Rice (Oryza sativa
L.) is a model species for monocotyledonous plants and cereals, which are the greatest source of food for the world’s population. With the great change in climate, rice is confronted with critical biotic and abiotic stresses. Plants have evolved complex signaling pathways to survive under multiple stresses, which are generally composed of receptors, secondary messengers, phytohormones, and signal transducers [1
]. Plant hormones are fundamentally involved in plant growth and development, and they play roles in adapting to the changing environment and in quick responses to multiple stresses. Jasmonic acid (JA), as a basic plant growth regulator, is widely present in higher plants and a natural compound that is produced in plants [2
]. As an important endogenous hormone, JA plays diverse roles in plant growth, seed germination, drought stress, pathogens, and insects’ defenses [3
Transcription factors (TFs) are triggers for gene expression and they play important regulatory roles throughout the plant life time, especially in plant growth, development, and responses to abiotic and biotic stresses. Being one of the largest families of transcriptional regulators, the basic region/leucine zipper motif (bZIP) transcription factors have been systematically characterized in many higher plants. There are 75 [12
] or 78 bZIP TFs in Arabidopsis [13
], 89 [14
] or 92 in rice [15
], 125 in maize [16
], and 247 in rapeseed [17
]. These bZIP TFs have been classified into 13 groups in Arabidopsis (A, B, C, D, E, F, G, H, I, J, K, M, S) [13
] and 11 in rice (I–XI),according to the DNA binding specificity and amino acid sequence similarities of bZIP domains [14
]. In Arabidopsis, group I is the subfamily with the third largest number of genes among all of the groups, containing 12 members [13
]. Among the 12 members, VIP1/AtbZIP51 is a VirE2-interacting protein and it has been well studied [18
]. It was thought to play an important role in Agrobacterium
-mediated T-DNA transfer [18
]. In the model of the “Trojan house hypothesis”, VIP1 serves as a bridge between VirE2 and nuclear importin α, which mediates the transport of the T-DNA strand to the plant nucleus [19
]. Moreover, the subcellular localization of VIP1 is affected by its own phosphorylation status and interaction with 14-3-3 [21
]. VIP1 is involved in other functions in addition to its role in Agrobacterium
-mediated transformation, including osmosensory signaling, low sulfur tolerance, metal-binding, touch response, Botrytis
and salt stress responses, the ABA response, and transcriptional regulation [22
]. VIP1 can bind to VRE (VIP1 response element: ACNGCT) or VRE similar sequences (AGCTGT/G, CAGCT) of promoters and control the expression of stress-related genes [23
]. In addition to VIP1, numerous members of the group I subfamily can interact with C58 VirE2 (AtbZIP52, AtbZIP69, PosF21/AtbZIP59, AtbZIP29, and AtbZIP30) and they are involved in osmosensory responses (PosF21/AtbZIP59, AtbZIP69, AtbZIP29, AtbZIP30, and AtbZIP52) and vascular development (AtbZIP18, AtbZIP29, AtbZIP30, AtbZIP52, PosF21/AtbZIP59, and AtbZIP69), revealing the functional redundancy among group I members [26
]. Moreover, AtbZIP29 has been defined to function in leaf and root development, PosF21/AtbZIP59 in auxin-induced callus formation and plant regeneration, DRINK ME/AtbZIP30 in growth and reproductive development regulation, and AtbZIP18 in pollen and male gametophyte development [6
Group IX (or B) of bZIPs in rice remains poorly described. This group represents the fourth largest subfamily, is very close to group I in Arabidopsis, and contains 11 members (OsbZIP25, OsbZIP30, OsbZIP35, OsbZIP36 OsbZIP61, OsbZIP68, OsbZIP75, OsbZIP76, OsbZIP78, OsbZIP81, and OsbZIP84); however, only two of them have been systematically studied [37
]. RF2a/OsbZIP75 functions in rice vascular development and they can bind to the Box II cis
-element of the promoter of rice tungro bacilliform virus (RTBV) to activate its expression [37
]. RF2b/OsbZIP30 can interact with RF2a and it is involved in the symptom development of rice tungro disease and vascular development [38
]. Transgenic rice plants overexpressing RF2a and RF2b present a tolerance to rice tungro virus replication and disease [40
) is a kind of soil bacterium and a pathogen. It is also a natural genetic engineer, which plays prominent roles in transferring genetic information into the eukaryotic genome [41
]. In our previous studies, we found that Agrobacterium
VirD5 could interact with Arabidopsis VIP1 and competitively inhibit the interaction between VIP1 and VBF. This competitive interaction could prevent T-DNA coat protein degradation in the plant cell nucleus [42
]. In addition, VirD5 could increase the Agrobacterium
infection efficiency, potentially by a competitive interaction with Arabidopsis VIP2 [41
]. In another work, our group demonstrated that a large maize fragment (164 kb) that contained a high content of repetitive sequences was successfully transferred into rice by Agrobacterium
-mediated transformation, but the transformation efficiency was very low [43
]. Thus, the application of large exogenous DNA fragments in rice is largely limited. Therefore, the elucidation of the mechanism underlying Agrobacterium
-mediated rice transformation may help to solve the problem.
