Perilipin1 inhibits Nosema bombycis proliferation by promoting Domeless- and Hop-mediated JAK-STAT pathway activation in Bombyx mori

ABSTRACT Lipid droplets (LDs) are dynamic organelles that participate in the regulation of lipid metabolism and cellular homeostasis inside of cells. LD-associated proteins, also known as perilipins (PLINs), are a family of proteins found on the surface of LDs that regulate lipid metabolism, immunity, and other functions. In silkworms, pébrine disease caused by infection by the microsporidian Nosema bombycis (Nb) is a severe threat to the sericultural industry. Although we found that Nb relies on lipids from silkworms to facilitate its proliferation, the relationship between PLINs and Nb proliferation remains unknown. Here, we found Nb infection caused the accumulation of LDs in the fat bodies of silkworm larvae. The characterized perilipin1 gene (plin1) promotes the accumulation of intracellular LDs and is involved in Nb proliferation. plin1 is similar to perilipin1 in humans and is conserved in all insects. The expression of plin1 was mostly enriched in the fat body rather than in other tissues. Knockdown of plin1 enhanced Nb proliferation, whereas overexpression of plin1 inhibited its proliferation. Furthermore, we confirmed that plin1 increased the expression of the Domeless and Hop in the JAK-STAT immune pathway and inhibited Nb proliferation. Taken together, our current findings demonstrate that plin1 inhibits Nb proliferation by promoting the JAK-STAT pathway through increased expression of Domeless and Hop. This study provides new insights into the complicated connections among microsporidia pathogens, LD surface proteins, and insect immunity. IMPORTANCE Lipid droplets (LDs) are lipid storage sites in cells and are present in almost all animals. Many studies have found that LDs may play a role in host resistance to pathogens and are closely related to innate immunity. The present study found that a surface protein of insect lipid droplets could not only regulate the morphological changes of lipid droplets but also inhibit the proliferation of a microsporidian pathogen Nosema bombycis (Nb) by activating the JAK-STAT signaling pathway. This is the first discovery of the relationship between microsporidian pathogen and insect lipid surface protein perilipin and insect immunity.

pathogen-infected cells, including bacteria, parasites, and viruses, proving that infectiondriven LD formation promotes innate immune activation and plays key roles in hostpathogen interactions (4).
LD-associated proteins, also known as perilipins (PLINs) are the most abundant proteins on the surface of LDs and regulate lipid storage and hydrolysis by binding to specific sites on the surface of LDs (5).PLINs were first discovered in the LDs of rat adipocytes by Greenberg in 1991 (6).To date, five different PLINs (PLIN1-5) have been identified in various human tissues (7)(8)(9)(10)(11).Both PLIN1 and PLIN2 are localized on the surface of LDs; however, they play opposite roles by recruiting different chaperone proteins from the cytoplasm to promote LD catabolism or synthesis, respectively.PLIN1 recruits hormone-sensitive lipase (HSL) to promote LD catabolism and reduce droplet size, whereas PLIN2 recruits adipose triglyceride lipase (ATGL) to promote LD synthesis and increase LD size (12).PLIN3, PLIN4, and PLIN5 are mainly localized in the cytoplasm and endoplasmic reticulum, and PLIN3 can compensate for PLIN2 in promoting LD stabilization when absent, as well as participate in intracellular transport to produce prostaglandin E2 (13).PLIN4 has been found to be associated with human adipocyte differentiation, whereas PLIN5 promotes LD stabilization and is involved in LD coupling to the mitochondria (14,15).
Microsporidia is a class of obligate intracellular parasitic eukaryotes that exist widely in nature and can infect almost all animals, including invertebrates and vertebrates (16,17).In the 1840s, Louis Pasteur studied the economically damaging silkworm disease pébrine, caused by infection by the microsporidium Nosema bombycis (Nb), which had caused great damage to European sericulture in the 19th century (18,19).The discov ery and naming of Nb are considered the beginning of microsporidia research (20).Microsporidia eject polar tubes to penetrate host cells at relatively short distances, and then the spore plasma is introduced into the host cytoplasm, where it metabolizes host nutrients for growth and reproduction (21).Microsporidian infection triggers a series of alterations in intracellular processes such as basic metabolism, ATP synthesis, protein and fat degradation, apoptosis, and autophagy (22)(23)(24).Some alterations may favor microsporidian proliferation, while others are pathways by which the host inhibits proliferation (25).Regarding intracellular lipid metabolism, the relationship between LDs and microsporidian proliferation remains unclear.We have previously found that Nb infection changes lipid metabolic homeostasis in B. mori and that Nb exploits the lipid metabolic system of silkworms to facilitate its own proliferation (26).In the present study, we investigated the characters of an LD surface protein plin1 and its effects on the proliferation of Nb in silkworms.We reveal a connection between microsporidia pathogens, perilipins, and insect immunity.

