Gene 728 (2020) 144288

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Gene
journal homepage: www.elsevier.com/locate/gene

Research paper

The dynamic transcriptome of pepper (Capsicum annuum) whole roots T

reveals an important role for the phenylpropanoid biosynthesis pathway in
root resistance to Phytophthora capsici
Ying Lia,1, Ting Yub,1, Tingquan Wua,c, Rui Wanga,c, Hengming Wanga, Hu Dua,c, Xiaowan Xua,
⁎
Dasen Xiea,c, XiaoMei Xua,c,
a
Vegetables Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, PR China
b
Agro-biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, PR China
c
Guangdong Key Laboratory for New Technology Research of Vegetables, Guangzhou, 510640, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Phytophthora root rot, caused by the soilborne oomycete pathogen Phytophthora capsici (Leon.), is a devastating
Capsicum annuum disease causing significant losses in pepper production worldwide. To uncover the mechanism of root-mediated
Phytophthora root rot resistance to P. capsici we elucidated the dynamic transcriptome of whole pepper roots of the resistant accession
Root-mediated resistance CM334 and the susceptible accession NMCA10399 after P. capsici infection at 0, 12 and 36 hpi using RNA-Seq
RNA-Seq
method. We detected that the roots of the resistant CM334 and the susceptible NMCA10399 had different
Secondary metabolites
transcriptional responses to P. capsici, suggesting the former activated a response to P. capsici earlier than the
latter. KEGG enrichment analysis showed the pathways involved in the synthesis of secondary metabolites were
those in which the most DEGs were enriched. Focusing on the gene regulation of phenylpropanoid biosynthesis-
related genes, we found genes related to the key enzyme phenylalanine ammonia-lyase (PAL) were activated
earlier with greater changes in the resistant accession than in the susceptible one. Moreover, genes related to
cinnamoyl-CoA reductase (CCR1) were also upregulated in resistant roots but downregulated with great folder
changes in susceptible roots. Briefly, we inferred that the phenylpropanoid biosynthesis pathway, especially
cinnamaldehyde and lignin derived from its branches, played significant roles in pepper root resistance to P.
capsici. These results provide new insight into root-mediated resistance to P. capsici in pepper.

1. Introduction
The soilborne oomycete plant pathogen Phytophthora capsici (Leon.)
is one of the most devastating pathogens to pepper production world-
wide (Leonian, 1922). In South China, which is characterized by high
temperature and humidity, Phytophthora blight on pepper becomes
especially serious. Depending on the point of infection, P. capsici may
cause several different disease syndromes, including root rot, stem rot,
fruit rot and foliar blight in pepper (Ristaino, 1990), and their genetic
mechanisms are not considered to be entirely the same (Sy et al., 2005).
For Phytophthora root rot, which usually causes plant death, two genetic
models have been reported, including control via a single dominant
gene (Walker and Bosland, 1999; Sy et al., 2005; Monroy-Barbosa and
Bosland, 2008; Xu et al., 2016) and quantitative trait loci (QTLs) acting
together (Ogundiwin et al., 2005; Quirin et al., 2005; Minamiyama
et al., 2007; Kim et al., 2008; Truong et al., 2012). However, no re-
sistant gene (QTL) for Phytophthora root rot has been cloned to date,
and the molecular mechanism of resistance to Phytophthora root rot in
pepper remains largely unknown.
With the exception of forward genetics, the analysis of the whole-
genome transcriptional response is likely the most effective approach to
uncover the mechanisms of resistance to P. capsici in pepper roots.
However, compared to studies of other crops, transcriptome studies in
peppers have been very limited until now. An analysis of whole-genome
transcriptional responses of pepper after P. capsici infection has not
been reported to date. Fortunately, a transcriptome profiling dataset of

Abbreviations: QTL, Quantitative trait loci; MAMP, Microbe-associated molecular pattern; GO, Gene ontology; LRR, Leucine-rich repeat; FPKM, Fragments Per
Kilobase of transcript sequence per Millions base pairs sequenced; DEG, Differential expression gene; KEGG, Kyoto encyclopedia of genes and genomes; RNA,
Ribonucleic acid; qRT-PCR, Quantitative real-time polymerase chain reaction
⁎
Corresponding author.
E-mail address: xiaomeixu@gdass.cn (X. Xu).
1
These authors have contributed equally to this work.

https://doi.org/10.1016/j.gene.2019.144288
Received 15 September 2019; Received in revised form 12 December 2019; Accepted 12 December 2019
Available online 14 December 2019
0378-1119/ © 2019 Elsevier B.V. All rights reserved.
Y. Li, et al.
pepper immune responses to multiple pathogens, including
Phytophthora infestans, pepper mottle virus and tobacco mosaic virus,
has been provided on the website (https://figshare.com/). These data
provide valuable resources for further investigation of the interaction
between pepper and these pathogens (Kim et al., 2018). Recently, a
time course transcriptomic study was performed on detached leaves
inoculated with P. infestans, and a subset of EAS/EAH gene family
members were identified as being induced upon P. infestans infection in
pepper (Lee et al., 2017). Transcriptome analysis has also been used to
identify disease resistance (R) genes in peppers. An example case in-
cludes the candidate Bs4C gene, an R gene from pepper that mediates
recognition of the Xanthomonas TALE protein AvrBs4, which was
identified using RNA-Seq (Strauss et al., 2012). Additionally, candidate
R genes against capsicum chlorosis virus were also identified using a
similar approach (Widana-Gamage et al., 2016).
