Plant Materials and Growth Conditions
Nicotiana benthamiana and its GFP-transgenic 16c line were used. The seeds were plated on nutrient soil and imbibed for 1 week at 25 ℃ under a 16-h-light/8-h-dark cycle. The seedlings were transferred individually into vials and kept at 25 ℃ under a continuous 16-h light/8-h dark cycle until reaching the 5-leaf stage (2-week-old N. benthamiana seedlings) for agrobacterial inoculation. In addition, BBTV-infected bananas maintained under natural conditions (20-30℃ and natural lighting) were harvested from the banana field at Fujian Agriculture and Forestry University.
Plasmid Construction and Viral Inoculations
The PVX:B4 and PVX:B4GFP constructs were assembled by cloning protein B4 and B4GFP fused/inserted between Sal I and Cla I sites of the PVX vector, respectively. The B4/B4GFP mutants were amplified using primers listed in Supplementary Table S1 and the corresponding PVX:B4 and PVX:B4GFP derivatives were obtained using a Fast Mutagenesis Kit V2 (Vazyme Biotech, Nanjing, China). All constructs were introduced into Agrobacterium tumefaciens strain GV3101 and cultured in Luria-Bertani (LB) broth overnight at 28 ℃. The inocula were transferred into fresh medium (1:50 dilution) and incubated for 6-8 h. Bacteria were pelleted and re-suspended in infiltration buffer (5 g/L glucose, 10 mmol/L MgCl2, 50 mmol/L MES-KOH, pH 5.7; including 100 μmol/L acetosyringone) until an OD600 value of 1.0 was obtained. Cultures of agrobacteria were maintained at room temperature for 1-2 h prior to infiltration into the lower epidermis of 14-day-old N. benthamiana leaves using 1-mL syringes without needles. Agrobacterium with PVX alone and infiltration buffer alone were used as negative controls. Seven-to-ten days post-infiltration, leaf tissues were harvested for total protein extraction. DNA extracted from BBTV-infected banana petioles was inoculated into the stem of N. benthamiana using a syringe, using DNA from healthy bananas as a control.
Fluorescence in situ Hybridization (FISH) Detection of BBTV
BBTV-infected banana petioles with (symptomatic) dark green streaks were sliced into thin sections. The samples were permeabilized in PBS (pH 7.4) containing 2% (v/v) Triton X-100 and 5% (v/v) β-mercaptoethanol for 4 h at 20 ℃. They were then fixed in PBS containing 1% Triton X-100 and 4% paraformaldehyde for 4 h. Post-fixation, samples were washed with hybridization solution three times, placed into a hybridization solution containing 1% β-mercaptoethanol and 30 μmol/l DNA fluorescent probe for 5 h, washed in 1× SSC three times, and washed twice in PBS (pH 7.4). Confocal microscopy was used to evaluate the samples for the presence of BBTV. The following FAM-labeled probe was used to visualize DNA4: B4 FAM-RC-Probe, 5′-FAM__CAGAAACCATTCGAAGAATAGTTTCACC-3′.
Protoplast Preparation of N. benthamiana Mesophyll Cells Infected by PVX:B4GFP
Approximately 7 days post-inoculation, newly developed leaves infected by chimeric PVX were harvested. The lower epidermis of tobacco leaves was separated/peeled using fine tweezers and tobacco mesophyll cells were placed into an enzyme solution containing 1% cellulase R-10 (Yakult Honsha, Tokyo, Japan), 0.25% macerozyme (Yakult Honsha), 0.4 mol/L mannitol, 10 mmol/L CaCl2, 20 mmol/L KCl, 0.1% bovine serum albumin, and 20 mmol/L MES (pH 5.8) for 1-2 h at room temperature (with constant agitation at ~30 rpm). The lysate was filtered using fiber sheets. The filtrate was centrifuged at low speed (~100 × g) for 5 min. The green pellet was rinsed three times using W5 buffer (150 mmol/L NaCl, 125 mmol/L CaCl2, 5 mmol/L KCl, 5 mmol/L glucose, 2 mmol/L MES pH 5.7) and re-suspended in W5 buffer. Approximately 20 µL of protoplast suspension was removed and placed onto sterile glass slides for inspection by microscopy (Nikon, Tokyo, Japan).
Preparation of Thylakoid Components from BBTV Petioles
The peeled BBTV petioles (BBTV-infected and healthy samples) were individually harvested and cut into small (1-2 cm) pieces. Cooled samples were macerated for several minutes in extracting buffer [0.15 mol/L Tris-Cl pH 8.0, 0.5% (w/v) NaSO3, 0.2% (W/V) PVP-K30], ground on ice for several minutes, and filtered using 100 meshes. Chloroplasts were isolated from filtrates using an extraction kit (Sigma-Aldrich, St. Louis, MO, USA), following the supplier's guidelines. Briefly, the macerates were centrifuged for 3 min at 200 × g to remove cellular debris. The supernatant was retained and centrifuged at 1000 × g for 7-10 min. This time, the supernatant was discarded and the green pellet was re-suspended in 1× CIB. The chloroplast extracts were loaded onto a discontinuous 40%-80% Percoll gradient. After centrifugation, the green layers containing chloroplasts were harvested to isolate thylakoid membranes.
