Molecular cloning and analysis of PGRP-L1 and IMD from silkworm Bombyx mori

Accepted Manuscript Molecular cloning and analysis of PGRP-L1 and IMD from silkworm Bombyx mori

Ming-Yue Zhan, Pei-Jin Yang, Xiang-Jun Rao PII: DOI: Reference:

S1096-4959(17)30160-4 doi:10.1016/j.cbpb.2017.10.002 CBB 10135

To appear in: Received date: Revised date: Accepted date:

19 July 2017 18 October 2017 19 October 2017

Please cite this article as: Ming-Yue Zhan, Pei-Jin Yang, Xiang-Jun Rao , Molecular cloning and analysis of PGRP-L1 and IMD from silkworm Bombyx mori. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Cbb(2017), doi:10.1016/j.cbpb.2017.10.002

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Molecular Cloning and Analysis of PGRP-L1 and IMD from silkworm Bombyx mori

Ming-Yue Zhan, Pei-Jin Yang, Xiang-Jun Rao*

SC

RI

PT

School of Plant Protection, Anhui Agricultural University, Hefei, Anhui 230036, China

NU

Running title: Analysis of silkworm PGRP-L1 and IMD

MA

ms. has 39 pages, 12 figures, 1 table

*Corresponding author:

Associate Professor

PT E

Department of Entomology

D

Xiang-Jun Rao, PhD

School of Plant Protection

Anhui Agricultural University

CE

Hefei, Anhui 230036 China Tel: + 86 055165786312

AC

[email protected]

1

ACCEPTED MANUSCRIPT Abstract Peptidoglycan is one of the major components of bacterial cell wall. The innate immune system of insects utilizes a group of peptidoglycan recognition proteins (PGRPs) for the recognition of specific peptidoglycans and activating immune signaling pathways. In Drosophila melanogaster, PGRP-LC and IMD (immune

PT

deficiency) are two important signaling molecules of the IMD pathway. Here we cloned

RI

and characterized PGRP-L1 and IMD from the domesticated silkworm Bombyx mori (BmPGRP-L1 and BmIMD). BmPGRP-L1 gene consists of five exons that encodes a

SC

polypeptide of 304 amino acids with a transmembrane region and an extracellular

NU

PGRP domain. The PGRP domain lacks key residues for the amidase activity. BmIMD cDNA encodes a polypeptide of 250 amino acids with a death domain. BmPGRP-L1

MA

and BmIMD were expressed in various tissues and induced by bacterial challenges. In addition, in vivo blocking of the PGRP domain by the antiserum or purified antibody

D

significantly reduced the expression of some antimicrobial peptide genes. The

PT E

extracellular region of BmPGRP-L1 bound to diaminopimelic acid-type and lysine-type peptidoglycans. Overexpression of full-length BmIMD in Drosophila Schneider 2 cells

CE

significantly induced three antimicrobial peptide genes. These results suggest that BmPGRP-L1 and BmIMD may be players in the IMD pathway of B. mori. This study

AC

provides a foundation for further studies on the functions of silkworm IMD pathway.

Key words: Insect immunity; Silkworm; PGRP; IMD.

2

ACCEPTED MANUSCRIPT 1. Introduction Peptidoglycan (PGN) is an essential component of the cell wall of gram-negative and gram-positive bacteria. Peptidoglycan is comprised of a backbone of β-1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) cross-linked by short stem peptides (Kurata, 2014). Most gram-negative bacteria have diaminopimelic

PT

acid (Dap)-type peptidoglycan, while gram-positive bacteria have lysine (Lys)-type

RI

peptidoglycan (Vollmer et al., 2008). Peptidoglycans are recognized by a group of immune receptors named peptidoglycan recognition proteins (PGRPs). PGRPs have at

SC

least one conserved PGRP domain homologous to the catalytic domain of N-

NU

acetylmuramyl-alanine amidases (Kurata, 2014; Royet et al., 2011). The first PGRP reported was BmPGRP-S1, a 19 kDa protein from the hemolymph of B. mori (Yoshida

MA

et al., 1996). PGRPs exist in most animals, but they are absent in lower metazoan or plants. Drosophila melanogaster has 13 PGRP genes encoding at least 19 products

D

(Werner et al., 2000). Anopheles gambiae has 7 PGRP genes. B. mori has 12 PGRP

PT E

genes (Tanaka et al., 2008). Yellow fever mosquito Aedes aegypti has 7 PGRP genes (Wang and Beerntsen, 2015). Mammals have four PGRP genes (Guan and Mariuzza,