Clone and sequence analysis of plin1
To explore the effects of Nb infection on silkworms, we previously examined the genes and proteins that were differentially expressed in the midgut and ovary of silkworm after Nb infection using transcriptomic and quantitative proteomic assays (23).In both data sets, plin1 (BGIBMGA013593 in SilkDB3.0) was differentially expressed.As such, we hypothesized that it may be related to Nb proliferation.Therefore, plin1 (BGIBMGA013593) was cloned from cDNA derived from the silkworm fat body, and the sequence was analyzed.The results showed that the coding region of plin1 was 1311 bp, encoding a protein containing 436 amino acids, which was consistent with PLIN1/LSD-1 from most Lepidoptera insects.A perilipin super family domain (69-369 aa, Accession:cl03851) of the plin1 protein could be found by NCBI Conserved Domains analysis (Fig. 1A and B).It has to be noted that NP_001040143.1 in NCBI and Q2F665_BOMMO in Uniprot are also annotated as the perilipin of silkworm.However, when compared with our cloned BGIBMGA013593, the NCBI-and Uniprot-annotated perilipins lack aa22-60 and aa222-225, respectively (Fig. 1B), and further experiments are needed to verify whether the shorter perilipin is indeed the isoform of plin1.According to the SilkDB3.0 genome database, the chromosomal location of plin1 is on chromosome 6 of the silkworm.The coding region consists of nine exons and eight introns.(Fig. 1C).Comparison of B.mori PLIN1 with that of other species showed that PLIN1 had higher identity with that of Pectinophora gossypiella, Hyposmocoma kahamanoa, Spodoptera frugiperda, and Manduca sexta (with 86%, 79%, 77%, and 82% identity, respectively).Also, the plin1 and plin2 from different insects were collected to investigate the phylogenetic relationships of the insect plins, and the result showed that the insect plins can be divided quite clearly into two main branches (Fig. S1).

Tissue and cellular distribution of plin1
To investigate the expression of plin1 in different tissues of silkworms, the expression of plin1 in the ovaries, midgut, testis, head, Malpighian tubule, hematocytes, silk glands, and fat body of third day fifth instar silkworm larvae was detected using qPCR.The results showed that the expression of plin1 was highest in the fat body and lowest in hematocytes (Fig. 2A), which was consistent with the expression in the SilkDB3.0 database.Plin1 is distributed in large quantities within the fat body, which is the primary site of LD storage and metabolism.
To confirm the cellular localization of plin1 in BmN cells, plin1 was fused and expressed using an eGFP-tag with the recombinant baculovirus vBac-eGFP-plin1.The results showed that the control virus-infected cells only expressed approximately 26 kDa of the eGFP protein, whereas in vBac-eGFP-plin1-infected cells, a band of 70 kDa was detected, consistent with the expected size of the eGFP-plin1 fusion protein, indicating that vBac-eGFP-plin1 was successfully constructed (Fig. 2B).The fluorescence signal of the eGFP-plin1 fusion protein was present mainly in the cytoplasm, which was also consistent with the distribution pattern of LDs that were primarily located in the cytoplasm (Fig. 2C).

