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Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis Jianxin Zhou a, 1, Wenwen Kong b, 1, Haiyan Zhao a, Rui Li a, Yujian Yang a, Jing Li a, * a b
College of Life Sciences, Northeast Agricultural University, Harbin, 150030, China College of Life Science and Oceanography, Shenzhen University, Shenzhen, 518060, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 September 2019 Accepted 25 September 2019 Available online xxx
Indole glucosinolates are known to play essential and diverse roles in Arabidopsis immunity to pathogens. However, a complete understanding of the function of these compounds in plant immunity remains unclear. In this study, we investigated the transcriptome profile in loss-of-function mutant of MYB51, the key transcription factor that controls the biosynthesis of indole glucosinolates. Upon treatment with flg22 (a 22-amino acid peptide derived from bacterial flagellin), the genes that responded in a MYB51-dependent manner were analyzed. The results suggested that MYB51 was possibly implicated in most resistance processes, including pathogen recognition, signal transduction and PR protein activation. Of note, several genes in the ethylene pathway and the WRKY family, including WRKY33, were induced by flg22 in a MYB51-dependent manner. WRKY33 and ethylene were demonstrated to be crucial regulators in plant immunity defense and are functionally upstream of MYB51 during MAMP triggered immunity (MTI). This result suggested a “positive feedback loop” between MYB51 and its upstream regulators. © 2019 Elsevier Inc. All rights reserved.
Keywords: Arabidopsis Transcriptome myb51 Glucosinolates flg22
1. Introduction Glucosinolates are nitrogen and sulfur containing secondary metabolites derived from amino acids. Depending on their amino acid precursors, they are classified as aliphatic, aromatic and indole glucosinolates [1]. In Arabidopsis, tryptophan derived indole glucosinolates and their metabolic products were found to be essential for the defense response to various biotic stresses, especially pathogens [2e7]. Therefore, these compounds have been widely investigated and the mechanisms by which indole glucosinolates are functional in pathogen resistance have been gradually revealed. Besides their direct antimicrobial activity [8], indole glucosinolates are also involved in triggering highly conserved immune responses in the plant kingdom. In Arabidopsis, the degradation of indole glucosinolate are essential for bacteria-triggered callose deposition [9,10], and can also modulate pathogen-induced hypersensitive programmed cell death (PCD) [11,12]. Indole glucosinolate breakdown product, indole-3-carbinol (I3C), can act as an auxin antagonist [13] and can affect production and localization of the auxin transporters [14]. In addition, indolic glucosinolates were reported
* Corresponding author. E-mail address: [email protected] (J. Li). 1 These authors have contributed equally to this work.
to be induced systemically at the uninfected tissue when challenged by bacterial pathogen and contributed to systemic acquired resistance (SAR) [15,16]. These studies all suggest that indole glucosinolate metabolism has a profound impact on pathogen resistance through various molecular mechanisms. Although the functions of indole glucosinolates and their breakdown products are attracting extensive attention, the global view of the exact role and precise mechanism of indole glucosinolates in immunity remains elusive. In this study, we used flg22 to simulate a pathogen-triggered immune response and compared these responses between wild type and an indole glucosinolate deficient mutant. The metabolism and regulation of indole glucosinolates have been clearly identified in Arabidopsis, and the MYB51, MYB34 and MYB122 transcription factors are known to be regulators of indole glucosinolates [17,18]. MYB122 is considered to play an accessory role in the biosynthesis of indole glucosinolates, while both MYB51 and MYB34 are important transcription factors that activate the production of indole glucosinolates. Since the indole glucosinolate biosynthesis intermediate product IAOx, is also a precursor of the predominate auxin IAA (indole-3-acetic acid), the metabolism of indole glucosinolates is closely related with IAA production. The biosynthesis of indole glucosinolates in both myb51 and myb34 is largely defective [17], however, MYB34 significantly affects the homeostasis of IAA
https://doi.org/10.1016/j.bbrc.2019.09.110 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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and in contrast, neither overexpression nor deficiency of MYB51 lead to altered IAA level [17,18]. To avoid pleiotropic effects and focus on obtaining information related to indole glucosinolate dependent immunity response, myb51 was chosen as the indole glucosinolate defective mutant. To provide a full view of indole glucosinolate functions during the Arabidopsis response to pathogen invasion, high-throughput Illumina sequencing was performed to characterize the transcriptomes in wild-type and myb51 plants. Analysis of MYB51 dependent flg22-response genes showed that MYB51 might participate in most of the pathogen resistance processes including pathogen recognition, signal transduction and activation of PR proteins. WRKY transcription factors and the ethylene (ET) signaling pathway have been demonstrated to be functionally upstream of MYB51 in response to flg22. Interestingly, the responses of these upstream regulatory genes to flg22 were impaired in myb51, suggesting that there might be a positive feedback loop among MYB51 and these important regulators. Our study shed light on the diversity and complexity of indole glucosinolate-mediated pathogen resistance. 2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis ecotype Columbia (Col-0) was used in this study. Seeds of wild type and a T-DNA insertion mutant for MYB51 (SALK_059771) were purchased from the Arabidopsis Biological Resource Center (ABRC). The seeds were sown on 1/2 Murashige and Skoog (1/2MS) basal medium under 24 C, a light regime of 16 h light and 8 h dark, 70% relative humidity and a constant illumination of 100 mmol m2 s1. 2.2. Genotyping and RT-PCR analysis of myb51 Genomic DNA was isolated using an EasyPure Plant Genomic DNA Kit (TransGen, Beijing, China). Primers for myb51 genotyping were 5’ - AAATTTTGGTCAAGAATCGGG-30 , 50 -AAAGGGGGTTGTTC TCAAGTG-30 and the T-DNA-specific primer LBa1 50 -TGGTTCACGT AGTGGGCCATCG-3’. Leaves from wild type and homozygous myb51 were harvested 12 d after germination. RNA was extracted using the E.Z.N.A. Plant RNA Kit (Omega Biotek, GA). First-strand cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). The expression level of MYB51 was assessed using the primer pair 50 ATGGTGCGGACACCGTGT-30 and 50 -TCATCCAAAATAGTTATCAA TTTCG-3’. 2.3. Treatment of flg22 Twelve-day-old seedlings of the wild type and myb51 were respectively incubated into 1/2MS liquid medium and 1/2MS liquid medium containing 1 mmol/L flg22 for 4 h (under 24 C and a constant illumination of 100 mmol m2 s1.). Each sample was treated and harvested in three independent biological replicates.
