Plasmids pCB-35S-R1 and pCB-35S-R2 containing a full-length CWMV RNA1 or RNA2 sequence behind a 35S promotor were produced as previously described . Partial sequences of the genes NbABA1 (accession number XM_016620556) and NbABA2 (accession number XM_016584441) were PCR-amplified using the primer pairs NbABA1-Fa with NbABA1-Rb and NbABA2-Fa with NbABA2-Rb, respectively. PCR products were digested using the restriction enzymes BamHI and SmaI (New England Biolabs, Ipswich, MA, USA), and the products were individually cloned into TRV-based pTRV2 to generate pTRV2:NbABA1 and pTRV2:NbABA2 vectors. PCR products used for plasmid construction were generated using KOD DNA polymerase (TOYOBO, Kita-ku, Osaka, Japan). PCR primers used in this study are listed in Additional file 10: Table S5.
A TRV-based VIGS system in N. benthamiana was described as previously with small modifications . pTRV2:NbABA1and pTRV2:NbABA2 vectors were individually transformed into A. tumefaciens strain GV3101 by electroporation. The agrobacterium cultures and A. tumefaciens strain GV3101 containing TRV RNA1 were grown overnight, pelleted, re-suspended in an induction buffer (1 M MgCl2, 10 mM MES, pH = 5.6, and 100 mM acetosyringone) and incubated for 3 h (h) at room temperature prior to leaf co-infiltration. Infiltrated leaves were collected at 7 days post agro-infiltration (dpai) and examined using RT-qPCR to confirm silencing of target-genes.
Plant material and CWMV inoculation
The original N. benthamiana seeds were kindly donated by Pro. Yule Liu (Tsinghua University, China). N. benthamiana plants were grown in a greenhouse at 22 °C with a 16/8 h (light/dark) photoperiod until CWMV inoculation at the four-leaf stage. CWMV inoculation was performed as previously described with minor modifications . The agrobacterium cultures carrying the recombinant binary constructs pCB-35S-R1 and pCB-35S-R2 were grown individually until approximately OD600 = 0.8. After centrifugation at 6000×g for 5 min, the supernatant was collected and re-suspended using an induction buffer (1 M MgCl2, 10 mM MES, pH 5.6, and 100 mM acetosyringone) for 3 h at room temperature. After this, the induction buffers containing pCB-35S-R1 or pCB-35S-R2 were mixed at equal volumes before leaf infiltration. All inoculated plants were grown in a constant-temperature incubator at 17 °C with a 14/10 h (light/dark) photoperiod. RT-qPCR and western blot were performed to confirm successful systemic infection at 14 dpi. Samples were collected for further analysis. Inoculation of wheat seedings with CWMV RNAs was performed as described previously . In brief, plasmids pCB-35S-R1 carrying CWMV RNA1 and pCB-35S-R2 carrying CWMV RNA2 were linearized for vitro transcription. Vitro transcripts of CWMV RNA1 and RNA2 in a molar ratio of 1:1 were mixed with an equal amount of excess inoculation buffer (0.1 M glycine, 0.06 M potassium phosphate, 1% bentonite, 1% sodium pyrophosphate, 1% celite, pH 8.5) and then inoculated into leaves of wheat seedings.
RNA extraction and RT-qPCR
Total RNA was isolated at 14 dpi using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). First strand cDNA was synthesized using a First Strand cDNA Synthesis Kit (TOYOBO, Kita-ku, Osaka, Japan). The RT-qPCR reaction was performed using an ABI7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with an AceQ qPCR SYBR Green Master Mix (Vazyme, China). Each treatment was performed using least three biological replicates with at least three technical replicates, each. Relative expression levels of ABA-related genes and CWMV CP were analyzed using the 2-ΔΔC(t) method as described previously . An actin gene was used as the internal reference for each reaction. These primers used in RT-qPCR are listed in Additional file 10: Table S5.
Northern blot analysis
Northern blotting was conducted as described previously . Briefly, 3 μg total RNA from each sample was loaded into a well of a 1.5% formaldehyde comprising agarose gel and separated by electrophoresis. Then, the separated RNAs were transferred onto Hybond-N+ membranes (Amersham Bioscience, Buckinghamshire, United Kingdom) and cross-linked for 2 h at 80 °C. CWMV genomic RNAs were analyzed with DIG-labeled DNA probes specific for the 3′-terminus of CWMV RNAs. The probe was made using a DIG High Prime DNA Labeling Kit II as instructed by the manufacturer (Roche, Basel, Switzerland). Finally, the blotting signal was detected with the Amersham Imager 600 (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).