Nb infection affected LD size and plin1 expression
Various pathogens have been found to promote the formation of LDs (27)(28)(29).To investigate the changes in LDs after Nb infection, the fat bodies from Nb-infected and -uninfected larvae were collected and stained to observe the morphology of the LDs.The results showed that LDs in the Nb-infected group were larger than those in the control group, suggesting that Nb infection may induce LD accumulation in silkworms (Fig. 3A through D).Previous studies have shown that LDs are associated with infections by pathogens, including viruses, bacteria, fungi, and protists (30).To verify the effect of LDs on Nb reproduction, we initially confirmed that the formation of LDs could be significantly inhibited in BmN cells by IBMX and isoproterenol, which are inhibitors of LD formation (31).Also, the copy number of Nb was significantly higher in the inhibitortreated groups at the early stage [24 hrs post-infection (h p.i.)] but was not significantly different at 48 h p.i. (Fig. 3E), suggesting that intracellular LDs may play a role in the early stages of Nb infection.To determine whether plin1 is associated with Nb infection, the expression of plin1 during Nb infection in the fat body and midgut was detected.The results showed that plin1 was slightly increased from 24 to 48 h p.i., massively increased 72 h p.i., and then decreased dramatically at 96 to 120 h p.i. in the fat body (Fig. 3F).In the midgut tissues, plin1 expression was increased significantly from 24 to 72 h p.i. and then decreased slowly at 96 h p.i. (Fig. 3G).The difference between infected fat bodies and the midgut may indicate that plin1 in the midgut is more sensitive to Nb infection.These results suggest a correlation between Nb infection, LD accumulation, and plin1 expression.

plin1 promotes LD accumulation and inhibits Nb proliferation
As LD surface proteins, PLINs maintain LD homeostasis.Studies have shown that PLIN1 can prevent lipases from approaching LDs, and when the energy is deficient, PLIN1 is phosphorylated to recruit lipase HSL, resulting in lipolysis (32).Plin1 expression leads to LD clustering (33).To investigate the effects of plin1 expression on the LDs in silkworm cells, we overexpressed plin1.A significant number of BODIPY-stained LDs were observed in plin1-overexpressing cells, indicating that plin1 promotes lipid synthesis or inhibits LD degradation (Fig. 4A and B).To confirm the relationship between plin1 and Nb prolifera tion, plin1 in BmN cells was knocked down (KD) by siRNAs.The results showed that the siRNA effectively reduced the expression of plin1 in the cells, and the copy number of Nb significantly increased after plin1 KD, indicating that reduction of plin1 favors prolifera tion of Nb (Fig. 4C and D).In contrast, plin1 overexpression caused a decrease in Nb copy number (Fig. 4E).The results of the overexpression and KD experiments confirmed that plin1 inhibits the proliferation of Nb.There are two perilipin genes in Drosophila, and PLIN1 plays a major role in the regulation of lipid metabolism, whereas PLIN2 plays a compensatory function when PLIN1 was missing (12).The NCBI database contains another gene known as perilipin of B. mori (plin2, accession number NM_001145332).We also investigated the effects of plin2 overexpression and knock down on Nb replication.plin2 had a modest effect on Nb proliferation (Fig. S2), suggesting that plin1 may play a dominant role in anti-Nb activity, whereas plin2 may act as a helper to plin1.