San Diego, CA), Illumina Miseq library construction was performed. To purify the mRNA from the total RNA, magnetic beads with oligo dT attached were used. Then, mRNA was cleaved by using fragmentation buffer. Using random hexamer primers, the first-strand cDNAs of the fragments were synthesized. Then, doubled-stranded cDNA was transformed by using RHase H and DNA polymerase I. A paired-end library was constructed from the double-stranded cDNA with a Genomic Sample Prep Kit (Illumina). Fragments of desirable lengths were purified with the QIA quick PCR (Qiagen) Extraction Kit, end repaired, and linked with sequencing adapters. Unsuitable fragments were removed with AMPureXP beads, followed by PCR amplification, and the sequencing library was constructed. The established library was then sequenced with the Illumina Hiseq 2000 platform (Shanghai Personal Biotechnology Cp., Ltd. Shanghai, China). 2.6. Sequence analysis Raw reads from the samples were first processed through inhouse Perl scripts. In this step, clean reads were obtained by removing adaptor contamination reads containing poly-N and lowquality reads from raw data. Then, the Arabidopsis TAIR10 genome was used as a reference and the reference genome index was built using Bowtie2. The clean reads were aligned to the reference genome using Tophat v2.0.9 and HTSeq2 was used to count the read number mapped to each gene. The RPKM value of each gene was calculated based on the length of the gene and reads count mapped to this gene with the edger program. 2.7. Clustering and annotation of differentially expressed genes (DEGs) DEG analysis was conducted using DESeq2 with the criteria change fold 2 and p-value 0.05. Gene ontology (GO) annotation was performed by alignment between genes and the GO database. GO classification of DEGs was performed by using the R package “clusterProfiler”. GO enrichment was analyzed in the categories of biological process. Pathway enrichment was performed by using MapMan. To identify the TFs from the DEGs, we searched the DEGs against the Arabidopsis TF gene list which was downloaded from the Plant Transcription Factor Database (http://plntfdb.bio.unipotsdam.de/v3.0/) [19]. For the DEG volcano plot, clustering and heatmap plotting, the R packages “ggplot2”, “gplots” and “clusterProfiler” were used. 2.8. qRT-PCR verification of DEGs Twelve-day-old seedlings of wild type and myb51 were treated with flg22. Isolation of total RNA and synthesis of first-strand cDNA was performed as described above. Twelve MYB51 dependent flg22-response genes from the WRKY family and the ET signaling pathway were selected for qRT-PCR analysis. The selected genes and primers used for qRT-PCR were listed in Table S1 qRTePCR analyses were performed using SYBR Green Master Mix on an ABI 7500 sequence detection system.
2.4. Total RNA extraction 3. Results Isolation of total RNA was performed as described above. High quality RNA with a 28 S:18 S greater than 1.5, absorbance 260/280 ratios between 1.8 and 2.2 and absorbance 260/230 ratios higher than 2.0 was used for library construction and sequencing. 2.5. Strand-specific cDNA library construction and sequencing According to the instructions from the manufacturer (Illumina,
3.1. Illumina sequencing and assembly First, the myb51 was confirmed to be homozygous and have no detectable transcripts for MYB51 (Fig. S1). RNA samples from 12day-old wild-type seedlings without flg22 treatment (represented as WT), wild type with flg22 treatment (represented as WT þ flg22), myb51 without flg22 treatment (represented as
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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Table 1 Statistical summary of cDNA sequences of samples. Samples
Raw Reads
Clean Reads
Mapped Reads
Unigenes
Mapped Percent (%)
WT myb51 WT þ flg22 myb51 þ flg22
63,344,558 66,268,194 72,675,626 83,224,090
45,250,494 47,172,134 52,202,996 59,359,844
42,611,717 44,715,380 49,126,338 56,264,002
22,538 22,665 22,891 22,843
94.17% 94.79% 94.11% 94.78%
Fig. 1. Gene expression profiles in wild type and myb51 upon treatment of flg22. (A) Clustering analysis based on the FPKM Z-score of each gene and GO enrichment for each cluster genes. (B) Hub genes identified from DEGs in WT upon WT þ flg22; (C) Hub genes identified from DEGs in myb51 upon myb51 þ flg22; Red dots represent up-regulated hub genes and green dots represent down-regulated hub genes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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myb51), and myb51 with flg22 treatment (represented as myb51 þ flg22) were used to generate cDNA libraries, yielding 45,250,494, 47,172,134, 52,202,996 and 59,359,844 clean reads for WT, myb51, WT þ flg22 and myb51 þ flg22, respectively (Table 1). The Arabidopsis genome from TAIR10 (https://www.arabidopsis. org/index.jsp) was used as the reference to assemble the clean reads; more than 94% clean reads were mapped for all four datasets (Table 1). The distribution of these mapped reads on the chromosomes was shown in Fig. S2. High-throughput RNA sequencing yielded 22,538, 22,665, 22,891 and 22,843 unigenes for WT, myb51, WT þ flg22 and myb51 þ flg22, respectively (Table 1). 3.2. Analysis of DEGs between WT and myb51 To understand the MYB51-mediated network under normal growth conditions, we evaluated global changes in gene expression between WT and myb51. RPKM (Reads Per Kilobase per Million mapped reads) were used to calculate gene expression levels and DEGs were filtered out with the criteria change fold 2 and pvalue 0.05. To determine the number of DEGs in WT and myb51, log2-fold-change values were plotted against negative log10 (pvalue) to generate a volcano plot (Fig. S3A). A total of 681 DEGs containing 257 down-regulated and 424 up-regulated genes were determined in myb51 compared to the wild type (Table S2). To obtain the functional categories of these DEGs, a functional
enrichment analysis with MapMan was performed. As shown in Fig. S3B, besides “Not assigned”, the most enriched categories (“BINs” in MapMan terms) were “RNA processing”, “Protein”, “Signaling”, “Miscellaneous enzyme families”, “Stress”, “Hormones”, “Transport”, “Development”, “Cell wall” and “Secondary metabolism”. Although myb51 does not present any significant morphologic phenotype, MapMan enrichment analysis (Fig. S3B) demonstrated that the mutation of MYB51 had a profound effect on many biological processes even under normal growing conditions since the expression of genes related to transcription, protein metabolism, hormone and signaling were largely altered. As expected, stress and secondary metabolism processes were significantly affected. The DEGs enriched in “development” and “cell wall” indicated a possible role of MYB51 in plant development, especially the formation of the cell wall. 3.3. Analysis of flg22-response DEGs in wild type and myb51 To investigate the genes response to flg22 in WT and myb51, a comparison between flg22-treated and untreated samples was performed. In total, 1200 (910 up-regulated and 290 downregulated) and 895 (725 up-regulated and 170 down-regulated) DEGs were identified from WT þ flg22 versus WT and myb51 þ flg22 versus myb51, respectively (Figs. S4AeB and Table S3). Compared to wild type, the response to flg22 in myb51
Table 2 Differently expressed hub genes in response to flg22 in wild type and myb51. Gene ID
Gene Name
Hub genes up-regulated in WT AT2G38470 WRKY33 AT1G80840 WRKY40 AT5G25930 AT5G25930 AT1G27730 STZ AT4G29780 AT4G29780 AT1G07160 AT1G07160 AT1G74360 AT1G74360 AT3G52400 SYP122 AT3G55980 SZF1 Hub genes down-regulated in WT AT3G50820 PSBO2 AT5G66570 PSBO1 AT4G02770 PSAD-1 AT1G06680 PSBP-1 AT1G54780 TLP18.3 AT1G52230 PSAH2 AT4G05180 PSBQ-2 AT5G64040 PSAN AT5G54270 LHCB3 Hub genes up-regulated in myb51 AT1G74360 AT1G74360 AT5G25930 AT5G25930 AT1G07160 AT1G07160 AT5G52640 HSP90.1 AT1G19020 AT1G19020 AT5G57220 CYP81F2 AT2G40180 PP2C5 AT3G12580 HSP70 AT2G18690 AT2G18690 Hub genes down-regulated in myb51 AT1G06680 PSBP-1 AT3G50820 PSBO2 AT5G64040 PSAN AT1G52230 PSAH2 AT3G08940 LHCB4.2 AT5G54270 LHCB3 AT3G54890 LHCA1 AT1G61520 LHCA3 AT1G54780 TLP18.3
Gene description WRKY DNA-binding protein 33 WRKY DNA-binding protein 40 kinase family with leucine-rich repeat domain-containing protein salt tolerance zinc finger nuclease Protein phosphatase 2C family protein Leucine-rich repeat protein kinase family protein syntaxin of plants 122 salt-inducible zinc finger 1 photosystem II subunit O-2 PS II oxygen-evolving complex 1 photosystem I subunit D-1 photosystem II subunit P-1 thylakoid lumen 18.3 kDa protein photosystem I subunit H2 photosystem II subunit Q-2 photosystem I reaction center subunit PSIeN light-harvesting chlorophyll B-binding protein 3 Leucine-rich repeat protein kinase family protein kinase family with leucine-rich repeat domain-containing protein Protein phosphatase 2C family protein heat shock-like protein CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase cytochrome P450, family 81, subfamily F, polypeptide 2 phosphatase 2C5 heat shock protein 70 transmembrane protein photosystem II subunit P-1 photosystem II subunit O-2 photosystem I reaction center subunit PSIeN, chloroplast, putative/PSIeN, putative (PSAN) photosystem I subunit H2 light harvesting complex photosystem II light-harvesting chlorophyll B-binding protein 3 chlorophyll a-b binding protein 6 PSI type III chlorophyll a/b-binding protein thylakoid lumen 18.3 kDa protein
The highlighted are the shared hub genes in wild type and myb51 upon flg22 treatment.