CE

2007). Some PGRP domains preserve key residues for the catalytic activity, while others have lost the activity but still bind to peptidoglycans. Drosophila PGRP can be

AC

categorized into the short-type and long-type. Short-type PGRP have signal peptides; long-type PGRP may be extracellular, intracellular or transmembrane proteins (Werner et al., 2000). For the humoral immunity of D. melanogaster, the production of antimicrobial peptides is mainly regulated by the Toll and IMD pathways (Anderson, 2000; Kaneko and Silverman, 2005; Romeo and Lemaitre, 2008). The Toll pathway mediates the recognition of fungi and gram-positive bacteria (Valanne et al., 2011). Binding of 3

ACCEPTED MANUSCRIPT circulating receptors to β-1,3-glucan and Lys-type peptidoglycan activates a protease cascade to trigger the cleavage of Spätzle, which binds to the transmembrane receptor Toll (Morisato and Anderson, 1994; Schneider et al., 1994). The IMD pathway is responsible for mediating responses to gram-negative bacteria and viruses (Costa et al.,

PT

2009; Kaneko et al., 2004; Lemaitre et al., 1995; Myllymäki et al., 2014). Dap-type peptidoglycans from gram-negative bacteria can be directly recognized by PGRP-LC

RI

(Choe et al., 2002; Gottar et al., 2002). PGRP-LC is a transmembrane receptor with

SC

multiple isoforms that mediate the recognition of different forms of Dap-type peptidoglycans (Choe et al., 2002; Gottar et al., 2002; Ramet et al., 2002). PGRP-

NU

LCa/LCx heterodimer recognizes the monomeric Dap-type peptidoglycan. PGRP-

MA

LCx/LCx homodimer recognizes the polymeric form (Kaneko et al., 2004; Mellroth et al., 2005; Stenbak et al., 2004). Binding of peptidoglycans to PGRP-LC induces its

D

dimerization and the recruitment of death domain-containing proteins IMD, FADD and

PT E

the caspase DREDD to form a complex. IMD is similar to mammalian receptorinteracting protein 1 (RIP1) (Georgel et al., 2001; Lemaitre et al., 1995). DREDD is polyubiquitinated by ubiquitin E3 ligase DIAP2 and cleaves IMD between D30 and

CE

A31, exposing the IAP-binding motif (IBM) AAPV. The cleaved IMD binds DIAP2

AC

via its BIR2/3 domains. In conjunction with the E2-ubiquitin-conjugating enzymes UEV1a, Bendless (Ubc13), and Effete (Ubc5), IMD is K63-polyubiquitinated (Paquette et al., 2010). The polyubiquitinated IMD activates downstream kinases TAK1 (TGF beta-activated kinase), which phosphorylates IKK-β/γ. NF-kappa B transcription factor Relish precursor is cleaved to activate expression of effector genes (Dushay et al., 1996; Ganesan et al., 2011; Ip et al., 1993; Reichhart et al., 1993).

4

ACCEPTED MANUSCRIPT So far, only a few homologs of IMD and PGRP-LC have been identified in Arthropods. Two IMD homologs were identified from Chinese whiteshrimp Fenneropenaeus chinensis and red swamp crayfish Procambarus clarkia (Feng et al., 2014; Lan et al., 2013). PGRP-LA, -LB, -LC, -LD and –LE have been identified in the yellow fever mosquito, Aedes aegypti (Wang and Beerntsen, 2015) and the red flour

PT

beetle, Tribolium castaneum (Koyama et al., 2015). PGRP-LC of the malaria mosquito

RI

Anopheles gambiae mainly senses Gram-negative infection and activates the Relish

SC

homolog REL2 (Meister et al., 2009). A genome-wide analysis of genes and gene families involved in innate immunity of B. mori showed that there are 6 PGRP genes

NU

(BmPGRP-L1 to L6) coding for long-type PGRPs ranging from 246 to 325 amino acids (aa) (Tanaka et al., 2008). BmPGRP-L1 to L5 are located on chromosome 1. Only

MA

BmPGRP-L1 and BmPGRP-L4 have transmembrane regions. BmPGRP-L2, -L3 and L5 have signal peptides. BmPGRP-L6 lacks signal peptides. RNAi of BmPGRP-L1

D

showed that it is related to the expression of some antimicrobial peptide genes (Yang

PT E

et al., 2016). BmPGRP-L6 is involved in the activation of E. coli or E. colipeptidoglycan mediated antimicrobial peptide promoter activation. BmPGRP-L6 binds

