Virologica Sinica www.virosin.org https://doi.org/10.1007/s12250-019-00094-4 www.springer.com/12250 (0123456789().,-volV)(0123456789().,-volV) RESEARCH ARTICLE Cleavage of the Babuvirus Movement Protein B4 into Functional Peptides Capable of Host Factor Conjugation is Required for Virulence 1,2 1,2 3 2 1,2 Jun Zhuang • Wenwu Lin • Christopher J. Coates • Pengxiang Shang • Taiyun Wei • 1,2 1,2 Zujian Wu • Lianhui Xie Received: 15 October 2018 / Accepted: 18 January 2019 Ó Wuhan Institute of Virology, CAS 2019 Abstract Banana bunchy top virus (BBTV) poses a serious danger to banana crops worldwide. BBTV-encoded protein B4 is a determinant of pathogenicity. However, the relevant molecular mechanisms underlying its effects remain unknown. In this study, we found that a functional peptide could be liberated from protein B4, likely via proteolytic processing. Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects, including dwarfism and sterility, in plants. The released protein fragment targets host proteins, such as the large subunit of RuBisCO (RbcL) and elongation factor 2 (EF2), involved in protein synthesis. Therefore, the peptide released from B4 (also a precursor) may act as a non-canonical modifier to influence host–pathogen interactions involving BBTV and plants. Keywords Banana bunchy top virus (BBTV) Á Movement protein B4 Á Functional peptide Á Pathogenicity Introduction genus Babuvirus in the family Nanoviridae, naturally infects only species in the Musaceae family and is a severe Single-stranded (ss) DNA viruses (such as geminiviruses) threat to commercial banana production (Wu and Su 1990). are among the most successful groups of plant pathogens The BBTV genome is made up of at least six circular (Hanley-Bowdoin et al. 2013). Mastreviruses and bego- ssDNA components, all of which exhibit rolling-circle moviruses in Geminiviridae are transmitted by leafhoppers replication (Burns et al. 1995). BBTV genomic DNA and whiteflies, respectively, and can infect monocots and components are displayed as a single open reading frame dicots (Hanley-Bowdoin et al. 2013). Aphid-borne Banana (ORF) and a 50 intergenic region including the origin of bunchy top virus (BBTV), an ssDNA virus belonging to the rolling-circle replication, similar to geminiviral compo- nents, exemplified by DNA component 4 (Burns et al. 1995). The BBTV DNA 4 protein can functionally restore Jun Zhuang and Wenwu Lin have contributed equally to this work. the movement of the CMV-Fny-DMP mutant (Cucumber mosaic virus-Fny isolate with the deletion of MP) (Sun Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12250-019-00094-4) contains supplemen- et al. 2002). Protein B4 is capable of redirecting DNA 6 tary material, which is available to authorized users. gene products, which preferentially translocate into the nucleus, to the cell periphery (Wanitchakorn et al. 2000). & Jun Zhuang Therefore, BBTV DNA 4 and 6 gene products are con- [email protected] sidered analogous to movement protein (MP) and nuclear & Lianhui Xie shuttle protein (NSP), respectively (herein referred to as [email protected] proteins B4 and B6). As previously reported, geminivirus 1 State Key Laboratory of Ecological Pest Control for Fujian NSP and MP orchestrate the ingress/egress of viral DNAs and Taiwan Crops, Fujian Agriculture and Forestry into and out of the nucleus and mobility between cells University, Fuzhou 350002, China (Rojas et al. 2001; Fondong 2013; Zorzatto et al. 2015). 2 Institute of Plant Virology, Fujian Agriculture and Forestry However, BBTV MP and NSP do not display University, Fuzhou 350002, China detectable sequence identity/similarity to bipartite gemi- 3 Department of Biosciences, College of Science, Swansea niviral counterparts. University, Swansea SA2 8PP, Wales, UK 123 Virologica Sinica Uniquely, BBTV movement protein B4 contains a single 14-day-old N. benthamiana leaves using 1-mL syringes transmembrane (TM) motif adjacent to the N-terminal without needles. Agrobacterium with PVX alone and domain, thereby facilitating membrane insertion (Zhuang infiltration buffer alone were used as negative controls. et al. 2016). The single N-terminal TM motif along with Seven-to-ten days post-infiltration, leaf tissues were har- lateral charged residues is likely equivalent in function to a vested for total protein extraction. DNA extracted from signal peptide sequence used to guide proteins through the BBTV-infected banana petioles was inoculated into the secretory pathway. Thus, the functional cleavage of protein stem of N. benthamiana using a syringe, using DNA from B4, i.e., the removal of the N-terminal TM domain by healthy bananas as a control. proteases, may be required for its activation. In the present study, we confirmed the membrane-bound subcellular Fluorescence In Situ Hybridization (FISH) localization