International Journal of
Molecular Sciences
Article
Strigolactones Modulate Salicylic Acid-Mediated Disease
Resistance in Arabidopsis thaliana
Miyuki Kusajima 1,2 , Moeka Fujita 2 , Khamsalath Soudthedlath 3 , Hidemitsu Nakamura 1 , Koichi Yoneyama 4 ,
Takahito Nomura 4 , Kohki Akiyama 5 , Akiko Maruyama-Nakashita 3 , Tadao Asami 1 and Hideo Nakashita 2, *
Citation: Kusajima, M.; Fujita, M.;
Soudthedlath, K.; Nakamura, H.;
Yoneyama, K.; Nomura, T.;
Akiyama, K.;
Maruyama-Nakashita, A.; Asami, T.;
Nakashita, H. Strigolactones
Modulate Salicylic Acid-Mediated
Disease Resistance in Arabidopsis
thaliana. Int. J. Mol. Sci. 2022, 23, 5246.
https://doi.org/10.3390/
ijms23095246
Academic Editors: Maurizio Battino,
Dilantha Fernando and
Vicent Arbona
Received: 29 December 2021
Accepted: 6 May 2022
Published: 8 May 2022
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Attribution (CC BY) license (https://
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4.0/).
Int. J. Mol. Sci. 2022, 23, 5246. https://doi.org/10.3390/ijms23095246
1 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan;
kusajima@g.ecc.u-tokyo.ac.jp (M.K.); ahide87@g.ecc.u-tokyo.ac.jp (H.N.); asami@g.ecc.u-tokyo.ac.jp (T.A.)
2 Department of Bioscience and Biotechnology, Fukui Prefectural University, Fukui 910-1195, Japan;
pt-fujita@fpu.ac.jp
3 Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 819-0395, Japan;
khamsalath.soudthedlath.682@s.kyushu-u.ac.jp (K.S.); amaru@agr.kyushu-u.ac.jp (A.M.-N.)
4 Center for Bioscience Research and Education, Utsunomiya University, Tochigi 321-8505, Japan;
yone2000@sirius.ocn.ne.jp (K.Y.); tnomura@cc.utsunomiya-u.ac.jp (T.N.)
5 Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 599-8531, Japan;
akiyama@biochem.osakafu-u.ac.jp
* Correspondence: nakashita@fpu.ac.jp; Tel.: +81-776-61-6000
Abstract: Strigolactones are low-molecular-weight phytohormones that play several roles in plants,
such as regulation of shoot branching and interactions with arbuscular mycorrhizal fungi and
parasitic weeds. Recently, strigolactones have been shown to be involved in plant responses to abiotic
and biotic stress conditions. Herein, we analyzed the effects of strigolactones on systemic acquired
resistance induced through salicylic acid-mediated signaling. We observed that the systemic acquired
resistance inducer enhanced disease resistance in strigolactone-signaling and biosynthesis-deficient
mutants. However, the amount of endogenous salicylic acid and the expression levels of salicylic acid-
responsive genes were lower in strigolactone signaling-deficient max2 mutants than in wildtype plants.
In both the wildtype and strigolactone biosynthesis-deficient mutants, the strigolactone analog GR24
enhanced disease resistance, whereas treatment with a strigolactone biosynthesis inhibitor suppressed
disease resistance in the wildtype. Before inoculation of wildtype plants with pathogenic bacteria,
treatment with GR24 did not induce defense-related genes; however, salicylic acid-responsive defense
genes were rapidly induced after pathogenic infection. These findings suggest that strigolactones
have a priming effect on Arabidopsis thaliana by inducing salicylic acid-mediated disease resistance.
Keywords: Arabidopsis; disease; phytohormones; strigolactones; salicylic acid; systemic acquired
resistance; GR24; strigolactone biosynthesis inhibitor; ethylene
1. Introduction
Plants are exposed to a range of biotic and abiotic stress factors, such as pathogen
attack, insect herbivory, and environmental stress. To adapt to such adverse conditions,
plants have evolved unique defense mechanisms that are regulated by phytohormones
such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA).
These phytohormones contribute to biotic stress responses through a complex network
involving synergistic and antagonistic interactions, although SA- and JA/ET-mediated
defense responses are important for the resistance against biotrophic and necrotrophic
pathogens, respectively [1–6].
In plant immunity, pathogen-associated molecular patterns (PAMPs) are first rec-
ognized by receptors on the surface of plant cells, activating a basal resistance called
PAMP-triggered immunity (PTI). Virulent pathogens have developed effector proteins
that suppress this PTI mechanism of host plants; however, resistant plants have acquired
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022, 23, 5246 2 of 14

a strong immune mechanism, called effector-triggered immunity (ETI), which overcomes