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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was much less sensitive, indicating the importance of MYB51 in flg22-triggered immunity. K-means clustering of all genes was performed, with each gene getting an FPKM Z-score value, revealing 4 large clusters of distinct expression profiles, each associated with distinct functions based on the GO analysis of the “Biological Process” sub-groups (Fig. 1A). As shown in Fig. 1A, in both wild type and myb51, pathogen resistance associated
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processes (cluster 1 and cluster 2) were activated and photosynthesis and carbohydrate-related processes (cluster 3 and cluster 4) were repressed upon treatment of flg22. (Fig. 1A). Distinctive expression patterns especially in cluster 2 can be observed in the two genotypes without flg22 treatment (Fig. 1A). The expression level of genes in cluster 2, which are mainly pathogen resistance genes, was significantly lower in myb51 than in wild type under
Fig. 2. Identification of MYB51 dependent flg22-response genes. (A) An overview of the DEGs in myb51 compared to WT and DEGs in WT and myb51 upon treatment with flg22. (B) Number of DEGs in WT and myb51 upon treatment with flg22. (C) GO enrichment analysis of the MYB51 dependent flg22-response genes. The size of each circle represents the fold enrichment of the GO terms. The color density indicates the significance of the GO terms [elog10(p-value)].
Fig. 3. MYB51 dependent flg22-response genes enriched in biotic-stress. Red squares represent up-regulated and blue squares represent down-regulated genes upon treatment with flg22. The grey circles represent no MYB51 dependent flg22-response genes involved in this process. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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normal growing conditions. This indicated that these genes were largely affected by MYB51 and might be involved in constitutive disease resistance, which is formed before pathogens attack. To elucidate the different response of myb51 and the wild type to flg22, STRING networks (https://string-db.org/) of DEGs in WT þ flg22 versus WT and myb51 þ flg22 versus myb51 were constructed respectively by using Cytoscape 3.7 (https://cytoscape. org/). Top hub genes were identified and compared between WT þ flg22 versus WT and myb51 þ flg22 versus myb51 (Fig. 1BeC). Among the up-regulated hub genes, which are mostly defensive genes, 6 of the 9 genes were different between myb51 and wild type, while among the down-regulated hub genes, which are mainly photosynthesis related genes, 3 of the 9 genes were different (Table 2). Interestingly, WRKY33, WRKY40 which have been demonstrated to play key roles in modulating flg22-induced MTI [20], were found to be the hub genes in response to flg22 in the wild type but not in myb51 (Fig. 1BeC), indicating that the function of these important pathogen defense associated transcription factors are closely related with MYB51 in defensive responses.
3.4. Analysis of MYB51 dependent flg22-response DEGs To discover the specific DEGs which respond to flg22 treatment
in a MYB51 dependent manner, an overview of the DEGs in the wild type and myb51 in response to flg22 was generated (Fig. 2AeB). In total, 482 DEGs were filtered out as MYB51-dependent flg22 response genes, which were only differentially expressed in wild type but not in myb51 upon treatment with flg22 (Fig. 2B and Table S4). The GO term “Biological Process” enrichment analysis showed that the most enriched terms of these MYB51 dependent flg22-response genes were “response to organonitrogen compound”, “response to chitin”, “intracellular signal transduction”, “systemic acquired resistance”, “response to salicylic acid”, “programmed cell death” as well as “regulation of immune system process”. (Fig. 2C). Most of the enriched biological processes were closely related with defense against pathogens, and some of the processes, such as “systemic acquired resistance” and “program cell death”, have been demonstrated to be triggered by indole glucosinolates in response to pathogens. In addition, as expected, some of the indole glucosinolate biosynthetic related genes including CYP79B2, ATSOT16, CYP83B1 and NIT4 were enriched in MYB51 dependent flg22-response genes (Table S4). Indole glucosinolate biosynthesis mediated by MYB51 is required for flg22-induced callose accumulation. Not surprisingly, eight callose biosynthetic genes, CML37, BCS1, ATSZF1, AOC3, ATCBL1, ATFC-I, CAD1 and APD5 were also enriched (Table S4). These results confirmed that the MYB51 dependent flg22-response genes we obtained can provide
Table 3 Selected MYB51 dependent flg22-response genes. Gene ID R AT1G72930 AT2G20142 AT2G32140 AT5G36910 signaling AT3G28930 MAPK AT1G01560 PR-proteins AT1G66090 AT1G72900 AT1G72920 AT1G72940 AT3G13650 AT5G41740 AT5G58120 AT1G13609 Ethylene AT4G11280 AT1G28370 AT2G44840 AT3G23230 AT4G34410 AT5G47230 AT5G51190 AT1G68765 ERF AT1G12610 AT1G33760 AT1G63030 AT1G64380 AT1G72360 AT1G79700 AT4G32800 WRKY AT2G38470 AT2G40750 AT2G46400 AT3G56400 AT4G23810 AT4G31550
Gene Name
Gene description
TIR AT2G20142 AT2G32140 THI2.2
toll/interleukin-1 receptor-like protein Toll-Interleukin-Resistance (TIR) domain family protein transmembrane receptor thionin 2.2
AIG2
AIG2-like (avirulence induced gene) family protein
MPK11
MAP kinase 11
AT1G66090 AT1G72900 AT1G72920 AT1G72940 AT3G13650 AT5G41740 AT5G58120 AT1G13609
Disease resistance protein (TIR-NBS class) Toll-Interleukin-Resistance (TIR) domain-containing protein Toll-Interleukin-Resistance (TIR) domain family protein Toll-Interleukin-Resistance (TIR) domain-containing protein Disease resistance-responsive (dirigent-like protein) family protein Disease resistance protein (TIR-NBS-LRR class) family Disease resistance protein (TIR-NBS-LRR class) family Defensin-like (DEFL) family protein
ACS6 ERF11 ERF13 TDR1(ERF98) RRTF1 ERF5 ERF105 IDA
1-aminocyclopropane-1-carboxylic acid (acc) synthase 6 ERF domain protein 11 ethylene-responsive element binding factor 13 ethylene-responsive factor redox responsive transcription factor 1 ethylene responsive element binding factor 5 Integrase-type DNA-binding superfamily protein Putative membrane lipoprotein
DDF1 AT1G33760 DDF2 AT1G64380 ERF73 WRI4 AT4G32800
member of the DREB subfamily A-1 of ERF/AP2 transcription factor family member of the DREB subfamily A-4 of ERF/AP2 transcription factor family member of the DREB subfamily A-1 of ERF/AP2 transcription factor family member of the DREB subfamily A-6 of ERF/AP2 transcription factor family member of the ERF subfamily B-2 of ERF/AP2 transcription factor family AP2/ERF-type transcriptional activator that specifically controls cuticular wax biosynthesis in Arabidopsis stems. member of the DREB subfamily A-4 of ERF/AP2 transcription factor family
WRKY33 WRKY54 WRKY46 WRKY70 WRKY53 WRKY11
