Deletion of a gene encoding an amino acid transporter in the midgut membrane causes resistance to a Bombyx parvo-like virus

Abstract

Bombyx mori densovirus type 2 (BmDNV-2), a parvo-like virus, replicates only in midgut columnar cells and causes fatal disease. The resistance expressed in some silkworm strains against the virus is determined by a single gene, nsd-2, which is characterized as nonsusceptibility irrespective of the viral dose. However, the responsible gene has been unknown. We isolated the nsd-2 gene by positional cloning. The virus resistance is caused by a 6-kb deletion in the ORF of a gene encoding a 12-pass transmembrane protein, a member of an amino acid transporter family, and expressed only in midgut. Germ-line transformation with a wild-type transgene expressed in the midgut restores susceptibility, showing that the defective membrane protein is responsible for resistance. Cumulatively, our data show that the membrane protein is a functional receptor for BmDNV-2. This is a previously undescribed report of positional cloning of a mutant gene in Bombyx and isolation of an absolute virus resistance gene in insects.

Bombyx densovirus is characterized by a very narrow host range and tissue specificity. Four unlinked mutations, nsd-1 (1), Nid-1 (2), nsd-2 (3), and nsd-Z (4), which were originally discovered in different Bombyx strains, control nonsusceptibility to infection by two distinctly different types of virus, BmDNV-1 and -2 (or Z). Previously, both BmDNV-1 and -2 had been assigned to Densovirinae in Parvoviridae. BmDNV-2 was recently excluded from the family Parvoviridae, because, in contrast to the accepted characters for the group, its genome is split into two molecules and contains its own DNA polymerase motif (5). Although the mutations have been mapped with phenotypic or DNA markers, neither of them has been isolated yet (reviewed in ref. 6). The identification of these genes will provide important information for understanding the host and tissue specificity and infection mechanisms of the corresponding densoviruses. Because nsd-2 was well mapped with flanking DNA markers, and significant Bombyx genome information had accumulated, we undertook map-based cloning of the gene.

Results

Chromosome Walking and Fine Mapping of nsd-2.

For the identification of a candidate region for nsd-2, chromosome walking was started from three EST markers shown to be closely linked with the mutation by RFLP mapping (3) (Fig. 1A), then elongated up- and downstream to generate additional markers closer to nsd-2 by BAC library screening (7). A diagram of the physical and genetic maps generated in the walk is shown in Fig. 1. A BAC contig of ≈5 Mb was constructed that covered the up- to downstream boundaries of the nsd-2 linked region (Fig. 1B). By genetic linkage analysis using this physical map, the nsd-2 linked region was narrowed to between the T7 end of BAC clone 034C07 and the SP6 end of BAC clone 001K10 [supporting information (SI) Datasets S1 and S2]. The region was ≈400 kb in length and covered by four BAC clones, 022C09, 067M09, 021B10, and 059I11 (Fig. 1C).

Fig. 1.

Chromosome walking and mapping of nsd-2. Mapping of nsd-2 on linkage group 17. (A) RFLP map of nsd-2, modified from the previous report (3). EST markers are shown above the map, whereas the distance between the markers and nsd-2 is shown in centiMorgan units (cM) below the map. EST markers m237, m274, and m134, which showed no recombination with nsd-2 in the initial study involving 49 male informative backcross (BC₁) progeny, served as the starting point for the walk (3). (B) BAC contig covering three EST markers. Each line shows a BAC clone. A dotted line indicates the result of linkage analysis using 206 BC₁ progeny surviving virus treatment to narrow the region linked to nsd-2. (C) Minimum BAC tiling path for the region closely linked to nsd-2. The arrowhead indicates the locus where the deletion was found specifically in resistant strain J150. (D) PCR amplification of genomic DNA from resistant and susceptible strains at the region indicated by the arrowhead in C. (Left) PCR patterns. (Right) The position of primer sets in the resistant strain J150(R) and the susceptible strain No. 908(S): (1) the forward and reverse primers encompass the deletion (dotted line), (2) the forward primer includes the specific intact sequence in J150, and (3) the forward primer is within the deletion. (M) λ and φX174 DNA digested individually with HindIII and HaeIII and mixed as molecular markers; lanes 1, J150; 2, J124; 3, No. 902; 4, No. 908; 5, J203; 6, C124; 7, B; 8, p50T; 9, C108T; 10, B. mandarina. 1–3, resistant; 4–10, susceptible.