plin1 promotes JAK-STAT immune pathway
Previous studies have shown that LDs are the primary organelles for lipid storage in eukaryotic cells and that they also possess antimicrobial properties (34).In Drosophila, perilipin expression is associated with bacterial-induced activation of the IMD pathway (35).Therefore, we hypothesized that plin1 may inhibit Nb proliferation by affecting the immune pathway in silkworms.To investigate this, we examined the effects of plin1 expression on key genes of the IMD, JAK-STAT, and TOLL pathways in silkworms.The results showed that among the 11 tested genes, only Domeless and Hop from the JAK-STAT pathway were significantly upregulated (Fig. S3 and S4).RT-qPCR analysis demon strated a significant increase in the expression of Domeless and Hop following Nb infection (Fig. 5A and D).To verify the effect of plin1 on Domeless and Hop, we examined their expression in plin1-overexpressing and KD cells.The results showed that the expression of Domeless and Hop decreased after plin1 KD (Fig. 5B and E) and increased after its overexpression (Fig. 5C and F).To test whether Domeless or Hop has a direct effect on Nb proliferation, we also examined the influence of Nb copy number after Domeless or Hop RNAi.The result showed that the number of Nb copies increases significantly when Domeless or Hop was decreased by siRNAs (Fig. S6).Furthermore, we inhibited the JAK-STAT pathway with a JAK inhibitor.The results showed that blocking the JAK-STAT pathway led to an increase in Nb proliferation in both normal cells and plin1-OE cells (Fig. 5G and H), indicating that the JAK-STAT pathway is involved in the process of resistance against Nb proliferation in silkworms.

