathway (Figs. 1C and 4A).
WT and fmo1 plants treated with N-OH-Pip accumulated the
highest levels of ICS1 and PR1 mRNA in treated lower and
untreated upper leaves, compared with plants treated with Mock
or Pip (Fig. 4C). Similar trends were observed for ALD1,
SARD4, PR2, PR5, and SAG13 mRNAs (Fig. 4C and SI Ap-
pendix, Fig. S5). N-OH-Pip–triggered accumulation of ALD1 and
SARD4 mRNA suggests that N-OH-Pip regulates ALD1 and
SARD4 transcription via a positive feedback loop. Accumulation
of Pip in leaves of WT and fmo1 plants treated with N-OH-Pip
implies that N-OH-Pip induction of ALD1 and SARD4 tran-
scription leads to increased synthesis of Pip in leaves. In addition,
N-OH-Pip treatment increased FMO1 mRNA levels in WT
leaves but not the fmo1 mutant (Fig. 4C). Our inability to detect
transcript for this fmo1 mutant is consistent with it being a null
mutant (16, 18). This implies that N-OH-Pip also positively
regulates FMO1 transcription via a positive feedback loop. No-
tably, induction of SAR gene expression was greater in N-OH-
Pip–treated plants compared with Pip-treated plants (Fig. 4C
and SI Appendix, Fig. S5), demonstrating that N-OH-Pip is a
more bioactive SAR molecule compared with Pip.
(local) leaves per plant were inoculated with 10 mM MgCl
(Mock) or a 5 × 10 cfu/mL suspension of Pst avrRpt2, an avir-
2
6
ulent strain. Two days later, the untreated upper leaves of each
5
plant were challenged with Mock or a 1 × 10 cfu/mL suspension
of Psm ES4326, a virulent strain. The titer of Psm ES4326 in the
infected upper leaves was determined at 3 dpi to quantify the
impact of Pip and N-OH-Pip treatment on the level of disease
resistance. An overview of the SAR experiment is shown in Fig. 5.
Before challenging upper leaves with Psm ES4326, lower
leaves infected with Pst avrRpt2 were yellow (chlorotic) and
collapsed by 2 dpi (Fig. 5B). We noticed that these symptoms
were significantly delayed in WT and fmo1 plants irrigated with
N-OH-Pip (Fig. 5B). Pip irrigation reduced leaf symptom de-
velopment in WT plants to a lesser degree, but not in fmo1 plants
(Fig. 5B). Metabolic profiling revealed that N-OGlc-Pip and m/z
100 were present in Pst avrRpt2-infected leaves of WT plants
irrigated with Pip or N-OH-Pip, and fmo1 plants irrigated with
N-OH-Pip (SI Appendix, Fig. S6). These results indicate that N-
OH-Pip treatment reduces symptom development of Arabidopsis
leaves in an FMO1-independent manner. The delay of symptom
development caused by N-OH-Pip and Pip could be attributed to
Collectively, these data demonstrate that exogenous treat-
ment of N-OH-Pip by leaf infiltration is sufficient to activate
metabolite production and SAR-associated gene transcription.
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Fig. 4. N-OH-Pip induces expression of SAR marker
genes and metabolites in treated lower leaves and
untreated upper leaves of Arabidopsis Col-0 WT
and fmo1 plants. (A) Proposed pathway of metabolite
and gene induction in N-OH-Pip–associated SAR. In
this study, FMO1 is hypothesized to convert Pip to
N-OH-Pip and unknown enzymes convert N-OH-Pip to
N-OGlc-Pip and m/z = 100.075. Pip biosynthesis re-
quires ALD1 activity and is partially dependent on
SARD4 activity (13, 21, 22). N-OH-Pip treatment leads
to the accumulation of camalexin and ALD1, SARD4,
FMO1, ICS1, and PR2 mRNAs. ICS1 converts chorismic
acid to isochorismic acid, leading to SA production. SA
is required for camalexin production in during SAR
(
31), and SA induces the expression of NPR1. NPR1 is a
transcription factor that regulates expression of PR1
and other defense-associated genes. In plants, SA-
Glucoside (SA-Glc) is thought to be nontoxic storage
form of SA (40). (B) Ion abundance of Pip, N-OGlc-Pip,
m/z = 100.075, camalexin and SA-Glc in leaves. Three
lower leaves of Col-0 WT and fmo1 were infiltrated
2 2
with 10 mM MgCl (Mock) or 10 mM MgCl contain-
ing 1 mM Pip or 1 mM N-OH-Pip. After chemical in-
cubation for 48 h, three treated lower (L) leaves and
three untreated upper (U) leaves of WT and fmo1
were analyzed by metabolite profiling and qPCR (C).