WRKY WRKY WRKY WRKY WRKY WRKY
DNA-binding protein 33 DNA-binding protein 54 DNA-binding protein 46 DNA-binding protein 70 family transcription factor DNA-binding protein 11
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valuable information to better understand the function of indole glucosinolates in MTI. For an overview of the biotic stress responses of these MYB51 dependent flg22-response genes, MapMan was used to perform pathway enrichment (Fig. 3). Four R genes which code for proteins recognizing specific pathogen effectors, a signaling protein coding gene AIG2 (a virulence induced gene), a MAP kinase gene MPK11 and eight PR-protein encoding genes were identified (Fig. 3 and Table 3). Furthermore, “Hormone signaling”, “Cell wall”, “Proteolysis” and “Transcription Factors”, were also enriched. This indicated that MYB51 possibly participates in most pathogen resistance processes including pathogen recognition, signal transduction and multiple defense reactions. It is noteworthy that several genes in the WRKY family, ERF family and ET signaling pathway were induced upon flg22 treatment in a MYB51-dependent way (Fig. 3). WRKY transcription factors are the major regulators of immune responses and signaling involving MTI, especially WRKY18, WRKY33 and WRKY40 which can directly regulate indole glucosinolate metabolism by binding to the promoters of related genes including MYB51 [20]. The ET signaling pathway also plays an active role in plant defense against pathogens. During flg22-induced callose deposition, ET signaling
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transduction is required and is functionally upstream of MYB51 [10]. Interestingly, as shown in Fig. 3, the induction of six WRKY genes including WRKY33, six ERF genes as well as eight genes in the ET pathway upon treatment of flg22 was found to be dependent on MYB51. To validate whether the results from the transcriptome analysis can reflect the real expression profile of these genes, six WRKY genes and six ET pathway genes were selected to perform quantitative RT-PCR (Fig. 4). To better reflect the dynamic responses of the above genes upon flg22 treatment, the expression levels of these genes were determined after 1 and 4 h treatment of flg22. Consistent with the results of the transcriptome analysis, the WRKY genes including WRKY33, WRKY53, WRKY46, WRKY54 and WRKY70 were significantly induced upon flg22 treatment in the wild type but not in myb51, as were the ET pathway genes ACS6, ERF5, ERF11, ERF13, ERF98 and ERF105. WRKY11 was the only gene of which the expression pattern was not completely consistent with that of transcriptome analysis. At 1 h of flg22 treatment, WRKY11 was induced in both wild type and myb51. At 4 h, the expression decreased in both wild type and myb51, but decreased more significantly in myb51. This suggested that the induction of WRKY11 upon flg22 treatment was dependent on MYB51 at the later stage (4 h).
Fig. 4. Expression of several WRKY genes and ethylene signaling related genes in response to flg22. (A) Expression of WRKY genes in response to flg22; (B) Expression of ethylene signaling related genes in response to flg22. Relative expression values are given in comparison with the WT (WT ¼ 1). Each bar represents the mean (±standard error) of three biological replicates. * and **, significantly different (Student’s t-test; * 0.01 < P < 0.05, **P < 0.01) from the 0 h.
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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The consistent expression profiling between the transcriptome and qRT-PCR indicated that our transcriptome analysis was reliable and it could be confirmed that the induction of certain WRKY genes and ET pathway genes in response to MAMP was dependent on MYB51. Since WRKY33 and the ET signaling pathway have been demonstrated to be functionally upstream of MYB51 in response to flg22, there might be positive feedback regulation among MYB51 and these important TFs.
Declaration of competing interest Authors declare that there is no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.110. Transparency document
4. Discussion Indole glucosinolates plays a variety of roles in plant immune responses, including systemic acquired resistance, hypersensitive response and callose accumulation [10,12,15]. The function of these metabolites seems to exceed our expectations. To more comprehensively understand the functions of these indolic secondary metabolites in plant immunity, high-throughput RNA-sequencing of wild type and myb51 following treatment with flg22 were performed. As shown in the analysis of MYB51-dependent biotic stress responsive genes, MYB51 is possibly involved in most pathogen resistance processes including pathogen recognition, signal transduction, and activation of MAPK and PR proteins. Of note, several genes in the WRKY family and ET signaling pathway were induced upon flg22 treatment in a MYB51-dependent way. The large WRKY family of transcription factors plays an important role in plant resistance to various stresses. Several WRKY transcription factors have been found to be key immune regulators that modulate the transcriptional reprogramming in response to various pathogens [21e23]. During flg22-triggered MTI, WRKY33, WRKY40 and WRKY18 directly target to MYB51 and other genes in the indole glucosinolate pathway to promote the biosynthesis of these secondary metabolites [20]. In Botrytis cinereaeinduced ethylene biosynthesis, WRKY33 activates the ET signaling pathway by inducing the expression of ACS2 and ACS6, two ET biosynthetic enzyme genes through direct binding to gene promoters [24]. Indole glucosinolate production mediated by MYB51 is required for flg22-induced callose accumulation. During this immune response, the ET pathway is functionally upstream of MYB51 [10]. In short, in response to pathogens, WRKY33 is functionally upstream of the ET pathway, and both WRKY33 and the ET signaling pathway play regulatory roles upstream of MYB51. However,in myb51, WRKY33 and the ET biosynthesis gene ACS6 were impaired in response to flg22. These results indicated that MYB51 had positive feedback on its upstream regulators. WRKY33, the ET pathway and MYB51 formed a “positive feedback loop”. In addition to WRKY33 and ACS6, WRKY46, WRKY53, WRKY54, WRKY70, WRKY11 and five ET response factor (ERF) genes, ERF5, ERF11, ERF13, ERF98 and ERF105 were also impaired in response to flg22 in myb51 (Fig. 4). Among these MYB51 dependent flg22responsive WRKYs and ERFs, WRKY53, WRKY54, WRKY70, WRKY 11, ERF5 and ERF11 have been demonstrated to have key regulatory roles in innate immune responses against various pathogens [22,23,25e29]. Indolic metabolite production mediated by MYB51 is implicated in diverse immune responses, which can be possibly explained by the positive effect of MYB51 on these key regulatory transcription factors in pathogen resistance.
