Fig 1.
BmSUH is a membrane-associated midgut protein.
(A) Expression patterns of BmSUH mRNA in B. mori expression data from SilkDB 3.0 (https://silkdb.bioinfotoolkits.net). (B) Expression profiles of BmSUH in the third day of fifth instar larvae (L5D3). Immunoblot analysis were performed using antibody against BmSUH and α-Tubulin as a loading control. Protein samples were isolated from brain (Br), silk gland (Sg), midgut (Mg), epidermis (EP), wing disc (Wi), Malpighian tube (Mt), fat body (Fb), integument (In), testis (Te), and ovary (Ov). (C) Subcellular localization of BmSUH in BmN cells. BmSUH was expressed in BmN cells by transfection. Antibody specific to BmSUH was used for immunofluorescence and green fluorescence (FITC) was observed as a marker. The cytoplasmic membrane was stained red using WGA-AF 594 and nuclei were visualized using 4’,6’-diamidino-2-phenylindole (DAPI, blue) counterstain.
Fig 2.
(A) Schematic description of the BmSUH gene and the sgRNA target site. Black and green squares represent the noncoding (UTRs) and coding parts of the transcript, respectively. The sgRNA targeting sites, S1 and S2, are located on the forward strand and reverse strand of the fifth exon, respectively. The sgRNA sequences are depicted in black and the corresponding PAM sequence is marked in red. The scissors image was obtained from the “Icon Library” of iSlide application under a CC0 1.0 Universal license. Copyright: https://en.islide.cc/copyright-notice. (B) Immunoblot validation of two types of BmSUH mutants. (C) Immunofluorescence staining of BmSUH (green) on midgut tissue sections of wild-type and BmSUH deficient silkworms. Silkworm specimens were obtained from L5D3. DAPI (blue) served as a nuclear dye. WT: wild-type; -8bp and -22bp: two BmSUH mutant lines.
Fig 3.
(A) The mortality analysis of fourth-instar WT and ΔBmSUH after being orally infected with the BmNPV at concentration of 1×103, 1×104, 1×105, 1×106, 1×107 and 1×108 OB/mL (n = 30). (B) The mortality of fifth-instar WT and ΔBmSUH after BV injection (n = 30). (C) The production of infectious BVs in the hemolymph of fourth-instar WT and ΔBmSUH after orally infected with 1×108 OB/mL BmNPV. The hemolymph was taken at designed time points and the BV titers were determined by TCID50 endpoint dilution assays. Each data point was determined from the average of at least three independent experiments. The data represents the mean ± SEM.
Table 1.
LC50 of fourth instar larvae after oral infection.
Table 2.
LT50 of fourth instar larvae after oral infection.
Table 3.
LD50 of fifth instar larvae after BV injection.
Fig 4.
Interruption of BmSUH reduced the production of virions and viral gene expression.
(A) TEM observations of virions in ΔBmSUH and WT larval midgut column epithelial cells. Electron-dense virogenic stroma (VS) and progeny nucleocapsids (black arrows) were observed both in the midgut of WT and ΔBmSUH larvae. The white triangles show intranuclear microvesicles (IM) and nucleocapsids associated with the membranous vesicular structures. PH, polyhedral. (B) Expression levels of different-phase viral genes, IE1, GP64, and VP39 in WT or ΔBmSUH larvae midgut after inoculated orally with 106 OBs (mean ± SEM). *p < 0.05 and **p < 0.01 by two-tailed Student t test.
Fig 5.
Identification of BmSUH interacting proteins by LC-MS/MS analysis of endogenous CO-IP products.
(A) SDS-PAGE gel image of samples after endogenous BmSUH immunoprecipitation. Whole-cell lysates of midgut tissue were used for immunoprecipitation (IP) with anti-BmSUH. The red triangle represented the specific bands. (B) Viral proteins detected by LC-MS/MS in 70 kDa and 100 kDa gels. The structural proteins of BmNPV were shown in the upper graph and the proteins that belonged to the functional categories of “binding” in GO analysis were showed in the lower graph. IP.1: the IP sample at 72 hpi; IP.2: the IP sample at 96 hpi.
Fig 6.
Co-immunoprecipitation analysis of interactions between different PIFs and BmSUH protein.
(A) Co-IP analysis of BmSUH in NPV-infected BmN cells. BmN cells were infected with recombinant viruses expressing BmSUH-His or co-infected with BmSUH-His and PIFs-Flag. Immunoprecipitation (IP) was performed using anti-BmSUH or anti-IgG antibody followed by immunoblotting (IB) with anti-PIF0, anti-PIF1, anti-PIF2 or anti-Flag antibody individually. (B) BmN cells that were infected or co-infected with recombinant viruses expressing BmSUH-His or/and a Flag-tagged PIFs were subjected to anti-Flag. Inputs and eluates were analyzed by immunoblotting with anti-His.
Fig 7.
The absence of BmSUH affects the interaction of ODV with midgut epithelium cells.
The amounts of labelled ODVR bound (solid line) and fused (dotted line) to the midgut epithelia of fourth-instar WT and ΔBmSUH were detected after larvae feeding of 3 μg of labelled ODVR from BmNPV and incubated with the designed time (n = 24, per time point). The data were analyzed using the GraphPad Prism 9 software. All data were shown as mean ± SEM.
Fig 8.
Proposed model of baculovirus oral infection in wild-type or BmSUH mutants.
BmSUH is widely distributed in the microvilli of Bombyx mori. After ODV virions traverse the peritrophic membrane, PIF complex binds to BmSUH and other host factors in the microvilli of epithelial midgut cells. Then the ODV virions fuse with microvilli, releasing nucleocapsids into the cytoplasm. In BmSUH mutants, PIF complex could not bind to BmSUH. The loss of BmSUH reduced the ODV entry and led to a lower efficiency of oral infection (the right part). The DNA clipart was obtained from the “Icon Library” of iSlide under a CC0 1.0 Universal license. The silkworm was drawn based on a clipart obtained from the “Icon Library” of iSlide under a CC0 1.0 Universal license. Copyright: https://en.islide.cc/copyright-notice.