In this study, we identified two rice homologues of the Arabidopsis VIP1 gene, OsbZIP81 and OsbZIP84, and functionally analyzed OsbZIP81. OsbZIP81 and OsbZIP84 both belong to group IX of bZIPs. OsbZIP81.1 and OsbZIP84 are typical transcription factors of the bZIP super family. OsbZIP81.1 may positively affect the JA levels of rice plant by directly targeting the genes in JA signaling and metabolism pathway, especially OsPIOX. In addition, OsbZIP81.2 can interact with Agrobacterium VirE2. To our knowledge, this is the first report on the interaction between rice proteins and Agrobacterium virulence proteins. Furthermore, we identified two pathogenesis-regulated (PR) proteins, PR10a/PBZ1 and RSOsPR10, and other stress response genes. These results suggest that OsbZIP81 may positive regulate JA levels and may play a role in pathogen resistance.
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
Rice Zhonghua 11 (ZH11; Oryza sativa ssp. japonica) was used as the wildtype. The OsbZIP81.1-overexpression line and the osbzip81 mutant were constructed in this study. Seedlings were grown under natural long-day conditions (approximately 14 h light/10 h dark) from June to September at Wuhan. Two-week-old seedlings were used for the abiotic and biotic treatments. For all of the samples, the shoots of the seedlings were harvested and frozen in liquid nitrogen for RNA isolation or immediately placed in 1% formaldehyde for chromatin isolation.
To generate the overexpression OsbZIP81.1 transgenic rice line, the genome fragment containing the full-length OsbZIP81.1 cDNA fragment amplified with the specific primers OsbZIP81.1-F/R and cloned into the binary expression vector pCAMBIA1301U-flag (driven by a maize ubiquitin promoter and fused to 3× flag tags at its C terminus) at the Kpn
I and Bam
HI sites. The constructed vector was introduced into rice ZH11 by Agrobacterium
-mediated transformation [92
4.2. Subcellular Localization Assay in Rice Protoplasts
The vector pM999-GFP was used to study the subcellular localization of OsbZIP81.1, OsbZIP81.2, and OsbZIP84. The full-length cDNA fragment was amplified from pGADT7-OsbZIP81.1 using the following specific primer pair: GFP-OsbZIP81.1-F/R (Supplementary Table S6
) and cloned into the pM999-GFP vector. The pM999-OsbZIP81.2-GFP and pM999-OsbZIP84-GFP were constructed in the same way.
For rice protoplast preparation and transformation, 14-day-old yellow seedlings of ZH11 (O. sativa
ssp. japonica) after germination in half-strength MS medium were used in this study. The protoplasts were isolated according to previous reports with little modifications [93
]. In general, the rice protoplasts were isolated by digesting the rice sheath strips in digestion solution (0.6 mol/L mannitol; 10 mmol/L MES, pH 5.7; 1.5% cellulose R-10; 0.75% macerozyme R-10; 0.1% BSA; 1 mmol/L CaCl2
) for 4 to 5 h at 28 °C at a speed of 50 rpm in a dark table concentrator. The protoplasts were then incubated in W5 solution (154 mmol/L NaCl; 125 mmol/L CaCl2
; 5 mmol/L KCl; 2 mmol/L MES, pH 5.7) at the 28 °C and 80 rpm for 10 min. The protoplasts were collected by centrifugation at 100× g
and 4 °C for 8 min. after filtering through 300-mesh filter (50 μm). The supernatant was removed and the pellet resuspended in another 4 mL W5 solution. The protoplasts were collected after another centrifugation at 100× g
and 4 °C for 8 min. and resuspended in MMG solution (0.6 mol/L mannitol; 15 mmol/L MgCl2
; 4 mmol/L MES, pH 5.7) to a final concentration of 1.0 × 107
. For transformation, 5 μL of each plasmid (5–10 μg) was pooled and gently mixed with 100 μL of protoplasts and 110 μL of PEG-CaCl2
solution (40% PEG33500, 0.6 mol/L mannitol, 100 mmol/L CaCl2
), and then incubated at 28 °C for 15 min. in the dark. Transformation was stopped by the addition of two volumes of W5 solution. The transformed protoplasts were then collected by centrifugation and then re-suspended in WI solution (0.6 mol/L mannitol; 4 mmol/L KCl; 4 mmol/L MES, pH 5.7). The transformed protoplasts were maintained in 12-well culture plates at 28 °C for 12–16 h in the dark. After incubation, the transformed protoplasts were collected by centrifugation at 100× g
for 8 min. and observed by fluorescence confocal microscopy (Leica Microsystems SP8, Wetzlar, Gemany).