In the comparison between resistant (J150) and susceptible (No. 908) strains using primers designed within the narrowed region, we found a critical region on the T7 end of BAC clone, 067M09. The primers constructed for the region gave no PCR products only in the resistant strain. Furthermore, it was found that the resistant strain had a deletion of ≈6 kb and an insertion of 34 bp in this region. In a more comprehensive PCR survey of other resistant and susceptible strains, including Bombyx mandarina, the most closely related living wild silkmoth having a putative common ancestor of B. mori, the long deletion and very short insertion were common to all of the resistant strains (Fig. 1D). The primers encompassing the long deletion gave size variation in susceptible strains, which is explained in detail in the next section.

Sequence Analysis and Expression of nsd-2 Candidate Gene.

One candidate gene was found in the annotation analysis used to search for expressed genes, proteins, and cDNAs in the deleted region. The full-length cDNA and corresponding genomic DNA sequences of this candidate were determined for strains No. 908 and J150 and compared to each other (Figs. S1–S4 and Dataset S3). The deletion in J150 corresponded to the region from exons 5–13 in No. 908 (Fig. 2). This structure agreed with the difference in PCR product size found in the genomic DNA (Fig. 1D) and messenger RNA (Fig. 3A), strongly suggesting that this gene is a candidate for nsd-2. The larger PCR products of lanes 6–9 in Fig. 1D-1 came from the insertion of >3 kb in the intron between exons 7 and 8 (data not shown). The amplicon of lane 10 was much smaller than amplicons generated from the other susceptibles (Figs. 1D-1 and 3). This could be explained by the presence of a deletion in the intron between exons 13 and 14 within to the primer sites located just after exon 13 and extending to the beginning of exon 14.

Fig. 2.

Schematic genome structure of nsd-2 and +^nsd-2 candidate. Relative position and size of exon/intron in the genome of susceptible No. 908 (Upper) and resistant J150 (Lower). The dotted line indicates the deletion in the genome of J150. The arrowheads show the start and stop codons.

Fig. 3.

Expression of nsd-2 detected by RT-PCR. (A) Expression of nsd-2 candidate transcripts purified from midgut of resistant and susceptible strains. Lanes 1, J150; 2, J124; 3, No. 902; 4, No. 908; 5, C124; 6, p50T; 7, B. mandarina. Primer sets are for nsd-2 and ribosomal 18S, a positive control. 1–3, resistant; 4–7, susceptible. (B) Tissue-specific expression of nsd-2 in No. 908 (1) and J150 (2). Lanes 1, silk gland; 2, foregut; 3, midgut; 4, hindgut; 5, Malpighian tubule; 6, fat body; 7, testis plus ovary; 8, central nervous system. (C) Stage-specific expression of nsd-2 in No. 908 (1) and J150 (2). (a) Lanes 1–3, embryo, day 0, 4 and 10; 4–6, whole body, day 0 from first to third-instar larva; 7–8, midgut, day 0 from fourth and fifth-instar larva; 9–10, pupal gut, day 0 and 7; 11, adult gut, day 0; (b) lanes 1–5, midgut, day 0, 1, 2, 3 and 4 of fourth-instar larva; 6–9, midgut, day 0, 2, 4, and 6 of fifth-instar larva.

In RT-PCR using total RNA isolated from eight different tissues, the transcript was detected only in the midgut (Fig. 3B). The tissue specificity of this gene transcription was consistent with the unique characteristic that BmDNV-2 is able to infect only midgut (8). For developmental stage specificity, the RNA of the candidate gene was detected in the later stages of embryo and throughout the larval stages except during a molt (Fig. 3C). These results support the idea that this gene is expressed only after organogenesis of the midgut is completed and when the midgut is functional for digestion and absorption.