pathogenic effectors [7]. Systemic acquired resistance (SAR), induced by SA, is a potent
innate immunity system in plants that is effective against a wide range of pathogens [8].
The induction of SAR in infected plants is accompanied by SA accumulation through
the increased expression of SA synthase, ISOCHORISMATE SYNTHASE 1 (ICS1), and
pathogenesis-related (PR) genes [9]. Further, a reduction of the SA-receptor protein NPR1
is reportedly important for SAR induction, to which an increase in total glutathione (GSH)
level contributes [10]. Additionally, probenazole and its derivative, 1, 2-benzisothiazol-
3(2H)-one-1, 1-dioxide (BIT), induce SAR by activating its signaling pathway upstream of
SA [11,12]. Furthermore, several studies have reported SAR as an effective strategy for dis-
ease control in plants [11–16], and priming of phytohormone-mediated disease resistance
has been reported as a different type of plant immunity system. Although only weak or no
expression at all of the major defense-related genes through SA- or JA-mediated signaling
pathways are observed in primed plants by interaction with arbuscular mycorrhizal (AM)
fungi [17,18], nonpathogenic bacteria [19,20], or treatment with certain chemicals [21], they
can still respond more rapidly and strongly to pathogenic infection to protect themselves.
Strigolactones (SLs) are carotenoid-derived metabolites that act as germination stimu-
lants on root parasitic weeds of the genus Striga [22]. Furthermore, SLs secreted by host
plants induce mycelial branching in rhizosphere AM fungi and stimulate symbiosis [23].
Recently, SLs have been reported to inhibit shoot branching [24,25]. Thus, SLs have been
categorized as a novel class of phytohormones.
Analysis using carotenoid-deficient mutants and carotenoid biosynthesis inhibitors
suggests that SLs are synthesized from carotenoids [26]. Moreover, several mutants, includ-
ing Arabidopsis more axillary growth (max) exhibiting increased shoot branching, have been
identified as SL biosynthesis mutants. Arabidopsis max3 and max4 mutants have mutations
in CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8, respectively, and their
phenotypes are restored upon treatment with synthetic SLs, indicating that they are SL-
deficient mutants [25]. Additionally, AtD27, which exhibits β-carotene isomerase activity
and converts all-trans-β-carotenes, is reportedly involved in SL biosynthesis. Furthermore,
carlactone (CL), an SL biosynthetic intermediate, is synthesized in plants from all-trans-
β-carotene using three different enzymes [27]. Mutant max1 is deficient in cytochrome
P450 oxygenase CYP711A, which converts CL into carlactonic acid in the SL biosynthetic
pathway [28]. Similarly, the shoot-branching mutant, SL-insensitive max2, lacks the F-box
protein that plays a role in recognizing SLs [29]. In addition, the SL-insensitive mutant d14
has a mutation in the gene encoding an α/β-hydrolyzing enzyme. After binding with SL,
the SL receptor D14 interacts with SCF (SKP1-CUL1-F-box) containing the F-box protein
MAX2, which transmits SL-mediated signals by degrading target proteins through the
ubiquitin–proteasome system [30]. The biological activities of SLs have been analyzed
using SL-related mutants and bioactive chemicals. Shoot branching is inhibited by the
synthetic SL-analog GR24, and it is improved by the SL-biosynthesis inhibitor TIS108,
which inhibits CYP711A activity [31].
SLs are also involved in plant responses to environmental stress, as in drought [32,33],
salt [34], and cold [35] resistance. As SLs are carotenoid derivatives that alleviate environ-
mental stress as ABA does, interactions between SL and ABA have been proposed. Thus,
for example, Arabidopsis SL biosynthesis-deficient (max3 and max4) and SL signaling-
deficient (max2) mutants exhibit low drought tolerance and are less sensitive to ABA [36].
However, some reports indicate that Arabidopsis max2 is less drought-tolerant and more
ABA-sensitive, whereas max3 and max4 are as tolerant as the wildtype (WT) [37]. Al-
together, these studies suggest that MAX2-mediated SL signaling interacts with ABA
signaling during stress tolerance.
Furthermore, recent studies suggest the specific role of SLs in plant responses to biotic
stress: Tomato SL biosynthesis-deficient mutant ccd8 exhibits increased susceptibility to
Botrytis cinerea and Alternaria alternata [38]. Similarly, rice SL signaling-deficient mutant d14
and SL biosynthesis-deficient mutant d17 are susceptible to Magnaporthe oryzae [39]. Further,
 to Botrytis cinerea and Alternaria alternata [38]. Similarly, rice SL signaling-deficient mu
d14 and SL biosynthesis-deficient mutant d17 are susceptible to Magnaporthe oryzae
Further, Arabidopsis max2, max3, max4, and d14 mutants are highly susceptible to
biotrophic virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000
Int. J. Mol. Sci. 2022, 23, 5246 [40,41]. The model compatible-interaction system between Arabidopsis and Pst, in w
3 of 14
the SA-mediated defense signaling is activated upon infection, has contributed to cla
ing the detailed mechanisms at work behind the plant immune system [42]. However
mechanismmax2,
Arabidopsis underlying the and
max3, max4, effects
d14 of SLs on
mutants areSA-mediated disease
highly susceptible resistance
to the biotrophicremains
virulent
clear. Therefore, to gainPseudomonas
bacterial pathogen syringae pv. tomato
a better understanding of DC3000
the role(Pst) [40,41].
of SL The model
in disease resistance
compatible-interaction system between Arabidopsis and Pst, in which the SA-mediated
aim of this study is to determine the effects of SLs on SA-mediated defense response
defense signaling is activated upon infection, has contributed to clarifying the detailed
Arabidopsis upon Pst infection.
mechanisms at work behind the plant immune system [42]. However, the mechanism
underlying the effects of SLs on SA-mediated disease resistance remains unclear. Therefore,
2. gain
to Results
a better understanding of the role of SL in disease resistance, the aim of this study
is to determine
2.1. Relationshipthebetween
effects ofSLs
SLsand
on SA-mediated
SA Signalingdefense responses
Pathway in Arabidopsis upon
in Arabidopsis
Pst infection.
To evaluate the effects of SLs on SAR induction, we examined SAR induction in
SLResults
2. biosynthesis-deficient mutants max1, max3, and max4 and in the SL signaling-defic
2.1. Relationship
mutant max2. between
SAR wasSLs induced
and SA Signaling
by BITPathway in Arabidopsis
applied using soil drench treatment to avoi
To evaluate
dilution duringthe effects of SLs on
dip-inoculation withSAR theinduction,
pathogen. weFurthermore,
examined SARtoinduction
avoid the ininfluen
the SL biosynthesis-deficient mutants max1, max3, and max4 and in
stomatal aperture on disease resistance, we covered plants with plastic wrap and m the SL signaling-
deficient mutant max2. SAR was induced by BIT applied using soil drench treatment to
tained high humidity for 12 h before and after pathogen inoculation.
avoid its dilution during dip-inoculation with the pathogen. Furthermore, to avoid the
Theofeffects
influence stomatalof BIT treatment
aperture on SAR
on disease induction
resistance, in theplants
we covered SL-related mutants
with plastic wrapwere ev
atedmaintained
and using a pathogenic
high humidity bacterial
for 12 hinoculation test,
before and after defenseinoculation.
pathogen gene expression profiling,
the estimation of endogenous SA content. Resistance to Pstmutants
The effects of BIT treatment on SAR induction in the SL-related was assessed by measu
were evalu-
ated using growth
bacterial a pathogenic
in thebacterial inoculation
leaf tissues. At test,
3 d defense gene expression
after inoculation, profiling,
leaves of alland
the SL-rel
the estimation
mutants of endogenous
treated SA content.> Resistance
with BIT exhibited to Pst bacterial
10-fold lower was assessed by than
titers measuring
those of the
bacterial growth in the leaf tissues. At 3 d after inoculation, leaves of all the SL-related mu-
treated (control) plants as well as WT (Col-0) plants (Figure 1). Reverse transcript
tants treated with BIT exhibited > 10-fold lower bacterial titers than those of the untreated
quantitative
(control) plantspolymerase
as well as WTchain
(Col-0)reaction (RT-qPCR)
plants (Figure 1). Reverseindicated that upon treatment w
transcription-quantitative
BIT, the expression
polymerase levels
chain reaction of PR1 (At2g14610),
(RT-qPCR) indicated that PR2
upon(At3g57260),
treatment withandBIT,PR5 (At1g75040) w
the expres-
upregulated in the SL-related mutants and the WT, compared to the control plants
sion levels of PR1 (At2g14610), PR2 (At3g57260), and PR5 (At1g75040) were upregulated in (Fi
the
2). SL-related
Moreover,mutants and the WT,
BIT treatment compared toupregulated
significantly the control plants
ICS1(Figure 2). Moreover,
(At1g74710) expression
BIT treatment significantly upregulated ICS1 (At1g74710) expression and increased the
increased the levels of free and total endogenous SA (free SA + SA glucoside) in the
levels of free and total endogenous SA (free SA + SA glucoside) in the SL-related mutants
related mutants and in the WT (Figures 2 and 3). These findings suggest that SAR ca
and in the WT (Figures 2 and 3). These findings suggest that SAR can be induced in SL
induced in SL
biosynthesis- andbiosynthesis- and mutants.
signaling-deficient signaling-deficient mutants.

