Abstract
Stripe rust (or yellow rust), which is caused by Puccinia striiformis
f. sp. tritici (Pst), is one of the most devastating wheat diseases
worldwide. The wheat cultivar Xingzi 9104 (XZ) is an elite wheat
germplasm that possesses adult plant resistance (APR), which is
non–race-specific and durable. Thus, to better understand the mechanism
underlying APR, we performed transcriptome sequencing of wheat
seedlings and adult plants without Pst infection, and a total of
157,689 unigenes were obtained as a reference. In total, 2,666, 783 and
2,587 differentially expressed genes (DEGs) were found to be up- or
down-regulated after Pst infection at 24, 48 and 120 hours
post-inoculation (hpi), respectively, based on a comparison of Pst- and
mock-infected plants. Among these unigenes, the temporal pattern of the
up-regulated unigenes exhibited transient expression patterns during
Pst infection, as determined through a Gene Ontology (GO) enrichment
analysis. In addition, a Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway analysis showed that many biological processes, including
phenylpropanoid biosynthesis, reactive oxygen species, photosynthesis
and thiamine metabolism, which mainly control the mechanisms of
lignification, reactive oxygen species and sugar, respectively, are
involved in APR. In particular, the continuous accumulation of reactive
oxygen species may potentially contribute to the ability of the adult
plant to inhibit fungal growth and development. To validate the
bioinformatics results, 6 candidate genes were selected for further
functional identification using the virus-induced gene silencing (VIGS)
system, and 4 candidate genes likely contribute to plant resistance
against Pst infection. Our study provides new information concerning
the transcriptional changes that occur during the Pst-wheat interaction
at the adult stage and will help further our understanding of the
detailed mechanisms underlying APR to Pst.
Introduction
Stripe rust (or yellow rust), which is caused by Puccinia striiformis
f. sp. tritici (Pst), is a common and damaging disease of wheat
(Triticum aestivum L.) that causes significant yield and grain quality
losses [[36]1–[37]4]. The plant innate immune response is highly
diverse in its capacity to recognize and respond to biotrophs, and most
plants are resistant to most plant pathogens [[38]5]. Accordingly, the
breeding of resistant cultivars is the most effective, economic and
environmentally favorable method for controlling diseases [[39]6].
Stripe rust resistance is broadly categorized as either all-stage
resistance (seedling resistance) or adult plant resistance (APR)
[[40]1]. Because that Pst rapidly evolves new races, cultivars with
seedling resistance to Pst usually become susceptible within a few
years after being popularized [[41]7, [42]8]. However, wheat cultivars
with APR usually remain resistant even after being popularized in large
areas for many years [[43]9, [44]10]. Wheat cultivar Xingzi 9104 (XZ)
is susceptible to Pst at seedling stage, which possesses non-race
specific to stripe rust at adult plant stage [[45]4, [46]11–[47]13].
However, its underlying protective mechanism is still unclear. Over the
past several years, the rapid and inexpensive next-generation
sequencing (NGS) method has resulted in high-throughput gene expression
profiling, genome annotation and the discovery of non-coding RNA
[[48]14, [49]15]. An increasing body of evidence suggests that
transcriptome sequencing using NGS technology provides high-resolution
data and is a powerful tool for studying global transcriptional
networks. Using this technology, we can successfully obtain a large
amount of sequence data that may represent the gene expression profile
of wheat under a particular condition. To the best of our knowledge,
the transcription profiling of the wheat cultivar XZ infected with
stripe rust has not yet been reported. Considering the agricultural
value of XZ, a comprehensive description of the genes expressed in XZ
during infection is necessary for the discovery of candidate
defense-related genes. In this study, we obtained a large number of
distinct sequences (designated as unigenes) from an equal mix of total
RNA from XZ at 0 hours post-inoculation (hpi) without Pst at the adult
plant stage (Ak-M-0) and total RNA from XZ at 0 hpi without Pst at the
seedling stage (Sk-M-0) using the Illumina NGS technology. In this
manuscript, we present the current understanding of the assembled and
annotated transcriptome sequences. Additionally, we provide information
obtained from a digital gene expression (DGE) system that was used to
compare the gene expression profiles of adult XZ plants at three
infection stages, namely 24, 48 and 120 hpi with Pst. This comparison
allowed us to elucidate the molecular mechanism and identify the
responsive genes underlying the complex XZ resistance to Pst. These
findings should facilitate the development of effective strategies for
the breeding of resistant “wheat” varieties to obtain a better control
of stripe rust.
Materials and Methods
Plant materials and Pst inoculation
Triticum aestivum cv. XZ and Pst pathotype CYR32 were used in the
study. For the preparation of plant samples, germinated seeds were
maintained at 4°C for 40 days for vernalization before being planted in
large 20-cm-diameter pots, which contained mellow soil. These pots,
which contained five seeds, were placed in a greenhouse at 20°C for
cultivation, where they received 16 hours of light a day. Topdressing
can be carried out with water during booting stage of wheat. For the
inoculation, the second leaf of wheat seedling plants at the two-leaf
stage and the flag leaf of wheat adult plants at the boot stage were
simultaneously inoculated with fresh Pst race CYR32 with a paintbrush
until whole surface became wet without run-off as described [[50]12].
Control plants (mock-inoculated plants) were treated with sterile
water. Inoculated and control leaves were collected at 0, 24, 48 and
120 hpi for RNA isolation. These time points were selected based on
microscopic studies [[51]12]. All of the samples were rapidly frozen in
liquid nitrogen and stored at -80°C. The remaining plants were rated in
terms of symptoms at 15 days post-inoculation (dpi). Three independent
biological replicates were performed for each time point.
RNA isolation and cDNA synthesis
The total RNA was isolated using lysis buffer from the RNeasy Plant
Mini Kit according to the manufacturer’s instructions (QIAGEN, Hilden,
Germany). DNA was removed using the TRIzol Reagent TURBO (Qiagen
RNase-Free DNase set) (Ambion) according to the manufacturer’s
instructions. The RNA integrity was confirmed by 1.0% agarose gel
electrophoresis, and the total RNA quantity was determined with a
NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA,
USA). First-strand cDNA was synthesized with 2.5 μg of total RNA using
the Reverse Transcription System (Promega, Madison, WI, USA) following
the manufacturer’s directions.
Illumina library construction and sequencing
The RNA of two samples (Ak-M-0 and Sk-M-0) was subjected to RNA-Seq
analysis at the Beijing Genomics Institute (BGI; Shenzhen, China). The
transcriptome library was prepared and sequenced using Illumina
HiSeq^TM2000 using paired-end technology. A fragmentation buffer was
used to cut the mRNA into short fragments (approximately 200 bp). These
short fragments were used as templates with random hexamer-primers to
synthesize the first-strand cDNA. The second-strand cDNA was
synthesized using buffer, dNTPs, RNaseH and DNA polymerase I. Short
double-stranded cDNA fragments were purified using a QIAquick PCR
purification kit (Qiagen) and resolved with EB buffer for end repair
prior to the addition of poly(A). The short fragments were then
connected with sequencing adaptors. We then selected suitable fragments
as templates via agarose gel electrophoresis and enriched them by PCR
amplification. A Solexa HiSeq^TM2000 sequencer was employed to sequence
the constructed libraries [[52]16].
