Abstract
Rice seedling blight, caused by Fusarium oxysporum, significantly
affects global rice production levels. Fluoro-substituted
benzothiadiazole derivatives (FBT) and chitosan oligosaccharide (COS)
are elicitors that can enhance plant resistance to pathogen infection.
However, there is a lack of information regarding FBT and COS used as
elicitors in rice seedlings blight. Therefore, the aim of this study
was to evaluate the effect of FBT and COS treatments on rice seedling
blight and elucidate the molecular mechanisms of the two elicitors for
inducing resistance using proteomic technique. Results indicated that
FBT and COS significantly reduced the disease incidence and index, and
relived the root growth inhibition caused by F. oxysporum (p < 0.05).
Biochemical analyses demonstrated that these two elicitors effectively
enhanced activities of defense enzymes. Moreover, the proteomic results
of rice root tissues disclosed more differentially expressed proteins
in diterpenoid biosynthesis pathway that were particularly stimulated
by two elicitors compared to the other pathways studied, resulting in
the accumulation of antimicrobial substance, momilactone. Findings of
this study could provide sound theoretical basis for further
applications of FBT and COS used as rice elicitors against seedling
blight.
Keywords: rice, Fusarium oxysporum, FBT, COS, momilactone
1. Introduction
Rice (Oryza sativa L.) is an important global cereal crops that
provides a stable food supply for more than 5 billion people worldwide.
Most Asian diets include rice as a main dish [[40]1]. Although global
rice demand is on the rise, its productivity is constrained by disease
outbreaks. One major fungal disease that occurs during the nursery and
field planting stages is the rice seedling blight. Grain yield losses
due to seedling blight infection ranges from 8 to 50% depending on
severity of the disease, stage of the crop at which it was infected by
the fungus, and overall environmental conditions [[41]2,[42]3,[43]4].
Although several species belonging to Fusarium, Rhizoctonia and
Rhizopus groups have been isolated from infected roots to cause
seedling blight [[44]5,[45]6,[46]7]. Among these fungus, the Fusarium
genus was regarded as the major pathogens of rice seedling blight in
China [[47]8,[48]9]. Thus, as a wide-ranging pathogens, Fusarium
oxysporum was usually selected to induce seedling blight by many
researchers [[49]10,[50]11].
Rice seedling blight is still treated using fungicides such as
imazalil, tolclofos-methyl, fenaminosulf, liturium, and hymexazol,
which are arguably effective against Fusarium oxysporum
[[51]12,[52]13]. Due to growing environmental and health concerns, the
need for eco-friendly alternatives to control this disease has become
imperative [[53]14,[54]15].
In response to pathogens infection, plants can amass an array of
formidable defense pathways using a variety of biological, chemical, or
physical agents, which are known as resistance inducers or elicitors
[[55]16,[56]17,[57]18]. An earlier study showed that treating host
plants with elicitors can increase secretion of defense-regulating
enzymes such as phenylalanine ammonia lyase (PAL), peroxidase (POD),
superoxide dismutase (SOD) and catalase (CAT) [[58]19]. While PAL
regulates host response to biotic and abiotic stress levels by
increasing phenolic acids and phytoalexin production via the
phenylpropanoid pathway, POD, SOD and CAT lower oxidative stress by
acting as ROS scavengers [[59]20]. Compared with other traditional
disease control methods, the elicitors not only prevent plant injury
but also reduce residue of chemicals in agricultural. It has features
of low pollution, long duration and minimal side effects on crop
quality. Therefore, it is an innovative approach to manage rice
seedling blight [[60]21,[61]22].
Fluoro-substituted benzothiadiazole derivatives (FBT) is a novel
elicitor similar to benzothiadiazole (BTH) structure, which was
invented in China [[62]23]. It can effectively induce high levels of
defense mechanisms in many kinds of vegetable crops against soil-borne
disease, as well as in the Chinese cabbage caused by Plasmodiophora
brassicae [[63]24]. Chitosan oligosaccharide (COS) derivatived from
chitosan, has recently been shown to be an ideal delivery material of
the elicitors regulating disease response and defense action
[[64]25,[65]26]. In comparison to chitosan, COS is easily water-soluble
and has good physio-chemical properties, qualifying it as a potential
plant bio-vaccine. Thus, it is of prime interest to agricultural
researchers [[66]27]. In spite of these promising features, detailed
molecular studies showing the use of FBT and COS in mitigating rice
seedling blight are scarce.