Bars (black, WT; gray, fmo1) represent the mean ±
STD of three biological replicates. (C) Relative ex-
pression of SAR marker genes in leaves: ALD1
(
AT2G13810), AGD2-like defense response protein 1;
SARD4 (AT5G52810), SAR deficient 4; FMO1
At1g19250), flavin-dependent monooxygenase 1;
ICS1 (At1g74710), isochorismate synthase 1; PR1
At2g14610), pathogenesis-related protein and
(
(
1
PR2 (At3g57260), β-1,3-glucanase 2. Bars (black, WT;
gray, fmo1) represent the mean ± STD of three bi-
ological replicates. The experiment was repeated
twice with similar results.
metabolite-induced defense priming in infected leaves or in-
herent bactericidal activity of the metabolites.
avrRpt2 in water-treated WT plants. Interestingly, N-OH-Pip
absorption by roots provided the strongest level of protection
(Fig. 5C). Psm ES4326 titers in Mock and Pst avrRpt2-inoculated
fmo1 plants were significantly less than those detected in water-
or Pip-treated fmo1 plants. Moreover, N-OH-Pip-induced pro-
tection in fmo1 plants was similar to that observed for Pst
avrRpt2-induced protection in WT plants (Fig. 5C). Collectively,
these data indicate that N-OH-Pip is a potent SAR-inducing
metabolite, and irrigation of plants with N-OH-Pip is also suf-
ficient to establish SAR in WT plants and complement the SAR
defect of fmo1 plants.
After challenging upper leaves with Psm ES4326, the titer of
Psm ES4326 in water-treated WT plants infected with Pst
avrRpt2 was lower than that in water-treated WT plants in-
oculated with Mock (Fig. 5C). These data indicate that Pst
avrRpt2 induces SAR under the conditions tested. By contrast,
Psm ES4326 titers were similar in water-treated fmo1 plants in-
oculated with Mock or Pst avrRpt2 (Fig. 5C). These data are
consistent with previous findings that fmo1 mutants are deficient
in SAR signaling (16, 18).
Irrigating plants with Pip and N-OH-Pip enhanced SAR, albeit
to different extents. Pip absorption by roots provided a moderate
but significant reduction in Psm ES4326 titer in Mock and Pst
avrRpt2-inoculated fmo1 plants (Fig. 5C). However, the level of
protection was less than SAR-induced protection elicited by Pst
N-OH-Pip Is Not Bactericidal. To rule out that N-OH-Pip is toxic to
bacterial growth in planta, we performed a minimum inhibitory
concentration (MIC) assay (33) to measure potential bactericidal
activity. Cultures of Psm ES4326, Pst DC3000, and Pst avrRpt2
Chen et al.
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Fig. 5. Root application of N-OH-Pip elicits local
defense and SAR in Arabidopsis Col-0 WT and fmo1
plants. (A) Experimental design of SAR assay. Each
Col-0 WT and fmo1 plant was treated with 10 mL of
water, 1 mM Pip, or 1 mM N-OH-Pip by root appli-
cation. One day later, three lower leaves of each
6
2
plant were infiltrated with 10 mM MgCl or a 5 × 10
cfu/mL suspension of Pst DC3000 expressing avrRpt2
Pst avrRpt2) in 10 mM MgCl . Two days later, one
upper leaf of each plant was inoculated with a 1 ×
(
2
5
1
0
cfu/mL suspension of Psm ES4326. The number of
Psm ES4326 in upper leaves was quantified 3 d later.
B) Phenotype of lower leaves inoculated with Pst
(
avrRpt2 after chemical treatment. Phenotypes of
four biological replicates were recorded 2 dpi with
Pst avrRpt2. Similar phenotypes were observed in
three independent experiments. (C) Titer of Psm
ES4326 in upper leaves of Col-0 WT (black bars) and
fmo1 (gray bars). Bars represent the mean ± STD of
three biological replicates. Asterisks denote the
significant differences between indicated samples
using a one-tailed t test (**P < 0.01; *P < 0.05; ns,
not significant). The experiment was repeated
three times with similar results.
were incubated with different concentrations (0–1 mM) of Pip,
N-OH-Pip, or SA and then grown at 28 °C for 36 h. SA was
included as a positive control because it inhibits multiplication of
Pseudomonas aeruginosa and Agrobacterium tumefaciens in vitro
WT and fmo1 leaves infected with Pst vector exhibited similar
electrolyte leakage (below 20%) when irrigated with water, Pip,
and N-OH-Pip (Fig. 6A), and no HR phenotypes were observed
for these infected plants at 5 hpi (Fig. 6B). WT plants irrigated
with water and infected with Pst avrRpt2 exhibited more elec-
trolyte leakage at 5 hpi than similarly treated fmo1 plants (Fig.