DISCUSSION
LDs are dynamic and ubiquitous organelles found in all cells ranging from prokaryotes to eukaryotes.In addition to modulating lipid and energy homeostasis, LDs respond to ER stress, oxidative stress, protein maturation, and turnover (36).LD accumulation in the cytoplasm of distinct host cell types has been reported after infection with Plasmodium berghei (37,38), Trypanosoma cruzi (39)(40)(41), Toxoplasma gondii (42)(43)(44), and Leishmania major (45,46).For example, protozoan parasites (such as Leishmania amazonensis and Plasmodium chabaudi) are able to induce LD genesis in non-immune and immune cells that not only accumulate in the host cytoplasm but also relocate around and move into parasitophorous vacuoles (47,48).Salmonellosis induces a time-dependent increase in LD formation in macrophages, and the inhibition of diacylglycerol O-acyltransferase 1 and cytosolic phospholipase A2 significantly reduces intracellular bacterial proliferation (49).Chronic hepatitis C virus (HCV) infection also triggers lipid accumulation in human liver-derived cells (50).In the present study, we found that Nb infection caused the accumulation of LDs in the fat body of silkworms, which is consistent with most patho genic infections, indicating that the accumulated LDs are an important site of antago nism between the infectious agent and the host.
As LD surface proteins, PLINs regulate the formation and decomposition of LDs (51,52).Perilipin is also closely associated with pathogenic infections.PLIN1 can respond to IMD activation and alter morphological changes in LDs to mitigate the immune response against bacterial infections in Drosophila (35).In mice infected with Pseudomonas aeruginosa, upregulation of PLIN2 leads to the accumulation of LDs in lung tissues, which ultimately triggers the release of COX-2-mediated anti-inflammatory cytokines to achieve immune effects (53).In hepatocytes, PLIN2 is necessary for the translocation of HCV nuclei and NS5A proteins to LDs and the formation of functional, low-density HCV viral particles (54).Perilipin KD in shrimp inhibited the proliferation of Vibrio parahaemo lyticus by increasing the production of reactive oxygen species (55).Mycobacterium leprae infection induces perilipin expression to facilitate lipid accumulation within the phagosome and creates a suitable environment for intracellular survival within macro phages (56).In a parasitic infection model, increased levels of PLIN2 were observed in the liver of pregnant mice infected with Plasmodium berghei NK65 (57).Studies have suggested that PLINs interact directly with cytosolic bacteria.The Dictyostelium homolog of mammalian perilipin binds to the cytosolic bacterium Mycobacterium marinum (58).In the current study, we found that plin1 inhibited the replication of Nb.All data confirmed a complex relationship between PLINs and infectious diseases.
Previous transcriptomic data have indicated that Nb infection activates immune pathways such as IMD, Toll, and JAK-STAT in silkworms (25).We found that Domeless and HOP from the JAK-STAT pathway were induced by Nb infection or plin1 expression, suggesting that plin1 may inhibit Nb replication by activating the JAK-STAT pathway.Blocking the JAK-STAT pathway with an inhibitor also confirmed that the JAK-STAT pathway is utilized by silkworms to inhibit Nb proliferation.Previous studies have shown that the JAK-STAT pathway is a functional approach to the mammalian interferon system (25,59) and plays an important role in fat body function and the regulation of lipid and glucose metabolism (60).In insects, the JAK-STAT pathway promotes the production of other proteins, including stress-response proteins and cytokines in the fat body.Furthermore, JAK-STAT signaling stimulates the production of antimicrobial peptides to resist infection (61).Domeless, first identified in Drosophila melanogaster, is a unique receptor involved in the invertebrate JAK/STAT pathway (3,62).Domeless KD significantly increases the replication of Drosophila melanogaster sigma virus (63).Tu et al. found that Domeless KD significantly increased the pathogenicity of Beauveria bassiana against the leaf beetle Plagiodera versicolora (64).In the shrimp Marsupenaeus japonicus, Vibrio infection induced the expression and secretion of peroxiredoxin 4, which could bind to Domeless to abrogate the activation of the JAK/STAT pathway (65).In silkworms, Domeless overexpression upregulates the expression of JAK/STAT pathway-related genes, promotes proliferation, and inhibits apoptosis (66).Hop is a tyrosine kinase in the JAK-STAT pathway, which has been found to autophosphorylate and phosphorylate Domeless in Drosophila.A point mutation in Drosophila Hop causes constitutive activation of the JAK/STAT pathway (67) and mutant Drosophila for Hop ws more susceptible to viral infection compared to the wild-type (68).Dengue virus infection levels can increase in mosquitoes after Hop interference (69).In this study, we revealed that Nb infection induced massive expression of plin1, followed by activation of the JAK-STAT pathway by promoting the expression of Domeless and Hop.It is worth noting that plin1 may not directly act as a transcription factor to promote the expression of Domeless and Hop, as its effect on these genes may be mediated by other proteins.As antimicrobial peptides responded to Nb infection and the JAK-STAT signaling pathway may regulate their expression, we investigated the effects of plin1 on silkworm antimicrobial peptides (70).The results showed the expression of gloverin 2-4 was significantly increased after plin1 overexpression (Fig. S5), suggesting the ultimate effectors of plin1 against Nb prolifera tion may be gloverins.
In conclusion, the present study showed that silkworm plin1 inhibits Nb cell proliferation by promoting the JAK-STAT pathway through the increased expression of Domeless and Hop.The hypothetical anti-Nb pathway of plin1 in silkworms can be summarized as follows: when the signal of Nb infection is transmitted to the nucleus, the transcription of plin1 is activated, which not only promotes the aggregation of LDs but also enhances the expression of Domeless and Hop.The activation of JAK-STAT promotes the expression of antimicrobial peptides, such as gloverins, which inhibit Nb proliferation (Fig. 6).These results provide new insights into the complex relationship between microsporidian pathogens, LD surface proteins, and insect immunity.

Silkworm culture and Nb challenge
Silkworm strain P50 was provided by the Sericultural Research Institute of the Chinese Academy of Agricultural Sciences, Zhengjiang.The larvae were fed with fresh mulberry leaves twice a day.The oral infection of Nb to silkworm was performed as follows: the first day fifth instar larvae were divided into two groups.For the Nb treatment group, fresh mulberry was immersed in 1 × 10 7 spores per mL of Nb solution, air-dried at room temperature, and then fed to the silkworms for 4 hrs.Control larvae were fed dried mulberry immersed in distilled water.Tissues from the midgut, testis, fat body, and so on were collected from the third day fifth instar larva of B. mori P50.

Total RNA extraction and genomic DNA extraction
The RNA isolation total RNA extraction reagent (R401-01, Vazyme, Nanjing, China) was used for total RNA isolation from silkworm tissues or BmN cells.mRNA was reverse-tran scribed into cDNA with HiScript IIQ RT SuperMix for qPCR (R222-01, Vazyme, Nanjing, China).For the copy number of Nb detections, genomic DNA from infected silkworms or BmN cells was extracted using the DNA Kit (Code No.9765, TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0,Japan) according to the instruction manual.