The aim of this study was to evaluate the effect of FBT and COS
treatments on F. oxysporum and elucidate the underlying molecular
mechanisms by which they induce resistance against rice seedling blight
using proteomics. It is anticipated that these findings will give
further insights into the theory and application for FBT and COS as
elicitors inducers for treating rice seedling blight.
2. Materials and Methods
2.1. Plant Growth Conditions
Rice (Oryza sativa L.) seeds (‘Qijing 2’), highly susceptible to
blight, were obtained from the Rice research laboratory, Qiqihar branch
of Heilongjiang Academy of Agricultural Sciences. Firstly, seed
surfaces were disinfected with 70% ethanol for 1 min to achieve surface
disinfection, subsequently incubated in 25 °C incubator until they
sprouted to 5 mm, and finally washed with water and planted in 16
cm-diameter holes. All cultivations were cultivated in soil in the
greenhouse of day 26 °C/night 20 °C with a 16 h light/8 h dark regime.
The light intensity was 6000 LX.
2.2. Pathogen Inoculation
Standard pathogenic strain of F. oxysporum FO2016038 was provided by
the Institute of Rice Research of the Northeast Agricultural
University, Harbin, China. The pathogen was cultured on potato dextrose
agar (PDA) medium at 4 °C. Conidial suspensions of the pathogen were
prepared by flooding the 7-day-old culture dishes and incubated at 25
°C with sterile distilled water containing 0.1% Tween-20. The resulting
zoospore concentration was adjusted to 1 × 10^6 spores/mL with sterile
distilled water using a hemocytometer to prepare the inoculum.
2.3. Elicitor Treatment and Sampling
Rice seedlings at 2-leaf stage were treated with 10 mL of one of three
following elicitors by surface spraying: (1) sterile distilled water as
the control (CK), (2) 50 mg/L FBT (1,2,3-benzothiadiazole-7-carboxylic
acid-2,2,2-trifluoroethyl ester, purchased from Shanghai Taihe Chemical
Co., Ltd., Shanghai, China) solution or (3) 100 mg/L COS (molecular
mass: 1500–3000 Da, purchased from Hainan Zhengye Zhongnong High-tech
Co., Ltd., Hainan, China) solution. After 2 days, they were then
inoculated with 10 mL of conidial suspension (1 × 10^6 spores/mL) by
root-dip technique. Afterwards, tissue samples were immediately
preserved in liquid nitrogen and stored at −80 °C until further
analysis.
2.4. Efficiency of FBT and COS to Elicit Resistance against Seedling Blight
Disease incidence, disease index and root growth were determined at 1,
2, 3, 4, 5, 6 and 7 d after inoculation of conidial suspension. There
are three replicates with 30 plants per replicate, and the experiment
was conducted thrice. The disease investigation was performed as
previously described by Wang et al. (Wang et al., 2009) ([67]Table S1).
The following formulas were used to determine the disease incidence and
disease index, respectively:
[MATH:
Disease incidence (%) = No
. of diseased
plants No. of
total investigated
plants × 100% :MATH]
[MATH:
Disease index
= ΣNo. of diseased <
mi>plants × grade<
mtext> No. <
/mtext>of total inv
estigated plants ×
the highest grade × 100 :MATH]
Water on the surface of rice root was absorbed by filter paper, then it
was dried at 105 °C for 15 min and baked to constant weight at 70 °C to
determine the root dry weight. Root length, root surface area and root
volume were measured with Epson root scanner and analyzed by WinRHIZO
software.
2.5. Effect of FBT and OCT on Enzymatic Activity of Root
2.5.1. Superoxide Dismutase (SOD) Assay
The activity of SOD was determined by measuring the inhibition of
nitroblue tetrazolium (NBT) as earlier described by Beauchamp and
Fridovich [[68]28]. Briefly, fresh sample (0.5 g) was taken and 5 mL of
50 mM pre-cooled potassium phosphate buffer (pH 7.0) was added to
extract the crude SOD enzyme solution. Afterwards a reaction mixture (9
mL) was prepared using the following −50 mM phosphate buffer (pH 7.8),
13 mM methionine, 75 μM NBT, 0.1 mM EDTA, 150 μL enzyme extract and 2
μM riboflavin which was added at the end. 3 mL of this mixture was
poured in a tube, stirred and placed 30 cm below two 15 W fluorescent
lamps. Reaction was induced by switching on the lamps for 15 min and
stopped by switching off the lamps and placing a black cloth over the
reaction tube. The control reaction mixture had no color. Reaction
mixture absorbance was read at 560 nm. One unit of activity was defined
as the amount of enzyme required to inhibit 50% of the NBT reduction
rate in the controls containing no enzymes.