Figure1.1.Effects
Figure Effects of BIT
of BIT treatment
treatment on growth
on growth of Pseudomonas
of Pseudomonas syringae pv.syringae pv. tomato
tomato DC3000 DC3000 in Arabi
in Arabidopsis
sis leaf
leaf tissues.
tissues. Three-week-old
Three-week-old plants plants were with
were treated treated
water with water(–)(control)
(control) or BIT (1(–) or BIT(+)
mg/pot) (1 mg/po
usingthe
using thesoil
soil drenching
drenching method
method 3 d before
3 d before inoculation
inoculation with Pst (2 with
× 10Pst (2 × 105 Leaves
5 CFU/mL). CFU/mL).
were Leaves
homogenizedatat
homogenized 3d3 post-inoculation,
d post-inoculation, andnumber
and the the number
of CFUofwas CFU was estimated
estimated using
using their growththeir growt
nutrient
on nutrient broth
brothagar
agarplates.
plates. Values aremeans
Values are means±±SE SEfrom
from sixsix replicates,
replicates, eacheach
withwith
threethree
leaves.leaves. Si
icant differences
Significant between
differences betweenthe
thecontrol andBIT-treated
control and BIT-treated leaves
leaves are indicated
are indicated by * (unpaired
by * (unpaired t-test, t-tes
p < 0.05). Different letters indicate significant differences between accessions of the same treatment
(Tukey’s multiple comparison test, p < 0.05).
 Int. J. Mol. Sci. 2022,Int.
23,J.xMol.
FORSci. 2022,
PEER 23, x FOR PEER REVIEW
REVIEW 4 of 14 4 of

Int. J. Mol. Sci. 2022, 23, 5246 4 of 14

0.05). Different
0.05). Different letters letters indicate
indicate significant significant
differences differences
between between
accessions of theaccessions of the same treatm
same treatment
(Tukey’s multiple(Tukey’s multiple
comparison test, comparison
p < 0.05). test, p < 0.05).

Figure 2. Figure
Expression
2. Expression 2.defense-related
Expression ofgenes
ofofdefense-related defense-related
genes inin genes
BIT-treated
BIT-treated in BIT-treated
plants.
plants. Leaves
Leaves of plants. Leaves ofplants
of three-week-old
three-week-old three-week-old
plants pla
were sprayed
were sprayed with were
with sprayed
water
water with(–)
(control)
(control) water
(–)or (control)
or11mMmMBIT BIT (–)
(+) orand
and
(+) 1collected
mM BIT (+)
collected and
after collected
3 d.
after 3Transcriptafter
d. Transcript 3 d.
levels Transcript
were
levels were levels w
normalized usingnormalized
normalizedusing theexpression
the expressionusingofthe
of UBQ2
expression
UBQ2 (At2g36170)
of UBQ2
(At2g36170) in in
(At2g36170)
thethe same
same
in theRelative
samples.
samples.
same samples.
Relative mRNA mRNA Relative mRNA lev
levels
levels among
among treatments among treatments
are presented. are presented.
Values are meansValues± SE (nare means
= 3). ± SE (ndifferences
Significant = 3). Significant
betweendifferences
the between
treatments are presented. Values are means ± SE (n = 3). Significant differences between the control
controlplants
control and BIT-treated and BIT-treated plants
are indicated by *are indicated
(unpaired by * p(unpaired
t-test, t-test, p <letters
< 0.05). Different 0.05).indicate
Different letters indic
and BIT-treated
significant plants
differences are indicated
significant
between by between
differences
accessions* (unpaired t-test,
of the accessions
same p of
< 0.05).
treatment Different
the(Tukey’s
same letters
treatment
multiple indicate
(Tukey’s
comparison significant
multiple
test, comparison t
differences
p < 0.05). between accessions
p < 0.05). of the same treatment (Tukey’s multiple comparison test, p < 0.05).

Figure 3.
Figure 3. Accumulation
Accumulation ofofAccumulation
free
freeand totalof
andtotal SA free
SA and total SA
ininBIT-treated
BIT-treated in BIT-treated
plants. Plants
plants. plants.
were
Plants Plants
treated
were with
treated were treated with wa
water
with water
(control) (–) or 1 (control)
mM BIT (–) or
(+). 1 mMwere
Leaves BIT (+). Leaves3were
harvested d harvested
after 3 dand
treatment, afterfree
treatment,
and and
total SA free
(freeand total SA (f
(control) (–) or 1 mM BIT (+). Leaves were harvested 3 d after treatment, and free and total SA
SA + SA glucoside) SA + levels
SA glucoside) levels were
were quantified usingquantified using high-performance
high-performance liquid chromatograp
liquid chromatography.
(free SA + SA glucoside) levels were quantified using high-performance liquid chromatography.
Significant differences between control and BIT-treated plants are indicated by * (unpaired t-test,
p < 0.05). Different letters indicate significant differences between accessions of the same treatment
(Tukey’s multiple comparison test, p < 0.05).
 (Tukey’s multiple comparison test, p < 0.05).