CE

to Dap- and Lys-type peptidoglycans (Tanaka and Sagisaka, 2016). So far, most studies on the IMD pathway have been performed in D. melanogaster, with only limited studies

AC

in silkworms, which is an important resource insect (Xia et al., 2004). We first report the cloning of full-length cDNAs of BmPGRP-L1 and BmIMD. The sequence features and phylogenetic relationships were analyzed. The tissue distribution and transcriptional responses to gram-negative and gram-positive bacteria were analyzed. By overexpressing full-length or deletion constructs in Drosophila Schneider 2 (S2) cells, the expression of some antimicrobial peptide genes was elevated. Binding

5

ACCEPTED MANUSCRIPT of recombinant BmPGRP-L1 to Dap-type and Lys-type peptidoglycans was examined by ELISA. 2. Materials and methods 2.1 Insects, bacteria and materials Silkworm (Dazao) larvae were reared with fresh mulberry leaves at 25±1°C,

PT

photoperiod 12L:12D, and 75±5% relative humidity. Escherichia coli DH5α (E. coli,

RI

HM01), BL21(GZEC-3), Staphylococcus aureus (S. aureus, RCB1010), Bacillus

SC

subtilis (B. subtilis, MPF_80) and S2 cells were kindly provided by Dr. Erjun Ling in Shanghai Institutes for Biological Sciences (SIBS). Peptidoglycans from E. coli K12

NU

(PGN-EK) and S. aureus (PGN-SA) were purchased from InvivoGen (San Diego, CA, USA). For the oral feeding assay, silkworm larvae were starved for 12 hours before

MA

feeding. Mulberry leaves were cut into 2x2 cm squares and soaked in PBS (137 mM NaCl, 12 mM Phosphate, 2.7 mM KCl, pH 7.4) or bacteria (4x106 cfu/μL) overnight.

D

For the bacterial injection assay, 5th instar larvae were injected with heat-killed bacteria

PT E

(106 cfu/larva). For the S2 cell stimulation assay, 106 live bacteria were washed, resuspended in PBS and mixed with S2 cells in 6-well plates for 6h before collecting

CE

cells for analysis. Whole worms and the midgut (for bacterial feeding assay) and the fat body (for bacterial injection assay) were collected. To extract membrane protein from

AC

larval tissues, 5th instar larvae were dissected on ice. Tissues were collected in microcentrifuge tubes and immediately homogenized in RIPA buffer (10 mM Tris-Cl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1mM PMSF, pH8.0). The mixture was centrifuged at 4 °C, 12,000g for 10 min. The supernatant was loaded on SDS-PAGE gels for western blotting. 2.2 RNA isolation, cDNA synthesis and qPCR analysis

6

ACCEPTED MANUSCRIPT Tissues (cells) were homogenized in RNAiso plus for extraction of total RNA (TaKaRa Biotechnology, Dalian, China). cDNA was synthesized with the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, MA, USA). qPCR was performed (95 °C 30s, 40 cycles of 95 °C 5s, 60 °C 30s) on BioRad CFX96 with Thunderbird SYBR qPCR mix (Toyobo, Osaka, Japan). The expression level of target

PT

genes was normalized to ActinA3 (for B. mori) and RP49 (for D. melanogaster). The

RI

relative expression levels were calculated by the 2(-∆∆CT) method, where ΔΔCT=

SC

(CTtarget– CTreference)treated –(CTtarget– CTreference)control. 2.3 Bioinformatic analysis gene

information

was

obtained

NU

The

from

the

KAIKObase

(http://sgp.dna.affrc.go.jp/KAIKObase/). The nucleotide and protein sequences were

MA

analyzed with DNAMAN 6.0 (Lynnon Corporation, Quebec, Canada). The conserved motifs were predicted by SMART (http://smart.embl-heidelberg.de/). Signal peptides

sites

were

PT E

glycosylation

D

were predicted by SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). N- and Opredicted

(http://www.cbs.dtu.dk/services/NetNGlyc/)

by and

NetNGlyc NetOGlyc

1.0 4.0

CE

(http://www.cbs.dtu.dk/services/NetOGlyc/). Potential phosphorylation sites were

AC

predicted by NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/). Potential ubiquitination sites were predicted by UbPred (http://www.ubpred.org/). Neighborjoining trees were constructed in MEGA6 (Tamura et al., 2013). 2.4 Expression and purification of the extracellular region of BmPGRP-L1 A cDNA fragment encoding the extracellular region (105-304) of BmPGRP-L1 was amplified (with primer set BmPGRP-L1ecto-F/BmPGRP-L1ecto-R) and subcloned into NdeI-XhoI sites of pET-30a vector (Novagen, Madison, WI, USA). A C-terminally 6xHis tagged recombinant protein was generated after the induction by 7