2.5.2. Peroxidase (POD) Assay
POD activity was measured by following the method of Hammerschmidt et
al. [[69]29]. Fresh samples (0.5 g) were taken and 5 mL of 10 mM
pre-cooled potassium phosphate buffer (pH 6.9) was added to extract the
crude POD enzyme solution. Then, a reaction mixture consisted of 0.25%
v/v guaiacol in 10 mM potassium phosphate buffer (pH 6.0) containing
100 mM hydrogen peroxide of which 3 mL was subsequently used. The crude
enzyme (10 μL) was added to initiate the reaction and POD activity was
evaluated using a spectrophotometer at a wavelength of 470 nm. The
results were expressed on fresh weight basis as units (U) g^−1. One
unit of POD activity was defined as the amount of enzyme that causes an
increase of 0.01 in the absorbance per minute at 470 nm.
2.5.3. Catalase (CAT) Assay
The method of Bailly et al. [[70]30] was used to determine CAT
activity. Briefly, fresh samples (0.5 g) were taken and 5 mL of 100 mM
pre-cooled potassium phosphate buffer (pH 7.0) was added to extract the
crude CAT enzyme solution. The reaction mixture contained 3 mL of
phosphate buffer along with 40 µL crude CAT extract was initiated by
adding 40 µL of 10 mM H[2]O[2]. The activity of CAT was measured using
a spectrophotometer (Hitachi U 2000, Tokyo, Japan) at 240 nm. CAT
activity was expressed in terms of the change in absorbance at 240 nm
in the linear phase of the slope (D240 min^−1 g^−1 fresh weight).
2.5.4. Phenylalanine Ammonia-Lyase (PAL) Assay
The PAL activity was determined as earlier described by Beaudoin-Eagan
and Thorpe [[71]31]. Briefly, PAL enzyme was extracted with 5 mL of 25
mM pre-cooled Tris HCl buffer from 0.5 g fresh samples. Afterwards, 100
µL of the extracted enzyme was mixed with 900 µL of 50 mM
L-Phenylalanine and 100 mM Tris HCl buffer solution (pH 8.01). Reaction
was initiated by placing this mixture in a water bath at 40 °C for 2 h
and stopped using 60 µL of 5 N HCl. The results were expressed on fresh
weight basis as units (U) g^−1. One unit of PAL acivity was defined as
the amount of enzyme that causes an increase of 0.01 in the absorbance
at 290 nm in 1 min.
2.6. Quantitative Analysis of Global Proteome
2.6.1. Protein Sample Preparation
The detailed description of protein sample preparation including
trypsin Digestion, tandem mass tags (TMT) labeling and HPLC
fractionation can be found in the [72]Supplementary Materials.
2.6.2. Liquid Chromatography Tandem Mass Spectrometry (LC−MS/MS) Analysis
Details of this procedure is available in the [73]Supplementary
Materials. Briefly, peptides were dissolved in 0.1% FA, directly loaded
onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo
Scientific, Waltham, MA, China). Peptide separation was performed using
a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo
Scientific). The peptides were subjected to NSI source followed by
tandem mass spectrometry (MS/MS) in Q ExactiveTM plus (Thermo
Scientific) coupled online to the UPLC. The mass spectrometry
proteomics data have been deposited to the ProteomeXchange Consortium
([74]http://proteomecentral.proteomexchange.org) via the iProX partner
repository [[75]32] with the dataset identifier PXD014979.
2.6.3. Protein Identification and Screening of Differentially Expressed
Proteins (DEPs)
The resulting MS/MS data were processed using Maxquant search engine
software (v.1.5.2.8). Tandem mass spectra were searched against
Phaffia_rhodozyma database concatenated with reverse decoy database.
The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to
identify enriched pathways by a two-tailed Fisher’s exact test to test
the enrichment of the DEPs against all identified proteins. Correction
for multiple hypothesis testing was carried out using standard false
discovery rate control methods. The pathway with a corrected p-value <
0.05 was considered significant. These pathways were classified into
hierarchical categories according to the KEGG website. Cluster
membership (Pearson algorithm) was constructed by a heat map using the
“heatmap.2” function from the “gplots” R-package.