By NCBI-tBLASTx search, the cDNA sequence of the +^nsd-2 candidate from strain No. 908 showed high homology with two amino acid transporter genes of Manduca sexta (GenBank accession nos. AF006063 with 63–77% and AF013963 with 62–76% identities) (9, 10). Also the topology prediction method, SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui), predicted a membrane protein that encompasses 12 putative transmembrane domains (Fig. 4). In contrast, the translated protein of J150 could contain only the first three-pass transmembrane domains, because a significant portion of its coding region was deleted (Fig. 2). From these results, it was suggested that the deleted membrane protein structure is crucial for the infection of BmDNV-2. The homologous amino acid transporters of Manduca sexta transport specific amino acids (9, 10). Whether the nsd-2 candidate functions as an amino acid transporter is still unknown. However, it is clear that the nsd-2 candidate is not essential for the silkworm, because the resistant strains that lack large portions of the internal sequence are healthy, with no detectable effect on development or other processes.

Fig. 4.

Hypothetical secondary structure of NSD-2. Two putative N-glycosylation sites (“Y”) are indicated between transmembrane domains 3 and 4. The gray region indicates the deletion in NSD-2. The secondary structure is based on the topology prediction method, SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui); the style of the diagram is based on a 12-pass membrane protein reported in M. sexta (10).

Restoration of Virus Susceptibility by Germ-Line Transformation of +^nsd-2 Gene to the Resistant Strain.

To demonstrate that the candidate gene we identified was responsible for virus resistance in the nsd-2 mutant, we transformed resistant silkworms with the +^nsd-2 candidate sequence using the GAL4-UAS system to drive expression of the transgene and tested their susceptibility after inoculation with BmDNV-2 at the first or fourth instar. After virus inoculation of the transgenic progeny of the four types of transgenic strains used for making the final construct (described in Materials and Methods), only the silkworms of the GAL4/UAS line (Fig. 5A) showed a notable susceptibility phenotype, whereas the other lines grew up without any symptoms (Fig. 5B and Dataset S4). The GAL4/UAS line silkworms without BmDNV-2 inoculation did not change their growth (Fig. 5C). Using PCR to confirm that the transgene was inserted into the genome in the transgenic silkworms, the transgene-specific PCR product was detected in the GAL4/UAS and GAL4 lines (Fig. 5D-1). To confirm whether BmDNV-2 could multiply in the midgut of transgenic animals, BmDNV-2 DNA was detected by PCR by using virus-specific primers. Only the GAL4/UAS lines had viral DNA (Fig. 5D-2). Moreover, by using RT-PCR, the +^nsd-2 transcript was detected only in the GAL4/UAS line (Fig. 5D-3). These results indicated that the candidate gene we identified is nsd-2 itself, the virus resistance gene, and the wild-type membrane protein expressed by the allele, +^nsd-2, is required for infection by BmDNV-2.

Fig. 5.

Plasmid constructs for transgenic silkworm and viral inoculation tests. (A) Physical maps of activator and effector constructs. (Upper) The activator construct of pBacMCS[BmA3-GAL4-3xP3-DsRed2]. It contains a full ORF of yeast transcriptional factor GAL4 under the control of the BmA3 promoter and the marker gene, DsRed2, under the control of the 3xP3 promoter. (Lower) The effector construct of pBacMCS[UAS-+^nsd-2(No. 908)-SV40-3xP3-EGFP]. It contains a DNA-binding motif, UAS, for GAL4, linked to the ORF of the targeted gene, +^nsd-2, and the marker gene, EGFP, under the control of the 3xP3 promoter. Arrows indicate piggyBac inverted terminal repeats. (B) Transgenic silkworms of the GAL4/UAS line, GAL4 line, UAS line and wild type after the viral inoculation test. (C) Comparison of the GAL4/UAS line after exposure to BmDNV-2 at day 0 of first instar (Left) and unexposed control (Right). The larvae started to die from 7 days after inoculation (at second to third instar). The pictures were taken at the fourth instar. (D) PCR and RT-PCR analysis of the transgene. (1) PCR analysis of the introduced gene from four transgenic lines. (2) Detection of BmDNV-2 from the midgut of four transgenic lines with primers based on the virus genome sequence. (3) Expression pattern of the introduced gene, +^nsd-2, from the midgut of four transgenic lines. Lanes 1–4, GAL4/UAS, GAL4, UAS lines and wild type, respectively; P, virus inoculation solution as a positive control; P1 and P2, midgut of J150 and No. 908 as positional markers; N, 0.5× TE (pH 8.0) as a negative control template. The white arrowheads indicate the PCR products of +^nsd-2.