ACCEPTED MANUSCRIPT IPTG (isopropyl-β-d-thiogalactoside) in BL21 cells. Briefly, BL21 cells were grown in LB (Luria-Bertani) medium to OD = 0.6. 1mM IPTG was used to induce protein expression for 6 hours at 37°C, 200rpm. Bacteria were collected, resuspended in PBST (PBS+ 0.1% v/v Tween-20) and sonicated for a total of 5 min. The inclusion body was collected, dissolved in buffer B (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, pH8.0)

PT

and mixed with Ni-NTA (Nickel-nitrilotriacetic acid) slurry in a gravity-flow column.

RI

After two washes with buffer C (same with buffer B except for pH6.3), the

SC

recombinant protein was eluted by buffer D (same with buffer B except for pH5.9). The elutions containing BmPGRP-L1 were pooled and dialyzed in succession in

NU

refolding buffers (20 mM Tris-HCl, 150 mM NaCl, 2 mM GSH, 0.02 mM GSSG, 10% glycerol, pH7.4) containing 6 M, 4 M, 2 M urea and finally in TBS (20mM Tris and

MA

150mM NaCl, pH7.4) with 10% glycerol. The purified BmPGRP-L1 was used to immunize a rabbit to collect antiserum (Huabio, Hangzhou, China). Proteins were

D

separated on 15 % SDS-PAGE gels and stained with coomassie brilliant blue, or

PT E

transferred to PVDF (polyvinylidene difluoride) membranes, blotted with the antibody (1:1,000) or anti-6xHis IgG (Huabio, Hangzhou, China) and goat anti-rabbit IgG-HRP

CE

(1:10,000, Santa Cruz Biotech, Dallas, Texas, USA). Western blotting bands were

AC

visualized with the DAB staining kit (Boster Biotech, Wuhan, China). 2.5 Cloning of full-length BmIMD by 3’ RACE A cDNA fragment encoding a death domain-containing IMD-like protein with an incomplete 3’ end was identified in GenBank (XM_004921474). Primers were designed based on the fragment to clone the full-length cDNA. Briefly, the oligo(dT)containing adapter primer (AP) was annealed to mRNA. AP was extended to the 5’ end of mRNA using SuperScript II Reverse transcriptase (Thermo Fisher Scientific, MA, USA). RNA template was degraded by RNase H. A user-designed Gene-Specific 8

ACCEPTED MANUSCRIPT Primer (GSP) and the Universal Amplification Primer (UAP) were used for PCR amplification of cDNA. The primary PCR product was used for a further amplification using Abridged Universal Amplification Primer (AUAP) and the ‘nested’ GSP. The full-length cDNA of BmIMD was deposited in GenBank. 2.6 Full-length and deletion constructs for expression in S2 cells

PT

Full-length and deletion constructs of BmPGRP-L1 and BmIMD were cloned from

RI

cDNA and subcloned into KpnI-XbaI sites of pAc5.1/V5-His B vector (Thermo Fisher

SC

Scientific, MA, USA). BmPGRP-L1 constructs were generated with a C-terminal FLAG tag. The primer sets used for subcloning were as follows: BmPGRP-

NU

N1/BmPGRP-C1 for full-length BmPGRP-L1 (313 amino acids); BmPGRPN1/BmPGRP-C2 for BmPGRP-L1-N (133 amino acids); BmPGRP-N2/BmPGRP-C1

MA

for BmPGRP-L1-C (253 amino acids). BmIMD constructs were generated with a Cterminal V5 and 6xHis tag. The primer sets used for subcloning were as follows:

D

BmIMD-N1/BmIMD-C1 for full-length BmIMD (281 amino acids); BmIMD-

PT E

N1/BmIMD-C2 for BmIMD-N (182 amino acids); BmIMD-N2/BmIMD-C1 for

CE

BmIMD-C (132 amino acids). All primers were listed in Table 1. 2.7 Antiserum and antibody blocking assay

AC

To purify antibodies from antisera, antisera were filtered through a 0.22 μm filter and mixed 1:1 with the binding/wash buffer (0.15 M NaCl, 20 mM Na2HPO4, pH7.0). The diluted antisera were mixed with protein A agarose for one hour at 4 °C (Sangon Biotech, Shanghai, China). Antibodies were eluted with the elution buffer (0.1 M Glycine, pH3.0) and neutralized by the neutralization buffer (1M Tris-HCl, pH8.5). For the in vivo blocking assay, 50 μL BmPGRP-L1 antiserum or 10 μg purified PGRP-L1 antibody was injected into each 5th instar worm. The rabbit preimmune serum or 9