Plant roots are usually the first organs to be infected by soilborne
pathogens. Recently, an increasing number of reports have focused on
the molecular mechanisms of root-mediated resistance in plants and
demonstrated that plant roots also have an immune system that func-
tions to protect the plant against soilborne pathogens. In Arabidopsis
roots, tissue-specific microbe-associated molecular pattern (MAMP)
responses and potential pathogen-encoded mechanisms were activated
to block MAMP-elicited signaling pathways (Millet et al., 2010). In
tomato, grafting of resistant rootstocks to susceptible scions was suc-
cessfully used to manage bacterial wilt caused by Ralstonia solana-
cearum, which indicated that roots also appear to trigger a defense re-
sponse to the pathogen (McAvoy et al., 2012; Rivard et al., 2012). In
addition, using whole root transcriptomic analysis, it was revealed that
roots mediate resistance to R. solanacearum through genome-wide
transcriptomic changes resulting in strong activation of defense genes
and alteration of auxin pathways (French et al., 2018). Similar studies
have also been performed in wild potato roots of resistant and sus-
ceptible genotypes against R. solanacearum and found that gene func-
tion was primarily downregulated during development, and those in the
Gene Ontology (GO) category ‘biotic stress’ were mainly upregulated in
both genotypes after infection (Zuluaga et al., 2015). In contrast, in a
time course of peanut root infection with R. solanacearum, the expres-
sion of many defense genes, including leucine-rich repeat (LRR) kinases
and R genes, were downregulated in both resistant and susceptible
roots, and carbohydrate metabolism was repressed after infection in
roots of both resistant and susceptible peanut plants but was more
strongly inhibited in resistant plants (Chen et al., 2014). All of these
reports suggested that the mechanisms of root-mediated resistance may
differ among different plant species.
Plant secondary metabolites, including terpenoids, phenylpropa-
noids, and nitrogen-containing substances, are low molecular mass
compounds derived from multiple pathways based on different bio-
synthetic substrates and pathways (Makkar et al., 2007) and function as
defense molecules against pathogens or competing plants or as signal
molecules such as hormones and attractants for pollinators or seed
dispersing animals (Makkar et al., 2007). Cinnamaldehyde, one of the
numerous secondary metabolites, is a natural antimicrobial that has
been found to have potent antifungal activities against a wide variety of
fungi and has recently been applied as an efficient bioagent for con-
trolling plant disease in agriculture (Chen and Dai, 2012; Hu et al.,
2013; Wu et al., 2017; Duan et al., 2018). It was reported that cinna-
maldehyde enabled the inhibition of bacterial growth by regulating
genome-wide transcriptional changes in bacteria (Visvalingam et al.,
2013) and markedly inhibited the growth of P. capsici by stimulating
transient Ca2+ efflux via Michael addition (Hu et al., 2013). Moreover,
cinnamaldehyde might exhibit its antifungal activity by interfering with
the synthesis of the cell wall of Geotrichum citri-aurantii and therefore
may damage cell wall permeability and integrity (OuYang et al., 2019).
In addition to cinnamaldehyde, lignin is another important secondary
metabolite involved in plant disease resistance. Lignin is a complex
phenolic compound synthesized through phenylpropanoid metabolism
2
Gene 728 (2020) 144288
and is one of the components of the plant cell wall (Pusztahelyi et al.,
2015). Increasing the content of lignin, which is a physical barrier for
plant defense against pathogens, can enhance the ability of the plant
cell wall to prevent fungal invasion and enzyme-induced degradation
(Sattler and Funnell-Harris, 2013). Pomar et al. (2004) observed that
the total stem lignin content increased in pepper plants after inocula-
tion with Verticillium dahliae and suggested that lignification may be a
mechanism through which pepper could restrict the growth of V. dah-
liae hyphae in the xylem (Pomar et al., 2004). Moreover, inoculation
with V. dahliae also induced a significant increase in the total amount of
lignin in tomato roots in both susceptible and resistant lines, although
at earlier times in the latter (Gayoso et al., 2010). Xu et al. (2011)
reported that lignin metabolism has a central role in resistance to the
wilt fungus V. dahliae in cotton, detecting an increased level of ex-
pression of lignin synthesis-related genes after inoculation in both re-
sistant and susceptible cotton plants but with greater fold change and
earlier activation in the resistant line.
In this study, the first global analysis of the dynamic transcriptome
of resistant and susceptible pepper roots after P. capsici infection at 12
and 36 h post inoculation (hpi) (0 hpi as mock-inoculated control) was
performed using the RNA-Seq method. We analyzed the responsive
genes in resistant and susceptible accessions independently and com-
pared the responses between these two accessions. KEGG enrichment
analysis of DEGs was performed and phenylpropanoid biosynthesis
pathway was found to play important roles in root resistance to P.
capsici. Gene expression of key genes on this pathway was analyzed and
verified by qRT-PCR. These related results provided further insight into
root-mediated resistance in plants and might help in developing pepper
varieties with resistance to P. capsici.
2. Materials and methods
2.1. Plant growth and inoculation with P. capsici
The plant materials are described in more detail in the reference
(Reeves et al., 2013). Briefly, CM334, a landrace from Mexico, is known
to have the highest resistance to P. capsici, and NMCA10399, the P.
capsici-susceptible accession, has been found to be susceptible to all P.
capsici isolates tested to date (Oelke et al., 2003; Sy et al., 2008). All
plant materials were grown in plastic pots filled with plant growth soil
mix (Floragard, Germany) and kept in a growth room at 26 ± 2 °C
with a relative humidity of 70% under a 14-h light and 10-h dark
photoperiod.
At the six-leaf stage, the plants were inoculated with P. capsici iso-
late Byl4 which was isolated from infected pepper plants in 2012 in
Baiyun field, Guangzhou, Guangdong Province, China (Xu et al., 2016).
Twelve hours before inoculation, the plants grown in plastic pots were
watered sufficiently, and then the root-shoot soil line was injected with
2 ml spore suspensions of isolate Byl4, at a concentration of 5 × 104
zoospores/ml, as described by (Xu et al., 2016) with a small number of
modifications. The inoculated plants were first kept in darkness at 28 °C
and 80% humidity for 24 h and then grown under the same conditions
as mentioned above.
2.2. Sampling and RNA extraction
The whole roots from 4 plants of each genotype (CM334 and
NMCA10399) were sampled at 0 h, 12 h, and 36 h after P. capsici in-
oculation. Roots from these 4 plants were pooled for each genotype at
each targeted timepoint in each replicate. Three independent duplicates
were performed. Harvested roots were ground into powder using a
mortar and pestle under liquid nitrogen. Approximately 120 mg of
powder from each sample was used to extract total RNA using the
GenElute™ Total RNA Purification Kit (Sigma-Aldrich, USA) according
to the manufacturer’s instructions. RNA degradation and contamination
were monitored on 1% agarose gels. An ND-2000C spectrophotometer
Y. Li, et al.
(NanoDrop, USA) was used to determine the absorbance ratios at 260/
280 nm and at 260/230 nm to quantify and assess the purity of the RNA
samples. All 18 qualified samples (2 genotypes × 3 timepoints × 3
duplicates) were submitted to Novogene Bioinformatics Technology Co.