Funding This work was supported by the National Natural Science Foundation of China (NSFC) 31370334 and Natural Science Foundation of Heilongjiang Province C2017031.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis Jianxin Zhou a, 1, Wenwen Kong b, 1, Haiyan Zhao a, Rui Li a, Yujian Yang a, Jing Li a, * a b
College of Life Sciences, Northeast Agricultural University, Harbin, 150030, China College of Life Science and Oceanography, Shenzhen University, Shenzhen, 518060, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 September 2019 Accepted 25 September 2019 Available online xxx
Indole glucosinolates are known to play essential and diverse roles in Arabidopsis immunity to pathogens. However, a complete understanding of the function of these compounds in plant immunity remains unclear. In this study, we investigated the transcriptome profile in loss-of-function mutant of MYB51, the key transcription factor that controls the biosynthesis of indole glucosinolates. Upon treatment with flg22 (a 22-amino acid peptide derived from bacterial flagellin), the genes that responded in a MYB51-dependent manner were analyzed. The results suggested that MYB51 was possibly implicated in most resistance processes, including pathogen recognition, signal transduction and PR protein activation. Of note, several genes in the ethylene pathway and the WRKY family, including WRKY33, were induced by flg22 in a MYB51-dependent manner. WRKY33 and ethylene were demonstrated to be crucial regulators in plant immunity defense and are functionally upstream of MYB51 during MAMP triggered immunity (MTI). This result suggested a “positive feedback loop” between MYB51 and its upstream regulators. © 2019 Elsevier Inc. All rights reserved.
Keywords: Arabidopsis Transcriptome myb51 Glucosinolates flg22
1. Introduction Glucosinolates are nitrogen and sulfur containing secondary metabolites derived from amino acids. Depending on their amino acid precursors, they are classified as aliphatic, aromatic and indole glucosinolates [1]. In Arabidopsis, tryptophan derived indole glucosinolates and their metabolic products were found to be essential for the defense response to various biotic stresses, especially pathogens [2e7]. Therefore, these compounds have been widely investigated and the mechanisms by which indole glucosinolates are functional in pathogen resistance have been gradually revealed. Besides their direct antimicrobial activity [8], indole glucosinolates are also involved in triggering highly conserved immune responses in the plant kingdom. In Arabidopsis, the degradation of indole glucosinolate are essential for bacteria-triggered callose deposition [9,10], and can also modulate pathogen-induced hypersensitive programmed cell death (PCD) [11,12]. Indole glucosinolate breakdown product, indole-3-carbinol (I3C), can act as an auxin antagonist [13] and can affect production and localization of the auxin transporters [14]. In addition, indolic glucosinolates were reported
* Corresponding author. E-mail address: [email protected] (J. Li). 1 These authors have contributed equally to this work.
to be induced systemically at the uninfected tissue when challenged by bacterial pathogen and contributed to systemic acquired resistance (SAR) [15,16]. These studies all suggest that indole glucosinolate metabolism has a profound impact on pathogen resistance through various molecular mechanisms. Although the functions of indole glucosinolates and their breakdown products are attracting extensive attention, the global view of the exact role and precise mechanism of indole glucosinolates in immunity remains elusive. In this study, we used flg22 to simulate a pathogen-triggered immune response and compared these responses between wild type and an indole glucosinolate deficient mutant. The metabolism and regulation of indole glucosinolates have been clearly identified in Arabidopsis, and the MYB51, MYB34 and MYB122 transcription factors are known to be regulators of indole glucosinolates [17,18]. MYB122 is considered to play an accessory role in the biosynthesis of indole glucosinolates, while both MYB51 and MYB34 are important transcription factors that activate the production of indole glucosinolates. Since the indole glucosinolate biosynthesis intermediate product IAOx, is also a precursor of the predominate auxin IAA (indole-3-acetic acid), the metabolism of indole glucosinolates is closely related with IAA production. The biosynthesis of indole glucosinolates in both myb51 and myb34 is largely defective [17], however, MYB34 significantly affects the homeostasis of IAA
https://doi.org/10.1016/j.bbrc.2019.09.110 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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and in contrast, neither overexpression nor deficiency of MYB51 lead to altered IAA level [17,18]. To avoid pleiotropic effects and focus on obtaining information related to indole glucosinolate dependent immunity response, myb51 was chosen as the indole glucosinolate defective mutant. To provide a full view of indole glucosinolate functions during the Arabidopsis response to pathogen invasion, high-throughput Illumina sequencing was performed to characterize the transcriptomes in wild-type and myb51 plants. Analysis of MYB51 dependent flg22-response genes showed that MYB51 might participate in most of the pathogen resistance processes including pathogen recognition, signal transduction and activation of PR proteins. WRKY transcription factors and the ethylene (ET) signaling pathway have been demonstrated to be functionally upstream of MYB51 in response to flg22. Interestingly, the responses of these upstream regulatory genes to flg22 were impaired in myb51, suggesting that there might be a positive feedback loop among MYB51 and these important regulators. Our study shed light on the diversity and complexity of indole glucosinolate-mediated pathogen resistance. 2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis ecotype Columbia (Col-0) was used in this study. Seeds of wild type and a T-DNA insertion mutant for MYB51 (SALK_059771) were purchased from the Arabidopsis Biological Resource Center (ABRC). The seeds were sown on 1/2 Murashige and Skoog (1/2MS) basal medium under 24 C, a light regime of 16 h light and 8 h dark, 70% relative humidity and a constant illumination of 100 mmol m2 s1. 2.2. Genotyping and RT-PCR analysis of myb51 Genomic DNA was isolated using an EasyPure Plant Genomic DNA Kit (TransGen, Beijing, China). Primers for myb51 genotyping were 5’ - AAATTTTGGTCAAGAATCGGG-30 , 50 -AAAGGGGGTTGTTC TCAAGTG-30 and the T-DNA-specific primer LBa1 50 -TGGTTCACGT AGTGGGCCATCG-3’. Leaves from wild type and homozygous myb51 were harvested 12 d after germination. RNA was extracted using the E.Z.N.A. Plant RNA Kit (Omega Biotek, GA). First-strand cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). The expression level of MYB51 was assessed using the primer pair 50 ATGGTGCGGACACCGTGT-30 and 50 -TCATCCAAAATAGTTATCAA TTTCG-3’. 2.3. Treatment of flg22 Twelve-day-old seedlings of the wild type and myb51 were respectively incubated into 1/2MS liquid medium and 1/2MS liquid medium containing 1 mmol/L flg22 for 4 h (under 24 C and a constant illumination of 100 mmol m2 s1.). Each sample was treated and harvested in three independent biological replicates.