DGE library construction and sequencing
The RNA of six samples (Ak-M-24, Ak-M-48, Ak-M-120; Ak-I-24, Ak-I-48
and Ak-I-120) was prepared for DGE library construction and sequencing
at the Beijing Genomics Institute (BGI; Shenzhen, China). In brief, the
mRNA was purified using oligo (dT) magnetic beads. The first- and
second-strand cDNA were synthesized after the mRNA was bound to the
beads. While on the beads, the double-stranded cDNA was then digested
with the anchoring restriction enzyme NlaIII to remove all fragments
other than the 3’-most CATG fragment attached to the oligo bead, and
the GEX adapter 1 was added to the new 5’end. The junction of GEX
adapter 1 and the CATG site was recognized by MmeI. This enzyme cuts 17
bp downstream of the CATG site, thus producing 17-bp cDNA sequence tags
with GEX adapter1. After removing the 3’ fragments through magnetic
bead precipitation, GEX adapter 2 was ligated to the 3’ end of the cDNA
tag. Tags flanked by both adapters were enriched by PCR using the GEX
PCR primers 1 and 2 (Illumina) according to the manufacturer’s
instructions. The PCR products were purified with a 12% PAGE gel. The
purified cDNA tags were sequenced on an Illumina cluster station and
genome analyzer (Illumina) following the manufacturer’s instructions
[[53]17, [54]18].
Raw read cleaning, assembly and sequence annotation
Prior to assembly, raw reads were obtained from the original sequence
data, and the reads were filtered using a Perl script dynamic-Trim.pl
[[55]19] to remove the adaptor sequences, empty reads, short reads
(<25bp), reads with an N ratio greater than 10%, and low-quality
sequences, all of which negatively affect the bioinformatics analysis.
We then generated clean reads in the FASTQ format. All of the clean
reads were mapped to our transcriptome reference database, allowing no
more than a 2-bp mismatch using the software SOAP aligner/soap2. The
results revealed the distribution and location of the clean reads in
the reference genome. De novo assembly of the high-quality reads was
performed using SOAP de novo and yielded unigenes. The unigenes of two
samples were spliced and processed by clustering software to obtain the
longest possible non-redundant unigene. Finally, the generated unigenes
were analyzed by a BlastX alignment search (E-value<10^−5) against the
protein databases NR, Swiss-Prot, KEGG (Kyoto Encyclopedia of Genes and
Genomes), and GO (Gene Ontology), and the best aligning result was used
to determine the sequence unigenes direction [[56]16, [57]20].
Statistical analysis of gene expression levels
Gene expression was calculated from the number of reads mapped to the
reference sequence [[58]21]. The expression level was calculated using
the RPKM method (reads per kb per million reads) with the following
formula:
[MATH:
RPKM=106CNL/<
msup>103 :MATH]
where RPKM (A) is the expression of gene A, C is the number of reads
uniquely aligned to gene A, N is the total number of reads uniquely
aligned to all of the genes, and L is the number of bases on gene A.
The RPKM method eliminates the influence of different gene lengths and
sequencing discrepancies during the calculation of gene expression such
that the calculated gene expression levels can be directly compared
among samples. If there is more than one transcript for a gene, the
longest is used to calculate its expression level and coverage
[[59]22].
Evaluation of DGE libraries
We compared the differential expression of genes in each DGE library
(Ak-I-24 vs. Ak-M-24, Ak-I-48 vs. Ak-M-48 and Ak-I-120 vs. Ak-M-120)
using the method described by Audic and Claverie [[60]23]. The
correlation between the detected count numbers between parallel
libraries was assessed statistically by calculating Pearson’s
correlation (P) coefficient. In addition to the P value, the false
discovery rate (FDR) was used to determine the threshold P value in
multiple tests. Genes were classified as significantly differentially
expressed if they had a P-value less than 0.005, an FDR less than 0.001
and an absolute value of log[2]Ratio of at least 1 in sequence counts
across the libraries utilized in our study.
GO and KEGG pathway enrichment analysis
GO enrichment analysis provides all of the GO terms that are
significantly enriched in differentially expressed genes (DEGs)
compared with the genome background and filters the reads that
correspond to biological functions. This method first maps all DEGs to
GO terms in the database by calculating gene numbers for every term and
then uses a hypergeometric test to identify the significantly enriched
GO terms in the DEGs compared with the genome background. The KEGG
database was used to identify the significantly enriched metabolic
pathways or signal transduction pathways in the DEGs compared with
whole genome backgrounds. N is the number of all of the genes with a
KEGG annotation, n is the number of DEGs in N, M is the number of all
of the genes annotated to specific pathways, and m is the number of
DEGs in M.
Quantitative real-time PCR analysis
Quantitative real-time PCR (qRT-PCR) was performed to verify the
expression of 30 differentially expressed genes. To standardize the
data, the wheat translation elongation factor 1α subunit gene (TaEF-1α,
GenBank Accession Number [61]M90077.1) was used as an internal
reference for the qRT-PCR analysis. The qRT-PCR analysis was performed
in triplicate using the SYBR Green fluorescence dye in a 7500 Real-Time
PCR System (Applied Biosystems, Foster City, CA, USA). Each qRT-PCR
reaction (25 μL) included 2.5 μL of 10×Taq Buffer, 3.0 μL of MgCl[2]
(25 mM/L), 0.5 μL of dNTPs (10 mM/L), 0.4 μL of 50×SYBR Green (QIAGEN,
Hilden, Germany), 2.0 μL of cDNA, 0.5 μL of forward/reverse primers (10
μmol/L) and 0.3 μL of Taq DNA polymerase (TaKaRa Bio Inc., Japan). The
qRT-PCR data were analyzed using the comparative 2^-ΔΔCt method
[[62]24]. Mean and standard deviation were calculated with data from 3
independent biological replicates.
Histological observations and host response
Capped in vitro transcripts were prepared from linearized recombinant
plasmids containing the tripartite BSMV genome [[63]25] with the
mMessage mMachine T7 in vitro transcription kit (Ambion, Austin, TX,
USA) according to the manufacturer's guidelines. Three independent sets
of plants were used, and germinated seeds were maintained at 4°C for 40
days for vernalization before being planted in large pots. The
inoculation system contained 0.5 μL of RNAs for each in vitro
transcription reaction for α, β, and γ (γ-PDS, γ-gene) (phytoene
desaturase) and was gently rubbed on the surface with 25 μL of FES
buffer. The mixtures were inoculated on the surface of the second top
leaves of boot stage plants with a gloved finger [[64]26, [65]27], and
the plants were then incubated at 23±2°C. The virus phenotypes were
observed and photographed 13 days after virus inoculation. The flag
leaves were further inoculated with Pst pathotype CYR32, and samples
were excised at 0, 24, 48 and 120 hpi for histological observation and
qRT-PCR. The Pst infection phenotypes were recorded and photographed at
15 dpi. The samples for histological observation were fixed and
decolorized as previously described [[66]28]. The necrotic area in
wheat leaves was observed via the auto fluorescence of the attacked
mesophyll cells by Olympus BX-51 microscope (Olympus Corp., Japan) and
calculated by the cellSens Entry software (Olympus Corp., Japan). To
stain the infection structures of Pst in wheat leaves, wheat germ
agglutinin (WGA) conjugated to Alexa-488 (Invitrogen, Carlsbad, CA,
USA) was used as previously described [[67]29]. For microscopic
observations, stained segments were kept in 50% glycerol, and the
hyphal length was examined under blue light excitation (excitation
wavelength 450–480 nm, emission wavelength 515 nm) by Olympus BX-51
microscope (Olympus Corp., Japan) as previously described [[68]29]. To
detect host response, hydrogen peroxide accumulation was detected using
3, 3'-diaminobenzidine (DAB; Amresco, Solon, OH, USA) as previously
described [[69]29]. At least 30–50 infection sites were examined on
each of three randomly selected leaf segments for every treatment. A
high performance liquid chromatographic method was used to determine
the endogenous hormones salicylic acid (SA) content in flag leaves as
previously described [[70]30]. The lignin content of the flag leaves
was examined according to the method of Morrison with some
modifications as previously described [[71]31]. The chloroplast content
of the flag leaves was determined using the direct sopping extraction
method with mixing solution of alcohol and acetone (1:1 in volume) as
previously described [[72]32].