4.3. Dual Luciferase Transcriptional Activity Assay in Rice Protoplasts
The full-length CDS of OsbZIP81.1
was cloned into GAL4-DB via the Bam
RI sites as the effector (the primer sequences are listed in Supplementary Table S6
), and the 35S promoter driven luciferase gene (35S-GAL4-fLUC) and basic promoter driven fluorescent luciferase gene (GAL4-fLUC) were used as reporter to detect the transcriptional activation of OsbZIP81.1 and OsbZIP81.2. The promoter of OsPIOX was cloned into 190LUC via the Hind
HI. The internal reference vector was the luciferase gene (rLUC). Effectors, reporter, and internal reference plasmids were extracted while using the Qiagen plasmid Midi Kit, and the final concentration of these plasmids was approximately 1 μg/μL. Subsequently, 3 μg effectors, 3 μg reporters, and 0.5 μg internal reference plasmids were transformed into rice protoplasts by PEG mediated transformation. The transformation methods were performed, as described above (Section 4.2
). The transformed protoplasts were cultured in the dark for 12 h or overnight and then collected and detected while using the Dual-Luciferase Reporter Assay System kit (E1910, Promega (Beijing), Beijing, China). Supplementary Table S6
lists the primers used for these different genes.
4.4. ChIP Sequencing
Overexpressed OsbZIP81.1 rice under normal condition was used for ChIP-Seq analysis. ChIP was performed, as described previously, with some modifications [94
]. Briefly, leaf tissues from four-leaf-stage seedlings were immediately fixed after harvest in 1% formaldehyde under vacuum for 30 min and 1.5 g tissues were used for chromatin isolation. Isolated chromatin was sheared to approximately 200 bp using a supersonic instrument (Bioruptor Plus, DIAGENODE, Belgium), as follows: high power, cycle conditions 30/90 (On/Off times in s), 30 cycles. For ChIP-Seq, the DNA was immunoprecipitated by anti-flag antibody, as described previously, and the precipitated DNA was purified and solubilized in distilled water. For each library, three independent replicated samples were mixed together to generate the sequencing library, which was processed by Wuhan Igenebook Company.
ChIP-Seq data processing and analysis were performed as described by Zong [95
]. Briefly, raw sequencing reads from each library were mapped to the rice genome (RGAP ver. 7.0, http://rice.plantbiology.msu.edu/
) while using SOAP2 [96
], and only uniquely mapped reads were used for peak identification. The Model-based Analysis of ChIP-Seq (MACS) software was used to identify OsbZIP81.1-associated regions with default parameters [97
]. The .wig files of the MACS output were visualized using the Integrated Genomics Viewer [98
]. A gene was regarded as an OsbZIP81.1-bound gene if the promoter region of the gene (including 2 kb upstream of the transcription start site) had at least 1 bp overlapping with the peaks. We extracted 200 bp around the peak summits (100 bp upstream and 100 bp downstream) of each library and the 1000 highest q
-value peaks were subjected to MEME-ChIP (http://meme-suite.org/tools/meme-chip
) to identify the enriched motifs [49
]. For functional category analysis, KEGG pathway information was collected from the KEGG database [100
], and the functional category (Rice_japonica_mapping_merged_08 download) was collected from the Mapman web site [101
The ChIP product was analyzed by quantitative real-time PCR (the primer sequences are listed in Supplementary Table S6
) with a CFX96 Real-Time System (Bio-Rad). Three replicates of each sample were evaluated and the enrichment values were normalized to the input sample and Actin
was used as the reference gene. Supplementary Table S6
lists the primers used here.