In the absence of SAR induction, bacterial growth on the leaf tissues was significantly
higher in the mutant max2 than in WT plants, whereas bacterial growth in mutants max1,
max3, and max4 was intermediate between those of max2 and WT (Figure 1). This finding
Int. J. Mol. Sci. 2022, 23, 5246 5 of 14
suggested that SL-mediated signaling is partially involved in basal resistance against Pst.
Although ICS1 levels in BIT-treated plants did not differ between the SL-related mutants
and the WT, SA levels were altered among these plants, with max2 and max4 exhibiting
In the absence
the lowest of SAR
and highest induction,
levels bacterial
of both free growth
and total SA,on the leaf tissues
respectively was significantly
(Figures 2 and 3).
higher in the mutant max2 than in WT plants, whereas bacterial growth
Consistent with SA levels, the levels of expression of PR genes after BIT treatment in mutants max1,
max3, and max4 was intermediate between those of max2 and WT
were significantly lower in max2 than in WT (Figure 2). Thus, BIT-mediated activation of (Figure 1). This finding
suggested
SA biosynthesis that SL-mediated
and signaling signaling
pathways is partially
were weakerinvolved in basal
in max2 thanresistance
in WT, which against canPst.
be
Although ICS1 levels in BIT-treated plants did not differ between the
associated with high bacterial titer in the leaves of max2 (Figure 1). Additionally, the level SL-related mutants
and the WT, SA
of activation of levels were altered signaling
the SA-mediated among these plants,and
pathway max2 and
withdisease max4 exhibiting
resistance in other the SL-
lowest and highest levels of both free and total SA, respectively
related mutants was intermediate between those of max2 and WT (Figures 1 and 2). These (Figures 2 and 3).
Consistent
findings suggestwith that SA
the levels,
absencethe levels
of SL of expression
signaling affected ofthePR genes after signaling
SA-mediated BIT treatment path-
were significantly lower in max2 than in WT (Figure 2). Thus,
way and, consequently, the degree of disease resistance against Pst. Furthermore, BIT-mediated activation of
the dif-
SA biosynthesis and signaling pathways were weaker in max2 than in WT, which can be
ference between SL biosynthesis- and signaling-deficient mutants can be explained by the
associated with high bacterial titer in the leaves of max2 (Figure 1). Additionally, the level of
fact that the inhibition of SL-mediated signaling was stronger in the mutant max2, which
activation of the SA-mediated signaling pathway and disease resistance in other SL-related
is defective in the F-box protein MAX2, than in other biosynthesis-deficient mutants.
mutants was intermediate between those of max2 and WT (Figures 1 and 2). These findings
suggest that the absence of SL signaling affected the SA-mediated signaling pathway and,
2.2. Effects of the Synthetic SL Analog GR24 on SA-Mediated Disease Resistance
consequently, the degree of disease resistance against Pst. Furthermore, the difference
between To reveal the effectsand
SL biosynthesis- of SLs on SA-mediatedmutants
signaling-deficient diseasecan resistance, we examined
be explained by the fact thethat
ef-
fects of GR24, a synthetic SL analog, on resistance against Pst in
the inhibition of SL-mediated signaling was stronger in the mutant max2, which is defectiveArabidopsis. Treatment
of the
in WTF-boxplants with GR24
protein MAX2,improved theirbiosynthesis-deficient
than in other resistance against Pstmutants.
(Figure 4). Furthermore,
GR24-treated SLs biosynthesis-deficient mutants (max1, max3, and max4) exhibited re-
duced
2.2. bacterial
Effects growth in
of the Synthetic SLthe leaf GR24
Analog tissues oncompared
SA-Mediated to Disease
the untreated control plants. In
Resistance
contrast, GR24 treatment did not affect disease resistance in
To reveal the effects of SLs on SA-mediated disease resistance, we examined max2 (Figure 4). These results
the ef-
indicate that the activation of SL-mediated signaling by GR24 improved
fects of GR24, a synthetic SL analog, on resistance against Pst in Arabidopsis. Treatment disease resistance
in WT
of Arabidopsis.
plants with GR24 improved their resistance against Pst (Figure 4). Furthermore,
To determine
GR24-treated the role of disease resistance-related
SLs biosynthesis-deficient mutants (max1, max3, phytohormones in GR24-induced
and max4) exhibited reduced
disease resistance,
bacterial growth in induction of resistance
the leaf tissues compared against
to thePst was analyzed
untreated controlinplants.
SA-, JA-, and ET-
In contrast,
mediated
GR24 signaling
treatment did defective
not affectmutants npr1, jar1, in
disease resistance and ein2,(Figure
max2 respectively. GR24
4). These treatment
results indi-
induced
cate that disease resistance
the activation only in jar1, indicating
of SL-mediated signaling by thatGR24
SA and ET—butdisease
improved not JA—were
resistance in-
volved in
in Arabidopsis. GR24-induced disease resistance (Figure 4).

Figure4.4.Effects
Figure EffectsofofGR24
GR24treatment
treatmentonon
growth
growthof Pseudomonas syringae
of Pseudomonas pv. tomato
syringae DC3000
pv. tomato in Arabidop-
DC3000 in Ara-
sis leaf tissues.
bidopsis Three-week-old
leaf tissues. plants ofplants
Three-week-old WT and SL-related
of WT mutants (A)
and SL-related and SA-,
mutants (A)ET-,
andand
SA-,JA-related
ET-, and
JA-related
mutants (B)mutants (B) were
were treated treated
with 0.02%with 0.02 %
acetone acetone(–)
(control) (control) (–) or
or 10 µM 10 µM
GR24 (+) GR24 (+) by drenching
by drenching the soil
3 d before inoculation with Pst (2 × 105 CFU/mL). Leaves were homogenized 3 d post-inoculation,
and the number of CFUs was estimated from their growth on nutrient broth agar plates. Values are
means ± SE of six replicates, each with three leaves. Significant differences between control and
GR24-treated plants are indicated by * (unpaired t-test, p < 0.05). Different letters indicate significant
differences between accessions of the same treatment (Tukey’s multiple comparison test, p < 0.05).

To determine the role of disease resistance-related phytohormones in GR24-induced

disease resistance, induction of resistance against Pst was analyzed in SA-, JA-, and ET-
mediated signaling defective mutants npr1, jar1, and ein2, respectively. GR24 treatment
 the soil 3 d before inoculation with Pst (2 × 105 CFU/mL). Leaves were homogenized 3 d post-inoc-
ulation, and the number of CFUs was estimated from their growth on nutrient broth agar plates.
Values are means ± SE of six replicates, each with three leaves. Significant differences between con-
Int. J. Mol. Sci. 2022, 23, 5246 6 of 14
trol and GR24-treated plants are indicated by * (unpaired t-test, p < 0.05). Different letters indicate
significant differences between accessions of the same treatment (Tukey’s multiple comparison test,
p < 0.05).
induced disease resistance only in jar1, indicating that SA and ET—but not JA—were
involvedTo incharacterize
GR24-induced GR24-induced disease
disease resistance resistance,
(Figure 4). we examined physiological
changes in the GR24-treated WT plants. The level of expression
To characterize GR24-induced disease resistance, we examined physiological of SAR marker genes,
changes
namely PR1, PR2, and PR5, and ICS1 did not increase in GR24-treated
in the GR24-treated WT plants. The level of expression of SAR marker genes, namely plants, indicating
that PR2,
PR1, the mechanism
and PR5, and of GR24-induced diseaseinresistance
ICS1 did not increase GR24-treatedwas different from thatthat
plants, indicating of SAR
the
(Figure 5A).
mechanism of Furthermore,
GR24-inducedthe expression
disease levels
resistance was of PDF1.2from
different (At5g44420),
that of SAR the(Figure
JA-related
5A).
gene, and ERF1
Furthermore, the(At4g17500), the ET-related
expression levels of PDF1.2gene, were notthe
(At5g44420), significantly
JA-relateddifferent
gene, and between
ERF1
GR24-treated and untreated control plants (Figure 5A), indicating that
(At4g17500), the ET-related gene, were not significantly different between GR24-treated and JA- and ET-medi-
ated signaling
untreated controlpathways were not
plants (Figure activated
5A), in GR24-treated
indicating plants. Endogenous
that JA- and ET-mediated signalingaccumu-
path-
lationwere
ways of SAR-related
not activated metabolites was examined
in GR24-treated three days after
plants. Endogenous GR24 treatment.
accumulation The lev-
of SAR-related
els of free SA
metabolites wasand total SA three
examined (free SA
days+ SA glucoside)
after were notThe
GR24 treatment. influenced
levels ofby GR24
free SA treatment
and total
(Figure
SA 5B).+Although
(free SA an increase
SA glucoside) in influenced
were not GSH level is byimportant for SA-mediated
GR24 treatment (Figure 5B).signaling
Althoughin
SAR
an induction,
increase in GSHthe level
level of GSH inforGR24-treated
is important SA-mediatedplantssignalingwasinslightly but significantly
SAR induction, the level
oflower
GSHthan that of untreated
in GR24-treated plantscontrol plants.
was slightly butThese findingslower
significantly suggestthanthat theofmechanism
that untreated
control
underlyingplants. These findings
GR24-induced suggest
disease that was
resistance the mechanism
independentunderlying
of SA-, JA-, GR24-induced
or ET-mediated
disease
signaling resistance
pathways. was independent of SA-, JA-, or ET-mediated signaling pathways.