of protein B4, which coexists in the vascula- Detection of BBTV ture with DNA 4 components. We further identified a B4- derived cryptic peptide whose functional release is asso- BBTV-infected banana petioles with (symptomatic) dark ciated with BBTV pathogenicity. The cryptic peptide green streaks were sliced into thin sections. The samples appears to act as a non-canonical modifier by host (target) were permeabilized in PBS (pH 7.4) containing 2% (v/v) protein conjugation. Triton X-100 and 5% (v/v) b-mercaptoethanol for 4 h at 20 °C. They were then fixed in PBS containing 1% Triton X-100 and 4% paraformaldehyde for 4 h. Post-fixation, Materials and Methods samples were washed with hybridization solution three times, placed into a hybridization solution containing 1% Plant Materials and Growth Conditions b-mercaptoethanol and 30 lmol/L DNA fluorescent probe for 5 h, washed in 1 9 SSC three times, and washed twice Nicotiana benthamiana and its GFP-transgenic 16c line in PBS (pH 7.4). Confocal microscopy was used to eval- were used. The seeds were plated on nutrient soil and uate the samples for the presence of BBTV. The following imbibed for 1 week at 25 °C under a 16-h-light/8-h-dark FAM-labeled probe was used to visualize DNA4: B4 cycle. The seedlings were transferred individually into FAM-RC-Probe, 50-FAM__CAGAAACCATTCGAAGAA vials and kept at 25 °C under a continuous 16-h light/8-h TAGTTTCACC-30. dark cycle until reaching the 5-leaf stage (2-week-old N. benthamiana seedlings) for agrobacterial inoculation. In Protoplast Preparation of N. benthamiana addition, BBTV-infected bananas maintained under natural Mesophyll Cells Infected by PVX:B4GFP conditions (20 °C–30 °C and natural lighting) were har- vested from the banana field at Fujian Agriculture and Approximately 7 days post-inoculation, newly developed Forestry University. leaves infected by chimeric PVX were harvested. The lower epidermis of tobacco leaves was separated/peeled Plasmid Construction and Viral Inoculations using fine tweezers and tobacco mesophyll cells were placed into an enzyme solution containing 1% cellulase The PVX:B4 and PVX:B4GFP constructs were assembled R-10 (Yakult Honsha, Tokyo, Japan), 0.25% macerozyme by cloning protein B4 and B4GFP fused/inserted between (Yakult Honsha), 0.4 mol/L mannitol, 10 mmol/L CaCl2, Sal I and Cla I sites of the PVX vector, respectively. The 20 mmol/L KCl, 0.1% bovine serum albumin, and B4/B4GFP mutants were amplified using primers listed in 20 mmol/L MES (pH 5.8) for 1–2 h at room temperature Supplementary Table S1 and the corresponding PVX:B4 (with constant agitation at *30 rpm). The lysate was fil- and PVX:B4GFP derivatives were obtained using a Fast tered using fiber sheets. The filtrate was centrifuged at low Mutagenesis Kit V2 (Vazyme Biotech, Nanjing, China). speed (*100 9g) for 5 min. The green pellet was rinsed All constructs were introduced into Agrobacterium tume- three times using W5 buffer (150 mmol/L NaCl, faciens strain GV3101 and cultured in Luria–Bertani (LB) 125 mmol/L CaCl2, 5 mmol/L KCl, 5 mmol/L glucose, broth overnight at 28 °C. The inocula were transferred into 2 mmol/L MES pH 5.7) and re-suspended in W5 buffer. fresh medium (1:50 dilution) and incubated for 6–8 h. Approximately 20 lL of protoplast suspension was Bacteria were pelleted and re-suspended in infiltration removed and placed onto sterile glass slides for inspection buffer (5 g/L glucose, 10 mmol/L MgCl2, 50 mmol/L by microscopy (Nikon, Tokyo, Japan). MES-KOH, pH 5.7; including 100 lmol/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 123 J. Zhuang et al.: Babuvirus B4 Cleavage Determines Virulence Preparation of Thylakoid Components from BBTV Imaging and Microscopy Petioles B4GFP fluorescence was detected using a confocal laser The peeled BBTV petioles (BBTV-infected and healthy microscope Zeiss LSM 710 (Carl Zeiss, Oberkochen, samples) were individually harvested and cut into small Germany) equipped with a 409 C-Apochromat objective. (1–2 cm) pieces. Cooled samples were macerated for GFP expressed in tobacco leaves was detected by long several minutes in extracting buffer [0.15 mol/L Tris–Cl wave UV excitation at 488 nm and emission at 509 nm. pH 8.0, 0.5% (w/v) NaSO3, 0.2% (W/V) PVP-K30], Ultrathin sections of banana petioles and immuno-gold ground on ice for several minutes, and filtered using 100 particles were observed by transmission electron micro- meshes. Chloroplasts were isolated from filtrates using an scopy (Hitachi H-7650). extraction kit (Sigma-Aldrich, St. Louis, MO, USA), fol- The banana samples were harvested and cut into thin lowing the supplier’s guidelines. Briefly, the macerates (0.2 cm2) slices and fixed in PBS (pH 7.2) containing 4% were centrifuged for 3 min at 200 9g to remove cellular paraformaldehyde, 0.1% glutaraldehyde, 0.1% Triton debris.