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Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus

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Abstract

The chitinase gene of baculoviruses is expressed in the late phase of virus replication in insects and possesses high exo- and endochitinase activity, which can hydrolyze chitin in the body of the insect, thus promoting terminal host liquefaction. Alphabaculovirus viral chitinases (vChitA) have been well analyzed, but information regarding viral chitinases from betabaculoviruses is limited. Whole-genome sequencing of a Korean isolate of Pieris rapae GV (PiraGV-K) predicted a putative chitinase gene corresponding to ORF10. The PiraGV-K chitinase gene had a coding sequence of 1,761 bp, encoding a protein of 586 amino acid (aa) residues, including an 18-aa putative signal peptide. Time course induction pattern observed by SDS-PAGE and subsequent Western blot with anti-PiraGV-K chitinase antibody revealed the cleavage of the signal peptide from the intact chitinase. Edman sequencing analysis was further conducted to confirm the exact nature of the mature chitinase, and the N-terminal amino acid sequence (KPGAP) exactly matched the sequence following the signal peptide sequence. The transcriptomics of PiraGV-K chitinase in infected P. rapae larvae, examined by real-time PCR, revealed a significant 75-fold increase after four days of feeding with PiraGV-K-treated leaves, with a subsequent decline at the later stages of infection. Confocal microscopic analysis showed that PiraGV-K chitinase possibly exists as a secreted protein, with strong chitinase-specific signals in fat body cells and integument at four days postinfection. Furthermore, immunogold labeling and electron microscopy studies localized the PiraGV-K chitinase in the cytoplasm and sparsely within vacuolar structures in the fat body apart from the extensive aggregation in the cuticular lining of the integument.

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Acknowledgements

This work was supported by the Mid-Career Researcher Program through NRF grant funded by the MEST (No. 2010-0027694).

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Authors and Affiliations

  1. Division of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University, Gwangju, 500-757, Korea

    Seunghan Oh, Dong Hyun Kim, Bharat Bhusan Patnaik, Yong Hun Jo, Mi Young Noh, Hyo Jeong Lee & Yeon Soo Han

  2. Department of Wood Science and Landscape Architecture, Chonnam National University, Gwangju, 500-757, Korea

    Kwang Ho Lee

  3. Department of Biology, Soonchunhyang University, Asan, 336-745, Korea

    Kwang Ho Yoon & Wan-Jong Kim

  4. Hampyeong County Insect Institute, Hampyeong County Agricultural Technology Center, Hampyeong, 525-811, Korea

    Ju Young Noh & Heon Cheon Jeong

  5. Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University, 22 Soonchunhyangro, Shinchang-myeon, Asan, 336-745, Chungchungnam-do, Korea

    Yong Seok Lee

  6. Institute of Insect Science, Zhejiang-California International Nanosystem Institute, Zhejiang University, Hangzhou, 310058, China

    Chuan-Xi Zhang

  7. Division of Applied Bioscience and Biotechnology, Institute of Environmentally Friendly Agriculture (IEFA), College of Agriculture and Life Sciences, Chonnam National University, Gwangju, 500-757, Korea

    Yong-Su Song & Woo-Jin Jung

  8. Department of Medicine, Medical Research Institute, College of Medicine Chung-Ang University, Seoul, 156-756, Korea

    Kisung Ko

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  1. Seunghan Oh
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Oh, S., Kim, D.H., Patnaik, B.B. et al. Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus. Arch Virol 158, 1701–1718 (2013). https://doi.org/10.1007/s00705-013-1649-z

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