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. 2018 May 9;8(1):7430.
doi: 10.1038/s41598-018-25388-7.

A single amino acid substitution in the Bombyx-specific mucin-like membrane protein causes resistance to Bombyx mori densovirus

Katsuhiko Ito  1   2   3 , Kurako Kidokoro  4 , Susumu Katsuma  5 , Hideki Sezutsu  6   7 , Keiro Uchino  6   7 , Isao Kobayashi  6   7 , Toshiki Tamura  6   7 , Kimiko Yamamoto  4   8 , Kazuei Mita  4   9 , Toru Shimada  5 , Keiko Kadono-Okuda  10   11
Affiliations

A single amino acid substitution in the Bombyx-specific mucin-like membrane protein causes resistance to Bombyx mori densovirus

Katsuhiko Ito et al. Sci Rep. .

Abstract

Bombyx mori densovirus type 1 (BmDV) is a pathogen that causes flacherie disease in the silkworm. The absolute nonsusceptibility to BmDV among certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. However, neither of these genes has been molecularly identified to date. Here, we isolated the nsd-1 gene by positional cloning and characterized the properties of its product, NSD-1. Sequence and biochemical analyses revealed that this gene encodes a Bombyx-specific mucin-like glycoprotein with a single transmembrane domain. The NSD-1 protein was specifically expressed in the larval midgut epithelium, the known infection site of BmDV. Sequence analysis of the nsd-1 gene from 13 resistant and 12 susceptible strains suggested that a specific arginine residue in the extracellular tail of the NSD-1 protein was common among susceptible strains. Germline transformation of the susceptible-type nsd-1 (with a single nucleotide substitution) conferred partial susceptibility to resistant larvae, indicating that the + nsd-1 gene is required for the susceptibility of B. mori larvae to BmDV and the susceptibility is solely a result of the substitution of a single amino acid with arginine. Taken together, our results provide striking evidence that a novel membrane-bound mucin-like protein functions as a cell-surface receptor for a densovirus.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Mapping of nsd-1 in linkage group 21. (A) SNP markers and linkage analysis. The upper panel indicates the positions of the SNP markers in BAC end sequences. Distances between the markers are shown in centiMorgans (cM). In the lower panel, the dotted arrows indicate the results of rough mapping of the region linked to nsd-1. (B) Fine mapping on the scaffold sequence. The dotted arrows indicate the results of linkage analysis, which was performed to narrow the nsd-1 region. The nsd-1 locus was located within an area of approximately 400-kb-long between 7,402,527 and 7,799,223 bp on Bm_scaf7. This region contains five predicted genes: BGIBMGA001390, 001596, 001389, 001388, and 001597. (C) RT-PCR analysis of predicted genes located within the nsd-1 region in resistant and susceptible strains. The internal control was 18S ribosomal RNA. Lane 1, C124 (resistant); 2, p50T (resistant); 3, J124 (susceptible); and 4, J150 (susceptible). A mixture of λ/HindIII and Φ×174/Hae III (New England Biolabs) was used as a DNA size marker.
Figure 2
Figure 2
Characterization of the NSD-1 protein. (A) Predicted NSD-1 polypeptide. The figure indicates the nonspliced form of NSD-1. Black and gray boxes indicate the transmembrane domain and the region corresponding to the intron in the spliced form of nsd-1, respectively. Green and blue boxes indicate the putative N-linked and O-linked glycosylation (mucin-like domain) sites, respectively. K (lysine) at 110 and G (glycine) at 118 indicate the amino acid residues derived from the resistant strain p50T, while E (glutamic acid) at 110 and R (arginine) at 118 are the amino acid residues derived from the susceptible strain J150. (B and (C) Western blot of recombinant NSD-1 in Sf9 cells using anti-His antibody (B) and anti-NSD-1 antibody (C). The membrane fraction was extracted from Sf9 cells and analyzed by western blot. The primary antibodies were anti-His antibody (1:3000) and anti-NSD-1 antibody (1:10,000), respectively. Lane 1, NSD-1-His-Res-nonspl-AcMNPV; 2, NSD-1-His-Sus-nonspl-AcMNPV; 3, Bac1-AcMNPV (negative control); 4, NSD-1-His-Res-spl-AcMNPV; 5, NSD-1-His-Sus-spl-AcMNPV. (D) Immunostaining of Sf9 cells infected with recombinant baculoviruses. The Sf9 cells were incubated with anti-NSD-1 antibody (1:100) followed by a secondary antibody labeled with AlexaFluor488 (1:500) (green) and counterstained with DAPI (1:1000) (red). DAPI staining (left); NSD-1 signals (center); merge (right). (E) Hypothetical membrane orientation of NSD-1 predicted by SOSUI (http://harrier.nagahama-i-bio.ac.jp/sosui/). The spliced form of NSD-1 lacks the gray region. The red filled circles indicate the positions of amino acids that vary between the resistant (p50T) and susceptible (J150) strains. The green and blue filled circles indicate the putative N-linked and O-linked glycosylation sites, respectively.
Figure 3
Figure 3
Expression profiles of nsd-1. (A) Tissue-specific expression of nsd-1 in a resistant strain, p50T. CNS, central nervous system; SG, silk gland; FB, fat body; HC, hemocyte; TES/OV, testis and ovary; FG, foregut; AMG, anterior midgut; MMG, middle midgut; PMG, posterior midgut; HG, hindgut; MT, Malpighian tubule. Primer sets were designed to amplify nsd-1 (upper) and ribosomal 18S (lower) as a positive control. (B) Stage-specific expression of nsd-1 in the resistant strain p50T. E, eggs at on days 0, 4, and 8; L, whole larvae at on day 0 (first to fifth instar); P, whole pupae at on days 0 and 5; A, whole adult at on day 0. (C) Immunohistochemical analysis of NSD-1 in the larval anterior midgut. The upper panels show immunofluorescence visualization of NSD-1. The lower panels show control experiments with pre-immune serum. The right panels are zoomed-in images of the dotted boxes in the corresponding left panels. The sections were incubated with anti-NSD-1 antibody (1:200) or pre-immune serum followed by a secondary antibody labeled with AlexaFluor488 (1:200) (green) and counterstained with DAPI (1:1000) (blue). Scale bar: 200 μm.
Figure 4
Figure 4
Expression and immunohistochemical analysis in transgenic silkworms. (A) RT-PCR of BmDV-derived transcripts and the + nsd-1 transgene in the midgut of transgenic silkworms. Transgenic silkworms carrying the nonspliced form of the susceptibility gene + nsd-1 [glutamic acid (E) at 110 and arginine (R) at 118], the spliced form of + nsd-1 (E at 85 and R at 93), and the point mutated spliced form of + nsd-1 [lysine (L) at 85 and R at 93] were generated. K (blue) and R (red) indicate the amino acid residue conserved in resistant and susceptible strains, respectively. −/−, −/G, U/−, and U/G indicate the wild-type, GAL4, UAS, and GAL4/UAS lines, respectively. The midgut was dissected from day 3 fifth instar following exposure to BmDV on day 0 of the fourth instar stage. RT-PCR was performed for 30 cycles with primer sets for + nsd-1, the NS1 gene of BmDV, and ribosomal 18S as a positive control. (B) Immunohistochemical analysis of BmDV in the midgut of transgenic silkworms expressing the nonspliced form of the susceptibility gene + nsd-1. Sections were incubated with anti-BmDV capsid antibody (1:100) followed by a secondary antibody labeled with AlexaFluor488 (1:200) (green) and counterstained with DAPI (1:1000) (red). DAPI staining (left); BmDV signal (center); merge (right). Scale bar: 200 μm.
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