(
34, 35). Similar to previous studies, 1 mM SA inhibited the
multiplication of Psm ES4326, Pst DC3000, and Pst avrRpt2 in
vitro (SI Appendix, Fig. S7). However, neither Pip nor N-OH-Pip
inhibited Psm ES4326, Pst DC3000, and Pst avrRpt2 growth in
vitro. These data indicate that Pip and N-OH-Pip are not
bactericidal.
6A). HR symptoms were not observed at 8 hpi (Fig. 6B); how-
ever, full leaf collapse occurred by 10 hpi. Compared with water
irrigation, Pip irrigation resulted in a significant increase in
electrolyte leakage from WT leaves infected with Pst avrRpt2
(
(
Fig. 6A), with most (7/10) of the leaves undergoing HR by 8 hpi
Fig. 6B). No significant changes were detected for similarly
N-OH-Pip–Treated Arabidopsis Plants Exhibit a Faster Hypersensitive
Response. Next, we investigated if N-OH-Pip enhances ETI in
local, infected leaves by performing electrolyte leakage and HR
assays. WT and fmo1 plants were irrigated with water, 1 mM Pip,
or 1 mM N-OH-Pip. One day later, six to seven leaves of each
treated fmo1 leaves. Notably, N-OH-Pip irrigation resulted in a
significant increase in electrolyte leakage from both WT and
fmo1 leaves infected with Pst avrRpt2 (Fig. 6A). HR phenotypes
for the N-OH-Pip–irrigated plants appeared faster and were
more severe than those observed for the Pip-irrigated plants
(Fig. 6B). Taken together, these results show that N-OH-Pip
treatment stimulates AvrRpt2-elicited HR in plants compared
8
plant were inoculated with a 3 × 10 cells per mL suspension of
Pst DC3000 carrying an empty vector (Pst vector) or expressing
avrRpt2 (Pst avrRpt2). Ion leakage was quantified at 5 hpi (Fig.
6A), and HR symptoms were recorded at 8 hpi (Fig. 6B).
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with water and Pip treatment, demonstrating that N-OH-Pip
treatment also affects ETI.
Discussion
FMO1 Produced Metabolite N-OH-Pip Activates SAR. FMO1-
dependent regulation of defense signaling has emerged as a crit-
ical determinant of the establishment and amplification of SAR in
Arabidopsis (16, 31, 32). The SAR deficiency of fmo1 mutants
could be due to a failure to produce a priming signal, to recognize
defense signal(s), or to transmit defense signal(s) from infected
tissue to uninfected tissue. Our discovery that FMO1 produces N-
OH-Pip, a bioactive metabolite that is mobile in plants and
confers pathogen resistance in systemic tissues, reveals that the
key biological function of FMO1 is to produce the priming
signal for SAR. The accumulation of N-OH-Pip–derived me-
tabolites (N-OGlc-Pip and m/z 100) in untreated, distal leaves
of fmo1 plants following infiltration of N-OH-Pip in local leaves
indicates that one or more of these metabolites are transported
during SAR.
N-OH-Pip establishment of SAR in Arabidopsis is character-
ized by the induction of transcriptional and metabolic programs
in systemic plant tissue. Application of N-OH-Pip to local leaves
resulted in the robust activation of several SA-dependent and
SA-independent SAR marker genes in distal leaves (Fig. 4C and
SI Appendix, Fig. S5). This is consistent with reports showing that
FMO1 is required for SA-dependent and SA-independent de-
fenses (31, 32). N-OH-Pip also activated transcription of the N-
OH-Pip biosynthetic genes (ALD1, SARD4, and FMO1) in both
local and systemic leaves (Fig. 4C), highlighting the importance
of transcriptional feedback regulation for amplifying N-OH-Pip–
dependent signaling during SAR.
Remarkably, disease symptoms in Arabidopsis leaves were
dramatically reduced in leaves of bacterially infected plants
treated with N-OH-Pip (Figs. 3C and 5B). We speculated that N-
OH-Pip might directly affect bacterial growth and/or the ability
of bacteria to deliver T3S effectors to plant cells. However,
N-OH-Pip was not toxic to pathogen growth in culture (SI Ap-
pendix, Fig. S7) and did not inhibit the activation of ETI in re-
sistant Arabidopsis plants (Fig. 6). These data suggest that
N-OH-Pip stimulates or enhances plant-specific processes in
infected leaves to prevent their collapse and yellowing. What
these physiological processes are remains to be determined.