Figure5.5.Analysis
Figure Analysisofofdefense-related
defense-relatedgene
geneexpression
expressionand
andmetabolite
metaboliteaccumulation
accumulationininArabidopsis
Arabidopsis
thaliana treated with GR24. Three-week-old plants were treated with 0.02% acetone (control) (–) or
thaliana treated with GR24. Three-week-old plants were treated with 0.02% acetone (control) (–)
10 µM GR24 (+) using the soil drenching method, and leaves were collected 3 d after treatment. (A)
or 10 µM GR24 (+) using the soil drenching method, and leaves were collected 3 d after treatment.
Expression levels of PR1, PR2, PR5, ICS1, PDF1.2, and ERF1 in GR24-treated plants. Transcript levels
(A) Expression
were levels
normalized usingof PR1, PR2, PR5, of
the expression ICS1,
UBQ2PDF1.2,
in theand
sameERF1 in GR24-treated
samples. plants.
Relative mRNA Transcript
levels of each
levels were normalized using the expression of UBQ2 in the same samples. Relative mRNA
gene among the treatments are presented. Values are means ± SE (n = 4). (B,C) Accumulation levels of
of free
each gene among the treatments are presented. Values are means ± SE (n = 4). (B,C) Accumulation
of free and total SA and GSH in GR24-treated plants. The levels of free and total SA (free SA + SA
glucoside) and GSH were quantified using high-performance liquid chromatography. Significant
differences between control and GR24-treated plants are indicated by * (unpaired t-test, p < 0.05).
 and total SA and GSH in GR24-treated plants. The levels of free and total SA (free SA + SA glucoside)
and GSH were quantified using high-performance liquid chromatography. Significant differences
Int. J. Mol. Sci. 2022, 23, 5246
between control and GR24-treated plants are indicated by * (unpaired t-test, p < 0.05). 7 of 14

To determine the mechanism underlying GR24-induced resistance against Pst, de-

fenseTo determinewere
responses the mechanism underlying
investigated GR24-induced
by analyzing resistanceupon
gene expression against Pst, defense
pathogen inocula-
responses were investigated by analyzing gene expression upon pathogen inoculation.
tion. Time-course analysis indicated that PR1 was significantly upregulated in GR24-
Time-course
treated analysis
plants, indicated
compared withthat PR1 plants,
control was significantly
at 16 andupregulated in GR24-treated
20 h post inoculation, whereas
plants, compared with control plants, at 16 and 20 h post inoculation, whereas similar
similar levels were maintained up to 24 h (Figure 6). This finding suggests that the defense
levels were maintained up to 24 h (Figure 6). This finding suggests that the defense system
system was rapidly activated in the GR24-treated plants, which should contribute to the
was rapidly activated in the GR24-treated plants, which should contribute to the increased
increased resistance against Pst. Conversely, the expression of PDF1.2 after Pst infection
resistance against Pst. Conversely, the expression of PDF1.2 after Pst infection was not
was not upregulated
upregulated but downregulated
but downregulated due to activation
due to activation of SA-mediated
of SA-mediated signaling signaling
(Figure 6).(Fig-
ure 6). Therefore, GR24-induced disease resistance showed a priming effect
Therefore, GR24-induced disease resistance showed a priming effect that activated thatmajor
activated
major defense pathways, such as the SA-mediated defense pathway,
defense pathways, such as the SA-mediated defense pathway, after infection. after infection.

6. Expression
Figure 6.
Figure Expressionofof PR1
PR1 PDF1.2
andand after after
PDF1.2 pathogen infection
pathogen in GR24-treated
infection plants. Three-week-
in GR24-treated plants. Three-
week-old plants were treated with 0.02% acetone (control) or 10 µM GR24 by the
old plants were treated with 0.02% acetone (control) or 10 µM GR24 by drenching soil 3 d before
drenching the soil 3 d
inoculation
before with Pstwith
inoculation 107(2
(2 ×Pst CFU/mL). Leaves Leaves
× 107 CFU/mL). were collected 16, 20, and
were collected 16,2420,
h after Psthinoculation.
and 24 after Pst inocu-
Closed Closed
lation. circle, water-treated control plants;
circle, water-treated closed
control square,
plants; GR24-treated
closed square, plants. Significant
GR24-treated differences
plants. Significant
between control
differences and GR24
between treatments
control and GR24 aretreatments
indicated by indicated t-test,
are* (unpaired p < 0.05). t-test, p < 0.05).
by * (unpaired

2.3. Effects
2.3. Effects of
of the
the SL
SLBiosynthesis
BiosynthesisInhibitor TIS108
Inhibitor on on
TIS108 Disease Resistance
Disease Resistance
Pathogen inoculation assays and gene expression analyses indicated that GR24 primed
Pathogen inoculation assays and gene expression analyses indicated that GR24
plants against Pst infection, suggesting that SLs positively regulated disease resistance. This
primed plants against Pst infection, suggesting that SLs positively regulated disease re-
was further supported by the fact that in-planta Pst growth was higher in max mutants than
sistance. This was further supported by the fact that in-planta Pst growth was higher in
in WT plants (Figure 1). However, max mutants, with the absence of SLs from germination,
max
may mutants
have some than
sideineffects
WT plants (Figure
on the stress 1). However,
response systemsmax mutants,
during with the absence of
their growth.
SLs from germination, may have some side effects on the stress
To verify the contribution of endogenous SLs to the resistance against Pst, response systems
we exam- during
their
ined growth.
the effects of the SL-biosynthesis inhibitor TIS108 on gene expression, metabolite
To verifyand
accumulation, the disease
contribution of endogenous
resistance. The expressionSLslevels
to theofresistance
SAR-related against
genesPst,
PR1,we PR2,exam-
ined
PR5, the
andeffects of the SL-biosynthesis
ICS1; JA-related gene PDF1.2;inhibitor TIS108gene
and ET-related on gene
ERF1expression, metabolite
were not affected by ac-
TIS108 treatment
cumulation, (Figure 7A).
and disease Endogenous
resistance. levels of free
The expression SA, of
levels total SA, and GSH
SAR-related were
genes notPR2,
PR1,
significantly
PR5, different
and ICS1; between
JA-related thePDF1.2;
gene TIS108-treated and untreated
and ET-related genecontrol
ERF1 plants (Figure
were not 7B). by
affected
Treatment with as low as 1 µM TIS108 significantly decreased disease
TIS108 treatment (Figure 7A). Endogenous levels of free SA, total SA, and GSH were not resistance in WT
plants compared
significantly to thatbetween
different of the untreated control plants,
the TIS108-treated andsuggesting
untreated that endogenous
control plants SLs(Figure
contribute to Pst resistance (Figure 8).
7B). Treatment with as low as 1 µM TIS108 significantly decreased disease resistance in
WT plants compared to that of the untreated control plants, suggesting that endogenous
SLs contribute to Pst resistance (Figure 8).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 14

