Plant material and viral inoculation
Plants (Nicotiana benthamiana) were grown in a growth chamber with a 16 h day length at 28°C. Six-week-old plants were mechanically inoculated with 500 ng of BaMV on each leaf. Virus- and mock-inoculated leaves were harvested on day 1, 3, 5 or 7 post-inoculation (dpi).
Plant mRNAs isolation
Total RNA was extracted from 3 g of leaves. The leaves were ground to powder with liquid nitrogen and mixed with 6 ml of STE buffer (100 mM Tris-HCl, pH 8.0, 100 mM NaCl and 10 mM EDTA), 660 μl of 10% SDS and 180 μl of 100 mg/ml bentonite. The mixture was centrifuged at 12000 rpm for 10 min at 4°C (Sigma model 3MK centrifuge) after three times of phenol/chloroform extraction. Total RNA in the supernatant was ethanol precipitated, stored at -80°C, and subjected to poly(A) RNA isolation by using oligo(dT)-coupled paramagnetic beads. Briefly, 100 μl of the total RNA (75 μg) were heated at 65°C for 2 min to disrupt secondary structure and then placed on ice. About 200 μl of Dynabeads Oligo (dT)25 (Dynal A.S., Oslo, Norway) were washed twice with 100 μl of binding buffer (20 mM Tris-HCl pH 7.5, 1.0 M LiCl, 2 mM EDTA) and resuspended in 100 μl of binding buffer. The beads were incubated with total RNA for 3-5 min at room temperature, washed twice with 200 μl of washing buffer (10 mM Tris-HCl pH 7.5, 0.15 M LiCl, 1 mM EDTA) and resuspend in 10 μl of deionized water to elute the mRNA.
For the first-strand cDNA synthesis, the 20-μl reaction containing 750 ng of mRNA, 30 pmole of Oligo (dT)40, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 1 mM dNTP,10 mM DTT, and 1 μl of 200 U/μl SuperScript® III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA)was incubate at 42°C for 90 min. Following removal of the mRNA by alkaline lysis, the cDNA was ethanol precipitated, washed, dried, and dissolved in 10 μl of deionized water. The second-strand cDNA was synthesized in a 10-μl reaction containing 4 μl of first-strand cDNA, 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 7.5 mM DTT, 10 mM dNTP, and 2.5 units of Klenow polymerase (New England Biolabs, Beverly, MA, USA) at 25°C for 30 min. The enzyme was inactivated with 100 mM EDTA for 20 min at 75°C.
cDNA-AFLP was carried out using the AFLP® Expression Analysis Kits (LI-COR Biosciences, Lincoln, NE, USA), according to the protocols provided by the manufacturer. Double-strand cDNA was sequentially digested with TaqI at 65°C for 2 hours and with MseI at 37°C for another 2 hours. After inactivation of the restriction enzymes at 80°C for 20 min, 9 μl of the adapter mixture containing the adapters (TaqI adapters: 5'GCGCGCCGTAGACTGCGTAC 3', 5'CGGTACGCAGTCTACGGCGCGC3', MseI adapters: 5'GGCCGCCGATGAGTCCTGAG3', 5'TACTCAGGACTCATCGGCGGCC3'), 0.4 mM ATP, 10 mM Tris-HCl pH 7.5, 10 mM Mg(OAc)2, 50 mM KOAc, and 6 Weiss units of T4 DNA ligase (New England Biolabs) were added to the restriction digestion mixture and incubated at 20°C for 2 hours.
Subsequently, twenty cycles of pre-amplification were carried out in a 20-μl reaction containing 2.5 μl of 30-fold diluted cDNA template, 100 pmole each of TaqI primer (5'GTAGACTGCGTAC3') and MseI primer (5'GATGAGTCCTGAG3'), 0.25 mM dNTP, 1.5 mM MgCl2, and 5 units of Taq DNA polymerase (Promega, Madison, WI, USA). The PCR thermal cycling consisting of 20 cycles of 94°C for 30 sec, 56°C for 1 min, and 72°C for 1 min was performed on a GeneAmp PCR system 9600 instrument (Applied Biosystems, Foster city, CA, USA). The amplification products (i.e. the secondary template) were diluted 300 folds and subjected to selective amplification. The reaction contained 6 μl of Taq DNA polymerase working mix (20 mM Tris-HCl pH 8.4, 1.5 mM MgCl2, 100 mM KCl and 0.75 unit Taq DNA polymerase), 2 μl of the secondary template, 2 μl of MseI primer, and 0.5 μl of IRDye™ 700-labeled TaqI primer. The amplification conditions are as follows: 13 cycles of 94°C for 30 sec, 65°C for 30 sec (temperature increment reduction of 0.7°C per cycle), and 72°C for 1 min, followed by 23 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 1 min. Samples were denatured at 95°C for 5 min after the addition of stop solution (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol) and separated on a 6.5% KBPlus™ gel. Labeled DNA fragments were visualized and recorded by the automatic DNA Sequencer LI-COR 4300 (LI-COR Biosciences)
Isolating and sequencing the differentially expressed cDNA fragments
The bands of interest, namely the transcript-derived fragments TDF, were marked on the Odyssey™ Scanner (LI-COR Biosciences), cut out with a sterile razor blade, and soaked in 10 μl of TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA). Following a series of freeze-thaw steps, the cDNA fragments were leached out from the gel by centrifugation at the top speed of a microfuge for 20 min at 4°C. Re-amplification of the cDNA fragments was carried out under the same conditions of the pre-amplification step. The PCR products were separated on a 5% polyacrylamide gel and cloned into pGEM®-T Easy vector (Promega). DNA sequencing was conducted using the Simultaneous Bi-directional Sequencing (SBS™) method (LI-COR) on a Global IR2 System (LI-COR). DNA sequence homology search within the GenBank® database was performed using BLAST .