ACCEPTED MANUSCRIPT BmActin3 antibody was injected similarly as controls. After one hour, 2 μL E. coli or B. subtilis (106 cfu/μL) was injected into the antiserum-injected larvae. 5 μg PGN-EK or PGN-SA was injected into the antibody-injected larvae. After three hours, midgut and fat body were collected for qPCR analysis of antimicrobial peptide genes. The

injected or BmActin3 antibody-injected larvae, respectively.

RI

2.8 ELISA

PT

relative expression levels were calculated by normalizing to the preimmune serum-

SC

Binding of the extracellular region of BmPGRP-L1 to peptidoglycans was analyzed by plate ELISA. Peptidoglycan from E. coli K12 (PGN-EK) and S. aureus

NU

(PGN-SA) were diluted to 40 μg/mL with pyrogen-free water. ELISA plates (Costar Corning, NY, USA) were coated with 2 μg/well of peptidoglycans and air dried.

MA

Uncoated wells were used as negative controls. Coated and uncoated wells were washed twice with TBS+0.1% tween-20, then blocked with TBS+10 mg/mL BSA for 2h at

D

37ºC. After 3 washes with TBS + 0.1% tween-20, different amounts of recombinant

PT E

BmPGRP-L1(ecto) were added and incubated 2h at 37 °C. 6xHis tagged recombinant EGFP was added as control. Plates were washed 3 times with TBS + 0.1% tween-20

CE

and incubated with rabbit polyclonal anti-6xHis antibody (Huabio, Hangzhou, China)

AC

(1:2,000 in TBS+1 mg/mL BSA, 100 μL/well) for 2h at 37 ºC. Plates were washed 3 times again and incubated with goat anti-rabbit IgG conjugated to HRP (Santa Cruz Biotech, Dallas, Texas, USA) (1:3,000 in TBS+1 mg/mL BSA, 100 μL/well). After 3 washes, the EL-TMB Chromogenic Reagent kit (Sangon Biotech, Shanghai, China) was used for color development. Absorbance at 450 nm was read on the Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific, MA, USA). 2.9 Statistical analysis 10

ACCEPTED MANUSCRIPT Data were plotted in GraphPad Prism 5 (GraphPad, San Diego, CA, USA). Statistical analyses were performed by student’s t-tests (for 2 groups) or one-way ANOVA (for ≥ 3 groups). Data were represented as means from three replicates ± SD. 3. Results 3.1 Sequence analysis, alignments and phylogenetic trees

PT

Based on a genome-wide analysis of genes and gene families involved in innate

RI

immunity in Bombyx mori, we identified the full-length cDNA of BmPGRP-L1

SC

(Accession numbers: KT907183; BGIBMGA000583-TA) (Tanaka et al., 2008). The cDNA contains an ORF of 912 nucleotides that encodes a 304 amino acids polypeptide

NU

(Fig. 1A). BmPGRP-L1 gene is about 13 kb on chromosome 1 (KAIKOGA018564) (Fig. 1B). The domain structure is similar to DmPGRP-LC with a cytosolic region, a

MA

transmembrane region and a PGRP domain in the extracellular region. However, the cytosolic region of BmPGRP-L1 (81 aa) is about 200 amino acids shorter than that of

D

DmPGRP-LCx (291 aa). The RHIM (RIP homotypic interaction motif) sequence was

PT E

not found in the cytosolic region of BmPGRP-L1. In DmPGRP-LC and PGRP-LE, the RHIM region is critical for signal transduction but dispensable for interacting with IMD.

CE

The PGRP domains of BmPGRP-L1 and DmPGRP-LCx showed an identity level of 41%. Four N-glycosylation sites (N42, N53, N109 and N136) and three O-

AC

glycosylation sites (T52, S126 and T 142) were predicted. The phylogenetic analysis reveals that BmPGRP-L1 is grouped with Lepidopteral homologs. BmPGRP-L1, -L3 and –L5 are paralogs derived from gene duplication events. The orthologs of BmPGRPL1 are identified in Lepidopteral species, such as Plutella xylostella, Helicoverpa armigera, Amyelois transitella, Papilio machaon and Papilio xuthus, but not in Drosophila, Anopheles and Tribolium. These Lepidopteral homologs originated from a common ancestor (Fig. 2A). Two of the five critical residues for Zn2+ binding and the 11