We also found that N-OH-Pip–treated leaves displayed
enhanced resistance in SAR assays (Fig. 5C) and are highly
sensitized for ETI (Fig. 6). This suggests that the level of N-
OH-Pip in tissues plays a critical role in establishing the mag-
nitude and timing of the disease resistance response. Consistent
with our findings, overexpression of FMO1 in Arabidopsis
resulted in enhanced basal resistance to the oomycete Hyalo-
peronospora parasitica and P. syringae pv. DC3000 (Pst), as well
as enhanced resistance to Pst avrRpt2 (17). In contrast, loss of
FMO1 function resulted in enhanced susceptibility to Pst and
H. parasitica (17, 18).
Fig. 6. AvrRpt2-elicited HR phenotype occurs earlier in Arabidopsis Col-
WT and fmo1 plants treated with N-OH-Pip compared with water or Pip.
0
Col-0 WT and fmo1 plant pots were treated with 10 mL of water, 1 mM Pip,
or 1 mM N-OH-Pip by root application. Twenty-four hours later, leaves of
8
Col-0 WT and fmo1 plants were infiltrated with a 3 × 10 cells per mL sus-
pension of Pst DC3000 with an empty vector (Pst vector) or avrRpt2 (Pst
avrRpt2). (A) Percent ion leakage of inoculated leaves at 5 hpi. Bars (black,
WT; gray, fmo1) represent the mean ± STD of nine randomly inoculated
leaves of three WT or fmo1 plants. Asterisks denote the significant differ-
ences between indicated samples using a one-tailed t test (**P < 0.01; *P <
0
.05). (B) HR phenotypes of inoculated leaves at 8 hpi. Fraction refers to
number of leaves showing HR of 10 randomly inoculated leaves. Experi-
ments were repeated three times with similar results.
Biochemistry of FMO1 and Chemistry of Pip Hydroxylation. Flavo-
protein monooxygenases (FMOs) catalyze incorporation of one
atom of molecular oxygen onto a nucleophilic substrate (36).
Similar to plant cytochromes P450 that activate and transfer
oxygen, the FMO family of genes has greatly expanded in plants,
suggesting they play critical roles in metabolism and fitness. In
Arabidopsis, 29 genes encode FMO-like proteins, compared with
only 5 in animals (37). These proteins have been divided into
three clades: Clade 1 includes FMO1 and a pseudogene. Clade
genes after pathogen challenge (SI Appendix, Fig. S9). Our dis-
covery that the FMO1 product N-OH-Pip is a bioactive SAR
metabolite is further evidence that FMOs have a privileged role
in plant defense metabolism. Moreover, it predicts that direct
products of FMO1 homologs in other plants may be novel nat-
ural products involved in defense priming.
Numerous nonproteinogenic amino acids have been described
in biological systems (38), yet N-OH-Pip has not previously
been reported. It remains a mystery why N-oxidation of pipecolic
acid, a metabolite produced in both plants and bacteria, has
evolved as a key step in generating an active signaling molecule
for SAR. The hydroxylamine functionality bears resemblance
to the hydroxamic acids required for binding iron in bacterial
2
includes the YUCCA group of 11 enzymes, which have been
well-studied and are associated with auxin biogenesis. Clade
has 16 enzymes, including an enzyme that S-oxygenates glu-
3
cosinolates, molecules critical for pathogen defense (37). Of
these, FMO1 is notably one of the most overexpressed metabolic
Chen et al.
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siderophores, and intriguingly, some bacterial siderophores are
now thought to be involved in signaling (39). Further studies are
required to determine if the hydroxylamine substituent of N-OH-
Pip has a functional role in its transport, perception, and/
or metabolism.
highlights the possibility for translating a chemical or metabolic
engineering approach to prime or enhance disease resistance in
plants under pathogen pressure.
Materials and Methods
Results from our transient expression experiments in N.
benthamiana indicate that N-OH-Pip may be unstable in planta
as FMO1-dependent levels of N-OH-Pip decreased from 28 to
Bacterial Strains and Growth Conditions. P. syringae pv. maculicola strain
ES4326 (Psm ES4326), P. syringae pv. tomato DC3000 (Pst DC3000), P. syrin-
gae pv. tomato carrying pVSP61 + avrRpt2 (Pst avrRpt2), P. syringae pv.
tomato carrying pVSP61 vector (Pst vector), E. coli strain DH5 alpha, and A.
tumefaciens strain C58C1 pCH32 were used in this study. Growth conditions
for all bacterial strains are described in SI Appendix, Table S1.
4
8 h while levels of m/z 100 increased (SI Appendix, Fig. S1D).
We speculate that O-glycosylation of N-OH-Pip could serve as a
stabilization mechanism and that N-OGlc-Pip may be the pri-
mary storage form of the molecule in Arabidopsis. This proposed
process is reminiscent of SA glycosylation and storage of SA-Glc
in response to pathogen infection (40). While we cannot de-
finitively rule out that m/z 100 is a product of extraction, its
presence could indicate a passive or active attenuation strategy.
For example, the level of N-OH-Pip could be controlled via
conversion to N-OGlc-Pip for storage or m/z 100 for de-
activation. Future work should investigate the precise roles of N-
OH-Pip and its derivatives in SAR.