To purify the thylakoid membrane, the chloroplast suspensions were subjected to ultrasonic treatment at 4 ℃. The homogenate was centrifuged at 10, 000 × g for 10-20 min. The green supernatants were loaded onto 15%-40% sucrose gradients and centrifuged in a swinging bucket rotor (type 32, Beckman) at 4 ℃ for 4 h at 110, 000g. The separated fractions were retained for SDS-PAGE and western blot analyses.
Tobacco stems were cut into 0.5 cm2 sections and fixed for 2 h in PBS (pH 7.2) containing 4% paraformaldehyde and 0.1% glutaraldehyde at room temperature. After fixation, the samples were washed three times using PBS. The samples were dehydrated through an ethanol gradient of 50%, 70%, 90%, and 100% for 1 h at -20℃. Post-dehydration, mixtures of the embedding agent LR-Gold and ethanol at 1:1 and 2:1 ratios were used to permeabilize the samples for 1 h at -20℃. The samples were embedded in complete LR-Gold for 1 h at -20℃. Samples were then transferred to LR-Gold containing 0.1% BENZIL for 3 h at -20℃. Finally, the samples were placed into thin-walled tubes with LR-Gold containing BENZIL and polymerization was achieved under UV light at 20 ℃ over a 7-day period. For immuno-electron microscopy, ultra-thin sections were blocked using 2% BSA for 30 min prior to labeling with the anti-GFP antibody and 10-nm gold particles conjugated to goat antibodies against rabbit IgG (GAR10; British Bifocals International, Cardiff, UK). The specificity of labeling was monitored by incubating non-infected tobacco samples with anti-GFP IgG.
Imaging and Microscopy
B4GFP fluorescence was detected using a confocal laser microscope Zeiss LSM 710 (Carl Zeiss, Oberkochen, Germany) equipped with a 40× C-Apochromat objective. GFP expressed in tobacco leaves was detected by long wave UV excitation at 488 nm and emission at 509 nm. Ultrathin sections of banana petioles and immuno-gold particles were observed by transmission electron microscopy (Hitachi H-7650).
The banana samples were harvested and cut into thin (0.2 cm2) slices and fixed in PBS (pH 7.2) containing 4% paraformaldehyde, 0.1% glutaraldehyde, 0.1% Triton X-100, and 2% β-mercaptoethanol at 26 ℃ for several hours. After fixation, the samples were dehydrated using a gradient of ethanol and then coated in embedding agent at 26 ℃ for several hours. The polymerizing patches were prepared for ultrathin slicing and stained in 1% uranyl acetate prior to observation by TEM.
Total Protein Extraction from BBTV-infected Stems
Approximately 20 g of 'green streak' sections from BBTV-infected banana petioles were peeled, flash frozen in liquid nitrogen, and ground into a fine powder. PBS pH 7.4 [containing 0.5% (w/v) NaSO3, 0.2% (W/V) PVP-30, and complete protease inhibitor (Roche, Basel, Switzerland)] was added to the sample prior to vortexing and homogenization in an ice bath. The sample was passed through a 100-mesh filter and subsequently centrifuged once at 10, 000 × g for 30 min and again at 40, 000 × g for 20 min at 4 ℃. The remaining supernatant was centrifuged at 150, 000 × g for 4.5 h at 4℃. The pellet was re-suspended in 500 µL of 50 mmol/L PBS, pH 7.4. The suspension was aliquoted (50 µL per vial) for co-immunoprecipitation (CoIP), and stored at -70℃. Total proteins from healthy bananas were extracted following the above procedure.
Approximately 0.5 g of PVX:B4GFP-infected N. benthamiana (GFP-expressing N. benthamiana as a control) leaves were homogenized using lysis buffer [50 mmol/L Tris-HCl pH 7.5, 5 mmol/L MgCl2, 150 mmol/L NaCl, 0.1% Tween 20, 5% (v/v) β-mercaptoethanol, and a protease inhibitor cocktail (Roche)] on ice. Lysates were centrifuged twice at 30, 000 × g at 4 ℃ for 20 min. Approximately 40 µL of supernatant was transferred to a 0.5-mL tube and 10 µL of 5× SDS sample buffer (CW Biotech) was added. The remaining supernatant was incubated with 20 µg of anti-GFP antibody at 16 ℃ for 0.5 h and then transferred to another column pre-filled with protein A/G agarose beads (Abmart). Next, the beads were rinsed thoroughly using a 6-fold column volume of the lysis buffer and 50 μL of 5× SDS sample buffer was used to suspend the beads after the washing step. Subsequently, all samples were boiled at 98 ℃ for 5 min to disrupt disulfide bonds and linearize the peptides. The treated protein samples were centrifuged at 14, 000 × g for 5-10 min, separated by 12%-15% SDS-PAGE, and transferred onto a PVDF membrane. Immunoblotting (IB) was performed using anti-B4 and anti-GFP antibodies (Genscript). Specifically, 1× TBST (50 mmol/L Tris-Cl pH 8.0, 150 mmol/L NaCl, 0.05% Tween 20) supplemented with 5 mmol/L MgCl2 was used for anti-B4 immunoblotting at 16 ℃.