Results
High-throughput RNA sequencing
The transcriptome sequencing of seedling and adult plants samples at 0
hpi resulted in a total of 66,666,668 and 63,954,974 reads,
respectively. The ratio of the Q20 sequencing value was greater than
91%, which indicated that the sequencing sufficiently captured most of
the expressed genes ([73]Table 1). Transcriptome de novo assembly was
conducted with the Short Oligonucleotide Analysis Package (SOAP)
program and resulted in 1,157,689 specific unigenes ([74]S1 Fig). For
the functional annotation and classification of the obtained unigenes,
we searched the annotated sequences for genes that were involved in the
cluster of orthologous group (COG) assignments ([75]S2 Fig). The
sequences could be categorized into 35 level-two functional groups,
which comprised three domains: ‘biological process’, ‘cellular
component’ and ‘molecular function’ ([76]S3 Fig). A G-test [False
Discovery Rate (FDR) < 0.001] of the RPKM-derived read counts with
multiple genetic differences greater than two-fold was performed to
detect the DEGs between the adult plant (Ak-M-0) and seedling (Sk-M-0)
stages and to identify the genes responsible for the development of
wheat ([77]S4 Fig). A total of 27,265 DEGs were obtained from this
analysis; of these, 13,977 and 13,288 unigenes were up- and
down-regulated at the adult plant stage respectively, which suggests
that these genes may be involved in the growth and development of the
host plant.
Table 1. Output statistics from sequencing.
Samples Total Reads Total Nucleotides Q20 percentage N percentage GC
percentage
AK-M-0 66,666,668 6,000,000,120 91.74 0.00 53.96
SK-M-0 63,954,974 5,755,947,660 91.57 0.00 54.32
[78]Open in a new tab
Total Nucleotides = Total Reads1 × Read1 size + Total Reads2 × Read2
size. The two libraries included non-inoculated adult plants at 0 hours
post-inoculation (hpi) (Ak-M-0) and non-inoculated seedling plants at 0
hpi (Sk-M-0).
DGE library sequencing and annotation
Based on the transcriptome sequence data, six DGE libraries were
constructed to identify the gene expression profiles of the XZ during
Pst infection at the adult plant stage. The six DGE libraries included
non-inoculated adult plants at 24 hpi (Ak-M-24), 48 hpi (Ak-M-48) and
120 hpi (Ak-M-120) and inoculated adult plants at 24 hpi (Ak-I-24), 48
hpi (Ak-I-48) and 120 hpi (Ak-I-120). All of the clean reads were
mapped to our transcriptome reference database. Each library generated
raw reads that ranged from 290 to 318 Mb, with reference genome
alignments greater than 84% ([79]S1 Table). Together, each library
generated clean reads that ranged from 5.78 to 6.34 million, and the
proportion of the total reads exceeded 98%, which indicates that the
transcriptional data were reliable ([80]S5 Fig). Saturation analyses of
the six DGE libraries were performed to determine whether the number of
detected genes continued to increase as the sequence quantity increased
(total tag number). The number of detected genes almost ceased to
increase at a sequence quantity of at least 600 million ([81]S6 Fig);
the coverage statistics were high ([82]S7 Fig). All of these findings
indicate that the sequencing data was accurate and reliable. A G-test
(FDR<0.001) of the RPKM-derived read counts with multiple genetic
differences greater than four-fold was performed to detect the DEGs
within these pairs, i.e., (Ak-I-24 vs. Ak-M-24), (Ak-I-48 vs. Ak-M-48)
and (Ak-I-120 vs. Ak-M-120), and to identify genes that are responsive
to Pst infection at the adult plant stage. Approximately 2,666, 786 and
2,587 DEGs were obtained at 24, 48 and 120 hpi, respectively.
Additionally, 1,198, 155 and 1,645 unigenes were down-regulated and
1,468, 628 and 942 unigenes were up-regulated at 24, 48 and 120 hpi,
respectively ([83]Fig 1). A total of 427 unigenes were up-regulated
between 24 and 48 hpi, whereas 48 unigenes were up-regulated between 48
and 120 hpi. In addition, 64 unigenes were up-regulated between 24 and
120 hpi, and 37 unigenes were up-regulated at all three sampled stages
([84]Fig 2). The regulated unigenes represented on the array were
significantly differentially expressed at 24, 48 and 120 hpi. We used
the gene cluster set to generate a tree that shows the similarities in
the relative gene expressions among the three time points ([85]Fig 3).
Fig 1. Statistical chart of DEGs during Pst infection at the adult plant
stage.
[86]Fig 1
[87]Open in a new tab
Differentially expressed genes were identified by filtering the
two-fold up-regulated and down-regulated genes with FDR≤10^−4. The bars
represent the number of up-regulated (black) and down-regulated (gray)
unigenes. The six DGE libraries included non-inoculated adult plants at
24 hours post-inoculation (hpi) (Ak-M-24), 48 hpi (Ak-M-48) and 120 hpi
(Ak-M-120), and inoculated adult plants at 24 hpi (Ak-I-24), 48 hpi
(Ak-I-48) and 120 hpi (Ak-I-120).
Fig 2. Statistical chart of DEGs up-regulated during Pst infection at the
adult plant stage.
[88]Fig 2
[89]Open in a new tab
Differentially expressed unigenes were identified by filtering the
two-fold up-regulated unigenes with FDR≤10^−4. The up-regulated
unigenes were obtained from the Ak-I-24 vs. Ak-M-24, Ak-I-48 vs.
Ak-M-48 and Ak-I-120 vs. Ak-M-120 comparisons. The six DGE libraries
included non-inoculated adult plants at 24 hours post-inoculation (hpi)
(Ak-M-24), 48 hpi (Ak-M-48) and 120 hpi (Ak-M-120), and inoculated
adult plants at 24 hpi (Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi
(Ak-I-120).
Fig 3. Cluster and heat map of DEGs of XZ at the adult plant stage.
[90]Fig 3
[91]Open in a new tab
The heat map shows the gene expression obtained by the clustering
affinity search technique. Each line refers to the data for one gene.
The color bar represents the log2 of fold change values and ranges from
green (−8) to red (8). The six DGE libraries included non-inoculated
adult plants at 24 hours post-inoculation (hpi) (Ak-M-24), 48 hpi
(Ak-M-48) and 120 hpi (Ak-M-120), and inoculated adult plants at 24 hpi
(Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi (Ak-I-120). A: The DEGs that
demonstrated at least a two-fold difference in each of the three
comparisons at the adult plant stage. B: The up-regulated unigenes that
demonstrated at least a four-fold change in each of the three
comparisons at the adult plant stage.
