Functional analysis of apple stem pitting virus coat protein variants

doi:10.1186/s12985-019-1126-8

. 2019 Feb 8;16(1):20.

doi: 10.1186/s12985-019-1126-8.

Functional analysis of apple stem pitting virus coat protein variants

Xiaofang Ma^{1

2

3

4}, Ni Hong^{1

2}, Peter Moffett³, Yijun Zhou⁴, Guoping Wang^{5

6}

Affiliations

¹ State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, People's Republic of China.
² Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, People's Republic of China.
³ Centre SÈVE, Département de Biologie, Université de Sherbrooke, 2500 Blvd. de l'Université, Sherbrooke, QC, J1K 2R1, Canada.
⁴ Jiangsu Academy of Agricultural Sciences, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Institute of Plant Protection, Nanjing, 210014, China.
⁵ State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, People's Republic of China. gpwang@mail.hzau.edu.cn.
⁶ Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, People's Republic of China. gpwang@mail.hzau.edu.cn.

PMID: 30736799
PMCID: PMC6368714
DOI: 10.1186/s12985-019-1126-8

Functional analysis of apple stem pitting virus coat protein variants

Xiaofang Ma et al. Virol J. 2019.

. 2019 Feb 8;16(1):20.

doi: 10.1186/s12985-019-1126-8.

Authors

Xiaofang Ma^{1

2

3

4}, Ni Hong^{1

2}, Peter Moffett³, Yijun Zhou⁴, Guoping Wang^{5

6}

Affiliations

¹ State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, People's Republic of China.
² Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, People's Republic of China.
³ Centre SÈVE, Département de Biologie, Université de Sherbrooke, 2500 Blvd. de l'Université, Sherbrooke, QC, J1K 2R1, Canada.
⁴ Jiangsu Academy of Agricultural Sciences, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Institute of Plant Protection, Nanjing, 210014, China.
⁵ State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, People's Republic of China. gpwang@mail.hzau.edu.cn.
⁶ Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, People's Republic of China. gpwang@mail.hzau.edu.cn.

PMID: 30736799
PMCID: PMC6368714
DOI: 10.1186/s12985-019-1126-8

Abstract

Background: Although the canonical function of viral coat protein (CP) is to encapsidate the viral genome, they have come to be recognized as multifunctional proteins, involved in almost every stage of the viral infection cycle. However, CP functions of Apple stem pitting virus (ASPV) has not been comprehensively documented. This study aimed to characterize the functions of ASPV CP and any functional diversification caused by sequence diversity of six ASPV CP variants and studied their biological, serological, pathogenic and viral suppressor of RNA silencing (VSR) functions.

Methods: Six ASPV CP variants that have previously been shown to belong to different subgroups were selected here to study their diversity functions. Agrobacterium mediated infiltration (Agroinfiltration) was used to express YFP-ASPV-CPs in Nicotiana. benthamiana and infect Nicotiana. occidental with PVX-ASPV-CPs in. Confocal microscopy was used to detect YFP-ASPV-CPs florescence. CPs expressed in Escherichia coli BL21 (DE3) were induced by IPTG.

Results: In this study, we showed that recombinant CPs expressed in Escherichia coli BL21 (DE3) had different levels of serological reactivity to three anti-ASPV antibodies used to detect ASPV. Furthermore, fusion CPs with YFP (YFP-CPs) expressed in N. benthamiana cells differed in their ability to form aggregates. We also showed that ASPV isolates that harbour these CPs induced different biological symptoms on its herbaceous host N. occidentalis. At the same time, we found that all six CPs when expressed in PVX vector showed similar VSR activity and produced similar symptoms in N. occidentalis, despite their differences in amino acids.

Conclusions: Different ASPV isolates induced different symptoms in N. occidentalis, however, ASPV CP variants expressed in PVX vector showed the same symptoms in N. occidentalis plants. Also, we showed that ASPV CP variants has the same level of VSR activity, but they have different abilities to aggregate in N. benthamiana.

Keywords: Aggregate; Apple stem pitting virus; CP variants; Coat protein; RNA silencing suppressor.

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Figures

**Fig. 1**
Symptoms induced by different isolates of Apple stem pitting virus (ASPV) on *Nicotiana occidentalis.* a Left, symptoms induced on pear leaves in an orchard in Hangzhou city, Zhejiang province, China. Right, healthy pear leaf. b Symptoms induced on *N. occidentalis* plants 14 days post inoculation (dpi), red arrows indicated typical symptoms in each photo. Pear isolates HB-HN1, HB-HN2, HB-HN6, HB-HN7, HB-HN9, and HB-HN10 were collected from an orchard in Wuhan city, Hubei province in China, Apple isolate HB-AP1 was collected from the same orchard. c Representative result of RT-PCR products to detect ASPV. NI, Non-Infected