Co-immunoprecipitation (CoIP) assay
Approximately 0.5 g of PVX:B4GFP-infected N. benthamiana (GFP-expressing N. benthamiana as a control) leaves was homogenized using lysis buffer [50 mmol/L Tris-HCl pH 7.5, 5 mmol/L MgCl2, 150 mmol/L NaCl, 0.1% Tween 20, 5% (v/v) β-mercaptoethanol, and a protease inhibitor cocktail (Roche)] on ice. Lysates were centrifuged twice at 30, 000 × g at 4 ℃ for 20 min. Approximately 40 µL of supernatant was transferred to a 0.5-mL tube and 10 µL of 5× SDS sample buffer (CW Biotech, Beijing, China) was added. The remaining supernatant was incubated with 20 µg of anti-GFP antibody at 16 ℃ for 0.5 h and then transferred to another column pre-filled with protein A/G agarose beads (Abmart, Shanghai, China). Next, the beads were rinsed thoroughly using a 6-fold column volume of the lysis buffer. Approximately 50 μL of 5× SDS sample buffer was used to suspend the beads after the washing step. Subsequently, all samples were boiled at 98 ℃ for 5 min to disrupt disulfide bonds and linearize the peptides. The treated protein samples were centrifuged at 14, 000 × g for 5-10 min, separated by 12%-15% SDS-PAGE, and transferred onto a PVDF membrane. Immunoblotting (IB) experiments were respectively performed using anti-B4 and anti-GFP antibodies (GenScript, Piscataway, NJ, USA). Specifically, 1× TBST (50 mmol/L Tris-Cl pH 8.0, 150 mmol/L NaCl, 0.05% Tween 20) supplemented with 5 mmol/L MgCl2 was used for anti-B4 immunoblotting at 16 ℃.
To capture the target protein fragments associated with the B4-derived cryptic peptide in infected banana, the total banana protein extracts separated by centrifugation at 40, 000 × g and the supernatant were used for CoIP at 16 ℃. Similarly, CoIP using anti-B4 IgG required the addition of appropriate amounts of Mg2+ into protein suspensions to a final concentration of 1.0 mmol/LM. Next, 20-30 µg of anti-B4 IgG was sequentially added and incubated at 16℃. The mixture was aspirated into columns pre-filled with protein A/G agarose beads (Abmart). The beads were gently rinsed by running buffer through the column and re-suspended in 5× SDS sample buffer.
Mass Spectrometry Detection
Protein bands were stained using Coomassie brilliant blue (CBB) R-250 staining and excised from gels for further analysis. The Coomassie-stained gel slices of target proteins were excised and applied to a 96-well plate, de-stained twice in 200 µL of 15 mmol/L potassium ferricyanide and 50 mmol/L sodium thiosulfate (1:1) and then dried twice with 200 μL of acetonitrile. The dried gels were incubated in a pre-chilled digestion solution (trypsin 12.5 ng/µL and 20 mmol/L NH4HCO3) for 20 min and incubated overnight at 37 ℃. Finally, the fragmented peptides were pelleted using an extraction solution (5% formic acid in 50% acetonitrile) and desiccated under a stream of N2.
The dried peptides were dissolved in solvent A (5% acetonitrile, 0.1% formic acid) and analyzed using a Triple-TOF 5600 system (AB SCIEX, Framingham, MA, USA). Briefly, peptides were separated on a reverse-phase column (ZORBAX 300SB-C18 column, 5 μm, 300 Å, 0.1 × 15 mm; MicroMass, Cary, MA, USA) using an Eksigent 1D PLUS system (AB SCIEX) at an analytical flow rate of 300 nL/min. The peptides were separated with a linear gradient from 5% to 40% of solvent B (0.1% formic acid/90% acetonitrile) over 2 h. Survey scans were acquired from 800 to 2500 Da with up to 15 precursors selected for MS/MS and dynamic exclusion for 20 s. For protein identification, MS/MS data were processed using MASCOT version 2.3.02 (Matrix Science, London, UK) and the Nicotiana subset of the NCBI sequence databases. Peptides with significance scores over the "identity threshold" were recorded and the sequence was confirmed by an artificial analysis according the b and y ions.