2.7. Determination of Momilactone in Rice Root
Momilactone was measured by following the method of Kato-Noguchi et al.
[[76]33]. Briefly, rice root (5 g fresh weight) was homogenized with 50
mL of 80% (v/v) aqueous methanol and the homogenate was filtered
through filter paper. The residue was homogenized again with 50 mL of
methanol and filtered. The two filtrates were combined and evaporated
in vacuo at 35 °C to give an aqueous residue. After evaporation, the
methanol fraction was dissolved in 50% aqueous methanol (2 mL, v/v) and
loaded onto a reverse-phase C18 Sep-Pak cartridge (Waters, Milford, MA,
USA). The cartridge was first eluted with 50% aqueous methanol (15 mL)
to remove impurities, and then with methanol (20 mL) to release
momilactone. Momilactone was quantified by measuring its peak height on
the chromatogram of High Performance Liquid Chromatography (HPLC) as
described by Kato-Noguchi et al. [[77]34].
2.8. Confirmation of the Infection-Responsive Expression Profiles by qRT-PCR
To validate the proteomic data results, qRT-PCR analysis was performed
on 9 proteomic samples. Several genes that were co-expressed in both
cultivars were analyzed by qRT-PCR at CK, FBT, and COS. The EASYspin
Plus kit (Aidlab, Beijing, China) was used to extract the RNA of all
samples following the manufacturer’s instructions, and 500 ng RNA was
used for cDNA synthesis using the SuperScript III First-Strand
Synthesis System for RT-PCR (Gene Denovo Biotechnology Co. Guangzhou,
China) using Oligo(dT)20 primer. qRT-PCR reactions were run on the ABI
PRISM^TM 7900HT Fast Real-Time PCR System (ABI) using SYRB^® GreenER^TM
qPCR SuperMix (Invitrogen, Carlsbad, CA, USA). Reverse transcription
was performed using 500 ng of RQ1 DNase (Promega, Madison, WI,
USA)-treated total RNA, oligo dT and SuperScript III reverse
transcriptase from the Invitrogen’s First-Strand cDNA Synthesis Kit
according to the manufacturer’s instructions. The first-strand cDNA
reaction was diluted 20 folds prior to qPCR and 5 μL of diluted cDNA
was used as the PCR template. Reverse transcriptase negative controls
were implemented for each PCR reaction to ensure that there is no
genomic DNA contamination. The primer sequences used were shown in
[78]Table S2. Ct values were determined based on two biological
replicates each with two technical replicates. Relative expression
levels of target genes were calculated using the ΔΔCt method [[79]35]
and with the housekeeping gene EF1α mRNA as an internal standard.
2.9. Statistical Analysis
All values obtained were expressed as mean ± standard deviation (SD).
All experiments were performed at least thrice using independent
assays. The statistical significance of data comparisons was determined
using one-way analysis of variance (ANOVA), followed by Duncan’s
multiple range test. Values of p < 0.05 were considered to be
statistically significant.
3. Results
3.1. Efficacy of FBT and COS on Control of Seedling Blight
As shown in [80]Figure 1, the disease incidence and index in the rice
roots of all groups were gradually increased following F. oxysporum
inoculation ([81]Figure 1). Compared with the control root, FBT and COS
significantly (p < 0.05) reduced the disease incidence and development
of disease symptoms caused by F. oxysporum.
Figure 1.
[82]Figure 1
[83]Open in a new tab
Effects of fluoro-substituted benzothiadiazole derivatives (FBT) and
chitosan oligosaccharide (COS) on disease incidence and disease index
caused by F. oxysporum. (A) disease incidence, and (B) disease index.
The values were the means of three replicates of three different
experiments. Values with different superscript letters were
significantly different at p < 0.05.
3.2. Effect of FBT and COS on Growth Status of Root
As shown in [84]Figure 2, there were no significant differences (p >
0.05) in root length, root surface area, root volume and root dry
weight between two treatments with the control during the first 2 days
after F. oxysporum incubation, respectively. However, after 4 d of
incubation of pathogen, FBT and COS significantly (p < 0.05) increased
the value of these parameters compared to the control root.
Figure 2.
[85]Figure 2
[86]Open in a new tab
Effects of FBT and COS on growth of root caused by F. oxysporum. (A)
root length, (B) root surface area, (C) root volume, and (D) root dry
weight. The values were the means of three replicates of three
different experiments. Values with different superscript letters were
significantly different at p < 0.05.