Regulated genes during Pst infection at the adult plant stage
To investigate the plant disease-resistance of XZ during Pst infection,
Fisher’s exact test in the Blast2GO software was used to explore the
statistically enriched GO terms of the up-regulated genes during Pst
infection at the adult plant stage compared with the entire
transcriptome background (P<0.05). The results of the up-regulated
unigenes identified from the Ak-I-24 vs. Ak-M-24 comparison were as
follows. In the ‘cellular component’ category, proteins involved with
cytoplasmic membrane-bounded vesicles, the chloroplastic endopeptidase
Clp complex, the glyoxysome and the cell wall were highly enriched. In
the ‘molecular function’ category, proteins involved in phenylalanine
ammonia-lyase activity, heme binding and pyruvate and phosphate
dikinase activity were highly enriched, and 78 compounds were also
enriched. In the ‘biological process’ category, proteins involved in
the SA catabolic process, cinnamic acid biosynthetic process and
L-phenylalanine catabolic process were highly enriched, and an
additional 97 compounds were also enriched ([92]S1 File). The results
from the up-regulated unigenes identified from the Ak-I-48 vs. Ak-M-48
comparison and the Ak-I-120 vs. Ak-M-120 comparison are as follows
([93]S1 File). Thirty-six categories were enriched in the Ak-I-24 vs.
Ak-M-24 and Ak-I-48 vs. Ak-M-48 comparisons ([94]Fig 4). Only one
category was enriched in the Ak-I-48 vs. Ak-M-48 and Ak-I-120 vs.
Ak-M-120 comparisons, whereas 36 categories were enriched in the
Ak-I-24 vs. Ak-M-24, Ak-I-48 vs. Ak-M-48 and Ak-I-120 vs. Ak-M-120
comparisons ([95]Fig 4).
Fig 4. Statistical chart of enrichments in ‘biological processes’ during Pst
infection at the adult plant stage.
[96]Fig 4
[97]Open in a new tab
The leaves of adult plants were inoculated with Pst CYR32. The panels
represent the transcriptional changes in ‘biological processes’
obtained from the Ak-I-24 vs. Ak-M-24, Ak-I-48 vs. Ak-M-48 and Ak-I-120
vs. Ak-M-120 comparisons. The six DGE libraries included non-inoculated
adult plants at 24 hours post-inoculation (hpi) (Ak-M-24), 48 hpi
(Ak-M-48) and 120 hpi (Ak-M-120), and inoculated adult plants at 24 hpi
(Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi (Ak-I-120).
KEGG pathways are differentially expressed in response to Pst
To explore the biochemical pathways in which the up-regulated DEGs
identified in the Ak-I-24 vs. Ak-M-24, Ak-I-48 vs. Ak-M-48 and Ak-I-120
vs. Ak-M-120 comparisons are involved in XZ wheat, a pathway analysis
utilizing the KEGG pathway database was conducted with an E-value
cutoff of < 0.05. Interestingly, the KEGG pathway analysis showed that
45, 42 and 34 significantly enriched pathways were identified at the
three infection stages, namely 24 hpi, 48 hpi and 120 hpi, respectively
([98]S2 File). These up-regulated DEGs were found to be involved in
many biological processes related to systemic symptom development,
including thiamine metabolism, purine metabolism, phenylpropanoid
biosynthesis, novobiocin biosynthesis and photosynthesis. An additional
15 significantly enriched pathways were identified at the three adult
plant stages. Fourteen significantly enriched pathways were only
identified from the Ak-I-24 vs. Ak-M-24 comparison, whereas 5
significantly enriched pathways were only identified in the Ak-I-48 vs.
Ak-M-48 comparison, and 7 significantly enriched pathways were only
identified from the Ak-I-120 vs. Ak-M-120 comparison.
Comparison of digital gene expression values with qRT-PCR analysis results
To evaluate the reliability of our RNA-Seq and DGE analysis, 30
unigenes, which presented a wide range of expression levels and
patterns under Pst infection, were selected for qRT-PCR analysis. These
unigenes were selected based on their homology to genes that are known
to play a role in pathogenesis/defense, reactive oxygen species (ROS)
burst, secondary metabolism or that encode hypothetical or unknown
proteins. The corresponding primers were designed and are listed in
[99]S3 File. As shown in [100]Table 2, the results agreed well with the
DGE patterns. In addition, the unigenes involved in
pathogenesis-related proteins (wheat11332_refgene, wheat10510_refgene
and wheat59172_refgene) were strongly up-regulated as early as 24 hpi
and afterward presented relatively up-regulated expression. The
transcripts of wheat31335_refgene and wheat12328_refgene were increased
by 3.53 to 21.09 -fold, respectively. The unigenes of
wheat57563_refgene, wheat12902_refgene, wheat75137_refgene,
wheat75952_refgene and wheat10297_refgene, which are related to ROS
burst, presented up-regulated expression at 24, 48 and 120 hpi.
However, 6 unigenes with putative functions associated with thiol
methyltransferase 2, ATP synthase subunit alpha, chloroplastic,
protease Do-like 2, ribonuclease1 and a hypothetical protein were
down-regulated as early as 24 and 48 hpi. The remaining 15 unigenes
showed significantly higher expression patterns, which suggests that
the selected unigenes may play an active role during the interaction
between wheat and Pst.
Table 2. Verification of DGE-Seq results by qRT-PCR.
Gene Name Annotation (BLASTX) Relative gene expression by qRT-PCR ^(2−
ΔΔCt) Gene expression of Illumina (Log2^(T_RPKM/C_RPKM))
24hpi 48hpi 120hpi 24hpi 48hpi 120hpi
wheat8730_refgene beta-fructofuranosidase 2.02±0.31 3.21±0.52 3.09±0.43
1.10 1.50 1.55
wheat84218_refgene thaumatin-like protein 20.99±5.55 6.88±1.01
6.02±0.77 5.59 2.16 1.37
wheat7284_refgene beta-1,3-glucanase 30.22±3.53 7.09±1.11 8.77±1.92
4.32 1.72 1.66
wheat72566_refgene predicted protein 3.83±0.89 2.01±0.54 5.76±0.32 1.12
2.15
wheat33491_refgene predicted protein 33.23±3.09 7.42±2.09 10.09±3.78
5.18 1.62 2.82
wheat31335_refgene Cell wall-associated hydrolase 5.88±1.73 3.53±2.01
3.86±0.22 1.15 1.77
wheat12328_refgene class I chitinase 21.09±3.22 4.54±0.35 9.78±2.98
4.12 1.03 3.12
wheat121909_refgene Glutathione S-transferase 2 9.88±1.78 1.09±0.88
5.54±1.63 2.59 2.09
wheat10297_refgene root peroxidase 3.09±0.77 9.09±3.02 2.00±1.09 1.24
2.40
wheat14242_refgene hypothetical protein -7.33+1.11 -2.99±0.98 4.87±0.86
-2.57 -1.10 1.17
wheat8458_refgene predicted protein 5.32±1.99 7.09±2.01 -1.12±0.79 1.60
1.16 -1.46
wheat13698_refgene unnamed protein product 4.09±6.99 2.44±5.00
2.56±2.01 2.06 1.82 1.47
wheat8254_refgene Ribonuclease 1 -7.09±1.22 -5.66±0.99 -9.08±1.33 -2.34
-1.64 -3.52
wheat7959_refgene 3-beta-hydroxysteroid-dehydrogenase 11.03±2.09
6.08±0.99 15.88±2.75 3.65 2.46 3.49
wheat75952_refgene lipoxygenase 1.1 3.21±0.94 4.00±0.86 -12.09±3.02
1.51 1.41 -3.33
wheat75846_refgene putative thiol methyltransferase 2 -22.09±3.33
-5.86±1.03 5.99±1.21 -4.12 -1.77 1.19
wheat7310_refgene MAP kinase 4.32±0.67 3.98±0.99 -5.00±1.81 1.52 1.36
-1.48
wheat70819_refgene ATP synthase subunit alpha, chloroplastic -2.74±0.97
-3.03±1.02 -4.23±0.56 -1.29 -1.08 -1.72
wheat66652_refgene Protein WIR1A 8.23±1.83 9.09±0.86 7.99±1.24 2.79
2.43 2.24
wheat59172_refgene pathogenesis-related 5 24.35±3.80 8.79±2.43
5.79±0.75 4.73 2.38 1.60
wheat57862_refgene hypothetical protein F775_31113 6.08±1.00 3.08±0.69
7.99±1.80 2.33 1.88 2.36
wheat57563_refgene NAD(P)H-dependent oxidoreductase 1 5.09±0.84
6.32±0.79 5.09±0.69 1.24 1.43 2.03
wheat11332_refgene pathogenesis-related protein 1 13.98±2.11 4.09±0.34
5.89±0.44 3.97 1.90 1.76
wheat10510_refgene pathogenisis-related protein 1.1 7,67±0.68 3.90±0.74
4.10±1.02 2.94 1.49 1.46
wheat37392_refgene Annexin D1 3.09±0.99 1.09±0.88 2.10±0.99 1.57
wheat12902_refgene class III peroxidase 8.09±0.56 1.07±1.10 1.90±0.79
2.93
wheat75137_refgene Peroxidase 12 155.88±9.38 1.03±0.33 0.67±0.44 13.94
wheat31306_refgene predicted protein 3.99±0.52 -0.33±0.22 0.24±0.13
1.89
wheat36302_refgene hypothetical protein F775_32419 -0.77±0.25
-0.18±0.03 4.09±0.51 1.13
wheat12266_refgene Protease Do-like 2 -0.42±0.11 -0.33±0.47 3.08±0.39
1.00
[101]Open in a new tab
Functional analysis of six candidate genes in the resistance response to Pst
Based on the GO and KEGG pathway enrichment results, we speculated that
six DEGs may play a role in the APR of XZ during Pst infection.