ACCEPTED MANUSCRIPT amidase activity are absent in the PGRP domain of BmPGRP-L1 (Fig. 2B). Therefore, the PGRP domain of BmPGRP-L1 does not have catalytic activities and it may serve as an immune receptor. The cDNA coding for partial BmIMD was identified from GenBank (accession

PT

number: XM_004921474; BGIBMGA003655-TA). We performed 3’ RACE to get the full-length cDNA (accession number: MF374784). The ORF of BmIMD contains 750

RI

nucleotides that encodes a 250 amino acids polypeptide with a death domain at the C-

SC

terminus. Six O-glycosylation sites (T17, S72, S82, S83, S127 and T158) and four polyubiquitination sites (K92, K132, K147 and K199) were predicted (Fig. 3A).

NU

BmIMD is similar to DmIMD (similarity: 45.8, identity 25.3%). Phylogenetic analysis

MA

reveals that Lepidoptera, Diptera and Coleoptera IMDs formed three groups. BmIMD is tightly grouped with the Lepidopteral orthologs (Fig. 3B). Cleavage of DmIMD is critical for activating TAK1, so the potential caspase cleavage site was analyzed by

PT E

D

comparing the amino acid sequences of different IMDs. The cleavage site between D and A is conserved for all the IMDs. The IBM motif is also conserved in different IMDs

CE

(Fig. 3C).

3.2 Protein expression in E. coli and S2 cells

AC

The extracellular region of BmPGRP-L1 was expressed in E. coli in the insoluble form. The recombinant protein migrated to about 24 kDa on the staining gel. Both PGRP-L1 antibody and HisTag antibody detected the recombinant protein at the same position. A faint band of about 45 kDa was detected by PGRP-L1 antibody. BmActin3 antibody detected recombinant BmActin3 at about 45 kDa (Fig. 4A). Full-length and deletion constructs of BmIMD and BmPGRP-L1 were subcloned into the pAc5.1/V5His B vector for constitutive expressions driven by the Drosophila Ac5 promoter (Fig. 4B). Most overexpressed proteins migrated to positions larger than the predicted 12

ACCEPTED MANUSCRIPT molecular weight, which is probably due to post-translational modifications (Fig. 4C). This is consistent with another report (Choe et al., 2005). 3.3 Tissue distribution analysis The tissue distribution was analyzed by qPCR and western blot. BmPGRP-L1 was expressed mainly in epidermis, fat body and midgut. BmIMD was mainly expressed in

PT

epidermis, testis and ovary (Fig. 5A). Western blot using PGRP-L1 antibody showed

RI

weak bands midgut, fat body and hemocytes (Fig. 5B).

SC

3.4 Expression profiles induced by microbial challenges

The mRNA changes of BmPGRP-L1 and BmIMD in the bacteria-challenged larvae

NU

were analyzed. For the midgut of bacteria-fed larvae, BmPGRP-L1 mRNA increased

MA

around 20-fold 12 hours after feeding of E. coli or S. aureus. BmIMD also increased significantly at some time points, the induction level was lower than 3-fold.

D

Antimicrobial peptide genes CecA and Glo1 showed significant inductions (Fig. 6).

PT E

These data indicate that BmPGRP-L1 and BmIMD may be involved in immune signaling in the midgut.

CE

Fat body is an important antimicrobial peptide-producing tissue for insects. Injection of bacteria into the body cavity can significantly induce immune-related genes

AC

in the fat body (Rao and Yu, 2010). Injection of S. aureus caused a significant induction of BmPGRP-L1 after 6 hours. BmIMD didn’t show significant inductions by either bacteria. Glo1 and CecA showed significant inductions to around 10 and 30-fold, respectively (Fig. 7). For whole larvae, after feeding for 6 hours, E. coli induced BmPGRP-L1 to around 3-fold, while BmIMD decreased about 2-fold. FADD and DREDD, another two

13

ACCEPTED MANUSCRIPT signaling molecules of the IMD pathway, didn’t show drastic changes. Leb3 and CecA showed notable inductions after 6 hours (Fig. 8). 3.5 PGRP-L1 in vivo blocking assay The extracellular region of PGRP-L1 was blocked by PGRP-L1 antiserum or preimmune serum, then the induction of antimicrobial peptide genes by bacteria were

PT

measured to obtain the relative expression level. The results showed that blocking of

RI

BmPGRP-L1 severely affected the induction of Gloverin1 by both bacteria in midgut

SC

and fat body. Gloverin2 was affected to a lesser extent, except for the midgut by B. subtilis. CecropinA and Moricin were both negatively affected (Fig. 9). Similarly,

NU

PGRP-L1 was blocked by purified PGRP-L1 antibody or BmActin3 antibody and induced by PGN-EK or PGN-SA. The inductions in BmActin3 antibody-blocked

MA

midgut were significantly higher than those in the PGRP-L1 antibody-blocked larvae (Fig. S). These data suggest that blocking of the PGRP domain affected the immune

PT E

D

signaling in vivo.