Ltd. (Beijing, China) for RNA-Seq. Before sequencing, the exact RNA
concentration was further measured using a Qubit® 2.0 Fluorometer
(Life Technologies, CA, USA), and RNA integrity was accurately as-
sessed using an Agilent 2100 Bioanalyzer system (Agilent Technologies,
CA, USA). All samples had an RNA integrity number (RIN) of at least
7.2, which means they qualified for sequencing (Schroeder et al., 2006).
2.3. cDNA library construction and sequencing
Eighteen samples of RNA from the roots of the resistant and sus-
ceptible accessions, with 9 samples per genotype, were used to con-
struct the cDNA libraries. For each sample, the mRNA was enriched by
magnetic beads with Oligo (dT) combining the ployA tail of mRNA
through A-T complementary pairing. Then, the mRNA was broken into
short fragments after adding the fragmentation buffer. The prepared
mRNA was used as the template, and the random hexamer bases were
used as primers to synthesize one chain of the cDNA, and the second
chain of the cDNA was synthesized by adding the buffer, dNTPs and
DNA polymerase I. The synthesized double-chain cDNA was purified
using AMPure XP beads, the ends were repaired, and the A tail was
appended and connected to the sequencing adaptors. Then, AMPure XP
beads were again used for the fragment size selection, and finally, PCR
enrichments were conducted to obtain the final cDNA libraries.
The libraries were preliminarily quantified using Qubit 2.0 and were
diluted to 1 ng/µl to check the inserted fragment length (insert size)
using the Agilent 2100. After determining that the insert size met the
expectations, the Q-PCR method was used for the accurate quantifica-
tion of the concentration of the libraries, and a concentration higher
than 2 nM was considered adequate. The 18 qualified cDNA libraries
were pooled together and sequenced on an Illumina HiSeq 2500 plat-
form at Novogene Bioinformatics Technology Co. Ltd. (Beijing, China)
using 150 bp paired-end sequencing. The raw paired-end reads were
submitted to the Sequence Read Archive of the NCBI (accession
number: SRP217055).
2.4. RNA-Seq data analysis
For the raw reads, which contained the adaptor sequences, those
with a proportion of N of more than 10% and with a proportion of low-
quality bases (Qphred ≤ 20) higher than 50% were removed. The clean
reads were aligned to the pepper reference genome of CM334 (http://
peppergenome.snu.ac.kr/) using the HISAT software with the default
parameters (Kim et al., 2015). HTSeq with the union model (Anders
et al., 2015) was used to calculate the FPKM values (expected number
of Fragments Per Kilobase of transcript sequence per Millions base pairs
sequenced) to obtain the gene expression level (Trapnell et al., 2010).
Then, DESeq (Anders and Huber, 2012) was performed to detect dif-
ferentially expressed genes (DEGs), and DEGs were considered sig-
nificant at Padj ≤ 0.05 (P ≤ 0.05 and FDR ≤ 0.05) and a relative
change threshold of 1.8-fold (log2fold change ≥ 0.8 or log2fold
change ≤ −0.8). Finally, KEGG pathway enrichment analysis of DEGs
was carried out using KOBAS (Xie et al., 2011), and Venn diagrams
were plotted in the R environment (v3.1.2) (http://www.r-project.org).
2.5. Validation of gene expression by qRT- PCR
The expression profiles of 18 out of 42 DEGs assigned to the phe-
nylpropanoid biosynthesis pathway (Figure S1) were validated using
qRT-PCR. Nine genes, including UBP5 (CA09g09640), UBP13
(CA11g15560), UBC38 (CA07g13540), IF1C (CA12g20290), IF4G
(CA01g27330), EFTS (CA07g07780), ubiquitin carboxyl-terminal hy-
drolase 3-like (Novel07992), E3 ubiquitin-protein ligase (Novel02297),
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Gene 728 (2020) 144288
and actin-related protein 2/3 complex subunit 3-like (Novel04650),
were selected from non-DEGs in the RNA-Seq dataset as candidate in-
ternal reference genes. Primers were designed using Primer Premier 5.0
(Premier Biosoft International) according to the coding sequences
(CDS) of each gene on the pepper reference genome of CM334 (http://
peppergenome.snu.ac.kr/) or in the RNA-Seq dataset. Information on
the primers is listed in Table S1. Complementary DNA was synthesized
using the same total RNA preparations used for Illumina sequencing
with RevertAidTM First Strand cDNA Synthesis Kit (Thermo Scientific,
MA, USA). qRT-PCR was performed with three technical replicates on a
StepOnePlusTM Real-Time PCR System (Applied Biosystems) with 25 μl
final volumes containing 2 μl of cDNA, 1 μl of each primer (2.5 μM),
8.5 μl of DNase- and RNase-free water and 12.5 μl of 2× SYBR® Select
Master Mix (Applied Biosystems), with the following amplification
protocol: 95 °C for 5 min followed by 40 cycles at 95 °C for 15 s and
60 °C for 30 s. The expression stability of the 9 candidate internal re-
ference genes was evaluated using NormFinder software (Andersen
et al., 2004), and the gene with the most stable expression was selected
as the internal reference gene. The relative expression levels of the 18
selected DEGs normalized to the expression level of the internal re-
ference control were calculated using the 2−ΔΔCt method (Livak and
Schmittgen, 2001), and coefficient analysis was carried out to evaluate
the correlation between the qRT-PCR and the RNA-Seq results (Benesty
et al., 2009).