Plant Materials and Growth Conditions. A. thaliana ecotype Col-0 (WT) and
fmo1 (SALK_026163) plants were grown in a controlled growth chamber
2
(22 °C, 80% relative humidity, 103 μmol/m per s of light intensity) on a 10-h
light/14-h dark cycle. Genotypes were confirmed by PCR using FMO1 gene-
specific primers and a T-DNA–specific primer (SI Appendix, Table S2). Four- to
5
-wk-old adult plants were used for SAR assays, qRT-PCR, and metabolite
profiling. For seedling hydroponics experiments, Arabidopsis seeds were ster-
ilized by suspending seeds in 50% ethanol for 1 min, washing three times in
sterile water, suspending in 50% bleach for 10 min, and washing three more
times in sterile water. Seeds were resuspended in 1× Murashige–Skoog (MS)
medium with vitamins (PhytoTechnology Laboratories) (pH 5.7) and vernalized
for 48 h at 4 °C. Seeds (15 ± 1) were placed into 3 mL of MS medium + 5 g/L
sucrose in wells of a six-well microtiter plate. Plates were sealed with Micro-
Presence of N-OH-Pip Biosynthesis Pathway in Other Plant Species.
Because SAR is a broad-spectrum defense strategy shared among
many plants, we mined available plant genomes to determine the
prevalence of enzymes involved in N-OH-Pip biosynthesis. We
tabulated the best BLAST hit between the Arabidopsis SARD4,
ALD1, and FMO1 proteins and those of sequenced plant
genomes, and found that >94% of species analyzed had
FMO1 homologs with greater than 50% amino acid identity (SI
Appendix, Fig. S8). All plants within the Brassicaceae that we
evaluated have enzymes with >88% amino acid identity to
Arabidopsis SARD4, ALD1, and FMO1, and these plants con-
tained the top five homologs to the Arabidopsis FMO1. In
Arabidopsis, the closest homolog to FMO1 has only a 74% amino
acid identity, and this protein originates from a likely pseudogene
of FMO1 that is missing a large internal coding region. The next
best BLAST hit has only a 26% amino acid identity to FMO1. The
scarcity of FMO1 homologs in Arabidopsis suggests that the ho-
mologs in other plants (and especially those of the Brassicaceae)
may have the same function. While the N-OH-Pip pathway may be
broadly conserved, it is possible that this pathway may have arisen
independently in the Brassicaceae and other metabolites may
mediate defense signaling outside this plant family.
2
pore tape (3M) and grown in a chamber at 50% humidity, 22 °C, and 100 μmol/m
per s photon flux under a 16-h light/8-h dark cycle. After 1 wk of growth, spent
medium was replaced with 3 mL of fresh MS medium + 5 g/L sucrose and plants
were grown for one additional week. N. benthamiana plants were grown in soil
on a growth shelf with a 16-h light cycle for 5 wk.
Elicitation Methods for Seedling Hydroponics Experiments. For bacterial elici-
tation, Pst was grown on LB agar plates at 30 °C. A single colony was picked
and grown to exponential phase in liquid LB at 30 °C. Cells were then
centrifuged at >13,000 × g and resuspended in sterile 1× MS media to an
OD600 of 0.2. Seventy microliters of the Pst suspension was added to each
seedling-containing well for elicitation. Plants were elicited 24 h before
sample harvest.
Plant Extraction for Metabolite Profiling. Arabidopsis seedlings were lyophi-
lized to dryness and ground using a ball mill (Retsch MM 400). A single 5-mm
steel ball was added to each sample and samples were shaken at 25 Hz for
2
min. Samples were resuspended in 10 mL of 80:20 MeOH:H
wet tissue and heated at 65 °C for 10 min. Samples were filtered through
.45-μm polytetrafluoroethylene (PTFE) filters and analyzed via LC-MS. Adult
leaf tissue was flash frozen and ground in liquid nitrogen using a mortar and
pestle. Samples were resuspended in 10 mL of 80:20 MeOH:H O per gram
wet tissue and heated at 65 °C for 10 min. Extracts were filtered through
.45-μm PTFE filters and analyzed via LC-MS.
2
O per gram
0
2
Is N-OH-Pip a Long-Distance Mobile Signal for SAR? The molecule
that initiates systemic defense signaling is still up for debate (7,
0
1
4). It is not clear if there is one molecule or a variety of mol-
LC-MS Methods. N-OGlc-Pip, SA-Glc, and camalexin were measured using
reverse-phase chromatography on an Agilent 1260 HPLC coupled to an
Agilent 6520 Q-TOF ESI mass spectrometer as previously described (24). A
ecules that orchestrate broad-spectrum plant disease resistance.