3.3. Effect of FBT and COS on Enzymatic Activities of Root
3.3.1. Superoxide Dismutase (SOD) Assay
Root samples with and without elicitors had SOD activity. At all
observed time intervals, SOD activity was significantly (p < 0.05)
higher in FBT and COS-treated roots than that in control. In all root
samples, the general trend was that SOD activity initially increased
but subsequently decreased and peaked at day 4 of pathogen incubation
([87]Figure 3A).
Figure 3.
[88]Figure 3
[89]Open in a new tab
Effects of FBT and COS on activities of superoxide dismutase (SOD) (A),
peroxidase (POD) (B), catalase (CAT) (C), and phenylalanine ammonia
lyase (PAL) (D) in root. Data are expressed as the mean of triplicate
assays. The values were the means of three replicates of three
different experiments.
3.3.2. Peroxidase (POD) Assay
Similar to the activity of SOD, the results of POD activity showed that
all root groups with and without elicitors had POD activity, and it was
significantly (p < 0.05) higher in FBT and COS-treated roots compared
to control at all the observed time intervals. In FBT-treated roots,
POD activity gradually increased and peaked at day 3, whereas, it
appeared as a bimodal curve in COS-treated roots. Furthermore, POD
activity in FBT-treated roots was higher than that in COS-treated roots
at all tested time points except day 3 ([90]Figure 3B), indicating that
FBT had a better effect than COS throughout the study test time.
3.3.3. Catalase (CAT) Assay
All roots with or without elicitors showed CAT activity. At all tested
time points, CAT activity was significantly (p < 0.05) higher in FBT
and COS-treated roots compared to the control root. The maximum CAT
activity was found at 4 days in all the tested roots. Moreover, it was
1.33 and 1.26 fold higher in FBT and COS-treated roots than that in the
control roots, respectively ([91]Figure 3C). These findings were
inconsistent with the results of peroxidase assay.
3.3.4. Phenylalanine Ammonia-Lyase (PAL) Assay
Similar to previous assays, PAL activity occurred in all root
categories with or without elicitors. Results showed that PAL activity
was triggered by FBT and COS in root after inoculation of F. oxysporum,
which was significantly (p < 0.05) higher than that in the control root
([92]Figure 3D). These results indicated that the two elicitors could
alleviate the rice seedling blight by regulating phenylpropanoid
pathway.
3.4. Proteomic Characteristics of All Samples
To study variations in protein regulation in root samples induced by
the two elicitors to resist F. oxysporum, TMT labling LC-MS/MS
proteomic approach was used to measure the protein expression in rice
roots after F. oxysporum inoculation. As shown in [93]Figure 4, high
correlations were observed among three replicates in the experimental
and control groups with each other (R^2 > 0.85), but correlation within
treatment groups were lower. This suggested that protein expression
profiles between biological replicates was consistent, and different
protein expression profiles were likely in response to different kinds
of elicitors.
Figure 4.
[94]Figure 4
[95]Open in a new tab
Heatmap showing Pearson correlation coefficients from all quantified
proteins between each pair of samples.
3.5. Differential Expression and Biological Pathway Enrichment Analysis
Here, 922 and 1323 differentially expressed proteins (DEPs) were
identified in FBT and COS-treated roots, respectively, compared with
those in the control root after F. oxysporum inoculation. Moreover, 501
up-regulated proteins and 421 down-regulated proteins were identified
between the FBT-treated root and the control root ([96]Figure 5A), a
total of 677 up-regulated genes and 646 down-regulated genes were found
between the COS-treated root and the control root ([97]Figure 5B).
Figure 5.
[98]Figure 5
[99]Open in a new tab
Volcano plots of Differentially Expressed Proteins (DEPs) in FBT-(A)
and COS-(B) treated roots. DEPs were selected by adjusted p-value <
0.05. The x-axis shows the fold change in protein expression, and the
y-axis shows the statistical significance of the differences. Grey dots
indicate proteins without significantly differential expression; red
dots denote significantly up-regulated proteins in FBT and COS-treated
roots compared to the control root; and blue dots mean significantly
down-regulated proteins in FBT and COS-treated roots compared to the
control root.