3.6 Expression of BmPGRP-L1 and BmIMD in S2 cells The mRNA changes of Metchikowin, Drosomycin, DiptericinA and DiptericinB in

CE

S2 cells in response to bacteria were first examined. Both types of bacteria significantly

AC

induced these antimicrobial peptide genes. The highest induction level was 15-fold (Fig. 10A). Full-length or deletion constructs of PGRP-L1 and IMD were overexpressed in S2 cells to analyze relative mRNA changes of Drosomycin, DiptericinA and DiptericinB (Gross et al., 1996; Hedengren et al., 2000; Zhang and Zhu, 2009). Overexpression of BmPGRP-L1-N and –C caused significantly induced target genes. Overexpression of BmIMD-FL induced all three target genes (Fig. 10B). These results indicate that BmPGRP-L1 and BmIMD can stimulate Drosophila antimicrobial peptide genes. 14

ACCEPTED MANUSCRIPT 3.7 Binding of recombinant BmPGRP-L1(ecto) to Dap- and Lys-type peptidoglycans To determine if the PGRP domain of BmPGRP-L1 can bind to Dap- and Lys-type peptidoglycans. Plate ELISA assays were performed for E. coli-PGN and S. aureus-

PT

PGN. Peptidoglycans were immobilized to interact with recombinant BmPGRP-L1. An increase in OD450 indicates binding of BmPGRP-L1 to the ligands. The results showed

RI

that recombinant BmPRRP-L1 can bind both types of peptidoglycans in a dose-

SC

dependent manner. The recombinant EGFP can not bind to either peptidoglycan (Fig.

AC

CE

PT E

D

MA

NU

11).

15

ACCEPTED MANUSCRIPT 4. Discussion The domain structure of BmPGRP-L1 is similar to DmPGRP-LC, which are both type II transmembrane proteins with an N-terminal cytosolic region, a single-pass TM region and a C-terminal extracellular region with a PGRP domain (Choe et al., 2005). The cytosolic region of BmPGRP-L1 is shorter than DmPGRP-LC and the RHIM motif

PT

is absent. In addition, our coimmunoprecipitation assay failed to detect the interaction

RI

between BmPGRP-L1 and BmIMD (data not shown). Drosophila PGRP-LC have three splicing forms: PGRP-LCa, PGRP-LCx and PGRP-LCy, which have identical cytosolic

SC

regions but different extracellular regions (Choe et al., 2002). Dimerization of PGRP-

NU

LCa and PGRP-LCx enables Drosophila to recognize monomeric peptidoglycans (Chang et al., 2006; Chang et al., 2005). Notably, BmPGRP-L1 to L5 are all type II

MA

transmembrane PGRPs and their cytosolic regions (ranging from 25 to 94 amino acids) are all shorter than that of DmPGRP-LC (291 amino acids) (Tanaka et al., 2008). We

D

speculate that either there are longer splicing transcripts, or PGRPs with shorter

PT E

cytosolic regions have different signaling mechanisms. All the IMD-like proteins from different insect species are all about 250 amino

CE

acids long. The caspase cleavage site and the IBM motif are conserved completely (Fig.

AC

3C). This suggests that the cleaving and activating mechanism also may be conserved. DmIMD is K63-polyubiquitinated at K137 and K153 by two E2 enzymes Ubc5 and Ubc13-Uev1a, in conjunction with the E3 ligase Diap2. Ubiquitinated IMD activates TAK1, which phosphorylates IMD to promote proteasomal degradation of IMD to restore homeostasis (Chen et al., 2017). BmDredd is the homologue of human caspase8. Overexpression of BmDredd in BmN-SWU1 cells induced apoptosis (Wang et al., 2016). BmIMD may be cleaved by BmDredd. Of note, the Toll, IMD, MAPK-JNK-p38

16

ACCEPTED MANUSCRIPT and JAK-STAT pathways are intact and operative in Manduca sexta and Bombyx mori (Cao et al., 2015; Tanaka et al., 2008). BmPGRP-L1 and BmIMD was significantly induced in the midgut of bacteria-fed larvae. In the fat body and whole larvae, mRNA levels didn’t change dramatically. This