Several mobile metabolites (MeSA, AzA, G3P, and DA) are
known to prime resistance in uninfected tissues (7, 14) and some
require DIR1 (a lipid transport protein) (10–12, 41), suggesting
that DIR1 is required for the accumulation and/or transport of
molecules that induce SAR. Notably, AzA, DA, and Pip require
FMO1 for the initiation of SAR (10, 12, 13), highlighting the
possibility that FMO1 may produce one of the elusive long-
distance mobile signals critical for SAR. Our work demon-
strates that the product of FMO1, N-OH-Pip, is necessary and
sufficient to initiate SAR in local and systemic tissue, making it a
prime candidate for a long-distance SAR signal. It remains to be
determined how and where in the plant the N-OH-Pip signal is
perceived to initiate SAR.
5
-μm, 2 × 100 mm Gemini NX-C18 column (Phenomenex) was used for sep-
aration. The mobile phases were A [water with 0.1% formic acid (FA)] and B
acetonitrile (ACN) with 0.1% FA] and the following gradient was imple-
mented at a flow rate of 0.4 mL/min (percentages indicate percent B): 0–
[
3
5
0 min (3–50%), 30–45 min (50–97%), 45–50 min (97%), 50–51 min (97–3%),
1–55 min (3%). The MS was run in positive ion mode with conditions as
used previously (24). For MS/MS analysis, a fragmentor voltage of 10 V was
used with an m/z window of 1.3.
Pip, N-OH-Pip, and hypothesized N-hydroxy piperideine were measured
using hydrophilic interaction chromatography on an Agilent 1290 UHPLC
coupled to an Agilent 6545 Q-TOF ESI mass spectrometer. A 130-Å, 1.7-μm,
2.1 mm × 50 mm Acquity UPLC BEH Amide column (Waters) was used for
separation. The mobile phases were A (water, 10 mM ammonium formate,
0
.125% FA) and B (95% ACN, 10 mM ammonium formate, 0.125% FA), and
the following gradient was implemented at a flow rate of 0.6 mL/min
percentages indicate percent B): 0–1.5 min (100%), 1.5–6 min (100–40%), 6–
min (40%), 8–8.5 min (40–30%), 8.5–9.5 min (30–100%), 9.5–12 min
100%). The MS was run in positive ion mode with the following parameters:
In conclusion, the discovery of N-OH-Pip as a mobile signal
provides critical insight into how plants use small molecules to
resist the spread of infection. This work provides an opportunity
to address fundamental open questions in SAR biology, in-
cluding the mechanism of transport, signal perception, and signal
attenuation once a heightened defense response is no longer
required. The sensitivity of plants to N-OH-Pip treatment also
(
8
(
mass range: 30–1,700 m/z; drying gas: 250 °C, 12 L/min; nebulizer: 10 psig;
capillary: 3,500 V; fragmentor: 140 V; skimmer: 65 V; octupole 1 RF Vpp:
750 V; 333.3 ms/spectrum. For MS/MS analysis, a fragmentor voltage of 10 V
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was used with an m/z window of 1.3 for N-OH-Pip and a fragmentor voltage
of 20 V with an m/z window of 1.3 was used for hypothesized N-hydroxy
piperideine. The initial 0.5 min of each run was sent to waste to avoid salt
contamination of the MS.
performed to determine the relative expression. The fold change of each value
was normalized to the value of MgCl -treated local leaves of WT.
2
SAR Assay. SAR bacterial growth assays were performed as described (13) with
slight modification. Each plant pot was drenched with 10 mL of water, 1 mM
L-(-)-Pipecolinic acid (Pip) (Oakwood) or 1 mM N-OH-Pip. After 24 h, three
N-OH-Pip Chemical Synthesis. N-OH-Pip was synthesized from L-pipecolic acid
(
1
98% purity; Oakwood Chemical) using a modified protocol (42). To begin,
0.1 g of L-pipecolic acid was added to a cooled solution of 4.93 g of 88%
lower leaves of each plant were infiltrated with 10 mM MgCl
2
or a 5 ×
6
1
0 cfu/mL suspension of Pst avrRpt2 in 10 mM MgCl . Two days later, one
2
pure KOH (1 equivalent). Acrylonitrile (5.58 mL; 1.1 equivalents) was then
added drop wise to the solution over 5 min. The solution was stirred for 1.5 h
in an ice bath and another 1.5 h at room temperature. Then, the pH of the
solution was adjusted to 6.6 with 12 M HCl to quench the KOH. A rotary
evaporator was used to evaporate the solvent. Four hundred milliliters of
acetone was added to the residue, and the solution was brought to a boil.