Discussion

In this study, by positional cloning using Bombyx genome information (11–15), we carried out the isolation of nsd-2, a putative transporter gene, and showed that a deletion corresponding to 9 of 12 predicted transmembrane domains confers resistance to BmDNV-2. This is a previously undescribed report of the isolation of a mutant gene in the silkworm by this strategy. Moreover, as far as we know, this is a previously undescribed report of the isolation of a mutant gene that causes absolute resistance to a virus in insects.

Infection mechanisms have been reported in human parvovirus, B19 (16), and canine and feline parvoviruses (17). The relationship between each virus and its receptor is very specific; specific amino acids both on the viral surface and on the exposed domain of the receptor are important for binding (17). From these reports, we think the complete membrane protein functions as a receptor for BmDNV-2, and the site that the virus recognizes as a target is present in the deleted portion of the membrane protein, nsd-2. It is still not clear how BmDNV-2 interacts with the membrane protein. It would be of great importance to determine the recognition sites of BmDNV-2 and nsd-2. In Densovirinae, many viruses are known among diverse insect groups such as B. mori, Junonia coenia, and Galleria mellonella in Lepidoptera; Aedes aegypti in Diptera; and Periplaneta fuliginosa in Blattodea, as well as in crustaceans such as lobster and crab (18). There are many reports about the relationship between densoviruses and their host range or tissue specificities (8). However, the factors controlling host and tissue specificity are still unknown. nsd-2 is a previously undescribed report of a critical host factor required for the infection of an insect virus, including Densoviruses. Although BmDNV-2 has been excluded from Densovirinae, it has distinctive pathological characters in common with BmDNV-1, such as an infective tissue of midgut columnar cell nuclei and the existence of well defined resistant and susceptible Bombyx strains. The discovery of nsd-2 will contribute to the analysis of the infection mechanism in other densoviruses. What are the targets for the other densoviruses that can infect many tissues, like J. coenia DNV or have a broad host range like J. coenia and A. aegypti DNV? The answers to these questions must be of great interest.

B. mandarina, which is considered to have a common ancestor to B. mori (6), is susceptible to BmDNV-2 (Figs. 1D and 3A). Chinese, Indian, and Japanese native (ancient) strains also tend to be susceptible; however, many genetically improved Japanese strains are resistant (19, 20). Therefore, this form of DNV-2 resistance may be an acquired mutation through silkworm breeding improvement. In contrast, considering that the susceptibility to DNV-1 is reversed in being present in genetically improved strains (19), DNV-2 is likely to be a native disease of Bombyx, whereas the origin of DNV-1 is likely to be from pathogens of other insects. With the availability of the complete genome sequence of B. mori (12, 13), identification of other BmDNV resistance genes, Nid-1, nsd-1, and nsd-Z is expected in the near future. A study of the relationship between the gene described here and the other BmDNV resistance genes is of interest, in part because it can reveal new findings on the infection mechanism of BmDNV. The results of our study will contribute to research not only on insect pathogenic viruses important to sericulture and for agricultural pest control but also for other viruses transmitted by insects (21). Although most virus receptors are essential for their hosts, it is of interest that, apparently, this one is not. In addition to the present gene, we found another similar gene that has no relationship with resistance (unpublished data). Although it is not known whether the second gene takes over a role in amino acid transport, having an extra copy might accelerate the mutation of the other and lead to its becoming resistant. The recently reported rapid emergence of field resistance to Baculovirus in codling moth (22) is consistent with this kind of scenario.

Materials and Methods

Silkworm Strains and Viral Inoculation.

Strains resistant to BmDNV-2 were J 150, J124, and No. 902 [National Institute of Agrobiological Sciences (NIAS) origin]; susceptible strains were No. 908, J203, C124 (NIAS), p50T, C108T (University of Tokyo), and B (Hokkaido University, Hokkaido, Japan). B. mandarina (Tsukuba native) was also used as a closely related susceptible species. For linkage analysis, single-pair backcrosses (BC₁) between J150 females and F₁ males (J150 female × No. 908 males) were used.