The pathways that were enriched in the treated roots compared to the
control roots are shown in [100]Figure 6. FBT-treated roots had 150
DEPs out of 922 and these were assigned to 9 biological pathway
annotations: ‘diterpenoid biosynthesis’, ‘biosynthesis of secondary
metabolites’, ‘phenylpropanoid biosynthesis’, ‘glutathione metabolism’,
‘tyrosine metabolism’, ‘terpenoid backbone biosynthesis’,
‘alpha-linolenic acid metabolism’, ‘cutin, suberine and wax
biosynthesis’ and ‘sulfur metabolism’ ([101]Figure 6A). On the other
hand, out of 1323 DEPs identified in COS-treated roots, 187 were
assigned to 14 enrichment pathways: ‘diterpenoid biosynthesis’,
‘photosynthesis’, ‘phenylpropanoid biosynthesis’,
‘photosynthesis-antenna proteins’, ‘cutin, suberine and wax
biosynthesis’, ‘glutathione metabolism’, ‘alpha-Linolenic acid
metabolism’, ‘glyoxylate and dicarboxylate metabolism’, ‘AGE-RAGE
signaling pathway in diabetic complications’, ‘zeatin biosynthesis’,
nitrogen metabolism’, ‘valine, leucine and isoleucine’, and ‘tyrosine
metabolism’ ([102]Figure 6B). Interestingly, the ‘diterpenoid
biosynthesis’ pathway, having the smallest Q value, was shown to be
significantly enriched in both FBT and COS treatments compared to the
other pathways. Furthermore, ‘diterpenoid biosynthesis’,
‘alpha-Linolenic acid metabolism’, ‘phenylpropanoid biosynthesis’,
‘cutin, suberine and wax biosynthesis’ and ‘tyrosine metabolism’ were
shared by the two groups, implying that FBT and COS may have the same
molecular mechanisms in the induction of resistance in rice seedling
blight.
Figure 6.
[103]Figure 6
[104]Open in a new tab
Bubble plot of KEGG pathway enrichment for DEPs in FBT-(A) and COS-(B)
treated roots. The rich factor is calculated as the DEP number divided
by the base number of any given pathway. Dot size denotes the number of
proteins and dot color denotes the range of –log10 p value, and a lower
–log10 p value indicates greater pathway enrichment.
3.6. Specific Pathway of Diterpenoid Biosynthesis Analysis
Compared to the control root, the diterpenoid biosynthesis pathway in
the COS-treated roots contained eight DEPs ([105]Figure 7). These
include two ent-copalyl diphosphate synthases [EC:5.5.1.13]
(Os02t0571100-01 and Os02t0570900-00), an ent-cassa-12,15-diene
synthase [EC:4.2.3.28] (Os02t0570400-01), a ent-kaurene oxidase
[EC:1.14.14.86] (Os06t0569500-01), two ent-cassa-12,15-diene
11-hydroxylases [EC:1.14.14.112] (Os02t0569900-01 and Os02t0569400-01),
a sandaracopimaradiene/labdatriene synthase [EC:4.2.3.29 4.2.3.99]
(Os12t0491800-01), a syn-copalyl-diphosphate synthase [EC:5.5.1.14]
(Os04t0178300-02), a 9-beta-pimara-7,15-diene oxidase [EC:1.14.14.111]
(Os04t0178400-01), and a momilactone-A synthase [EC:1.1.1.295]
(Os04t0179200-01). In addition, a stemar-13-ene synthase [EC:4.2.3.33]
(Os11t0474800-01) and a syn-pimara-7,15-diene synthase [EC:4.2.3.35]
(Os04t0179700-01) were found in the FBT-treated treated roots compared
to the control root ([106]Figure 7B). These results show that all DEPs
were upregulated in both treatments.
Figure 7.
[107]Figure 7
[108]Open in a new tab
Illustration of KEGG pathway of diterpenoid biosynthesis in COS-(A) and
FBT-(B) treated roots. Red signifies up-regulated proteins in FBT and
oligochitosan-treated roots compared to control root.
In order to confirm the prediction made by the proteomic changes, the
level of momilactone in rice root in all groups was measured by HPLC.
As shown in [109]Figure 8, compared with the control group, FBT and COS
treatment significantly (p < 0.05) increased the concentrations of
momilactone in the roots. Furthermore, it in the FBT-treated roots was
higher than that in the COS-treated roots. This result was consistent
with the profile of proteomics.
Figure 8.
[110]Figure 8
[111]Open in a new tab
Concentrations of momilactone in rice root. The values were the means
of three replicates of three independent experiments. Values with
different superscript letters were significantly different at p < 0.05.
