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Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus

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Abstract

In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 ‘weak’ HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN.

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Acknowledgments

This research was supported by Golden Seed Project Vegetable Seed Center (213002-04-2-WTc11), Ministry of Agriculture, Food and Rural Affairs, Korea. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture (USDA); USDA is an equal opportunity provider and employer.

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Authors and Affiliations

  1. Chungnam National University, Daejeon, Republic of Korea

    Jae-Yeong Han, Jinsoo Chung, Jungkyu Kim, Eun-Young Seo & Hyoun-Sub Lim

  2. Floral and Nursery Plants Research Unit, USDA-ARS, USNA, Beltsville, MD, 20705, USA

    James P. Kilcrease & John Hammond

  3. Electron and Confocal Microscopy Unit, USDA-ARS, BARC, Beltsville, MD, 20705, USA

    Gary R. Bauchan

  4. Plant Systems Engineering Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon, 305-806, Republic of Korea

    Seungmo Lim

  5. Biosystems and Bioengineering Program, University of Science and Technology, Daejeon, 305-350, Republic of Korea

    Seungmo Lim

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  1. Jae-Yeong Han
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Fig. 1

Turnip mosaic virus (TuMV) pathogenicity in Raphanus sativus, showing mottle or mild mosaic with each isolate; pictures were taken at 20 days post inoculation (d.p.i). Supplementary material 1 (TIFF 8197 kb)

Fig. 2

Phylogenetic tree based on nucleotide sequences of Turnip mosaic virus (TuMV) HC-Pro genes, constructed with MEGA 6.0 using Maximum-Likelihood method with 1000 bootstrap replicates. The HC-Pro of thirty-three non-recombinant TuMV isolates for comparison were obtained from GenBank; the label indicates isolate name (for more details see Supplemental Table 1). The numbers at nodes are bootstrap values above 50 %; the scale bar indicates the number of nucleotide substitutions. The sequence of the Potato virus Y HC-Pro genes was used as the outgroup. World-B, Basal-B, Asian-BR, and Basal-BR indicate the previously established groupings of TuMV isolates (see text references [17, 18]). Supplementary material 2 (TIFF 4780 kb)

Fig. 3

Amino acid alignment (CLUSTALW) of HC-Pro of the three Korean isolates. Residues differing between isolates are highlighted, and numbered below the alignment. Fully conserved residues are marked below the sequence with an asterisk ( * ); conservation of strong groups by a colon (:); conservation of weak groups by a period (.); and residues with no consensus by a space ( ). Motifs that have been identified in the HC-Pro of various potyviruses are shown in grey shading; where identified motifs are adjacent, an underscore and/or overscore differentiates motifs (see text references [8, 10–13, 15, 36–39]). Dots above the sequence indicate every fifth residue (60 residues per line). Supplementary material 3 (DOCX 13 kb)

Fig. 4

Confocal images of pGDG:HC-Pro of three isolates agroinfiltrated to Nicotiana benthamiana together with: pGDR:Talin (a, b, c) or pGDR:Talin and pGD:p19 (d, e, f). Leaves were examined at 3 days after agroinfiltration. Red color indicates chloroplast autofluorescence, green color shows GFP-tagged HC-Pro, and white represents DsRed:Talin (pGDR:Talin). Bars indicate 20μm. Note obvious punctate aggregates in leaves infiltrated with pGDG:HC-Pro of R007 (a, d) and R041 (b, e), or less defined aggregates of R065 apparently associated with microfilaments (c, f). No difference in HC-Pro aggregation was observed in the presence (a, b, c) or absence (d, e, f) of pGD:p19, in contrast to observation with PVY (see text reference [32]). Supplementary material 4 (TIFF 21797 kb)

Fig. 5

Comparison of VSR activity (upper) and subcellular localization pattern (lower) between wild type HC-Pros and mutant HC-Pros. Left side of each panel is wild type HC-Pro and right side is HC-Pro mutant. (a) R007 HC-Pro; (b) R041 HC-Pro; (c), R065 HC-Pro. F = phenylalanine at amino acid position 395 and L = leucine at that position. To assay VSR activity (upper), Agrobacterium harboring pGD:TuMV HC-Pro was co-infiltrated to Nicotiana benthamiana with pGD:smGFP. For localization (lower), pGDG:TuMV HC-Pro was co-infiltrated with pGDR:Talin and pGD:p19 to N. benthamiana. Green color shows GFP:HC-Pro, red color indicates chloroplast autofluorescence, and white represents DsRed:Talin (pGDR:Talin). Bars indicate 20 µm. Note differences in aggregation: (a) between WT R007 HC-Pro (F395; obvious punctate aggregates) and mutant HC-Pro(F395L); (c) between WT R065 HC-Pro (L395; apparent association with filaments) and mutant HC-Pro (L395F; obvious punctate aggregates with less apparent filaments), whereas (b) both WT and mutant R041 HC-Pro produced obvious punctate aggregates throughout the cytoplasm. Supplementary material 5 (TIFF 20591 kb)

Table 1

Supplementary material 6 (DOCX 14 kb)

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Han, JY., Chung, J., Kim, J. et al. Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus . Virus Genes 52, 592–596 (2016). https://doi.org/10.1007/s11262-016-1330-1

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