Int. J. Mol. Sci. 2022, 23, 5246 8 of 14

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 14

(A) PR1 PR2 PR5 ICS1 PDF1.2 ERF1

3
(A)

expression
3 PR1 PR2 PR5 ICS1 PDF1.2 ERF1

(genes/UBQ2)
expression
2

(genes/UBQ2)
2

Relative
Relative
1
1

0
TIS1080 − + − + − + − + − + − +
TIS108 − + − + − + − + − + − +
(B)
(B) Free SA Total SA (C) (C) GSH
0.4
Free SA Total SA
1.5
GSH2.5
0.4 1.5 2.5
Total SA µg/g FW
FW

2.0
Total SA µg/g FW
µg/g FW

0.3 2.0
0.3

pmol/mgFW
pmol/mgFW
1.01.0
SA µg/g

1.5 1.5
0.2
0.2
Free SA

1.0 1.0
0.50.5
Free

0.1
0.1 0.5 0.5
0.0 0.0 0.0
0.0 0.0 0.0
TIS108 − + TIS108 − + TIS108 − +
TIS108 − + TIS108 − + TIS108 − +
Figure 7. Analysis of defense-related gene expression and metabolite accumulation in Arabidopsis
Figure 7. Analysis of defense-related gene expression and metabolite accumulation in Arabidopsis
Figure
thaliana7.treated
AnalysiswithofTIS108.
defense-related
Three-week-old gene plants
expression and metabolite
were treated with 0,02% accumulation in Arabidopsis
dimethyl sulfoxide
thaliana treated with TIS108. Three-week-old plants were treated with 0,02% dimethyl sulfoxide
(DMSO)treated
thaliana (–) or 10with
µM TIS108
TIS108.(+) using the soil drenching
Three-week-old plantsmethod, and leaves
were treated were
with collected
0,02% 3 d aftersulfoxide
dimethyl
(DMSO) (–)
treatment.
(DMSO) oror
(–)(A) 1010
µM µM TIS108
Expression
TIS108 (+)of(+)
levels usingusing
PR1, PR2, the
PR5,soil
the soil drenching
ICS1, PDF1.2,
drenching method,
and
method, ERF1 and
andinleaves leaves were
TIS108-treated collected 3 d
plants.
were collected 3 d after
after treatment.
Transcript levels (A)
were Expression
normalized levels
using of
thePR1, PR2,
expression PR5,
of ICS1,
UBQ2 inPDF1.2,
the sameand ERF1
samples.
treatment. (A) Expression levels of PR1, PR2, PR5, ICS1, PDF1.2, and ERF1 in TIS108-treated plants. in TIS108-treated
Relative
mRNA
plants. levels of each gene were
among the treatments
Transcript Transcript
levels levels
were normalized
normalized theare
using using presented.
the
expression Values
expression
of UBQ2 are means
of UBQ2 ± SE
in the
in the (n = 4).
same
same (B,C) Relative
samples.
samples. Relative
Accumulation of free and total SA and GSH in TIS108-treated plants. The levels of free and total SA
mRNAlevels
mRNA levelsofofeach
eachgene
geneamong
amongthe thetreatments
treatmentsarearepresented.
presented.Values
Valuesare means ±±SE
aremeans SE(n(n= =4).4).(B,C)
(B,C)
(free SA + SA glucoside) and GSH were quantified using high-performance liquid chromatography.
Accumulation
Accumulationofoffree freeand
andtotal
totalSA SAandandGSHGSHininTIS108-treated
TIS108-treatedplants.
plants.The
Thelevels
levelsofoffree
freeand
andtotal
totalSA SA
(free
(freeSASA+9+SA SAglucoside)
glucoside) andandGSHGSH were
were quantified
quantified using
usinghigh-performance
high-performance liquid
liquidchromatography.
chromatography.
10 a a
108 b
1097
10 a a
10 8 b
106
FW FW

10 7
105
CFU/gCFU/g

10 6
104
1053
10
1042
10
1031
10
1020
10
TIS108
1 0 1 10 (µM)
10
108.0 Effects of strigolactone biosynthesis inhibitor TIS108 on Pst growth in Arabidopsis leaf
Figure
tissues. Three-week-old plants were treated with 0.02% dimethyl sulfoxide (DMSO) (control) or 1
TIS108 0 1 10 (µM)
or 10 µM TIS108 by drenching the soil 3 d before inoculation with Pst (2 × 105 CFU/mL). Leaves were
homogenized 3 d post-inoculation, and the number of CFUs was estimated using their growth on
Effects ofstrigolactone
Figure8.8.Effects
Figure strigolactonebiosynthesis
biosynthesis inhibitorTIS108TIS108 onPstPstgrowth
growthininArabidopsis
Arabidopsisleaf leaf
nutrient broth agarofplates. Values are means ± SE ofinhibitor
six replicates, eachon
with three leaves. Different
tissues.
tissues. Three-week-old
Three-week-old plants
plants were
were treated
treated with
with 0.02% dimethyl sulfoxide
sulfoxide
letters indicate significant differences (Tukey’s multiple comparison test, p < 0.05). (DMSO)
(DMSO) (control)
(control)or
or1 1or
or1010µM
µMTIS108
TIS108bybydrenching
drenchingthethesoil
soil33ddbefore
beforeinoculation
inoculationwithwithPst (2×
Pst(2 1055CFU/mL).
× 10 CFU/mL).LeavesLeaveswere
were
homogenized
homogenized3 3ddpost-inoculation,
post-inoculation,andandthethenumber
numberofofCFUsCFUswaswasestimated
estimatedusing
usingtheir
theirgrowth
growthon on
nutrient
nutrientbroth
brothagar
agarplates.
plates.Values
Valuesare
aremeans
means±±SESEofofsix
sixreplicates,
replicates,each
eachwith
withthree
threeleaves.
leaves.Different
Different
letters
lettersindicate
indicatesignificant
significantdifferences
differences(Tukey’s
(Tukey’smultiple
multiplecomparison
comparisontest,test,pp<<0.05).
0.05).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 of 14
Int. J. Mol. Sci. 2022, 23, 5246 9 of 14