San Diego, CA), Illumina Miseq library construction was performed. To purify the mRNA from the total RNA, magnetic beads with oligo dT attached were used. Then, mRNA was cleaved by using fragmentation buffer. Using random hexamer primers, the first-strand cDNAs of the fragments were synthesized. Then, doubled-stranded cDNA was transformed by using RHase H and DNA polymerase I. A paired-end library was constructed from the double-stranded cDNA with a Genomic Sample Prep Kit (Illumina). Fragments of desirable lengths were purified with the QIA quick PCR (Qiagen) Extraction Kit, end repaired, and linked with sequencing adapters. Unsuitable fragments were removed with AMPureXP beads, followed by PCR amplification, and the sequencing library was constructed. The established library was then sequenced with the Illumina Hiseq 2000 platform (Shanghai Personal Biotechnology Cp., Ltd. Shanghai, China). 2.6. Sequence analysis Raw reads from the samples were first processed through inhouse Perl scripts. In this step, clean reads were obtained by removing adaptor contamination reads containing poly-N and lowquality reads from raw data. Then, the Arabidopsis TAIR10 genome was used as a reference and the reference genome index was built using Bowtie2. The clean reads were aligned to the reference genome using Tophat v2.0.9 and HTSeq2 was used to count the read number mapped to each gene. The RPKM value of each gene was calculated based on the length of the gene and reads count mapped to this gene with the edger program. 2.7. Clustering and annotation of differentially expressed genes (DEGs) DEG analysis was conducted using DESeq2 with the criteria change fold 2 and p-value 0.05. Gene ontology (GO) annotation was performed by alignment between genes and the GO database. GO classification of DEGs was performed by using the R package “clusterProfiler”. GO enrichment was analyzed in the categories of biological process. Pathway enrichment was performed by using MapMan. To identify the TFs from the DEGs, we searched the DEGs against the Arabidopsis TF gene list which was downloaded from the Plant Transcription Factor Database (http://plntfdb.bio.unipotsdam.de/v3.0/) [19]. For the DEG volcano plot, clustering and heatmap plotting, the R packages “ggplot2”, “gplots” and “clusterProfiler” were used. 2.8. qRT-PCR verification of DEGs Twelve-day-old seedlings of wild type and myb51 were treated with flg22. Isolation of total RNA and synthesis of first-strand cDNA was performed as described above. Twelve MYB51 dependent flg22-response genes from the WRKY family and the ET signaling pathway were selected for qRT-PCR analysis. The selected genes and primers used for qRT-PCR were listed in Table S1 qRTePCR analyses were performed using SYBR Green Master Mix on an ABI 7500 sequence detection system.
2.4. Total RNA extraction 3. Results Isolation of total RNA was performed as described above. High quality RNA with a 28 S:18 S greater than 1.5, absorbance 260/280 ratios between 1.8 and 2.2 and absorbance 260/230 ratios higher than 2.0 was used for library construction and sequencing. 2.5. Strand-specific cDNA library construction and sequencing According to the instructions from the manufacturer (Illumina,
3.1. Illumina sequencing and assembly First, the myb51 was confirmed to be homozygous and have no detectable transcripts for MYB51 (Fig. S1). RNA samples from 12day-old wild-type seedlings without flg22 treatment (represented as WT), wild type with flg22 treatment (represented as WT þ flg22), myb51 without flg22 treatment (represented as
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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Table 1 Statistical summary of cDNA sequences of samples. Samples
Raw Reads
Clean Reads
Mapped Reads
Unigenes
Mapped Percent (%)
WT myb51 WT þ flg22 myb51 þ flg22
63,344,558 66,268,194 72,675,626 83,224,090
45,250,494 47,172,134 52,202,996 59,359,844
42,611,717 44,715,380 49,126,338 56,264,002
22,538 22,665 22,891 22,843
94.17% 94.79% 94.11% 94.78%
Fig. 1. Gene expression profiles in wild type and myb51 upon treatment of flg22. (A) Clustering analysis based on the FPKM Z-score of each gene and GO enrichment for each cluster genes. (B) Hub genes identified from DEGs in WT upon WT þ flg22; (C) Hub genes identified from DEGs in myb51 upon myb51 þ flg22; Red dots represent up-regulated hub genes and green dots represent down-regulated hub genes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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myb51), and myb51 with flg22 treatment (represented as myb51 þ flg22) were used to generate cDNA libraries, yielding 45,250,494, 47,172,134, 52,202,996 and 59,359,844 clean reads for WT, myb51, WT þ flg22 and myb51 þ flg22, respectively (Table 1). The Arabidopsis genome from TAIR10 (https://www.arabidopsis. org/index.jsp) was used as the reference to assemble the clean reads; more than 94% clean reads were mapped for all four datasets (Table 1). The distribution of these mapped reads on the chromosomes was shown in Fig. S2. High-throughput RNA sequencing yielded 22,538, 22,665, 22,891 and 22,843 unigenes for WT, myb51, WT þ flg22 and myb51 þ flg22, respectively (Table 1). 3.2. Analysis of DEGs between WT and myb51 To understand the MYB51-mediated network under normal growth conditions, we evaluated global changes in gene expression between WT and myb51. RPKM (Reads Per Kilobase per Million mapped reads) were used to calculate gene expression levels and DEGs were filtered out with the criteria change fold 2 and pvalue 0.05. To determine the number of DEGs in WT and myb51, log2-fold-change values were plotted against negative log10 (pvalue) to generate a volcano plot (Fig. S3A). A total of 681 DEGs containing 257 down-regulated and 424 up-regulated genes were determined in myb51 compared to the wild type (Table S2). To obtain the functional categories of these DEGs, a functional
enrichment analysis with MapMan was performed. As shown in Fig. S3B, besides “Not assigned”, the most enriched categories (“BINs” in MapMan terms) were “RNA processing”, “Protein”, “Signaling”, “Miscellaneous enzyme families”, “Stress”, “Hormones”, “Transport”, “Development”, “Cell wall” and “Secondary metabolism”. Although myb51 does not present any significant morphologic phenotype, MapMan enrichment analysis (Fig. S3B) demonstrated that the mutation of MYB51 had a profound effect on many biological processes even under normal growing conditions since the expression of genes related to transcription, protein metabolism, hormone and signaling were largely altered. As expected, stress and secondary metabolism processes were significantly affected. The DEGs enriched in “development” and “cell wall” indicated a possible role of MYB51 in plant development, especially the formation of the cell wall. 3.3. Analysis of flg22-response DEGs in wild type and myb51 To investigate the genes response to flg22 in WT and myb51, a comparison between flg22-treated and untreated samples was performed. In total, 1200 (910 up-regulated and 290 downregulated) and 895 (725 up-regulated and 170 down-regulated) DEGs were identified from WT þ flg22 versus WT and myb51 þ flg22 versus myb51, respectively (Figs. S4AeB and Table S3). Compared to wild type, the response to flg22 in myb51
Table 2 Differently expressed hub genes in response to flg22 in wild type and myb51. Gene ID
Gene Name
Hub genes up-regulated in WT AT2G38470 WRKY33 AT1G80840 WRKY40 AT5G25930 AT5G25930 AT1G27730 STZ AT4G29780 AT4G29780 AT1G07160 AT1G07160 AT1G74360 AT1G74360 AT3G52400 SYP122 AT3G55980 SZF1 Hub genes down-regulated in WT AT3G50820 PSBO2 AT5G66570 PSBO1 AT4G02770 PSAD-1 AT1G06680 PSBP-1 AT1G54780 TLP18.3 AT1G52230 PSAH2 AT4G05180 PSBQ-2 AT5G64040 PSAN AT5G54270 LHCB3 Hub genes up-regulated in myb51 AT1G74360 AT1G74360 AT5G25930 AT5G25930 AT1G07160 AT1G07160 AT5G52640 HSP90.1 AT1G19020 AT1G19020 AT5G57220 CYP81F2 AT2G40180 PP2C5 AT3G12580 HSP70 AT2G18690 AT2G18690 Hub genes down-regulated in myb51 AT1G06680 PSBP-1 AT3G50820 PSBO2 AT5G64040 PSAN AT1G52230 PSAH2 AT3G08940 LHCB4.2 AT5G54270 LHCB3 AT3G54890 LHCA1 AT1G61520 LHCA3 AT1G54780 TLP18.3
Gene description WRKY DNA-binding protein 33 WRKY DNA-binding protein 40 kinase family with leucine-rich repeat domain-containing protein salt tolerance zinc finger nuclease Protein phosphatase 2C family protein Leucine-rich repeat protein kinase family protein syntaxin of plants 122 salt-inducible zinc finger 1 photosystem II subunit O-2 PS II oxygen-evolving complex 1 photosystem I subunit D-1 photosystem II subunit P-1 thylakoid lumen 18.3 kDa protein photosystem I subunit H2 photosystem II subunit Q-2 photosystem I reaction center subunit PSIeN light-harvesting chlorophyll B-binding protein 3 Leucine-rich repeat protein kinase family protein kinase family with leucine-rich repeat domain-containing protein Protein phosphatase 2C family protein heat shock-like protein CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase cytochrome P450, family 81, subfamily F, polypeptide 2 phosphatase 2C5 heat shock protein 70 transmembrane protein photosystem II subunit P-1 photosystem II subunit O-2 photosystem I reaction center subunit PSIeN, chloroplast, putative/PSIeN, putative (PSAN) photosystem I subunit H2 light harvesting complex photosystem II light-harvesting chlorophyll B-binding protein 3 chlorophyll a-b binding protein 6 PSI type III chlorophyll a/b-binding protein thylakoid lumen 18.3 kDa protein
The highlighted are the shared hub genes in wild type and myb51 upon flg22 treatment.