First-strand cDNA of RNA prepared from mock- or BaMV-inoculated N. benthamiana plants was synthesized with d(T)39 primer using SuperScrpt® III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Four sets of primers were used to confirm the expression profiles of four cDNA fragments identified by cDNA-AFLP, namely ACAG2-1, ACCT8-1, ACCT2-1, and ACCT13. The forward primers are (5'GAACAAAAAAATGGAGTTTTA3'), (5'CGAACTCCCAACTGGCTTTC3'), (5'CTCTGGAAAGGAGAGCAATGTC3'), and (5'GAACGCTTTGATGAGAATAGAGA3') and the reverse primers (5'GTCATTGCTCCTAATAAGGT3'), (5'CTCCTCCAGAAGCAAATAGTTTC3'), (5'CGAACAAATTGGTGTATCC3'), and (5'CTAACTCAACCGCAGCCTTT3'), respectively. PCR amplifications were performed using Taq DNA polymerase (Promega) with 28 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. PCR products were separated on a 5% polyacrylamide gel and visualized by EtBr staining.
Primer pairs for ACAG1 (forward, 5'GAGAAAATGAAGGAGAAGGCCC3'; reverse, 5'GCTCTGCCTTCTTCAATTGCTTCTT3') and ACAG8 (forward, 5'GAAGGAAGCTGTGAATGTGTCA3'; reverse, 5'TGGTTAAGTTCATACGGAAAGA3') were used to determine the knockdown efficiency of host genes by VIGS (virus-induced gene silencing). The actin primer pair (forward, 5'GTGGTTTCATGAATGCCAGCA3'; reverse 5'GATGAAGATACTCACAGAAAGA3') was used for normalization of RT-PCR data.
Virus-induced gene silencing (VIGS)
Two transcript-derived fragments (ACAG1 and ACAG8) were first cloned into pGEM-T Easy vector (Promega) and subcloned into the EcoRI site of the pTRV2 vector . The control plasmid pTRV2/mGFP was obtained by subcloning the KpnI-XhoI fragment containing the polyhistidine-tagged mGFP5-coding sequence  from pBI-mGFP1 into pTRV2. The pTRV2/ACAG1, pTRV2/ACAG8 and pTRV2/mGFP constructs were transformed into Agrobacterium tumefaciens C58C1 strain by electroporation.
For agroinfiltration, the A. tumefaciens C58C1 containing pTRV1, pTRV2/mGFP, pTRV2/ACAG1, or pTRV2/ACAG8 was cultured to OD600 = 1 at 30°C and subjected to induction in 150 μM acetosyringone and 10 mM MgCl2 for 2 h at room temperature. Subsequently, the pTRV2/mGFP-, pTRV2/ACAG1- or pTRV2/ACAG8-containing A. tumefaciens C58C1 was mixed with the pTRV1-containing A. tumefaciens C58C1 at a 1:1 (v:v) ratio. The 2nd and 3rd true leaves were infiltrated with the mixture at the four-leaf stage (seedlings with two cotyledons and two leaves). BaMV virion RNA (1 μg) was inoculated onto the 7th leaf when the plants were mature. Total RNAs and proteins were extracted from the leaves on 5 dpi for subsequent studies.
Total proteins of the leaves were extracted in 1x Laemmli buffer (2.5 mM Tris-HCl, pH 8.3, 250 mM glycine and 0.1% SDS) and incubated in boiling water for 5 min. Proteins separated by SDS-PAGE were subjected to Western blotting analysis using the polyclonal rabbit anti-BaMV coat protein antibody. The relative levels of the Rubisco large subunit (rbcL) in gels stained with Coomassie Blue were determined and used for the normalization of the Western blotting signals.