3. Discussion
3. Discussion
Various aspects of plant immunity are regulated by phytohormones such as SA, JA,
and ET. Previous
Various studies
aspects have immunity
of plant demonstrated the role ofby
are regulated SLsphytohormones
in disease resistance
such asusing
SA,
SLs-biosynthesis
JA, and ET. Previous and signaling
studies have mutants of rice and
demonstrated the Arabidopsis
role of SLs in[39,40].
diseaseThis study
resistance
demonstrated that SLs positively
using SLs-biosynthesis and signaling regulated
mutantsSA-mediated signaling during
of rice and Arabidopsis basal
[39,40]. defense
This study
demonstrated
responses but that
not SLs
SARpositively
induction. regulated
IncreasedSA-mediated signaling
disease resistance during
upon basal
GR24 defense
treatment
responses
proved but
to be not of
a case SAR induction.
priming Increased
of the plant immunedisease resistance
system, that is, upon GR24
a rapidly treatment
upregulated
proved to be
expression of adefense
case of genes
priming of the
upon plant immune
infection system,
(Figure 6). that is,
Moreover, thea reduction
rapidly upregulated
in disease
expressionupon
resistance of defense genes
treatment upon
with theinfection (Figure 6). Moreover,
SL biosynthesis-inhibitor, the reduction
TIS108, in disease
strongly supported
resistance
that upon treatment
SL-mediated signalingwith the SLaffected
positively biosynthesis-inhibitor,
SA-mediated disease TIS108,resistance
strongly (Figure
supported8).
that SL-mediated
These findings suggestsignaling
that positively
SLs play aaffected
positiveSA-mediated
role in plant disease
immunity, resistance (Figure
especially 8).
in the
These findingsdisease
SA-mediated suggestresistance.
that SLs play a positive
Similar rapidrole in plant immunity,
upregulation especially in
of defense-related the ex-
gene SA-
mediated was
pression disease resistance.
observed Similar primed
in plants rapid upregulation
by symbioses of defense-related
with microbesgene or expression
chemicals
[18,19,21]. In turn, this study is the first demonstration of a priming effect [18,19,21].
was observed in plants primed by symbioses with microbes or chemicals on plants by In
Fig. 9 turn, this study is the first demonstration of a priming effect on plants
exogenous and endogenous SLs; however, how SLs contribute to plant disease resistance
endogenous SLs; however, how SLs contribute
by exogenous and
in nature remains to be elucidated. Although to plant disease
whether resistance in
SLs-biosynthesis nature
can remains
be activated
to be
by elucidated.
pathogen attackAlthough whether
is unknown, SLs SLs-biosynthesis can be
probably affect some activated by
unidentified pathogen attack
mechanism along
is unknown, SLs probably affect some unidentified mechanism along
the pathway between PAMPs recognition and expression of defense-related genes, such the pathway between
PAMPs
as recognition and expression
SA-biosynthesis-related genes and of PRdefense-related genes, primed
genes, which enables such asplants
SA-biosynthesis-
to counter-
related genes and PR genes, which enables primed
attack pathogens more rapidly than unprimed plants (Figure 9). plants to counterattack pathogens more
rapidly than unprimed plants (Figure 9).
Resistance

SLs biosynthesis Pathogen

infection

PR genes
SLs SA
(GR24) PAMPs
Resistance

SCFMAX2
MAX2 Pathogen
infection
PR genes

SA

PAMPs

Time
Figure 9.
Figure Proposed model for
9. Proposed for modulation
modulationmechanism
mechanismofofSA-mediated
SA-mediateddefense
defensesignaling byby
signaling strigolac-
strigo-
lactones.
tones. SCF MAX2
SCF MAX2isisSKP1-CUL1-F-box
SKP1-CUL1-F-boxcomplex
complexcontaining
containingMAX2
MAX2asasF-box
F-boxprotein.
protein.

As SLs
As SLs comprise
comprise an an important
important class
class of
of phytohormones
phytohormones with with various
various roles
roles in
in plant
plant
growth and
growth and development,
development, therethere may
may bebe some
some side
side effects
effects on
on the
the physiology
physiology of of SL-related
SL-related
mutants due
mutants due to to the
the absence
absence of
of SLs.
SLs.Similarly,
Similarly, TIS108
TIS108 treatment
treatment should
should also
also have
have some
some side
side
effects, as it suppressed SL-mediated signaling. However, such effects did not
effects, as it suppressed SL-mediated signaling. However, such effects did not occur in the occur in the
short-time treatment with TIS108, which was supported by our finding that
short-time treatment with TIS108, which was supported by our finding that the expression the expression
of defense-related
of defense-related genes genesand
andthetheaccumulation
accumulation ofof
SASAwere notnot
were affected by TIS108
affected by TIS108treatment.
treat-
ment.In Arabidopsis SL biosynthesis- and signaling-defective mutants, as well as in WT
plants,
In aArabidopsis
SAR inducer SLimproved disease
biosynthesis- andresistance by activating
signaling-defective SA biosynthesis
mutants, as well asandin WTSA-
mediated signaling, suggesting that SLs do not affect SAR induction (Figure
plants, a SAR inducer improved disease resistance by activating SA biosynthesis and SA- 1). Further, and
consistently with a previous report [40], without SAR induction, these mutants were more
Int. J. Mol. Sci. 2022, 23, 5246 10 of 14

susceptible to Pst than WT plants (Figure 1), and, compared to other SL-related mutants, the
SL-signal-perception-defective mutant max2 exhibited a different phenotype. Furthermore,
regardless of SAR induction, the disease resistance and SA-mediated signaling processes,
such as PR gene expression and SA accumulation, were suppressed in max2 compared
to WT plants (Figure 1). In contrast, SL biosynthesis-related mutants exhibited such
processes at levels that were intermediate between those observed for max2 mutant and WT
plants (Figure 1). These findings suggest that SLs are important, at least for SA-mediated
disease resistance.
When the pathogen was inoculated via foliar spray, the levels of disease resistance
were lower in max2 than in the other SLs biosynthesis-deficient mutants, which was
attributed to the increased number of open stomata in the max2 mutant [40]. In this
study, to avoid the influence of stomatal opening and closing, pathogen inoculation was
performed by dipping the plants into the bacterial suspension after keeping 100% RH
for 12 h before and after inoculation. Thus, the differences in disease resistance and SA-
mediated signaling did not result from differences in stomatal opening (Figure 1). We
hypothesize that the mutations in MAX2 affected SL-mediated signaling by altering SL-
signal perception, thereby affecting the SA-mediated disease resistance. In this case, the
differences in SA-mediated signaling processes among SL-biosynthesis-defective mutants
would likely be owing to the different physiological effects of each specific mutation,
a plausible idea that warrants further research.
Treatment with GR24 failed to induce disease resistance in the mutant npr1, indicating
that an SA signal was involved in SL-mediated disease resistance (Figure 4), which is
consistent with the altered SA-mediated disease resistance in SL-related mutants (Figure 1).
However, SAR induction via the activation of SA-mediated signaling was observed in
SL-related mutants, suggesting that SLs are not directly involved in SA-mediated disease
resistance. Alternatively, a plausible mechanism involves the regulation of SA-mediated
signaling by SL during basal resistance, including PTI. However, further investigation is
necessary to confirm the operation of this mechanism.
Treatment with GR24 effectively induced disease resistance in jar1 but not in ein2,
indicating that ET but not JA was involved in SL-mediated disease resistance (Figure 4).
Further, some reports indicate that ET is involved in SL-mediated signaling; thus, for
example, SL-induced seed germination in Striga hermonthica (Delile) Benth. requires ET
biosynthesis and signaling [43]. Additionally, SL promotes ET accumulation in etiolated
Arabidopsis seedlings by upregulating the expression of 1-aminocyclopropane-1-carboxylic
acid oxidase (ACO) [44]. Similarly, SL promotes ET biosynthesis in Arabidopsis roots [45].
In this study, ET-responsive ERF1 was not induced in GR24-treated plant leaves (Figure 5),
suggesting that ET was not involved in SL-mediated priming.
In contrast, several other reports have underlined the importance of ET in SA-mediated
disease resistance; however, such findings remain controversial. In Arabidopsis, co-
treatment with an ET biosynthetic precursor (1-aminocyclopropane-1-carboxylic acid)
and SA upregulated PR1 relative to SA treatment alone [46]. Conversely, ein3 eil1, a double
mutant of ET-responsive transcription factors, constantly accumulated SA, indicating that
ET negatively affects SA-mediated signaling pathways [47]. Nonetheless, GR24-induced
disease resistance required ET-mediated signaling through EIN2 (Figure 5), suggesting that
ET improved SA-mediated disease resistance after pathogen infection of primed plants.
Nutrient stress, such as low phosphate or nitrogen, induces SL biosynthesis, which
alters SL-mediated responses in AM fungi symbioses, which effectively increase the soil
volume explored by plant roots in search for phosphorus. However, the trade-off between
growth and disease resistance is well known. In contrast to plants growing under soil
nutrient-rich conditions (i.e., excess phosphate or nitrogen for better growth), which are
more susceptible to diseases, plants growing under soil nutrient-poor conditions exhibit
higher levels of expression of defense-related genes after infection [48]. Overall, SL syn-
thesized under soil nutrient-poor conditions may not only promote symbiosis with AM
fungi but effectively prime plants for infection as well. This effect might be possible be-
Int. J. Mol. Sci. 2022, 23, 5246 11 of 14