3.7. Confirm Unigenes Expression Using Real-Time Quantitative Reverse
Transcription PCR
Quantitative real-time PCR (qRT-PCR) was performed to validate our
earlier obtained proteomics results. Here, eight DEPs, which are in the
diterpenoid biosynthesis pathway-ent-copalyl diphosphate synthases,
ent-cassa-12,15-diene synthase, ent-kaurene oxidase,
ent-cassa-12,15-diene 11-hydroxylases, sandaracopimaradiene/labdatriene
synthase, syn-copalyl-diphosphate synthase, 9-beta-pimara-7,15-diene
oxidase and momilactone-A synthase. After confirming our results, all
selected 8 DEPs exhibited similar expression patterns as observed in
proteomic data, demonstrating that proteomic results were accurate in
this study ([112]Figure 9).
Figure 9.
[113]Figure 9
[114]Open in a new tab
Verification of the proteomic results by qRT-PCR. White bar: proteomic
data for the proteins. Black bar: qRT-PCR results for the proteins. The
values were the means of three replicates of three different
experiments. (A) ent-copalyl diphosphate synthases, (B)
ent-cassa-12,15-diene synthase, (C) ent-kaurene oxidase, (D)
ent-cassa-12,15-diene 11-hydroxylases, (E)
sandaracopimaradiene/labdatriene synthase, (F) syn-copalyl-diphosphate
synthase, (G) 9-beta-pimara-7,15-diene oxidase, and (H) momilactone-A
synthase.
4. Discussion
In response to numerous pathogen attacks, plants amass a formidable
defense system by using a number of biotic and abiotic resources that
serve as elicitors, a defense strategy largely known as induced
resistance [[115]36,[116]37]. This type of defense strategy has
garnered tremendous research interests because it is not harmful to
humans and poses no threat to the environment. It is a safe approach in
combating a host of plant diseases [[117]38,[118]39]. Therefore, the
present study aims to elucidate the resistance-eliciting efficiency of
the FBT and COS treatments against rice seedling blight from
biochemical and molecular perspectives.
In this study, FBT and COS treatments significantly reduced both
disease incidence and index ([119]Figure 1). This suggests that FBT and
COS could be potential elicitors that increase rice resistance to F.
oxysporum attack. FBT and COS treatments also prevented root inhibition
caused by this fungus. ([120]Figure 2). In addition, there was a
corresponding increase in the activities of defense enzymes, such as
POD, SOD, CAT and PAL ([121]Figure 3). Among these enzymes, POD, SOD
and CAT are important components of antioxidant systems to develop a
broad range of defense responses to cope with pathogenic infections
[[122]40]. POD plays an important role in generating H[2]O[2] as part
of the defense response and confers resistance to a wide range of plant
pathogens [[123]41]. A previous study also found that POD is implicated
in the polymerization of monolignols into lignin and cell wall
reinforcements after pathogen attack [[124]42]. SOD can transform
superoxide radical anions (O^2−) to H[2]O[2] [[125]28], CAT can turn
H[2]O[2] into water in plant cell [[126]43]. PAL plays a critical role
in the phenylpropanoid pathway and in the response and regulation of
biotic and abiotic stresses [[127]44,[128]45]. Our results showed that
FBT and COS could increase the activities of POD, SOD, CAT and PAL
([129]Figure 3), these results were in line with previous
investigations. Similarly, it was reported that at all tested time
points, the activities of POD, SOD and CAT was significantly higher in
chitosan nano-treated seedlings compared to the pathogen-inoculated
seedlings in pearl millet cultivars [[130]26]. In a related study,
chitosan acted as an elicitor to increase CAT activity, giving improved
systemic resistance of potato tuber [[131]46]. The phenylpropanoid
biosynthesis pathway was also recently implicated in resistance
conferment among citrus fruit samples in response to three
elicitors—Salicylic acid, Pichia membranaefaciens and oligochitosan
[[132]25]. The above fingdings established the basis that FBT and COS
could be used as elicitors to promote increases in the potential
immunity of rice to ameliorate seedling blight.
In recent years, proteomic techniques have been increasingly useful in
gene screening protocols, functional genes discovery and understanding
complex molecular mechanisms in the ever-evolving plant-pathogen
interaction [[133]47]. The current study used the TMT-based proteomic
technique in order to mine some of the underlying molecular mechanisms
by which FBT and COS induce resistance against rice seedling blight.