Sequence and phylogenetic analysis
The characteristic domains and sequence of plin1 were obtained from the National Center for Biotechnology Information (NCBI) databases.MEGA software (71) (version 11.0.10) was used to construct the multiple sequence alignment and phylogenetic tree using the maximum likelihood (ML) method and genes from different species.The multiple sequence alignment was constructed based on the amino acid sequence of perilipin proteins.The phylogenetic tree was constructed based on the gene sequence of perilipin (the species accession numbers for comparison are shown in Table S2).

RT-qPCR
The samples from silkworm and BmN cell were collected at different times of injection with Nb or ddH 2 O (control group).After RNA extraction and cDNA synthesis, RT-qPCR was performed using ChamQ SYBR qPCR Master Mix (Q341-02, High ROX Premixed, Vazyme, Nanjing, China).The RT-PCR program was as follows: one cycle of pre-denatura tion for 30 s at 95°C, 40 cycles of 95°C for 10 s, and 60°C for 30 s.The relative mRNA level of genes was determined by the 2 -ΔΔCt method, using BmGADPH as an internal reference.All the primers are listed in Table S1.Nb-tubulin gene-specific primers were used to amplify the tubulin gene of Nb, as indicator of the genome copy number (26).A standard curve described by y = −2.3327x +38.202 (R 2 = 0.9557) was used to evaluate the copy number of Nb.Genomic DNA from infected silkworms or BmN cells (100 ng of each DNA sample) was detected with the following program: one cycle of pre-denaturation for 30 s at 95°C, 40 cycles of 95°C for 5 s, and 60°C for 30 s.

Gene cloning and vector construction
According to the nucleotide sequence of BGIBMGA013593-PA in SilkDB 3.0, primers (Table S1) were designed to amplify plin1 in silkworm cDNA by PCR.The PCR program was 95°C for 3 min, 35 cycles of 95°C for 15 s, 50°C for 15 s, and 72°C for 1 min, with a final stage of 72°C for 5 min.The plin1 gene was cloned into the plasmid pIZ-mCherry to obtain pIZ-mCherry-plin1 (72).The plin1 gene was cloned into the plasmid vBac-eGFP to obtain vBac-eGFP-plin1.EcoRI and XhoI restriction sites were used to clone into both of the abovementioned vectors.All the plasmid constructs were verified by sequencing.

LD staining and microscopy
The fat body or BmN cells with pIZT-mCherry-plin1 was collected and fixed in 4% paraformaldehyde fix solution (E672002, BBI, Sangon Biotech, Shanghai, China) for 10 min on ice.Fat bodies were then rinsed twice with PBS and incubated in PBS containing 1 µg/mL of BODIPY 493/503 (ajci70160, Amgicam, Shanghai, China) for 30 min, 1 µL/mL DAPI (D-9564, SIGMA) was added to stain nuclei for 5 min.After staining, fat bodies were rinsed three times with PBS.Stained samples were mounted in 80% glycerol (A100854, Diamond, Sangon Biotech, Shanghai, China) for microscopy analysis (73).All images were taken using an inverted fluorescence microscope and cellSens Standard software (OLYMPUS, BX53M, Japan).To quantify LD size and number in each cell form 30 fat body cells, the ImageJ analysis software was used to measure the size and number (74).

Treatment of cells with the LD inhibitor and JAK inhibitor
BmN cells were incubated in a medium containing 0.5-1mM IBMX (HY-12318, MCE, Nanjing, China) and 1 mM isoproterenol (HY-B0468, MCE, Nanjing, China) for 24 h and 48 h, respectively.When these two chemicals were used in combination, LDs were found to disperse in cells (31).BmN cells were treated with 0.5-1 mM JAK inhibitor (HY-18300, Filgotinib, MCE, Nanjing, China) to inhibit JAK synthesis (75).The copy number of Nb was detected after using the LD inhibitor and JAK inhibitor with RT-qPCR.