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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was much less sensitive, indicating the importance of MYB51 in flg22-triggered immunity. K-means clustering of all genes was performed, with each gene getting an FPKM Z-score value, revealing 4 large clusters of distinct expression profiles, each associated with distinct functions based on the GO analysis of the “Biological Process” sub-groups (Fig. 1A). As shown in Fig. 1A, in both wild type and myb51, pathogen resistance associated
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processes (cluster 1 and cluster 2) were activated and photosynthesis and carbohydrate-related processes (cluster 3 and cluster 4) were repressed upon treatment of flg22. (Fig. 1A). Distinctive expression patterns especially in cluster 2 can be observed in the two genotypes without flg22 treatment (Fig. 1A). The expression level of genes in cluster 2, which are mainly pathogen resistance genes, was significantly lower in myb51 than in wild type under
Fig. 2. Identification of MYB51 dependent flg22-response genes. (A) An overview of the DEGs in myb51 compared to WT and DEGs in WT and myb51 upon treatment with flg22. (B) Number of DEGs in WT and myb51 upon treatment with flg22. (C) GO enrichment analysis of the MYB51 dependent flg22-response genes. The size of each circle represents the fold enrichment of the GO terms. The color density indicates the significance of the GO terms [elog10(p-value)].
Fig. 3. MYB51 dependent flg22-response genes enriched in biotic-stress. Red squares represent up-regulated and blue squares represent down-regulated genes upon treatment with flg22. The grey circles represent no MYB51 dependent flg22-response genes involved in this process. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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normal growing conditions. This indicated that these genes were largely affected by MYB51 and might be involved in constitutive disease resistance, which is formed before pathogens attack. To elucidate the different response of myb51 and the wild type to flg22, STRING networks (https://string-db.org/) of DEGs in WT þ flg22 versus WT and myb51 þ flg22 versus myb51 were constructed respectively by using Cytoscape 3.7 (https://cytoscape. org/). Top hub genes were identified and compared between WT þ flg22 versus WT and myb51 þ flg22 versus myb51 (Fig. 1BeC). Among the up-regulated hub genes, which are mostly defensive genes, 6 of the 9 genes were different between myb51 and wild type, while among the down-regulated hub genes, which are mainly photosynthesis related genes, 3 of the 9 genes were different (Table 2). Interestingly, WRKY33, WRKY40 which have been demonstrated to play key roles in modulating flg22-induced MTI [20], were found to be the hub genes in response to flg22 in the wild type but not in myb51 (Fig. 1BeC), indicating that the function of these important pathogen defense associated transcription factors are closely related with MYB51 in defensive responses.
3.4. Analysis of MYB51 dependent flg22-response DEGs To discover the specific DEGs which respond to flg22 treatment
in a MYB51 dependent manner, an overview of the DEGs in the wild type and myb51 in response to flg22 was generated (Fig. 2AeB). In total, 482 DEGs were filtered out as MYB51-dependent flg22 response genes, which were only differentially expressed in wild type but not in myb51 upon treatment with flg22 (Fig. 2B and Table S4). The GO term “Biological Process” enrichment analysis showed that the most enriched terms of these MYB51 dependent flg22-response genes were “response to organonitrogen compound”, “response to chitin”, “intracellular signal transduction”, “systemic acquired resistance”, “response to salicylic acid”, “programmed cell death” as well as “regulation of immune system process”. (Fig. 2C). Most of the enriched biological processes were closely related with defense against pathogens, and some of the processes, such as “systemic acquired resistance” and “program cell death”, have been demonstrated to be triggered by indole glucosinolates in response to pathogens. In addition, as expected, some of the indole glucosinolate biosynthetic related genes including CYP79B2, ATSOT16, CYP83B1 and NIT4 were enriched in MYB51 dependent flg22-response genes (Table S4). Indole glucosinolate biosynthesis mediated by MYB51 is required for flg22-induced callose accumulation. Not surprisingly, eight callose biosynthetic genes, CML37, BCS1, ATSZF1, AOC3, ATCBL1, ATFC-I, CAD1 and APD5 were also enriched (Table S4). These results confirmed that the MYB51 dependent flg22-response genes we obtained can provide
Table 3 Selected MYB51 dependent flg22-response genes. Gene ID R AT1G72930 AT2G20142 AT2G32140 AT5G36910 signaling AT3G28930 MAPK AT1G01560 PR-proteins AT1G66090 AT1G72900 AT1G72920 AT1G72940 AT3G13650 AT5G41740 AT5G58120 AT1G13609 Ethylene AT4G11280 AT1G28370 AT2G44840 AT3G23230 AT4G34410 AT5G47230 AT5G51190 AT1G68765 ERF AT1G12610 AT1G33760 AT1G63030 AT1G64380 AT1G72360 AT1G79700 AT4G32800 WRKY AT2G38470 AT2G40750 AT2G46400 AT3G56400 AT4G23810 AT4G31550
Gene Name
Gene description
TIR AT2G20142 AT2G32140 THI2.2
toll/interleukin-1 receptor-like protein Toll-Interleukin-Resistance (TIR) domain family protein transmembrane receptor thionin 2.2
AIG2
AIG2-like (avirulence induced gene) family protein
MPK11
MAP kinase 11
AT1G66090 AT1G72900 AT1G72920 AT1G72940 AT3G13650 AT5G41740 AT5G58120 AT1G13609
Disease resistance protein (TIR-NBS class) Toll-Interleukin-Resistance (TIR) domain-containing protein Toll-Interleukin-Resistance (TIR) domain family protein Toll-Interleukin-Resistance (TIR) domain-containing protein Disease resistance-responsive (dirigent-like protein) family protein Disease resistance protein (TIR-NBS-LRR class) family Disease resistance protein (TIR-NBS-LRR class) family Defensin-like (DEFL) family protein
ACS6 ERF11 ERF13 TDR1(ERF98) RRTF1 ERF5 ERF105 IDA
1-aminocyclopropane-1-carboxylic acid (acc) synthase 6 ERF domain protein 11 ethylene-responsive element binding factor 13 ethylene-responsive factor redox responsive transcription factor 1 ethylene responsive element binding factor 5 Integrase-type DNA-binding superfamily protein Putative membrane lipoprotein
DDF1 AT1G33760 DDF2 AT1G64380 ERF73 WRI4 AT4G32800
member of the DREB subfamily A-1 of ERF/AP2 transcription factor family member of the DREB subfamily A-4 of ERF/AP2 transcription factor family member of the DREB subfamily A-1 of ERF/AP2 transcription factor family member of the DREB subfamily A-6 of ERF/AP2 transcription factor family member of the ERF subfamily B-2 of ERF/AP2 transcription factor family AP2/ERF-type transcriptional activator that specifically controls cuticular wax biosynthesis in Arabidopsis stems. member of the DREB subfamily A-4 of ERF/AP2 transcription factor family
WRKY33 WRKY54 WRKY46 WRKY70 WRKY53 WRKY11
WRKY WRKY WRKY WRKY WRKY WRKY
DNA-binding protein 33 DNA-binding protein 54 DNA-binding protein 46 DNA-binding protein 70 family transcription factor DNA-binding protein 11