The results of correlation coefficients showed that a good consistency
of protein expression profiles between biological replicates,
suggesting that our experiment designs and proteomic data were reliable
([134]Figure 4). A total of 922 and 132 DEPs were found in FBT and
COS-treated roots, respectively, compared with those in the control
roots after F. oxysporum inoculation, indicating that dfifferent kinds
of elicitors are likely in response to different protein expression
profiles ([135]Figure 5). In addition, the KEGG database (two-tailed
Fisher’s exact test) was used to assess DEP functions. Our results
showed that 14 and 9 enriched pathways were observed in FBT and
COS-treated roots after F. oxysporum inoculation ([136]Figure 6).
Based on Q value, the diterpenoid biosynthesis pathway was thus
selected for further study and results show that all DEPs enriched into
this pathway were up-regulated in FBT and COS-treated roots
([137]Figure 7), resulting in the increased secretion of momilactone
([138]Figure 8). As an allelochemical in rice and moss plants with a
19, 6 β-lactone structure [[139]48,[140]49], Momilactone (A and B) has
been proposed as environmentally-safe fungicidal and bacteriostatic
agents [[141]50]. Previous studies on its antifungal properties against
the highly destructive rice blast pathogen Piricularia oryzae
[[142]49,[143]51], as well as an integral part of the defense system of
the moss Hypnum plumaeforme [[144]52,[145]53,[146]54,[147]55], strongly
support the findings of the present study. In addition, momilactones A
and B significantly suppressed F. oxysporum and F. solani activities in
vitro [[148]56]. Although the biological importance of momilactones and
its defense role in rice and mosses have been abundantly reported in
the past, it was not until recently that the molecular basis for some
of these functionalities were unraveled
[[149]57,[150]58,[151]59,[152]60,[153]61]. This resulted in Momilactose
(A and B) being officially patented as non-chemical herbicides a little
over a decade ago, giving more credence to their value in green
agriculture research [[154]62]. The present study demonstrates that the
activation of the diterpenoid biosynthesis pathway plays an important
role in resistance induction against rice seedling blight using FBT and
COS treatments. More so, the terpenoid biosynthesis pathway can
regulate the synthesis of precusor substances of momilactone. While
analyses of FBT-treated roots showed DEPs upregulation in the terpenoid
biosynthesis pathway ([155]Figure S1), no upregulated proteins were
found in COS-treated root. This may be the reason why there were higher
resistance levels in the former compared to the latter. Finally, some
POD activities were up-regulated while others were down-regulated after
F. oxysporum inoculation ([156]Table S3). Both treatments elevated PAL
activities, but the expression level of PAL was not altered. The basis
for this divergence is unclear at this time and thus further studies
are recommended.
In summary, FBT and COS significantly reduced both the disease
incidence and index, improved the growth status of root, and enhanced
defense enzyme activities induced by F. oxysporum. Both elicitors
significantly induced the changes of proteins involved in diterpenoid
biosynthesis pathway, resulting in the increase of related enzyme
activities and the accumulation of the antimicrobial substance,
momilactone. It can be concluded that FBT and COS could be eco-friendly
elicitors for resistance against rice seedling blight, which could
significantly boost rice productivity to meet the nutritional needs of
a rising global population in China.
Supplementary Materials
The following are available online at
[157]https://www.mdpi.com/2223-7747/8/12/538/s1, Figure S1:
Illustration of KEGG pathway of terpenoid backbone biosynthesis in
oligochitosan-treated roots. Red means up-regulated proteins in FBT and
COS-treated roots compared to control root, Table S1: Grading standard
of rice seedlings blight, Table S2: Primer sequences of Protein for
RT-qPCR, Table S3: DEPs annotated as peroxidase in FBT and COS-treated.
[158]Click here for additional data file.^ (458.6KB, pdf)
Author Contributions
B.M. and J.Z. conceived the study and designed the project. B.M., C.L.,
J.H., K.T. and F.Z. performed the experiment, analyzed the data and
drafted the manuscript. J.W., M.Y. and Z.G. helped to revise the
manuscript. All authors read and approved the final manuscript.
Funding
This research was funded by the Scientific and Technological Project of
Qiqihar City of China (NYGG-201716) and the Natural Science Foundation
of Heilongjiang Province of China (C2017032). And the APC was funded by
C2017032.
Conflicts of Interest
The authors hereby declare that there was no conflict of interest in
the present study.
References