After several minutes of boiling, the solution was filtered, and a rotary
evaporator was used to remove approximately half of the solvent. Then, the
residue from the filtration and fresh acetone was added back into the
remaining filtrate, and the solution was brought to a boil, filtered, and set
on the rotary evaporator. The cycle of heating the solution and filtering was
repeated five times. Two hundred milliliters of filtrate from the fifth cycle
was then stored at −20 °C overnight to recrystallize.
5
upper leaf of each plant was inoculated with a 1 × 10 cfu/mL suspension of
Psm ES4326, and then plants were covered with a dome to maintain hu-
midity. The titer of Psm ES4326 in the upper leaves was quantified at 3 dpi by
homogenizing leaves discs in 1 mL of 10 mM MgCl
dilutions on NYGA medium with rifampicin (100 μg/mL), incubating plates at
8 °C for 2 d, and then counting bacterial colonies. Three plants were used
2
, plating appropriate
2
per condition, and the experiment was repeated more than three times.
Construction of FMO1 Mutants. The ORF of FMO1 lacking the stop codon was
amplified from Arabidopsis Col-0 WT cDNA by PCR using FMO1-specific
primers (SI Appendix, Table S2) and cloned into the pCR8/GW/TOPO vector
(
Life Technologies). Two alanine substitution mutants, FMO1(G17A/G19A)
and FMO1(G215A), were generated using pCR8/GW/TOPO-FMO1 as tem-
plate and fmo1 mutant primers (SI Appendix, Table S2). All constructs were
confirmed by DNA sequence analysis. WT and mutant FMO1 cDNAs were
subcloned into pEAQ-HT-DEST3 (45) to create C-terminal 6× His-tagged fu-
sion proteins. Plasmids were introduced into E. coli DH5 alpha and A.
tumefaciens C58C1 by heat shock transformation.
Crystallized product (2-cyanoethyl-pipecolic acid; 3.78 g) was mixed with
6
0 mL of MeOH and 4.80 g of 70% metachloroperoxybenzoic acid (mCPBA)
(
1 equivalent) was added to 20 mL of MeOH. The mCPBA solution was added
drop wise to the cooled 2-cyanoethyl-pipecolic acid slurry over 30 min. The
slurry was stirred for an additional hour in an ice bath, and as the reaction
progressed, the slurry dissolved into solution. Then, 300 mL of precooled
diethyl ether was added to the reaction and the mixture was stored at
−
20 °C overnight to recrystallize.
Crystallized product (2-cyanoethyl-pipecolic acid oxide; 0.5 g) was dis-
Transient Expression in N. benthamiana. Agrobacterium strains harboring the
pEAQ-gene constructs were grown on LB agar plates with the appropriate
antibiotics. After 48 h of growth, cells were removed from plates using an
inoculation loop and resuspended in 1 mL of LB. Cells were centrifuged at
4,000 × g for 5 min, the supernatant was removed, and cells were resus-
pended in 1 mL of Agrobacterium induction medium (10 mM MES buffer,
solved in 150 mL of acetone in a 250-mL flask with a short path distillation
head. Acetone was slowly distilled drop by drop for 3 h, and fresh acetone
was periodically added to keep the original volume. Then, a rotary evapo-
rator was used to evaporate the majority of the solvent and the remainder
was evaporated to dryness under reduced pressure.
2
10 mM MgCl , 150 μM acetosyringone, pH 5.7) and incubated at room
1
13
H and C NMR spectra were taken of a 25 mM solution of N-OH-Pip in D
2
O
temperature with shaking for 2 h. Cells were then diluted to a final OD600 of
0.3 in induction medium. In tests with supplemented Pip, cells were diluted
to a final OD600 of 0.3 in induction medium + 1 mM Pip. These solutions
were then infiltrated into the underside of N. benthamiana leaves (three
leaves per plant) using a needleless 1-mL syringe. Plants were grown on a
growth shelf with a 16-h light/8-h dark cycle for 28 or 48 h before sample
harvest for metabolic analysis and immunoblot. Total protein of each sample
was extracted from two leaf discs (1-cm diameter per disk) by using urea
buffer (8 M urea, 15% β-mercaptoethanol, 3× Laemmli buffer). Proteins
were separated by 12% SDS/PAGE analysis and transferred to a PVDF
membrane and visualized by Ponceau S red staining before immunoblot
analysis. FMO1-6×-His, FMO1(G17A/G19A)-6×-His, FMO1(G215A)-6×-His
proteins were visualized by chemiluminescence using anti-His (Qiagen),
peroxidase-conjugated secondary antibodies (Bio-Rad), and ECL reagent
with a Varian Inova 500 NMR spectrometer. The following parameters were
used for H NMR spectra: temperature: ambient; probe: 5-mm PFG switchable;
scan number: 16; receiver gain: 40; relaxation delay: 0; pulse width: 8; fre-
quency: 499.75 Hz. The following parameters were used for C NMR spectra:
temperature: ambient; probe: 5-mm PFG switchable; scan number: 120; re-
ceiver gain: 54; relaxation delay: 0.5; pulse width: 7; frequency: 125.67 Hz.