To obtain stable binary GAL4/UAS transgenic strains, the activator, enhancer trap strain of A3GAL4 (Fig. 5A), 52-2-1 was chosen and was able to direct transgene transcription in the midgut. The activator strain was made by the cross (52-2-1 × J124) × J124 to replace the +^nsd-2 gene of 52-2-1 with the homozygous nsd-2 gene of J124, because 52-2-1 was susceptible. For the effector strains, the effector construct (Fig. 5A) and the transposase-carrying helper plasmid, pHA3PIG (23), were injected into homozygous nsd-2 J124 preblastoderm embryos at a concentration of 0.2 mg/ml. After sibling selection with the marker, EGFP, the G₁ moths of the UAS line were crossed with the BC₁ moths of the GAL4 line to generate +^nsd-2-overexpressing GAL4/UAS lines. The progeny of this cross showed four different phenotypes in terms of eye color: both DsRed2- and EGFP-positive, GAL4/UAS line, [GAL4(+), UAS(+)]; only DsRed2-positive, GAL4 line, [GAL4(+), UAS(−)]; only EGFP-positive, UAS line, [GAL4(−), UAS(+)]; and neither DsRed2- nor EGFP-positive, wild type, [GAL4(−), UAS(−)].

The transgenic and wild-type strains and the related species, B. mandarina, were reared at 25°C. A BmDNV-2 inoculum was prepared, and larvae were treated as described (24). The newly hatched first instar or the newly ecdysed fourth instar larvae were fed mulberry leaves smeared with DNV-2 suspension at 10⁻² diluted supernatant of midgut homogenate from DNV-2-infected larvae.

Chromosome Walking and Positional Cloning.

Chromosome walking was performed by BAC high-density replica filter hybridization (7) with PCR-amplified probes designed with sequences derived from BAC clone ends, EST clones, and genome shotgun data. The new primers were designed with the end sequences of screened BAC clones that were most distal in aligned BAC clones. This approach was repeated to extend the BAC contig. For positional cloning, PCR, RFLP, and sequencing markers were generated for each position of the walked region, and the markers that showed polymorphism between the parents were used for linkage analysis of 206 BC₁ moths selected with virus inoculation from ≈500 individuals.

Isolation of Genomic DNA and Total RNA.

Genomic DNA was isolated from whole bodies of individual moths or fifth instar larvae as described (3), modified by homogenization with stainless steel beads instead of mortar and pestle with liquid nitrogen. Genomic DNA of individual transgenic silkworms was isolated from a small portion of the body using DNAzol (Invitrogen) according to the manufacturer's protocol. Day 1 fourth instar larvae were used for the isolation of total RNA from individual tissues, including silk gland, foregut, midgut, hindgut, Malpighian tubule, fat body, testis plus ovary, and central nervous system. In the isolation of total RNA from individuals of different stages, eggs of day 0, 4, and 10; whole body from day 0 of first to fourth instar larvae; midgut from days 1, 2, 3, and 4 of fourth instar; days 0, 2, 4, and 6 of fifth instar; days 0 and 7 of pupae; and day 0 of adults were used. Total RNA was purified with TRIzol (Invitrogen) according to the manufacturer's protocol. Full-length cDNAs were determined by using a SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer's protocol.

Annotation Analysis.

The resistant/susceptible strain-specific sequences that we found in the region narrowed by linkage analysis were subjected to analysis by using KAIKOGAAS (http://kaikogaas.dna.affrc.go.jp) for annotation and by KAIKO blast (http://kaikoblast.dna.affrc.go.jp) and NCBI blast (www.ncbi.nlm.nih.gov/BLAST) to look for homologs and othologs.

Plasmid Construction.

The effector plasmid construction (UAS-line) was generated by the following method. The susceptibility gene, +^nsd-2, was amplified by PCR from the cDNA clone using KOD-plus-DNA polymerase (TOYOBO). Both forward and reverse primers contained an XbaI site. The PCR product was subcloned to a pCR-Blunt II-TOPO vector (Invitrogen), and the subcloned product was digested with XbaI. The fragment obtained was ligated into pBacMCS-UAS.SV40-3xP3EGFP previously digested with Bln I. The PCR product and constructed plasmid, pBacMCS-UAS- +^nsd-2 (No. 908)-SV40-3xP3EGFP, were confirmed by sequencing.