3. Results
3.1. Phenotypes after infection with P. capsici
We used the resistant accession CM334 and the susceptible acces-
sion NMCA10399 for our analysis. CM334, known as the accession most
resistant to P. capsici, has displayed resistance to all P. capsici isolates
thus far (Oelke et al., 2003; Thabuis et al., 2004). NMCA10399 has been
susceptible to all P. capsici isolates tested to date (Oelke et al., 2003; Sy
et al., 2008). These materials including the inoculum Byl4 had pre-
viously been utilized in our study on genetic analysis and gene mapping
for resistance to P. capsici (Xu et al., 2016). As expected, the resistant
and susceptible accessions showed completely different phenotypes
7 days after inoculation. For the resistant CM334, there were no visible
lesions on the roots of plants. Moreover, the size of roots indicated that
the plant continued to grow as normal after inoculation. In contrast to
the resistant CM334, the roots of the susceptible NMCA10399 were
completely lesioned and began to rot 7 days after inoculation, and the
size of the roots suggested that their growth slowed after inoculation
(Fig. 1). Considering that changes in gene expression must precede the
appearance of the disease phenotype and our pre-experimental results,
we chose 12 hpi and 36 hpi for comparison with the 0 hpi mock control
for each genotype to identify the transcriptional responses in resistant
and susceptible accessions.
3.2. Summary of the transcriptome data
The root transcriptomes of two pepper accessions at targeted times
of 0, 12 and 36 hpi were sequenced and yielded an average of 53.8 M
clean reads and 8 G high-quality bases per sample after sequences were
filtered (Table S2). An average of 81% of clean reads of each sample
was uniquely aligned to the pepper reference genome (Table S3). FPKM
were used as the normalized expression values of genes. Approximately
40% of the genes of each sample whose FPKM was lower than 1 were
considered to be expressed at levels that were too low, while 9%, 21%,
20% and 9% of the genes of each sample were determined to belong to
the low expression (1 < FPKM ≤ 3), medium expression
(3 < FPKM ≤ 15), high expression (15 < FPKM ≤ 60), and ex-
tremely high expression (60 > FPKM) categories, respectively (Table
S4).
Y. Li, et al.
Fig. 1. Root architecture of the resistant accession CM334 and the susceptible
accession NMCA10399 at 0 and 7 days post inoculation (dpi) of P. capsici.
3.3. Sample correlations
Pearson's correlation coefficient was used to investigate the sample
correlation and biological duplication. As shown in Fig. 2A, the corre-
lation coefficients among the samples of CM334 at 0 h (S0334), 12 h
(S12334), and 36 h (S36334) were all higher than those between the
genotypes of CM334 and NMCA10399 at any timepoint after P. capsici
inoculation. Similarly, the correlation coefficients among the samples of
NMCA10399 at 0 h (S0339), 12 h (S12339), and 36 h (S36339) were all
higher than the correlation coefficients between S0334 and S0339,
S12334 and S12339, S36334 and S36339. The correlation analysis in-
dicated that the intra-genotype correlations were higher than the inter-
genotype correlations, implying that the intra-genotypes gene expres-
sion patterns were much more similar than the inter-genotypes pat-
terns. Moreover, the correlation analysis also showed that one group
with three biological duplications was highly relevant and proved the
feasibility of this experiment.
3.4. Transcriptional response of resistant and susceptible plant roots to P.
capsici infection
To identify DEGs, pairwise comparisons were independently made
4
Gene 728 (2020) 144288
between each timepoint and the 0 hpi (mock-inoculated control)
timepoint within the resistant and susceptible accessions. A total of 810
and 1110 DEGs were identified in the resistant CM334 at 12 and 36 hpi,
respectively, while a total of 291 and 2465 were identified in the sus-
ceptible NMCA10399 at these two timepoints, respectively (Fig. 2B). In
roots of the susceptible NMCA10399 at 12 hpi, 236 genes were sig-
nificantly upregulated and 55 were downregulated, while in the re-
sistant CM334 at that time point, 560 genes were significantly upre-
gulated and 250 were downregulated, which was a difference of more
than twice as many DEGs. In contrast, at 36 hpi, 1641 genes were
significantly upregulated and 824 genes were downregulated in
NMCA10399 roots, which were more than twice as many genes com-
pared with the 763 upregulated and 347 downregulated genes in
CM334 roots (Fig. 2B). From the results of differential expression
analysis, we found that after P. capsici infection, both the resistant and
the susceptible pepper accessions presented more DEGs (including up-
regulated and downregulated) at 36 hpi than at 12 hpi, indicating that
both plants continued to respond to P. capsici after 12 hpi. Additionally,
more than twice the number of DEGs was detected in the resistant ac-
cession (560 upregulated and 250 downregulated) compared with the
susceptible accession (236 upregulated and 55 downregulated) at
12 hpi, suggesting that the resistant accession CM334 activated the
response to P. capsici earlier than did the susceptible NMCA10399.
To better understand the response of resistant and susceptible ac-
cession roots to P. capsici infection, we performed Venn analysis of
DEGs. There was a total of 2154 DEGs (469 exclusively in the resistant
CM334, 1229 exclusively in the susceptible NMCA10399 and 456 in
both) upregulated at 12 and/or 36 hpi in resistant and/or susceptible
pepper roots after P. capsici inoculation (Fig. 2C). Among them, 123
DEGs were upregulated in both pepper accessions at all time points
(Fig. 2C), suggesting that these DEGs were the key genes involved in the
response to P. capsici after infection, including some DEGs were most
related to transcription factors and transport, such as ERF95 (ethylene-
responsive transcription factor), ERF98 (ethylene-responsive tran-
scription factor), CHX20 (cation/H(+) antiporter 20), RVE1 (protein
REVEILLE 1), ALMTA (aluminum-activated malate transporter 10),
COL5 (zinc finger protein CONSTANS-LIKE 5) and so on (Table 1); most
of these DEGs are involved in stress tolerance. Furthermore, in the roots
of the resistant CM334, 127 and 153 upregulated DEGs were specifi-
cally detected at 12 and 36 hpi, respectively. Compared to those of the
resistant roots, there was a major shift in the number of upregulated
DEGs in susceptible roots, from 14 to 1171 between 12 and 36 hpi,
indicating that a larger number of upregulated DEGs were induced to
respond to P. capsici infection at 36 hpi in the susceptible roots.