1
13
Chemical Treatment of Leaves for Bacterial Growth Assay. Leaf numbering was
performed according to refs. 43 and 44. Three lower leaves (leaf nos. 7–9) of
WT and fmo1 Arabidopsis plants (4- to 5-wk-old) were infiltrated with
1
0 mM MgCl
2 2
, 1 mM Pip in 10 mM MgCl , or 1 mM N-OH-Pip in 10 mM
MgCl . After 24 h, one untreated upper leaf (leaf nos. 11, 12, or 13) of each
2
5
plant was inoculated with a 1 × 10 cfu/mL suspension of Psm ES4326. The
inoculated plants were then covered with a dome to maintain humidity. The
titer of Psm ES4326 in the upper leaves was quantified at 3 dpi by homog-
(
GE Biosciences).
enizing leaves discs in 1 mL of 10 mM MgCl
on nutrient yeast glycerol medium supplemented with 1.5% wt/vol agar
NYGA) with rifampicin (100 μg/mL), incubating plates at 28 °C for 2 d, and
then counting bacterial colonies. Four biological repeats were performed
per treatment in two independent experiments.
2
, plating appropriate dilutions
Electrolyte Leakage and Hypersensitive Reaction Assays. Electrolyte leakage
and hypersensitive reaction (HR) assays were performed according to ref. 46.
Thirty- to 32-d-old WT and fmo1 plants were irrigated with 10 mL of water,
1 mM Pip, or 1 mM N-OH-Pip. One day later, six to seven random leaves of
(
8
each plant were inoculated with a 3 × 10 cells per mL suspension of Pst
DC3000 (vector) or Pst DC3000 (avrRpt2) and then incubated at room tem-
perature under lights. For electrolyte leakage assay, at 5 hpi, three leaf discs
Chemical Treatment of Leaves for qRT-PCR and Metabolite Profiling. Three
lower leaves (leaf nos. 7–9) of WT and fmo1 Arabidopsis plants (4- to 5-wk-
(
7 mm diameter) of each plant were pooled and floated in 20 mL of water in
Petri dishes. Five minutes later, the leaf discs were transferred to a 15-mL
tube containing 3 mL of water and incubated at room temperature for 1 h
with shaking. Conductivity of each sample before and after boiling was
measured using an electrical conductivity meter (Spectrum Technologies).
The percentage of electrolyte leakage was calculated as conductivity before
boiling/conductivity after boiling. For HR assays, leaf phenotypes of WT and
fmo1 for each condition were record at 8 hpi. Three plants were used per
condition, and the experiment was repeated three times.
old) were infiltrated with 10 mM MgCl
2 2
, 1 mM Pip in 10 mM MgCl , or 1 mM
N-OH-Pip in 10 mM MgCl . After 48 h, the three treated lower leaves and
2
three untreated upper leaves (leaf nos. 11–13) were harvested, pooled, re-
spectively, and then frozen in liquid nitrogen. Frozen tissue was pulverized
and divided into two aliquots: one for qRT-PCR and the other for metabolic
profiling. Three biological repeats were performed per treatment in two
independent experiments.
RNA Isolation and qRT-PCR. Total RNA was isolated from leaves using TRIzol
reagent (Invitrogen) according to the manufacturer’s instructions. Two micro-
grams of RNA were used to synthesize cDNA by oligo dT and reverse tran-
scriptase. For qRT-PCR, each cDNA sample was amplified with gene-specific
primers (SI Appendix, Table S2) using Green Taq DNA polymerase (GenScript)
with EvaGreen Dye (Biotium) and the MJ Opticon 2 (Bio-Rad). UBC21 (Ubiquitin-
Conjugating Enzyme 21; At5g25760) mRNA abundance was used to normalize
ACKNOWLEDGMENTS. We thank Russ Li and Russ Stabler for assistance pre-
paring synthetic N-OH-Pip and George Lomonossoff (John Innes Centre) for
providing pEAQ plasmid. This work was supported by an HHMI and Simons
Foundation Grant 55108565 (to E.S.S.), NIH DP2 Grant AT008321 (to E.S.S.),
National Science Foundation Graduate Research Fellowship DGE-1656518 (to
E.C.H.), National Science Foundation Grant IOS-1555957 (to M.B.M.), Bina-
tional Science Foundation Grant 2011069 (to M.B.M.), and Ministry of Sci-
ence and Technology of Taiwan Grant 105-2917-I-564-093 (to Y.-C.C.).
−ΔΔCt
the expression value in each sample. The comparative Ct method (2
) was
Chen et al.
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