Wheat37392_refgene and wheat12902_refgene were enriched in the
phenylalanine metabolism pathway, whereas wheat75137_refgene and
wheat31306_refgene were enriched in peroxidase activity and
nitric-oxide synthase activity, and wheat36302_refgene and
wheat12266_refgene were enriched in the chlorophyll biosynthetic
process and the photosynthesis process. The qRT-PCR analysis showed
that the six candidate genes were only up-regulated at the adult plant
stage ([102]Fig 5), and which showed that the six candidate genes were
to be positive regulators of the APR at the adult plant stage. So, the
candidate genes were only silenced in wheat at the adult plant stage
using the Barley stripe mosaic virus (BSMV)-based virus-induced gene
silencing (VIGS) system. The corresponding primers were designed and
are listed in [103]S2 Table. At the adult plant stage, all of the
BSMV-inoculated plants displayed mild chlorotic mosaic symptoms at 13
dpi, but no obvious defects in further leaf growth were observed
([104]Fig 6). We tested the silencing of the wheat PDS gene to confirm
whether our VIGS system functioned correctly and obtained typical
photo-bleaching at 15 dpi on the flag leaves of the plants inoculated
with BSMV:PDS ([105]Fig 6). To calculate the silencing efficiency of
VIGS, qRT-PCR assays were performed to examine the relative transcript
levels of the candidate genes in the flag leaves of plants infected
with CYR32. Compared with the BSMV:γ-infected leaves, the transcript
levels of candidate genes in knockdown plant were reduced from 59% to
71% at 24, 48 and 120 hpi ([106]Fig 7). After inoculation with CYR32,
hypersensitive reaction (HR) was observed in the flag leaves of the
control plants (mock- and BSMV-γ-infected) ([107]Fig 6). Phenotypic
changes relative to the control were observed on the flag leaves of
four candidate gene-knockdown plants (wheat37392_refgene,
wheat12902_refgene, wheat75137_refgene and wheat31306_refgene),
([108]Fig 6). On the contrary, no phenotypic changes were observed on
the wheat36302_refgene- and wheat12266_refgene-knockdown plants
([109]Fig 6). Furthermore, to determine wheather candidate genesis
involved in host resistance, we have assayed histological changes in
VIGS-silenced plants inoculated with Pst race CYR32 isolate. At 48 and
120 hpi with Pst race CYR32, we evaluated the necrotic area, as well as
the hyphal length. The histological observations showed that the
average of necrotic area per infection site decreased significantly at
120 hpi in knockdown wheat plants (wheat37392_refgene,
wheat12902_refgene, wheat75137_refgene and wheat31306_refgene) compared
with the control plant ([110]Fig 8A); and the hyphal length increased
significantly at 120 hpi ([111]Fig 8B). In addition, the content of
lignification and ROS decreased significantly at 120 hpi in knockdown
plant (wheat12902_refgene, wheat75137_refgene and wheat31306_refgene)
([112]Fig 8C and 8E); Wheat-37392_refgene or 12902_refgene -silenced
plants showed a decrease in content of SA at 24, 48 and 120 hpi
([113]Fig 8D); and the content of chloroplast did not changed ([114]Fig
8F). The VIGS assays were reproduced similarly in three independent
experiments.
Fig 5. Quantitative real-time polymerase chain reaction analysis of the
relative transcript levels of the six candidate unigenes induced by Pst
infection at seedling and adult stages.
[115]Fig 5
[116]Open in a new tab
The relative expression levels of the unigenes were calculated by the
comparative threshold method (2^–ΔΔCT) and were relative to that at the
0 hour time point. The results are presented as the means ± standard
errors of three biological replications. The different letters
represent significant differences [P≤0.05 according to analysis of
variance (ANOVA)].
Fig 6. Phenotypes of wheat leaves in candidate gene-knockdown plants
inoculated with Pst.
[117]Fig 6
[118]Open in a new tab
To perform a functional analysis of the candidate genes, virally
induced gene silencing (VIGS) was applied to adult plants. (A) No
change was found in mock-inoculated wheat leaves pre-inoculated with
FES buffer; mild chlorotic mosaic symptoms were observed on the flag
leaves of wheat at 13 days post-inoculation (dpi) with BSMV:γ;
photobleaching was evident on the first top leaves of plants 15 days
after infection with BSMV:PDS. (B) Mock-inoculated wheat flag leaves
treated with FES buffer and then challenged with Pst CYR32; the flag
leaves of knockdown plant (BSMV:γ, BSMV:wheat37392_refgene,
BSMV:wheat12902_refgene, BSMV:wheat75137_refgene,
BSMV:wheat31306_refgene, BSMV:wheat36302_refgene and
BSMV:wheat12266_refgene) challenged with Pst CYR32. Typical leaves were
observed and photographed at 15 dpi.
Fig 7. Relative transcript levels of candidate genes in candidate
gene-knockdown wheat leaves.
[119]Fig 7
[120]Open in a new tab
RNA samples were isolated from the flag leaves of wheat infected with
BSMV:γ, BSMV:wheat37392_refgene, BSMV:wheat12902_refgene,
BSMV:wheat75137_refgene, BSMV:wheat31306_refgene,
BSMV:wheat36302_refgene and BSMV:wheat12266_refgene at 0, 24, 48 and
120 hours post-inoculation (hpi) with Pst CYR32. The error bars
represent the variations among three independent replicates. The
different letters represent significant differences [P≤0.05 according
to analysis of variance (ANOVA)]. The relative gene expression levels
were quantified using the comparative threshold (2^-ΔΔCT) method and
compared with that of BSMV:γ.