**Fig. 2**
Analysis of recombinant CPs from different ASPV isolates by SDS-PAGE and western blot. a SDS-PAGE analyze of rCPs expressed in *E. coli*. Lane M: Protein Ladder; Lanes 1–6 and Lane EV (from left to right): protein extracts from *E. coli* transformed with vectors pET-HB-HN9–3, pET-HB-HN6–8, pET-LN-AP1–1, pET-HB-HN7–18, pET-HB-HN1–3, pET-YN-MRS-17 and the empty vector pET-28a (+), respectively. **b-d** Western blot analysis of rCPs by antibody PAb-HB-HN6–8, PAb-YN-MRS-17 and PAb-HB-HN9–3, respectively. Hybridization signals on western blots were quantified by Image J software (right column of B-D)

**Fig. 3**
ASPV CPs differ in their propensity to aggregate. Subcellular localization of YFP-ASPV-CPs upon transient expression in *Nicotiana. benthamiana* leaves. Confocal micrographs of *N. benthamiana* leaf cells expressing YFP-ASPV-CPs (as indicated at the top left of each image) were taken at 72 hpi (Scale Bar, 50 μm). a YFP channel. b TD channel. Red arrowheads indicate inclusions. The results shown are representative of three separate experiments. c Total protein was extracted from YFP-ASPV-CPs infiltrated leaf spots. Samples were subjected to anti-GFP immune-blotting. Ponceau staining are shown as a loading control

**Fig. 4**
ASPV CPs possess VSR activity. Local patches on *N. benthamiana* leaf were agroinfiltrated with 35S:mGFP5 in combination with either 35S:P19, 35S:P25, PVX (wt) or PVX-ASPV-CPs, respectively. a PVX-ASPV-CPs were infiltrated on the same leaf. b PVX-ASPV-CPs were infiltrated on different leaves. GFP signal was monitored by UV illumination at 4 days post infiltration (dpi) (a) and 6 dpi (b). c Total protein was extracted from leaf infiltration spots of (b). Samples were subjected to anti-GFP immune-blotting. Ponceau staining are shown as a loading control. All experiments were repeated 3 times and representative results are shown

**Fig. 5**
ASPV CPs induce symptoms on *N. occidentalis.* Five-week-old *N. occidentalis* plants were agroinfiltrated with PVX (wt) or PVX-ASPV-CPs and symptoms induced on these plants were photographed at 30 dpi

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References

1. Carstens EB. Ratification vote on taxonomic proposals to the international committee on taxonomy of viruses (2009) Arch Virol. 2010;155:133–146. - PMC - PubMed
1. Jelkmann W. Nucleotide sequences of apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex- and carlaviruses. J Gen Virol. 1994;75:1535–1542. - PubMed
1. Mathioudakis MM, Maliogka VI, Dovas CI, Vasilakakis M, Katis NI. First record of the apple stem pitting virus (ASPV) in quince in Greece. J Plant Pathol. 2006;88:225.
1. Mathioudakis MM, Maliogka VI, Katsiani AT, Katis NI. Incidence and molecular variability of apple stem pitting and apple chlorotic leaf spot viruses in apple and pear orchards in Greece. J Plant Pathol. 2010;92:139–147.
1. Wu Z, Ku H, Su C, Chen I, Jan F. Molecular and biological characterization of an isolate of apple stem pitting virus causing pear vein yellows disease in Taiwan. J Plant Pathol. 2010;92:721–728.

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[1] Carstens EB. Ratification vote on taxonomic proposals to the international committee on taxonomy of viruses (2009) Arch Virol. 2010;155:133–146. - PMC - PubMed

[2] Carstens EB. Ratification vote on taxonomic proposals to the international committee on taxonomy of viruses (2009) Arch Virol. 2010;155:133–146. - PMC - PubMed

[3] Jelkmann W. Nucleotide sequences of apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex- and carlaviruses. J Gen Virol. 1994;75:1535–1542. - PubMed

[4] Jelkmann W. Nucleotide sequences of apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex- and carlaviruses. J Gen Virol. 1994;75:1535–1542. - PubMed

[5] Mathioudakis MM, Maliogka VI, Dovas CI, Vasilakakis M, Katis NI. First record of the apple stem pitting virus (ASPV) in quince in Greece. J Plant Pathol. 2006;88:225.

[6] Mathioudakis MM, Maliogka VI, Dovas CI, Vasilakakis M, Katis NI. First record of the apple stem pitting virus (ASPV) in quince in Greece. J Plant Pathol. 2006;88:225.

[7] Mathioudakis MM, Maliogka VI, Katsiani AT, Katis NI. Incidence and molecular variability of apple stem pitting and apple chlorotic leaf spot viruses in apple and pear orchards in Greece. J Plant Pathol. 2010;92:139–147.

[8] Mathioudakis MM, Maliogka VI, Katsiani AT, Katis NI. Incidence and molecular variability of apple stem pitting and apple chlorotic leaf spot viruses in apple and pear orchards in Greece. J Plant Pathol. 2010;92:139–147.

[9] Wu Z, Ku H, Su C, Chen I, Jan F. Molecular and biological characterization of an isolate of apple stem pitting virus causing pear vein yellows disease in Taiwan. J Plant Pathol. 2010;92:721–728.

[10] Wu Z, Ku H, Su C, Chen I, Jan F. Molecular and biological characterization of an isolate of apple stem pitting virus causing pear vein yellows disease in Taiwan. J Plant Pathol. 2010;92:721–728.

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