A western blot assay was performed as described previously with minor modifications [6, 31]. Samples were individually ground in liquid nitrogen and were then homogenized in a protein extraction buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with Protease Inhibitor Cocktail Tablets (Roche, Basel, Switzerland; 1 tablet/50 mL buffer). After centrifugation at 16,000×g and 4 °C for 20 min, the supernatant was collected and was boiled for 10 min, after which proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis before transfer to nitrocellulose membranes. A CWMV CP-specific antibody was produced in-house.
Protein extraction for nanoliquid chromatography (LC)-tandem MS (LC-MS/MS) was conducted as described previously with minor modifications . Sample powder was collected and ground individually in liquid nitrogen. An amount of 0.1 g sample powder was transferred to extraction buffer (40 mM Tris-Cl, pH 8.5, 7 M urea, 2 M thiourea, 4%SDS, 1 mM PMSF, 10 mM DTT, and 2 mM EDTA), which was then vortexed thoroughly. The reaction was incubated on ice for 10 min. After centrifugation at 16,000×g and 4 °C for 20 min, the supernatant was mixed with the quadruple volume of cold acetone. The mixtures were incubated at − 20 °C overnight. After centrifugation at 16,000×g and 4 °C for 10 min, the supernatant was discarded, and the pellet was dried using a Speed-Vacuum concentrator. The dried pellet was dissolved using solution buffer containing 8 M urea and 100 mM triethylamonium bicarbonate (TEAB; pH 8.0) stored at − 80 °C. Protein concentration and quantification was assessed using an RC DC™ Protein Assay (Bio-Rad, Hercules, CA, USA).
The protein solution was mixed with 10 mM DTT for 30 min followed by incubation at 56 °C, after which 20 mM iodoacetamide was added, and the solutions were incubated at room temperature for 30 min. For trypsin digestion, protein samples were diluted five-fold using 100 mM TEAB. Trypsin was added at a ratio of 1:50 (mass ratio, trypsin: protein) for overnight digestion and at a ratio of 1:100 (mass ratio, trypsin: protein) for the second digestion step of 4 h. Approximately 150 μg sample was digested.
LC-MS/MS analysis was performed as described previously with minor modifications [61, 62]. In brief, the reaction mixtures were dissolved using 0.1% formic acid and then loaded on an reversed-phase analytical column with 15 cm length and 75 μm i.d. A gradient of solvent contains 0.1% formic acid in 98% acetonitrile was produced from 6 to 23% for 25 min, from 23 to 35% within 8 min, then rising to 80% in 3 min, and remaining at 80% for the final 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 Ultrahigh Liquid Chromatography-triple Quadrupole Mass Spectrometry (UPLC) system. The peptides were subjected to an NSI source followed by MS/MS in Q Exactive™ Plus (Thermo Fisher Scientific, Waltham, MA, USA) together with UPLC. The electrospray voltage was set at 2.0 kV. Full scanning was conducted with the m/z scan ranging from 350 to 1800. Peptides were then picked out for MS/MS with NCE setting at 28, and fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan and 20 MS/MS scans with 15 s dynamic exclusion. Automatic gain control was set to 5E4. Fixed first mass was set to 100 m/z.
Database search was conducted as previously described with minor modifications [61, 62]. The Maxquant search engine (v.184.108.40.206) was used to process the resulting MS/MS data. Then the tandem mass spectra were searched against the UniProt Nicotiana tabacum database (73,605 sequences; updated in May 2019). In the first and main search precursor mass and fragment mass had an initial mass tolerance of 20 ppm and 5 ppm, respectively. The search included variable modifications of methionine oxidation, and fixed modification of carbamidomethyl cysteine. Trypsin/P was specified as cleavage enzyme and a maximum of two miscleavages was allowed. The false discovery rate (FDR) was set to 0.01 for peptide and protein identifications. Protein quantification was performed with MaxQuant as previously described with minor modifications . P-values were corrected according to the FDR and were applied for statistical analyses to estimate differences between infected and control samples. Intensity-based absolute quantification in MaxQuant was performed on the identified peptides to quantify protein abundance. Differentially expressed proteins were filtered at a fold change of > 1.5 and an FDR P-value < 0.05.