Fig 8. Functional analysis of six candidate genes during the interaction
between XZ and stripe rust using the BSMV-mediated virus-induced gene
silencing system.
[121]Fig 8
[122]Open in a new tab
The second top leaves were pre-inoculated with BSMV:γ or recombinant
BSMV followed by inoculation with Puccinia striiformis f. sp. tritici
race CYR32. (A) Necrotic area, the average area was calculated by
DP-BSW software. wheat37392_refgene, wheat12902_refgene,
wheat75137_refgene or wheat31306_refgene -silenced plants showed a
significant decrease at 120 hours post-inoculation (hpi). (B) Hyphal
length, the length of IH was measured from the substomatal vesicle to
the apex of the longest infection hyphae, Wheat-37392_refgene,
wheat12902_refgene, wheat75137_refgene and wheat31306_refgene -silenced
plants showed a significant increase at 120 hpi. (C)
Wheat37392_refgene-silenced plants showed a significant decrease in the
content of lignin at 48 and 120 hpi. (D) Wheat- 37392_refgene or
12902_refgene -silenced plants showed a decrease in the content of SA
at 48 and 120 hpi, but no significant decrease between control and
silenced plants. (E) Significant decrease in ROS accumulation in
wheat-12902_refgene, wheat-75137_refgene and wheat-31306_refgene
-silenced plants at 120 hpi. (F) No difference in the content of
Chloroplast was observed between control and wheat-36302_refgene and
wheat 12266_refgene -silenced plants at 120 hpi. The error bars
represent the variations among three independent replicates. The
different letters represent significant differences [P≤0.05 according
to analysis of variance (ANOVA)].
Discussion
Transcripts exhibited transient expression patterns at the adult stage of
wheat resistance to Pst
In the present study, we generated a cDNA library through the Illumina
sequencing of mRNA isolated from leaves of adult plants of the wheat
cultivar XZ inoculated with Pst CYR32. A previous study showed that
when a sequencing throughput technique generates more than 2,000,000
tags, nearly all of the genes expressed in the sample can be identified
in the expression data [[123]33]. In our study, the number of clean
reads from each of the eight treatments ranged from 578 to 639 million;
therefore, the sequences should allow us to identify almost every gene
involved in the plant response to Pst infection. For the transcriptome
sequencing of seedling and adult plant samples at 0 hours
post-mock-infection, 157,689 specific unigenes were identified. The
transcription of genes in other species, such as Arabidopsis, maize,
barley and cucumber, also accounted for a large proportion
[[124]34–[125]36], which illustrates the importance of transcriptional
regulation during biological activities. The assigned GO terms were
summarized into three main GO categories: molecular function, cellular
component and biological process. Based on the Nr annotation, the
Blast2GO program was used to obtain the GO annotations. Under the
biological process category, cellular process (32.05%) was the largest
group, followed by metabolic process (32.01%). Under the cellular
component category, cell (34.02%) and cell part (34.02%) were the
largest groups, whereas in the molecular function category, binding
(45.07%) was the largest group, followed by catalytic activity
(40.84%). These similar aforementioned results showed that normal
biological activity was dependent on the regulation of biological
processes, which has also been shown through the transcriptome
sequencing of microorganisms, plants, animals and humans [[126]37,
[127]38]. The DGE technique, which is based on the computational
analysis of 21-bp tags derived from the 3’ ends of transcripts, has
been used to generate transcriptome profiles for various species
[[128]18]. In the present study, the DGE profiles of wheat during Pst
infection were based on the immediate direct application of the
transcriptome data from the Sk-M-0 and Ak-M-0 samples. The sequenced
tags of the six DGE libraries were then matched to 157,689 unigenes
from our transcriptome reference database. Therefore, we used the gene
cluster set to generate a tree that showed the similarities in the
relative gene expression among the three time points. We only
considered the transcript levels of unigenes that had increased or
decreased by at least two-fold in each of the comparisons. The results
indicated that the change in most of the unigenes was transient and
that the unigenes were only altered at one time point during Pst
infection. A few of the identified unigenes were long-lived, i.e.,
changes in their expression were sustained for two or three time points
([129]Fig 3). This result was consistent with the findings of a study
on the soybean response to Asian soybean rust controlled by the Rpp2
gene [[130]39] and of a study on the wheat response to Pst [[131]40].
Indeed, it has been proposed that there is a transcriptional peak in
the temporal pattern of transcript accumulation that occurs around the
time of fungal penetration [[132]40]. In the afore-mentioned study,
this peak occurred at 24 hpi, which is consistent with the timing
observed in our study. The 24-hpi time point reflects the haustorial
penetration by Pst at approximately 24 hpi [[133]12]. After this peak,
the differential expression sharply declines from 72 to 96 hpi and then
increases at approximately 72–168 hpi [[134]39]. In our study, we
observed a decrease in expression at 48 hpi, which is consistent with
the results of the previous study [[135]41], and then another increase
at 120 hpi. This result indicates that the regulation response is
differentially expressed ([136]Fig 3). The question of why the majority
of the up-regulated unigenes show a one-step-up and one-step-down
pattern then arises. It is possible that the marked reprogramming of
the transcriptome is connected to the three phases of Pst growth, which
include a penetration phase, a parasitic phase and a sporulation phase
[[137]42, [138]43]. A GO term enrichment analysis was then applied to
our data in the present study ([139]S1 File), even though this tool has
not been perfected and has limited coverage. Previous studies have
suggested that upon pathogen recognition, plants activate complex
signaling pathways that lead to a broad array of responses.
The phenylpropanoid pathway plays an important role during the early stage of
infection
Germlings of Pst penetrate the host cell and form a haustorium at 24
hpi [[140]12]. At 24 hpi, the over-represented GO categories among the
up-regulated unigenes were related to functions such as the
L-phenylalanine biosynthetic process, the cell-wall macromolecule
catabolic process, the SA catabolic process and the lignin catabolic
process. Furthermore, the KEGG pathway analysis showed that the
pathways of phenylpropanoid biosynthesis and phenylalanine metabolism
were highly enriched at 24 hpi ([141]S2 File). Previous studies have
identified SA, lignins, flavonoids, phytoalexins and coumarins as
secondary metabolic compounds that are produced by the phenylpropanoid
pathway [[142]44, [143]45]. The activation of this pathway has been
shown to be involved in or related to plant defense [[144]41, [145]46,
[146]47]. In our data for the ‘biological process’ category, proteins
associated with the L-phenylalanine catabolic process, the
L-phenylalanine biosynthetic process and the response to phenylalanine
were highly enriched at 24 and 48 hpi ([147]S1 File). The production of
these compounds relies on the conversion of phenylalanine to precursor
substances by various enzymes, including phenylalanine ammonia-lyase
and 4-coumarate-CoA ligase [[148]48], all of which were observed to be
enriched at 24 or 48 hpi ([149]S1 File). Seven refgenes were enriched
in the GO term L-phenylalanine biosynthetic process and were only
regulated at 24 hpi according to the RNA-Seq data ([150]S8 Fig). In the
present study, to further characterize the function of phenylalanine
metabolism during the wheat-Pst interaction, we used a knockdown
approach to determine the role of wheat37392_refgene and
wheat12902_refgene, which were found to be enriched in the
phenylalanine metabolism pathway at 24 hpi in the wheat-Pst interaction
at the adult plant stage. The histological observations of knockdown
plants were consistent with the host response experiments, and which
were also in parallel with the original hypothesis. The time point of
haustorium formation in the infection sites of adult plant leaves was
at 24 hpi [[151]12]. These histological observations suggested that
lignification in plants at the adult plant stage may arrest or retard
fungal penetration or haustorium formation. SA is a primary plant
defense hormone that is crucial for the activation of many plant
defenses, including the induction of systemic acquired resistance and
the HR [[152]49, [153]50]. In the present study, we also found that low
SA level reduced the accumulation of ROS in the knockdown plants
([154]Fig 8), which was associated with HR. It has been suggested that
the synthesis of SA occurs through two alternative pathways: the
shikimate pathway (SP) and the isochorismate synthase-dependent pathway
[[155]51–[156]54]. According to the data obtained for the ‘biological
process’ category, proteins involved with SP, systemic acquired
resistance and the SA-mediated signaling pathway were enriched at 24
hpi ([157]S1 File). It was evident that the Pst-induced accumulation of
SA by phenylalanine ammonia-lyase, which is an enzyme required for the
SP-dependent pathway production of SA, was highly enriched at 24 and 48
hpi ([158]S1 File). Additionally, the pathway of isochorismate
synthase-dependent was not significantly regulated. Therefore,
lignifications and SA maybe play significant roles in the APR to Pst
infection. Taken together, these data suggest that during the early
stage of the infection, the phenylpropanoid pathway plays an important
role in resistance to Pst, which hinges on the ability to induce a
broad defense response at the adult plant stage.