FIG 1
FIG 1 Cloning and sequence characteristics of plin1.(A) PCR of plin1 with cDNA from the fat body tissues of the silkworm.The gel image shows the DL5000 DNA marker and the 1,311 bp PCR product of plin1.(B) CDS and protein sequences of plin1.The amino acid sequence from 69 to 369 codes for the perilipin superfamily (accession: cl03851, shown in blue).The missing amino acids (aa22-60 and aa222-225) in NCBI-annotated perilipin were framed.(C) Intron and exon distributions of plin1 on chromosome 6 of the silkworm.

FIG 2
FIG 2 Expression of plin1 in silkworm tissues and BmN cells.(A) The relative expression of plin1 was measured in the tissues of third day fifth instar larva of the silkworm and normalized to the ovary.The experiment was repeated three times.(B) Western blotting analysis of eGFP-plin1 with anti-GFP antibody.lane 1: marker: 180 kDa, lane 2: vBac-eGFP-infected cells.lane 3: vBac-eGFP-plin1-infected cells.(C) Subcellular localization of plin1 in BmN cells.Error bars represent mean ± SD.One-way ANOVA or t-test, **P < 0.01; ****P < 0.0001.ns, not significant.

FIG 3
FIG 3 Effects of Nb infection on lipid droplet size and plin1 expression.(A) Fat body of uninfected and infected groups; CK and Nb +were collected at 72 h p.i.Samples were stained with BODIPY 493/503 (green) and DAPI (blue) for LD visualization; scale bar = 20 µm.(B) Statistics of LD size in the fat body from CK and Nb +silkworms.(C) IBMX and isoproterenol inhibit LD synthesis.BmN cells were stained with BODIPY 493/503 (green) and DAPI (blue) for LD visualization; scale bar = 20 µm.(D) Average number of LDs per cell in the IBMX-and isoproterenol-untreated and -treated cells.(E) Copy number of Nb after IBMX and isoproterenol treatment.(F and G) plin1 expression in the fat body and midgut of Nb-infected silkworms.Error bars represent mean ± SD.All the experiments were repeated three times.One-way ANOVA or t-test, *<I>P < 0.05, **P < 0.01, and ****P < 0.0001.ns, not significant.

FIG 5
FIG 5 plin1 enhances the anti-Nb effect of the JAK-STAT pathway by promoting Domeless and Hop expression.(A, D) Quantification of Domeless and Hop expression by RT-qPCR in BmN cells at 24, 48, and 72 hrs post-Nb infection.(B, E) Quantification of Domeless and Hop expression after knockdown of plin1 in BmN cells.BmN cells transfected with dsRNA-eGFP were used as a negative control.(C, F) Quantification of Domeless and Hop expression after overexpression of plin1 in BmN cells.BmN cells transfected with the empty vector were used as a negative control.(G) Quantification of Nb copy number by RT-qPCR after treatment with the JAK inhibitor at 24 and 48 h p.i. in BmN cells.BmN cells treated with DMSO were used as the negative control.(H) Quantification of Nb copy number by RT-qPCR after treatment with the JAK inhibitor and overexpression of plin1 in BmN cells.BmN cells treated with DMSO were used as the negative control.All the experiments were repeated three times.Error bars represent mean ± SD.One-way ANOVA or t-test, *<I>P < 0.05, **<I>P < 0.01, and ***<I>P < 0.001.ns, not significant.

FIG 6
FIG 6 Overview of plin1 inhibiting Nb proliferation in silkworms.(A-B) Accumulation of lipid droplets and expression of plin1 were induced by Nb infection.(C) Expression of Domeless and Hop is promoted by plin1, followed by activation of the JAK-STAT immune pathway.(D-E) plin1 promotes the expression of antimicrobial peptides gloverin 2 and gloverin 3, which inhibit the proliferation of Nb.