4.6. Random DNA Binding Selection Assay (RDSA)
RDSA was used to identify the motif(s) for OsbZIP84 binding. Purified GST-tagged OsbZIP81 protein was used in this experiment, and the experimental protocol was performed according to Dr. Wang [38
]. The primers used can be found in Supplementary Table S6
. After seven cycles, purified DNA was ligated into the pGEM-T easy vector (Promega, Cat. # A3600) and then transformed into the DH10B strain. The plasmids from monoclones were extracted and sequenced. Supplementary Table S6
lists the primers.
4.7. Electrophoretic Mobility Shift Assay
GST-fused OsbZIP81.1 and GST tag proteins were expressed and purified, as described above. Oligonucleotides were synthesized and labeled with a biotin-tag at their 5′ end by TSINGKE Biological Technology Company (Supplementary Table S6
). To generate the double-stranded oligos, an equal amount of the complementary single-stranded oligos was mixed and run using the following program: 95 °C for 1 min., 55 °C for 1 min., 72 °C 5 min., two cycles, and annealed by gradually cooling down to 4 °C. The LightShift Chemiluminescent EMSA Kit was used for the EMSA experiment (20148, Thermo Scientific) following the manufacturer’s instructions. The competition assay was performed, as follows. Unlabelled DNA was incubated with protein and other materials at room temperature (~25 °C). After 20 min., 2 μL biotin-labeled DNA was added and incubated at room temperature for 20 min. The reactions were then subjected to electrophoresis on 6% polyacrylamide gels running with 0.5× TBE buffer at 4 °C until the bromophenol blue dye had migrated approximately 2/3 to 3/4 down the length of the gel. The next steps were performed according to the instructions provided with the kit. Finally, the signals were detected with X-ray films (ChemiScope 5000Pro, CLiNX, Shanghai, China).
4.8. Yeast Two-Hybrid and Library Screening Assay
Full-length cDNA was amplified using specific primers. The obtained fragments were cloned into the pGBKT7 or pGADT7 vector (Clontech, Mountain View, CA, USA), depending on the different restriction sites. Supplementary Table S6
lists the primers and restriction sites that were used for these different genes. These plasmid pairs were used to cotransform the yeast strain AH109 according to the manufacturer’s instructions (Clontech). The transformed yeast cells were grown on SD medium lacking Leu and Trp (SD/-Leu-Trp) and then transferred to SD medium lacking Leu, Trp, Ade, and His, and were supplemented with 40 μg·mL−1
X-α-Gal (SD/-Leu-Trp-Ade-His + X-α-Gal).
For yeast two-hybrid screening, a library was constructed with rice seedling cDNA and kept in our laboratory was used (Y187 strain). The screening was performed using pGBKT7-OsbZIP81.2 (AH109 strain) with 5 mmol/L 3-Amino-1,2,4-triazole (3-AT). The mating procedures followed the YeastmakerTM Yeast Transformation System 2 User Manual (Clontech, Takara (Beijing), Beijing, China).