Compared to 2154 upregulated DEGs, there were a total of 1138
(298 in the resistant CM334, 728 in the susceptible NMCA10399 and
112 in both) downregulated DEGs, almost half the number of upregu-
lated DEGs, found at 12 and/or 36 hpi in resistant and/or susceptible
pepper roots (Fig. 2D). Among them, 26 DEGs, including mostly genes
related to enzymes, such as SBT17 (Subtilisin-like protease), THIC
(phosphomethylpyrimidine synthase), THI41 (thiamine thiazole syn-
thase 1), ZIP2 (zinc transporter 2), BZP06 (basic leucine zipper 6) and
so on (Table 2), were downregulated in the two pepper accessions at all
time points (Fig. 2D), suggesting that these DEGs were suppressed after
P. capsici infection. In addition, 54 and 130 downregulated DEGs were
exclusively detected in the roots of the resistant CM334 at 12 and
36 hpi, respectively. The same trend in the number of downregulated
DEGs from 10 to 708 was also observed in susceptible roots between 12
and 36 hpi (Fig. 2D), indicating that a larger number of genes were
suppressed in response to P. capsici infection at 36 hpi in the susceptible
accession NMCA10399. Briefly, the results stated above indicate that
resistant and susceptible plant roots respond to P. capsici infection
differently. In the resistant accession CM334, the response to P. capsici
infection was induced earlier than in the susceptible NMCA10399, with
many more upregulated/downregulated DEGs detected than those in
the susceptible NMCA10399 roots at 12 hpi. However, a more intense
Y. Li, et al.
Fig. 2. The sample correlations, differentially expressed genes (DEGs) and the Venn analysis of DEGs. (A) Pearson correlation between RNA-seq samples. R2: the
square of the Pearson correlation coefficients. (B) DEGs identified between two-group comparisons. (C) Venn analysis of upregulated DEGs. (D) Venn analysis of
downregulated DEGs.
response was initiated in the susceptible accession NMCA10399 at
36 hpi, with more than twice as many upregulated/downregulated
DEGs than those observed in the resistant CM334 roots (Fig. 2B).
3.5. KEGG functional annotations of DEGs
To better understand the pathways in which DEGs were involved in
response to P. capsici infection, KEGG enrichment analysis of DEGs was
performed. A total of 23 and 16 KEGG pathway categories were en-
riched by up- and downregulated DEGs, respectively, from four pair-
wise comparisons (Fig. 3). Among them, five KEGG pathways were
enriched in both up- and downregulated DEGs, including plant hor-
mone signal transduction and zeatin biosynthesis pathways, which
were considered to be involved in the defense response. Accordingly, 18
and 11 KEGG pathways were uniquely enriched in up- and
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Gene 728 (2020) 144288
downregulated DEGs, respectively (Fig. 3 and Table S5). For plant
hormone signal transduction, the classic defense hormone pathway was
uniquely significantly enriched in upregulated DEGs in roots of the
susceptible accession only at 12 hpi (P = 4.6 × 10−3) compared to that
in downregulated DEGs, which were significantly enriched in roots of
the resistant CM334 at both 12 (P = 2.8 × 10−2) and 36 hpi
(P = 4.3 × 10−4) (Fig. 3 and Table S5), suggesting that the resistant
and susceptible accessions may respond to P. capsici through different
signaling pathways. Unexpectedly, the plant-pathogen interaction
pathway was only present in the upregulated DEGs. Moreover, it was
exclusively significantly enriched in upregulated DEGs in roots of the
susceptible accession only at 36 hpi (P = 1.0 × 10−2) but not in re-
sistant roots at 12 or 36 hpi (Fig. 3 and Table S5). Notably, the KEGG
pathways enriched for the most DEGs are those involved in the synth-
esis of secondary metabolites. The top three enriched pathways were
Y. Li, et al. Gene 728 (2020) 144288

Table 1
Common upregulated DEGs in the two pepper accessions at any timepoint (top 20 listed).
Gene ID CM334-12hpi CM334-36hpi NMCA10399-12hpi NMCA10399-36hpi Gene name Description
vs vs vs vs
CM334-0hpi CM334-0hpi NMCA10399-0hpi NMCA10399-0hpi
CA03g05620 3.0843 3.9763 14.6 17.9 ERF95 Ethylene-responsive transcription factor ERF095
CA04g00510 5.8852 6.645 3.9045 7.6687 ERF98 Ethylene-responsive transcription factor ERF098
CA09g04800 6.0671 5.0844 3.4259 2.7923 CHX20 Cation/H(+) antiporter 20
CA02g18810 5.1835 4.796 5.8302 5.315 RVE1 Protein REVEILLE 1
CA01g00460 4.165 4.2066 5.2938 4.5388 ALMTA Aluminum-activated malate transporter 10
CA07g00270 2.9268 3.0803 5.2183 5.0655 COL5 Zinc finger protein CONSTANS-LIKE 5
CA03g25750 5.179 5.1985 3.7489 3.5176 Y5279 DUF21 domain-containing protein
CA03g19240 3.6012 3.3166 5.0677 4.7648 SIGE RNA polymerase sigma factor sigE
CA10g22110 4.9361 4.363 2.6639 3.0818 FKB65 Peptidyl-prolyl cis–trans isomerase FKBP65
CA12g19270 3.4323 2.9005 4.1197 4.8042 COL4 Zinc finger protein CONSTANS-LIKE 4
CA05g08190 4.3777 4.2222 4.7481 4.1848 CDF2 Cyclic dof factor 2
CA11g02380 2.9525 3.0137 3.6758 4.6787 BZP43 Basic leucine zipper 43
CA12g02080 4.0658 4.096 4.2583 4.6353 SKOR Potassium channel SKOR
CA06g04100 3.6436 3.9418 4.2986 4.5599 WTR14 WAT1-related protein
CA10g20370 4.4848 3.7259 3.9392 2.8681 ZOG Zeatin O-glucosyltransferase
CA02g04730 4.3211 4.1598 4.2914 4.102 NFYA7 Nuclear transcription factor Y subunit A-7
CA11g04440 2.0886 2.0732 1.9521 4.2994 NAC29 NAC transcription factor 29
CA12g01260 2.367 2.1231 3.0293 4.2401 TDT Tonoplast dicarboxylate transporter
CA05g16280 3.545 3.4406 4.1839 3.1897 UVR8 Ultraviolet-B receptor UVR8
CA01g16810 3.9813 3.8964 3.9002 4.0753 P2C34 Probable protein phosphatase 2C 34

secondary metabolite biosynthesis, phenylpropanoid biosynthesis and
phenylalanine metabolism pathways (Fig. 3A). Among them, the latter
two were considered to be major pathways leading to the synthesis of
secondary metabolites involved in plant defense against pathogens
(Widana-Gamage et al., 2016).