The continuous accumulation of reactive oxygen species contributed to APR to
Pst
ROS and nitric oxide (NO) are not only important as signaling
mechanisms for defense but also thought to regulate programmed cell
death through the establishment of a HR [[159]55, [160]56]. During the
development of a pathogen on adult plants, HR is not exhibited until 36
hpi in the inoculated leaves of the adult plants, and the host cells
become increasingly necrotic and begin to lose their original shape at
48 hpi [[161]12]. The analysis of the data revealed that some
enrichments related to the ROS or NO production systems, including
peroxidase activity and nitric-oxide synthase activity, which were
highly enriched at 24 hpi, were found to be involved in the defense
responses of wheat to Pst infection ([162]S1 File). The ‘response to
ozone’ and ‘response to hydrogen peroxide’ categories were highly
enriched at 24and 48 hpi ([163]S1 File). It was hypothesized that the
first phase of ROS generation at 24 and 48 hpi coincided with the
beginning of haustorium formation and subsequent HR in infected host
cells. Plants attempt to maintain a dynamic balance between ROS
generation and elimination through their own defense mechanisms. The
‘positive regulation of superoxide dismutase activity’ and ‘hydrogen
peroxide catabolic process’ categories were highly enriched at 120 hpi
([164]S1 File). A previous study offered similar evidence that the
second burst phase of ROS at 120 hpi coincides with an increasing
number of necrotic host cells and the formation of secondary hyphae
surrounding the infected plant cells [[165]12]. In this study,
knockdown of wheat75137_refgene and wheat31306_refgene by VIGS
decreased plant resistance to Pst associated with the low ROS
accumulation, where oxidative stress is generated to create toxicity
and kill pathogens at the infection unit. So, hyphal length increased
and the necrotic area decreased in the knockdown plants ([166]Fig 8).
Therefore, we speculated that these two candidate genes most contribute
to APR by modulating the ROS accumulation. Based on previous data and
our study, it could be hypothesized that the burst of ROS at different
phases coincides with the profiles of Pst development in infected
plants.
Photosynthesis was up-regulated during the later stage of infection
When responding to Pst infection, the wheat plant activated thousands
of genes in the early stage at 24 hpi, which may induce the expression
of key defense-related genes at 48 and 120 hpi through a synergistic
effect. This proposed hypothesis suggests that priming or core control
genes must trigger other downstream or defense-related genes through a
network of pathways. By 120 hpi, there is an increasing number of
necrotic host cells surrounding the attacked cells; in addition, the
encased haustorial body collapses and becomes necrotic, or the
haustorial body cannot expand and thus becomes necrotic [[167]12]. The
categories obtained from the GO enrichment analysis of the up-regulated
unigenes at 120 hpi were related to the chlorophyll biosynthetic
process, the photosystem II assembly process and photosynthetic
electron transport in photosystem I, photosynthesis, light harvesting
and the chloroplast relocation process ([168]S9 Fig). In our study, we
found that the unigenes involved in the glyoxylate cycle process were
clearly induced at 24 hpi. In terms of primary metabolism, the
metabolism of certain amino acids was also clearly altered. Several
earlier studies have shown that plants appear to switch off
photosynthesis locally during the early stages of the defense reaction
[[169]57]. The data obtained in this study detected no enrichments of
photosynthesis at 24 and 48 hpi, which is consistent with the results
of the aforementioned previous studies. This decrease was due not only
to the elimination of the green (photosynthetic) leaf area as a
consequence of the HR but also to an alteration in the host metabolism
[[170]58]. Photosynthesis has been reported to modulate plant defense
responses induced by pathogen infection [[171]59]. Interestingly, the
photosystem II assembly process, the photosynthetic electron transport
in the photosystem I process, photosynthesis, the light harvesting
process and the chlorophyll biosynthetic process were enriched at 120
hpi. Surprisingly, knockdown of candidate genes (wheat36302_refgene,
wheat12266_refgene) via VIGS system showed no differences compared with
the control plant. There may be two main reasons why experiments
failed. First, the candidate genes were only knockdown, not knockout,
the silencing efficiency of the candidate genes were 61% - 76% at each
time point. And second, sequence alignment with the T. aestivum cv.
Chinese Spring genome sequence showed that there were three and four
copies in the wheat genome, respectively. The starch and sucrose
metabolism pathway, which is related to the carbohydrate metabolism
process, was identified at 120 hpi. This proposed hypothesis is
consistent with previous results: a plant’s susceptibility to certain
diseases relatively depends on the sugar levels in the leaf tissues
[[172]60, [173]61]. Therefore, we speculate that the photosynthesis in
the adult XZ plant is associated with the enhanced resistance of wheat
to Pst.
Thiamine metabolism was potentially linked to adult plant resistance to Pst
The thiamine metabolism pathway was the most enriched KEGG pathway
during successive symptom phases ([174]S2 File). Plants contain a wide
range of vitamins that are essential not only for human metabolism but
also for plants because of their redox chemistry and role as enzymatic
cofactors in plant major metabolic pathways, including the oxidative
pentose phosphate pathway, acetyl-CoA synthesis, the tricarboxylic acid
cycle, the Calvin cycle, plant pigment biosynthesis, anaerobic
ethanolic fermentation and the branched-chain amino acid pathway
[[175]62, [176]63]. In a recent study, thiamine was shown to alleviate
the effects of several environmental stresses on Zea mays seedlings and
Arabidopsis thaliana, presumably by protecting the plant from oxidative
damage. Hence, it has been suggested that thiamine plays an indirect
role as an antioxidant in plants by providing NADH and NADPH to combat
oxidative stress [[177]64, [178]65]. Other reported phenomena suggest
that thiamine compounds may play an important role in the induction of
systemic acquired resistance by activating pathogenesis-related genes
in plant species against some fungal and bacterial infections [[179]66,
[180]67]. Thiamine is one of the products of the purine biosynthetic
pathway [[181]68], which was also found to be enriched through the KEGG
pathway analysis during the infection stage ([182]S2 File). We also
speculate that the thiamine metabolism and purine biosynthesis pathways
are involved in the APR to Pst.