4.9. BiFC Assay
The OsbZIP81.2 was cloned into the pSPYCE(M) vector and VirE2 was cloned into the Pspyne173 vector [102
]. Supplementary Table S6
lists the primers used for the vector construction. The two vectors were mixed and transformed into the rice protoplasts, as described above. After incubation in the dark for 16 h, the fluorescence was observed by confocal microscopy. The primers used can be found in Supplementary Table S6
4.10. Glutathione S-Transferase (GST) Pull-down Assay
For the GST pull-down assay, the full-length coding sequences of OsbZIP81.2
were cloned into pTXB3 and pGEX-6p-1 vectors, yielding CBD-OsbZIP81.2 and GST-VirE2, respectively. The constructed vectors were transferred into Escherichia coli
BL21 (DE3) cells for the expression of fusion proteins. Two purified proteins were mixed with equal volumes and incubated in 1 mL PBS buffer for 6 h at 4 °C. One-hundred microliters of Glutathione Sepharose 4B beads (GE Healthcare) were added into the protein mixture and incubated for another 2 h at 4 °C. The beads were washed five times with PBS buffer and the pulled proteins were eluted by boiling and further analyzed by immunoblotting using anti-GST (ABclonal) and anti-CBD (NEB). The primers that were used can be found in Supplementary Table S6
4.11. Multiple Stress Treatment
To detect the sensitivity of OsbZIP81 and other members of the same bZIP subfamily under multiple treatments, ZH11 rice plants were grown in the greenhouse with a 14-h-light/10-h-dark cycle. Two-week-old seedlings were treated with chemical or abiotic stress. Chemical treatments were conducted by spraying leaves with 0.1 mmol/L ABA, 0.1 mmol/L MeJA, 0.1 mmol/L SA, 10 μmol/L C2H4, 10 μmol/L NAA, and 10 μmol/L IAA, followed by sampling at 0, 3, 6, 12, and 24 h, or irrigating the plants with 20% PEG6000, followed by sampling at 0, 1, 5, 12, and 14 h. For cold and heat stress, the seedlings were transferred to a growth chamber at 4 or 42 °C and sampled at 0, 1, 3, 6, 12, and 24 h after treatment. Incubating two-week-old seedlings with 200 mmol/L NaCl solution, followed by sampling at 0, 1, 3, 6, 12, and 24 h after treatment performed salt stress. Two-week-old seedlings were placed in air without a water supply and sampled at 0, 1, 3, 6, 12, and 24 h. AS (100 μmol/L) alone or with Agrobacterium (EHA105 strain) were performed while using the rice callus and followed by sampling at 0, 1, 3, 6, 12, 24, 48, and 72 h after treatment. Every treatment was performed at least three times.
4.12. RNA-Seq and Data Analysis
RNA-Seq was used to identify the target genes of OsbZIP81.1 by integrating the analysis with the ChIP-Seq data. Total RNAs were extracted from four-leaf-stage rice seedlings using RNAiso Plus (Takara) reagent according to the user manual. For library construction, three independent replicated RNA samples were prepared and each 10 μg of total RNA was used for RNA-Seq by Novogene Company (Beijing, China). The libraries were then sequenced with an Illumina HiSeq 3000. The O. sativa genome (RGAP v. 7.0) was used as a reference. The gene expression levels were calculated by using the reads per kilo bases per million reads (RPKM) method. To identify the differentially expressed genes between the libraries, edgeR software was applied to identify DEGs. The fold change (|log2FC| ≥ 1) and p-value (p ≤ 0.05) were used as the indexes of statistical significance.
4.13. Real-Time qPCR
The total RNAs were isolated from rice seedlings using RNAiso Plus (Takara) reagent according to the manufacturer’s instructions. RNAs (2 μg) were used for cDNA synthesis with the PrimeScript RT reagent Kit with gDNA Eraser (Takara). SYBR Green Realtime PCR Master Mix (TOYOBO, Shanghai) was used for real-time PCR analysis with the CFX96 Real-Time System (Bio-Rad). Three technical replicates were evaluated for each sample and Actin
was used as the reference gene. The RT-qPCR profiles included the following steps: 94 °C for 3 min., followed by 45 cycles at 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. Supplementary Table S6
lists the primer sequences.
4.14. Quantification of Endogenous JA, MeJA, SA and ABA
Half gram of four-leaf-stage rice samples were used for measuring the contents of endogenous JA, MeJA, SA, and ABA. Wild type rice ZH11 was selected as the control and three OsbZIP81.1 overexpression transgenic rice plants were sampled and mixed as the experiment group. Every group contains at least three samples (ZH11-1, ZH11-2, ZH11-3; OX-OsbZIP81.1-1, OX-OsbZIP81.1-2, OsbZIP81.1-3). Harvested samples were sent to company (ProNetsBio, Wuhan, China) to measure the hormones by HPLC-MS/MS.
4.15. Accession Numbers and Data Availability
The sequence data from this article can be found in the RGAP data base (http://rice.plantbiology.msu.edu/
), under the following accession numbers: OsbZIP81
, LOC_Os11g06170; OsbZIP84
, LOC_Os12g06520; RSOsPR10
, LOC_Os12g36830; PBZ1
, LOC_Os12g36880; OsMADS1
, LOC_Os11g34450; Actin
, LOC_Os03g50855; OsLOX5
, LOC_Os02g10120; OsAOC
, LOC_Os03g32314; OsHI-LOX
, LOC_Os08g39840; OsPIOX
, and LOC_Os12g26290. The ChIP-Seq and RNA-Seq raw data are deposited in NCBI’S Sequence Read Archive (SRA) with accession code PRJNA510886.