3.6. Secondary metabolites synthesized through the phenylpropanoid
biosynthesis pathway played important roles in the response to P. Capsici
As mentioned above, the phenylpropanoid biosynthesis pathway
could lead to the synthesis of secondary metabolites involved in plant
defense against pathogens. One of the compounds synthesized through
this pathway was cinnamaldehyde, which is a natural antimicrobial
that has been found to have potent antifungal activities against a wide
variety of fungi (Bang et al., 2000; Cheng et al., 2008; Hu et al., 2013;
Wu et al., 2017; Duan et al., 2018; OuYang et al., 2019). There are 10
enzymes involved in the phenylpropanoid biosynthesis pathway
(Fig. 4). Among them, PAL is the first and most important enzyme,
which converts phenylalanine to cinnamic acid, the precursor of cin-
namaldehyde. Mapping DEGs to the pathway showed that two PAL-
related genes (CA05g20790 and CA12g15510) were all upregulated in
the resistant accession CM344 at 12 hpi and continued to show higher
expression at 36 hpi. In contrast, in the susceptible NMCA10399, only
one gene (CA12g15510) was upregulated at 36 hpi (Fig. 4 and Figure
S1). 4-coumarate-CoA ligase (4CL2) and cinnamoyl-CoA reductase
(CCR1) were the other key enzymes related to the synthesis of cinna-
maldehyde, and two DEGs were assigned to each enzyme. The expres-
sion of DEGs mapped to 4CL2 showed that one gene (CA03g21580) was

Table 2
Common downregulated DEGs in the two pepper accessions at any timepoint.
Gene ID CM334-12hpi CM334-36hpi NMCA10399-12hpi NMCA10399-36hpi Gene name Description
vs vs vs vs
CM334-0hpi CM334-0hpi NMCA10399-0hpi NMCA10399-0hpi
CA10g14810 −1.4467 −1.6759 −2.3562 −6.0878 SBT17 Subtilisin-like protease SBT1.7
CA06g07140 −5.707 −5.8391 −3.5328 −3.8671 ELF4 Protein EARLY FLOWERING 4
CA06g05410 −2.6945 −2.8697 −2.586 −4.7984 THIC Phosphomethylpyrimidine synthase
CA04g14140 −4.3552 −3.7509 −4.5629 −4.3666 GIGAN Protein GIGANTEA
Novel03040 −4.1931 −3.8017 −4.1099 −3.8849 – PREDICTED: protein GIGANTEA
CA06g04640 −4.0511 −3.9195 −2.7148 −1.6707 PCL1 Transcription factor LUX
CA07g19520 −2.4562 −3.1805 −3.276 −4.0251 THI41 Thiamine thiazole synthase 1
CA04g18690 −3.5286 −3.9464 −2.6011 −2.0211 – –
CA08g05800 −2.7055 −2.9099 −2.4984 −3.6588 NLTP Non-specific lipid-transfer protein
Novel04270 −3.3392 −3.609 −2.9249 −2.9529 – Uncharacterized protein
CA12g10560 −1.7317 −2.0028 −1.6808 −3.5997 SUT34 Probable sulfate transporter 3.4
CA07g19510 −1.5819 −1.9097 −1.8875 −3.5951 PME1 Pectinesterase 1
CA01g10700 −2.721 −2.6333 −3.3156 −3.5697 JMJ30 Putative lysine-specific demethylase JMJD5
CA02g03340 −2.3779 −3.3966 −2.3362 −2.1619 DNAJ Chaperone protein DnaJ
CA03g16270 −2.9494 −2.7021 −3.3594 −2.4117 – –
CA09g13840 −2.7648 −3.2868 −2.9826 −2.2651 COL9 Zinc finger protein CONSTANS-LIKE 9
Novel03250 −2.6056 −2.6118 −2.5833 −1.8049 – Uncharacterized protein
CA07g15940 −2.5423 −2.3762 −2.3162 −1.6828 HSPC2 Small heat shock protein C2
CA08g18480 −2.1785 −2.4473 −2.2832 −2.4125 ELF4 Protein EARLY FLOWERING 4
CA03g21270 −1.3879 −1.5319 −1.3189 −2.4138 SBT33 Subtilisin-like protease SBT3.3
CA07g20290 −1.7108 −1.7054 −1.3573 −2.1102 ZIP2 Zinc transporter 2
CA02g25790 −1.8116 −1.9877 −1.8654 −1.6254 SRK2I Serine/threonine-protein kinase SRK2I
CA12g07640 −1.2727 −1.1276 −1.8501 −1.6145 BZP06 Basic leucine zipper 6
CA08g12400 −1.5603 −1.7969 −1.4352 −1.1563 DNJB6 DnaJ homolog subfamily B member 6
CA01g06670 −1.3049 −1.3133 −1.4208 −1.7957 CLSY3 SNF2 domain-containing protein CLASSY 3
CA11g16570 −1.0851 −1.2199 −1.542 −1.6497 – –

6
Y. Li, et al.
Fig. 3. KEGG pathway enrichment analysis of DEGs. (A) KEGG pathways enriched in upregulated DEGs. (B) KEGG pathways enriched in downregulated DEGs. The
number in the box is the number of DEGs enriched in one pathway per sample.
upregulated in both the resistant CM334 and the susceptible
NMCA10399 at both timepoints but showed greater fold changes in the
resistant CM334, with the other (CA06g17350) was uniquely upregu-
lated in the resistant CM334 at 36 hpi (Fig. 4 and Figure S1). For the
DEGs assigned to CCR1, however, one (CA03g32090) was uniquely
upregulated in the resistant CM344 at 12 hpi, while the other
(CA03g32100) was drastically downregulated in the susceptible
NMCA10399 at 36 hpi (Fig. 4 and Figure S1). These results suggest that
resistant CM334 initiated cinnamaldehyde synthesis earlier and at
higher levels than did the susceptible NMCA10399 to defend against P.
capsici.