Conclusions
APR is agriculturally valuable because of its non-race specificity and
durability; thus, understanding its resistance mechanism is important.
In the present study, our work was carefully designed to capture the
transcript response that occurs in XZ through the selection of sampling
time points based on a quantitative estimation of Pst development.
Therefore, the data obtained in this study were generated by
transcriptome sequencing and digital expression profiling. It has been
suggested that the change in most unigenesis is transient and that
unigenes are only altered at one time point during Pst infection. In
addition, we revealed that the development of Pst was markedly
inhibited in adult plants at different infection stages by many
biological processes, such as phenylpropanoid biosynthesis, reactive
oxygen species, photosynthesis, and the thiamine metabolism pathway.
Therefore, this study provides a tool for elucidating the mechanisms of
APR that can be used in broader APR research.
Supporting Information
S1 Fig. Statistics of the assembly quality of the transcriptome
sequencing samples.
Transcriptome de novo assembly was performed with the short-reads
assembling program Trinity, which resulted in 159,931 unigenes of
Ak-M-0 and 109,606 unigenes of Sk-M-0. The two libraries included
non-inoculated adult plants at 0 hours post-inoculation (hpi) (Ak-M-0)
and non-inoculated seedling plants at 0 hpi (Sk-M-0). (A) The size
distribution of the unigenes of Ak-M-0. (B) The size distribution of
the gaps of Ak-M-0. (C) The size distribution of the unigenes of
Sk-M-0. (D) The size distribution of the gaps of Sk-M-0. (E) The size
distribution of all of the unigenes. (F) The size distribution of the
gaps of all of the unigenes. (G) The size distribution of the ESTs
obtained from the EST scan results. (H) The size distribution of the
proteins predicted from the CDS sequences.
(TIF)
[183]Click here for additional data file.^ (8.9MB, tif)
S2 Fig. COG functional classification of all of the unigenes.
A total of 157,689 unigenes showed significant homologies to genes in
the COG Nr database (E-value<10^−5) and were distributed into 25 COG
categories.
(TIF)
[184]Click here for additional data file.^ (9MB, tif)
S3 Fig. GO classification of all of the unigenes.
A total of 69,100 unigenes were assigned to GO term annotations using
BLAST2GO and then summarized into three main GO categories and 42
sub-categories (functional groups) using WEGO.
(TIF)
[185]Click here for additional data file.^ (5.6MB, tif)
S4 Fig. Distribution of DEGs between the seeding and adult plant
stages.
All of the DEGs were obtained from this analysis: unigenes (red
portion) that were up-regulated, unigenes (green portion) that were
down-regulated, and unigenes (blue portion) that were not regulated at
the adult plant stage. The two libraries included non-inoculated adult
plants at 0 hours post-inoculation (hpi) (Ak-M-0) and non-inoculated
seedling plants at 0 hpi (Sk-M-0).
(TIF)
[186]Click here for additional data file.^ (1.2MB, tif)
S5 Fig. Evaluation of the sequencing quality of the DGE samples.
Classification of raw reads of six DGE libraries, Ak-M-24, Ak-M-48,
Ak-M-120, Ak-I-24, Ak-I-48 and Ak-I-120. The six DGE libraries included
non-inoculated adult plants at 24 hours post-inoculation (hpi)
(Ak-M-24), 48 hpi (Ak-M-48) and 120 hpi (Ak-M-120), and inoculated
adult plants at 24 hpi (Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi
(Ak-I-120).
(TIF)
[187]Click here for additional data file.^ (5.2MB, tif)
S6 Fig. Analysis of the sequencing saturation of the DGE samples.
The saturation analyses of the six DGE libraries, Ak-M-24, Ak-M-48,
Ak-M-120, Ak-I-24, Ak-I-48 and Ak-I-120. The six DGE libraries included
non-inoculated adult plants at 24 hours post-inoculation (hpi)
(Ak-M-24), 48 hpi (Ak-M-48) and 120 hpi (Ak-M-120), and inoculated
adult plants at 24 hpi (Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi
(Ak-I-120).
(TIF)
[188]Click here for additional data file.^ (6.8MB, tif)
S7 Fig. Gene coverage statistics for the DGE samples.
The gene coverage statistics of the DGE samples in Ak-M-24, Ak-M-48,
Ak-M-120, Ak-I-24, Ak-I-48 and Ak-I-120. The six DGE libraries included
non-inoculated adult plants at 24 hours post-inoculation (hpi)
(Ak-M-24), 48 hpi (Ak-M-48) and 120 hpi (Ak-M-120), and inoculated
adult plants at 24 hpi (Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi
(Ak-I-120).
(TIF)
[189]Click here for additional data file.^ (4.3MB, tif)
S8 Fig. The expression patterns of wheat refgenes for ‘phenylpropanoid’
were determined by RNA-Seq.
The relative expression levels of eight candidate unigenes associated
with the phenylpropanoid biological process at the infection stage were
determined by RNA-Seq. The candidate unigenes were only expressed at 24
hours post-inoculation (hpi).
(TIF)
[190]Click here for additional data file.^ (6.2MB, tif)
S9 Fig. The expression patterns of wheat refgenes associated with the
‘chlorophyll biosynthetic process’ were determined by RNA-Seq.
Thirty-five candidate unigenes were activated during the chlorophyll
biosynthetic process. The relative expression level at the infection
stage was determined by RNA-Seq. The unigenes were only expressed at
120 hours post-inoculation (hpi).
(TIF)
[191]Click here for additional data file.^ (8.5MB, tif)
S1 File. Enrichment of the genes up-regulated during Puccinia
striiformis f. sp. Tritici infection.
Fisher’s exact test with the Blast2GO software was used to explore the
significantly enriched GO terms of the genes up-regulated during
Puccinia striiformis f. sp. Tritici infection. The data included the
‘Biological process’, ‘Molecular function’ and ‘Cellular component’
categories.
(XLSX)
[192]Click here for additional data file.^ (27KB, xlsx)
S2 File. The up-regulated DEGs in the Ak-I-24 vs. Ak-M-24, Ak-I-48 vs.
Ak-M-48 and Ak-I-120 vs. Ak-M-120.
Comparisons were categorized according to Kyoto Encyclopedia of Genes
and Genomes (KEGG) pathway analyses.
(XLSX)
[193]Click here for additional data file.^ (16.7KB, xlsx)
S3 File. qRT-PCR primer sequences for 30 DEGs.
(DOCX)
[194]Click here for additional data file.^ (15.3KB, docx)
S1 Table. Statistics of the DGE profiling sample map to the reference
genome.
This table includes the number of six samples of reads that were of
Total Reads, Total Base Pairs, Total Mapped Reads (sum of the highly
repetitive and high quality reads), Perfect match (mapped to over 100
locations), < = 2-bp mismatch, Unique match (mapped to 1 location) and
Multi-position match, respectively. The 6 DGE libraries included
non-inoculated adult plants at 24 hours post-inoculation (hpi)
(Ak-M-24), 48 hpi (Ak-M-48) and 120 hpi (Ak-M-120), and inoculated
adult plants at 24 hpi (Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi
(Ak-I-120).
(DOCX)
[195]Click here for additional data file.^ (17.3KB, docx)
S2 Table. Virus-induced gene silencing (VIGS) system primer sequences
for six candidate genes.
This table includes twelve pairs of primer sequences for six genes, six
pairs for BSMV-mediated gene silencing (S), and six pairs for qRT-PCR
(Q).
(DOCX)
[196]Click here for additional data file.^ (14.9KB, docx)
Acknowledgments