cause priming does not affect growth, while it effectively protects plants from pathogen
attack by accelerating the induction of SA-, JA-, or other phytohormone-mediated disease
resistance mechanisms.

4. Materials and Methods

4.1. Plant Material and Chemical Treatment
Arabidopsis thaliana Col-0 and seven mutants with the same ecotype, including max1-1 [24],
max2-1, max3-1, max4-1, npr1-2, jar1-1, and ein2-1, were used for the experiments [9].
Plants were grown in sterilized potting soil (Nihon hiryo, Fujioka, Japan) in plastic pots
(5 cm × 5 cm × 5 cm) inside a growth chamber under a 16:8 h light: dark photoperiod at
22 ◦ C and 60% RH.
BIT [11] solutions (1 mg/mL for soil drenching and 1 mM for spraying) were prepared
by dissolving the BIT powder in sterilized distilled water. GR24 (rac-GR24) and TIS108 [31]
solutions (1 mM) were prepared from 50 mM GR24 in acetone and 50 mM TIS108 in DMSO,
respectively, by diluting with sterilized distilled water. Three-week-old plants were treated
with chemicals as indicated in the Figures.

4.2. Pathogenicity Assays

Control and chemical-treated plants were inoculated with Pst as previously de-
scribed [49].

4.3. Estimation of SA Levels

Three-week-old plants were treated with chemicals for three days. Leaf samples (ca.
100 mg) were harvested and used for extraction and measurement of SA as previously
described [12]. Briefly, leaf tissues were homogenized in 1 mL of 90% methanol and
then extracted using 1 mL of 100% methanol. These extracts were pooled and dried at
40 ◦ C. The dried residues were extracted in 4 mL of water at 80 ◦ C for 15 min, and equal
aliquots (1 mL) were used for free and total SA estimation. One aliquot was extracted using
2.5 mL ethyl acetate–cyclohexane (1:1) following the addition of 50 µL of concentrated
HCl, and the upper layer was dried and dissolved in 1 mL of 20% methanol in 20 mM
sodium acetate buffer (pH 5). This solution was then subjected to high-performance liquid
chromatography (HPLC) analysis to determine free SA levels. Then, 1 mL of β-glucosidase
(Merck, Darmstadt, Germany) solution (3 units/mL) was added to the remaining aliquots
and incubated for 6 h. These aliquots were prepared for HPLC analysis using the above
procedure to determine total SA levels. SA analysis was performed using an Agilent 1220
Infinity LC (Agilent, Snata Clara, CA, USA) equipped with a ZORBAX Eclipse Plus C18
(4.6 × 150 mm; Agilent) and run with 20% methanol in 20 mM sodium acetate buffer (pH 5)
at a flow rate of 1 mL/min. SA was detected and fluorometrically quantified at 295 nm
excitation and 370 nm emission wavelengths.

4.4. GSH Measurement

Three-week-old plants were treated with chemicals for three days. Leaf samples (ca.
100 mg) were harvested and used for extraction and measurement of GSH as previously
described [50].

4.5. RT-qPCR
Gene expression profiles were determined using leaf samples. Total RNA isolation,
cDNA synthesis, and RT-qPCR were performed as previously described [49]. Gene-specific
primers used in this study are listed in Supplementary Table S1. Transcript levels were
normalized using the expression of UBQ2 in the same samples [51].

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms23095246/s1.
Int. J. Mol. Sci. 2022, 23, 5246 12 of 14

Author Contributions: Conceptualization, M.K. and H.N. (Hideo Nakashita); methodology, H.N.
(Hidemitsu Nakamura), A.M.-N. and H.N. (Hideo Nakashita); validation, M.K., M.F., K.Y., T.N., H.N.
(Hidemitsu Nakamura), K.A., A.M.-N., T.A. and H.N. (Hideo Nakashita); investigation, M.K., M.F.
and K.S.; resources, T.N., H.N. (Hidemitsu Nakamura) and T.A.; writing—original draft preparation,
M.K.; writing—review and editing, H.N. (Hidemitsu Nakamura), T.A. and H.N. (Hideo Nakashita);
visualization, M.K.; project administration, T.A. and H.N. (Hideo Nakashita); funding acquisition,
K.Y., T.N., K.A., T.A. and H.N. (Hideo Nakashita). All authors have read and agreed to the published
version of the manuscript.
Funding: This work received partial support for K.Y., T.N., K.A., T.A. and H.N. (Hideo Nakashita)
from the Ministry of Agriculture, Forestry, and Fisheries of Japan under the Science and Technology
Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry (27004A); also,
from Grant-in-Aid for JSPS Fellows 19J14665 for M.F., and from JSPS KAKENHI Grant Numbers
18K05656 for H.N. (Hideo Nakashita).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available on request.
Acknowledgments: We thank H. Takeuchi and A. Sugimoto (Fukui Pref. Univ.) for their kind
support in plant culture.
Conflicts of Interest: The authors declare no conflict of interest.

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