GO annotation was produced for protein sequences obtained from the UniProt-GOA database (www.http://www.ebi.ac.uk/GOA/). First, the ID of the identified protein was converted to a UniProt ID and was then mapped to GO IDs by protein ID. For identified proteins that were not annotated in the UniProt-GOA database, InterProScan software (v.5.14–53.0) (www.http://www.ebi.ac.uk/interpro) was used to annotate each protein’s GO function. Then, the proteins were classified by GO annotation according to three groups: biological processes, cellular components, and molecular functions.
Proteins in eukaryotic cells are localized in various cellular organelles, depending on what membrane structure they bind to. The main subcellular localization of eukaryotic cells includes the extracellular space, cytoplasm, the nucleus, mitochondria, peroxisomes, vacuoles, the Golgi apparatus, the endoplasmic reticulum, the cytoskeleton, nucleoplasm, the nuclear matrix, and the ribosome. We used Wolfpsort (v.0.2) (www.http://www.genscript.com/psort/wolf_psort.html), a subcellular localization predication software. Wolfpsort is an updated version of PSORT/PSORT II, which is used for predicting eukaryotic sequences.
The KEGG database was used for pathway annotation. First, the KEGG online tool KAAS was used to annotate the KEGG database description of proteins, after which the annotation result was mapped using the KEGG pathway database and the KEGG online tool KEGG mapper.
The domain functional descriptions of identified proteins were annotated using InterProScan (a sequence analysis application) based on a protein sequence alignment method, and the InterPro domain database was used. InterPro (http://www.ebi.ac.uk/interpro/) is a database that integrates diverse information on protein families, domains, and functional sites and makes it freely available to the public via web-based interfaces and services. Central to the database are diagnostic models, known as signatures, against which protein sequences can be searched to determine their potential function. InterPro can be utilized in large-scale analysis of whole genomes and meta-genomes as well as for characterizing individual protein sequences.
Functional enrichment analysis
Proteins were classified by GO annotation into three categories: biological processes, cellular compartments, and molecular functions. For each category, a two-tailed Fisher’s exact test was employed to test enrichment of DEPs against all identified proteins. The GO with a P value < 0.05 and false discovery rate <1% was considered significant.
The KEGG database was used to identify enriched pathways using a two-tailed Fisher’s exact test to test enrichment of DEPs against all identified proteins. Pathways with a corrected P value < 0.05 and false discovery rate <1% were considered significant. These pathways were classified into hierarchical categories according to the KEGG website.
For each category of proteins, InterPro (a resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites) database was searched, and a two-tailed Fisher’s exact test was employed to test enrichment of DEPs against all identified proteins. Protein domains with a P value < 0.05 were considered significant.
ABA and ABA inhibitor treatments (NDGA)
ABA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.2% ethanol to a final concentration of 100 μM. NDGA (Sigma-Aldrich, St. Louis, MO, USA), targeting 9-cis-epoxycarotenoid dioxygenase, was dissolved using 0.2% ethanol to 10 mM. N. benthamiana plants were treated with 100 μM ABA solution, 10 mM NDGA solution, or 0.2% ethanol solution (control). Treatment solutions were applied on the adaxial and abaxial sides of leaves until solution drops dripped off the leaves. After 12 h, pre-treated N. benthamiana leaves were inoculated with CWMV.
Analysis of ABA contents
Samples were collected from experimental plants for ABA extraction as described previously . Samples were then ground in liquid nitrogen and were mixed individually (200 mg leaf powder per sample) with 2H5-ABA (45 pmol). Two milliliters of methanol were added to each sample, which was then mixed, and the mixture was incubated overnight at − 20 °C. After centrifugation at 160,000×g and 4 °C for 20 min, the supernatant was collected and dried under nitrogen gas. The pellet was dissolved in 1 mL 5% ammonia solution and purified using Oasis MAX SPE columns (Waters, Milford, MA, USA) as the manufacturer’s instructions. Eluted ABA was dried under nitrogen gas, dissolved using 200 μL water/methanol mixture (20:80, v/v), and was then analyzed by Ultrahigh Liquid Chromatography-triple Quadrupole Mass Spectrometry (UPLC-MS/MS). Three independent biological replicates were used.