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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valuable information to better understand the function of indole glucosinolates in MTI. For an overview of the biotic stress responses of these MYB51 dependent flg22-response genes, MapMan was used to perform pathway enrichment (Fig. 3). Four R genes which code for proteins recognizing specific pathogen effectors, a signaling protein coding gene AIG2 (a virulence induced gene), a MAP kinase gene MPK11 and eight PR-protein encoding genes were identified (Fig. 3 and Table 3). Furthermore, “Hormone signaling”, “Cell wall”, “Proteolysis” and “Transcription Factors”, were also enriched. This indicated that MYB51 possibly participates in most pathogen resistance processes including pathogen recognition, signal transduction and multiple defense reactions. It is noteworthy that several genes in the WRKY family, ERF family and ET signaling pathway were induced upon flg22 treatment in a MYB51-dependent way (Fig. 3). WRKY transcription factors are the major regulators of immune responses and signaling involving MTI, especially WRKY18, WRKY33 and WRKY40 which can directly regulate indole glucosinolate metabolism by binding to the promoters of related genes including MYB51 [20]. The ET signaling pathway also plays an active role in plant defense against pathogens. During flg22-induced callose deposition, ET signaling
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transduction is required and is functionally upstream of MYB51 [10]. Interestingly, as shown in Fig. 3, the induction of six WRKY genes including WRKY33, six ERF genes as well as eight genes in the ET pathway upon treatment of flg22 was found to be dependent on MYB51. To validate whether the results from the transcriptome analysis can reflect the real expression profile of these genes, six WRKY genes and six ET pathway genes were selected to perform quantitative RT-PCR (Fig. 4). To better reflect the dynamic responses of the above genes upon flg22 treatment, the expression levels of these genes were determined after 1 and 4 h treatment of flg22. Consistent with the results of the transcriptome analysis, the WRKY genes including WRKY33, WRKY53, WRKY46, WRKY54 and WRKY70 were significantly induced upon flg22 treatment in the wild type but not in myb51, as were the ET pathway genes ACS6, ERF5, ERF11, ERF13, ERF98 and ERF105. WRKY11 was the only gene of which the expression pattern was not completely consistent with that of transcriptome analysis. At 1 h of flg22 treatment, WRKY11 was induced in both wild type and myb51. At 4 h, the expression decreased in both wild type and myb51, but decreased more significantly in myb51. This suggested that the induction of WRKY11 upon flg22 treatment was dependent on MYB51 at the later stage (4 h).
Fig. 4. Expression of several WRKY genes and ethylene signaling related genes in response to flg22. (A) Expression of WRKY genes in response to flg22; (B) Expression of ethylene signaling related genes in response to flg22. Relative expression values are given in comparison with the WT (WT ¼ 1). Each bar represents the mean (±standard error) of three biological replicates. * and **, significantly different (Student’s t-test; * 0.01 < P < 0.05, **P < 0.01) from the 0 h.
Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110
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The consistent expression profiling between the transcriptome and qRT-PCR indicated that our transcriptome analysis was reliable and it could be confirmed that the induction of certain WRKY genes and ET pathway genes in response to MAMP was dependent on MYB51. Since WRKY33 and the ET signaling pathway have been demonstrated to be functionally upstream of MYB51 in response to flg22, there might be positive feedback regulation among MYB51 and these important TFs.
Declaration of competing interest Authors declare that there is no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.110. Transparency document
4. Discussion Indole glucosinolates plays a variety of roles in plant immune responses, including systemic acquired resistance, hypersensitive response and callose accumulation [10,12,15]. The function of these metabolites seems to exceed our expectations. To more comprehensively understand the functions of these indolic secondary metabolites in plant immunity, high-throughput RNA-sequencing of wild type and myb51 following treatment with flg22 were performed. As shown in the analysis of MYB51-dependent biotic stress responsive genes, MYB51 is possibly involved in most pathogen resistance processes including pathogen recognition, signal transduction, and activation of MAPK and PR proteins. Of note, several genes in the WRKY family and ET signaling pathway were induced upon flg22 treatment in a MYB51-dependent way. The large WRKY family of transcription factors plays an important role in plant resistance to various stresses. Several WRKY transcription factors have been found to be key immune regulators that modulate the transcriptional reprogramming in response to various pathogens [21e23]. During flg22-triggered MTI, WRKY33, WRKY40 and WRKY18 directly target to MYB51 and other genes in the indole glucosinolate pathway to promote the biosynthesis of these secondary metabolites [20]. In Botrytis cinereaeinduced ethylene biosynthesis, WRKY33 activates the ET signaling pathway by inducing the expression of ACS2 and ACS6, two ET biosynthetic enzyme genes through direct binding to gene promoters [24]. Indole glucosinolate production mediated by MYB51 is required for flg22-induced callose accumulation. During this immune response, the ET pathway is functionally upstream of MYB51 [10]. In short, in response to pathogens, WRKY33 is functionally upstream of the ET pathway, and both WRKY33 and the ET signaling pathway play regulatory roles upstream of MYB51. However,in myb51, WRKY33 and the ET biosynthesis gene ACS6 were impaired in response to flg22. These results indicated that MYB51 had positive feedback on its upstream regulators. WRKY33, the ET pathway and MYB51 formed a “positive feedback loop”. In addition to WRKY33 and ACS6, WRKY46, WRKY53, WRKY54, WRKY70, WRKY11 and five ET response factor (ERF) genes, ERF5, ERF11, ERF13, ERF98 and ERF105 were also impaired in response to flg22 in myb51 (Fig. 4). Among these MYB51 dependent flg22responsive WRKYs and ERFs, WRKY53, WRKY54, WRKY70, WRKY 11, ERF5 and ERF11 have been demonstrated to have key regulatory roles in innate immune responses against various pathogens [22,23,25e29]. Indolic metabolite production mediated by MYB51 is implicated in diverse immune responses, which can be possibly explained by the positive effect of MYB51 on these key regulatory transcription factors in pathogen resistance.
Funding This work was supported by the National Natural Science Foundation of China (NSFC) 31370334 and Natural Science Foundation of Heilongjiang Province C2017031.
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Please cite this article as: J. Zhou et al., Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.110