Caffeoyl quinic acid, suggested to function in defense against pre-
dation and infection in plants (Faulds and Williamson, 1999; Jr.
Harrison et al., 2008), is a secondary metabolite of one of the branch
pathways of phenylpropanoid biosynthesis. Herein, we found that two
synthetase 5-O-(4-coumaroyl)-D-quinate 3′-monooxygenase (C98A2/
3)-related genes (Novel06712 and CA10g22120) were upregulated in
the resistant CM344 at 12 hpi, and the other three genes (CA10g22130,
CA10g22140 and CA10g22150) were upregulated in the susceptible
NMCA10399 at 36 hpi with much higher fold changes. Additionally, the
degrading enzyme of caffeoyl quinic acid, shikimate O-hydro-
xycinnamoyl transferase (HST)-related gene (CA09g18230), was ex-
clusively downregulated in susceptible NMCA10399 roots at 36 hpi but
not in resistant CM334 roots at any time point (Fig. 4 and Figure S1).
These results indicated that both the resistant CM344 and susceptible
NMCA10399 plants initiated the biosynthesis of caffeoyl quinic acid to
respond to P. capsici after infection, but the susceptible NMCA10399
activated biosynthesis more strongly and repressed its degradation at
the same time.
Capsaicin is a kind of phytoalexin found in solanaceous plants that
possesses antimicrobial and antifungal properties and induces the ex-
pression of defense-related genes to play roles in plant defense (Veloso
et al., 2014). It is also one of the compounds synthesized through the
other branch pathway of phenylpropanoid biosynthesis. Caffeoyl-CoA
O-methyltransferase (CAMT) is the key enzyme that converts caffeoyl-
CoA to feruloyl-CoA, which is the precursor of capsaicin. We observed
that the expression of two genes (CA02g14460 and CA02g14470)
mapped to CAMT were both upregulated in the susceptible
NMCA10399 at 36 hpi, but no such marked changes were observed in
the resistant CM334 at any time point (Fig. 4 and Figure S1), implying
that NMCA10399 might strengthen its defense against P. capsici by
synthesizing capsaicin.
In addition to actively attacking P. capsici by synthesizing the three
7
Gene 728 (2020) 144288
antimicrobials mentioned above, we observed that pepper plant roots
also impeded infection by P. capsici by strengthening ‘barriers’ through
the synthesis of lignin, which is the main component of the plant cell
wall and has been demonstrated to be involved in plant defense against
pathogens (Naoumkina et al., 2010; Xu et al., 2011; Guo et al., 2016). In
the phenylpropanoid biosynthesis pathway, 6 enzymes function in the
branch pathway of lignin synthesis, including PAL, trans-cinnamate 4-
monooxygenase (TCMO), 4CL2, CCR1, cinnamyl-alcohol dehy-
drogenase (CADH6) and peroxidase (PER), and 27 DEGs were mapped
to them. Among these DEGs, all were upregulated in the resistant
CM334 or/and susceptible NMCA10399 except one gene (CA03g32100)
assigned to CCR1 (Fig. 4 and Figure S1), suggesting that both the re-
sistant and susceptible accessions activated the synthesis of lignin to
recover the cell wall after P. capsici infection, but the CCR1-related gene
might play more crucial roles, resulting in the different resistance to P.
capsici between the resistant and susceptible genotypes.
3.7. Validation of RNA-Seq data by qRT-PCR analysis
The expression of 18 selected DEGs assigned to the phenylpropanoid
biosynthesis pathway was verified by qRT-PCR. After stability evalua-
tion, IF4G (CA01g27330) was considered to be the most stable and
selected as the internal reference gene. The fold change of gene ex-
pression at 12 hpi and 36 hpi was calculated using the 2−ΔΔCt method
(Livak and Schmittgen, 2001) (Tables S6 and S7). For RNA-Seq data,
the fold change of gene expression was calculated by dividing the FPKM
value at each timepoint in each genotype with the FPKM value of the
0 hpi mock control. Coefficient analysis showed that qRT-PCR data of
the 18 selected DEGs were significantly correlated with the RNA-Seq
results (r = 0.96, p < 0.001; Fig. 5), which indicated that the RNA-Seq
data in the present study were reliable and could support the tran-
scriptomic analysis presented above.
4. Discussion
Although Phytophthora blight is one of the most devastating diseases
to pepper production worldwide, an analysis of whole-genome tran-
scriptional responses of pepper after P. capsici infection has not been
reported to date. This report was the first global analysis of the dynamic
transcriptome of resistant and susceptible pepper roots after P. capsici
infection. In the present study, we found that a total of 810 and 1110
DEGs presented in resistant CM334 roots at 12 and 36 hpi, respectively,
compared with a total of 291 and 2465 DEGs in susceptible
Y. Li, et al. Gene 728 (2020) 144288

Fig. 4. The phenylpropanoid biosynthesis pathway. The yellow boxes represent enzymes, and the blue ovals represent the products. Black arrows indicate a direct
product, and dotted arrows indicate an indirect product. The equals sign indicates DEGs assigned to a given enzyme. The number in the box is the fold change of the
DEG, which is marked by red or yellow to represent the upregulated DEGs, or marked by green to represent the downregulated DEGs. Samples from left to right are
CM334_12hpi, CM334_36hpi, NMCA10399_12hpi, and NMCA10399_36hpi.

NMCA10399 roots at these two timepoints, respectively. This result
showed that both roots of resistant and susceptible accessions activated
defense responses to P. capsici after infection; however, the resistant
CM334 activated the response earlier than did the susceptible
NMCA10399 with more than twice as many genes differentially ex-
pressed at 12 hpi. Similar results have been reported in other plants in
previous studies. Using the same whole-genome transcriptional analysis
method, it was determined that defense-related gene activation oc-
curred earlier and was stronger in roots of resistant tomato plants after
R. solanacearum infection (French et al., 2018). In addition, Xu et al.
(2011) deduced phenylpropanoid metabolism was involved in the
cotton defense response after V. dahliae infection and both resistant and
susceptible cotton plants showed an increased level of expression of
lignin synthesis-related genes, but the increase was greater and faster in
the resistant line (Xu et al., 2011). All the points stated above suggest
that activation of the response in a timely manner is very important for
plant disease resistance.
Based on KEGG enrichment analysis, we observed that the plant-
pathogen interaction pathway was only present in the group of
upregulated DEGs. Moreover, it was exclusively significantly enriched
in upregulated DEGs in roots of the susceptible accession only at 36 hpi
but not in resistant roots at 12 or 36 hpi, which was unexpected. We
presumed that plant-pathogen interactions might not be the main way
to mediate resistance to P. capsici in pepper roots. In contrast, we ob-
served that pathways involved in the synthesis of secondary metabolites
were those that were enriched for the most DEGs. The important roles
of the phenylpropanoid biosynthesis pathway in defense against pa-
thogens have been demonstrated in previous studies (Naoumkina et al.,
2010). Therefore, we inferred that the pathway involved in secondary
metabolite synthesis, phenylpropanoid biosynthesis, might play a more
critical role than plant-pathogen interactions in root-mediated re-
sistance to P. capsici in pepper.
In the present study, secondary metabolite synthesis pathways
played significant roles in root-mediated resistance to P. capsici. We
focused our study on the phenylpropanoid biosynthesis pathway, which
was divided into four branch pathways synthesizing five secondary
metabolites, coumarine, cinnamaldehyde, lignin, caffeoyl quinic acid
and capsaicin. Among them, cinnamaldehyde (Bang et al., 2000; Cheng

8
Y. Li, et al.
Fig. 5. Coefficient analysis of gene expression levels obtained from RNA-seq
and qRT-PCR. Fold change indicates the gene expression fold change at 12 hpi
and 36 hpi compared to the 0 hpi mock control in the resistant CM334 and the
susceptible NMCA10399, respectively.
et al., 2008; Hu et al., 2013; Wu et al., 2017; Duan et al., 2018; OuYang
et al., 2019), lignin (Naoumkina et al., 2010; Xu et al., 2011; Guo et al.,
2016), caffeoyl quinic acid (Faulds and Williamson, 1999; Jr. Harrison
et al., 2008) and capsaicin (Veloso et al., 2014) have been shown to be
involved in plant defense against pathogens. PAL was the first enzyme
detected, plays an essential role in the phenylpropanoid pathway and
has been reported to be responsive to both biotic and abiotic stresses,
including pathogen attack (Xu et al., 2011), wounding, cold, and UV
light exposure (Huang et al., 2010). In this study, two genes
(CA05g20790 and CA12g15510) mapped to PAL were both upregulated
in resistant accessions with greater changes, and more importantly,
their activation time was earlier than that in susceptible accessions.
Similar results were also observed in tomato (Gayoso et al., 2010) and
cotton (Xu et al., 2011) defense responses to V. dahliae. In addition,
CCR1 is a common enzyme in the branch pathways of cinnamaldehyde
and lignin synthesis, and genes in its family have been reported to play
an important role in disease resistance in plants (Eynck et al., 2012).
Our results showed that genes assigned to CCR1 were also upregulated
in resistant roots but downregulated with great folder changes in sus-
ceptible roots. All of these results suggested that the most significant
difference between resistant and susceptible plants was whether the
plants could activate the involved pathways in a timely and efficient
manner.
In addition to potent antifungal activities against a wide variety of
pathogens, cinnamaldehyde has recently been reported to show great
potential in promoting lateral root formation, which is mediated by
endogenous hydrogen sulfide (H2S) in pepper plants (Xue et al., 2016).
In the present study, the resistant and susceptible accessions showed
completely different phenotypes 7 days after P. capsici inoculation. For
the resistant CM334 roots, there were no visible lesions, and the roots
grew normally with many more lateral roots developed than in the
susceptible accession. However, the roots of the susceptible
NMCA10399 were completely lesioned and began to rot at that same
time. Moreover, the size of the roots suggested that their growth slowed
down with almost no lateral root development after inoculation. The
development of lateral roots results in the branching of roots, which is
critical for the acquisition of water and nutrients by higher plants
(Benkova and Bielach, 2010). This is likely very important for plants to
recover quickly after pathogen infection. Therefore, it was suggested
that cinnamaldehyde could not only inhibit the zoospore germination
and radical mycelial growth of P. capsici (Hu et al., 2013) but also
promote the development of lateral roots to obtain more water and
9
Gene 728 (2020) 144288
nutrients to recover after pathogen infection, which mutually con-
tribute to disease resistance in pepper plants.
Understanding the mechanisms of root-mediated disease resistance
is an important step in developing crops with resistance to soilborne
pathogens. Our study suggested that phenylpropanoid compounds de-
rived from the phenylpropanoid biosynthesis pathway, especially cin-
namaldehyde and lignin, played important roles in pepper root defense
against P. capsici. Although the transcriptional analysis of these genes
from the multiple branches of the phenylpropanoid biosynthesis
pathway helped to better highlight these findings, many questions re-
main largely unknown. Further characterization of these genes is
needed and may provide candidates for genetic improvement of pepper.
Moreover, identification of transcription factors involved in the phe-
nylpropanoid pathway, especially in the cinnamaldehyde and lignin
synthesis branches, will provide new and further insights into root-
mediated resistance to P. capsici in pepper.
Funding
This research was funded by the project of National Natural Science
Foundation of China (Grant No. 31601750), the project of
China Scholarship Council (Grant No. 201808440093), the project of
Guangzhou Science and Technology (Grant No. 201710010055) and
the projects of Guangdong Science and Technology (Grant No.
2018A0505060520 and 2017B030314111).
CRediT authorship contribution statement
Ying Li: Resources, Supervision. Ting Yu: Software, Data curation,
Writing - original draft. Tingquan Wu: Writing - review & editing. Rui
Wang: Validation. Hengming Wang: Investigation. Hu Du: Validation.
Xiaowan Xu: Data curation. Dasen Xie: Writing - review & editing.
XiaoMei Xu: Methodology, Formal analysis, Data curation, Writing -
original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors are grateful to Paul W. Bosland of New Mexico State
University for providing experimental materials such as CM334 and
NMCA10399.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.gene.2019.144288.
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