PT

indicates that BmPGRP-L1 may be an important receptor in the midgut. The extracellular regions of PGRP-LC receptors are directly involved in recognizing

RI

peptidoglycans. Therefore, blocking of this region will affect interactions with

SC

pathogen-associated molecular patterns. Indeed, blocking of BmPGRP-L1 by the

NU

antiserum or antibody significantly decreased antimicrobial peptide gene inductions. Recombinant BmPGRP-L1 can bind both Dap- and Lys-type peptidoglycans. In

MA

Drosophila, Drosomycin is mainly regulated by the Toll pathway. However, the injection of Gram-negative peptidoglycan into the IMD pathway deficient mutants

D

(PGRP-LC∆E and key1) also significantly induced Drosomycin expression (Leulier et

PT E

al., 2003). A further study showed that in the presence of Spätzle (the Toll ligand) and gram-negative peptidoglycan (the PGRP-LC ligand), target genes of the Toll

CE

(Drosomycin) and IMD pathway (Diptericin and AttacinA) were activated synergistically (Tanji et al., 2007). It is possible that both gram-positive and gram-

AC

negative peptidoglycans can be recognized by BmPGRP-L1. E. coli and S. aureus both induced the production of antimicrobial peptide genes in S2 cells. But the induction level was less than 10-fold in most cases (Fig. 10A). The induction of Drosomycin was very low by gram-positive S. aureus. This was probably due to the lack of extracellular Toll pathway components in the medium of cell culture. Different Drosophila cell lines showed varied immune sensitivities (Samakovlis et al., 1992). Highly immune responsive S2* cells were used in previous studies (Helenius et 17

ACCEPTED MANUSCRIPT al., 2009; Kaneko et al., 2004; Kaneko et al., 2006). Therefore, an alternative cell line may be more appropriate for this assay. Overexpression of full-length IMD induced three antimicrobial peptide genes most strongly. However, overexpression of PGRPL1-N, but not full-length PGRP-L1 significantly induced target genes. This implies that BmIMD can be efficiently activated in S2 cells, but PGRP-L1 may be activated by a

PT

different mechanism.

RI

In conclusion, our data suggest that BmPGRP-L1 and BmIMD may be involved

SC

in immune signaling. It is not known which PGRP receptor interacts with BmIMD. Further studies are required to complete the catalog of PGRPs and uncover the functions

MA

NU

of the IMD pathway in silkworms.

Acknowledgements. The financial support provided by the National Natural Science

D

Foundation of China (31402017), Natural Science Foundation of Anhui Province

PT E

(1508085QC51) and Anhui Agricultural University (YJ2013-16) is greatly appreciated

AC

CE

and acknowledged. The authors declare no conflict of interest.

18

ACCEPTED MANUSCRIPT References Anderson, K.V., 2000. Toll signaling pathways in the innate immune response. Curr. Opin. Immunol. 12, 13-19. Cao, X., He, Y., Hu, Y., Wang, Y., Chen, Y.R., Bryant, B., Clem, R.J., Schwartz, L.M., Blissard, G., Jiang, H., 2015. The immune signaling pathways of Manduca

PT

sexta. Insect Biochem. Mol. Biol. 62, 64-74. Chang, C.I., Chelliah, Y., Borek, D., Mengin-Lecreulx, D., Deisenhofer, J., 2006.

RI

Structure of tracheal cytotoxin in complex with a heterodimeric pattern-recognition

SC

receptor. Science 311, 1761-1764.

Chang, C.I., Ihara, K., Chelliah, Y., Mengin-Lecreulx, D., Wakatsuki, S.,

NU

Deisenhofer, J., 2005. Structure of the ectodomain of Drosophila peptidoglycanrecognition protein LCa suggests a molecular mechanism for pattern recognition.

MA

Proc. Natl. Acad. Sci. USA 102, 10279-10284.

Chen, L., Paquette, N., Mamoor, S., Rus, F., Nandy, A., Leszyk, J., Shaffer, S.A., Silverman, N., 2017. Innate immune signaling in Drosophila is regulated by

D

transforming growth factor beta (TGFbeta)-activated kinase (Tak1)-triggered

PT E

ubiquitin editing. J. Biol. Chem. 292, 8738-8749. Choe, K.M., Lee, H., Anderson, K.V., 2005. Drosophila peptidoglycan recognition

CE

protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor. Proc. Natl. Acad. Sci. USA 102, 1122-1126.

AC

Choe, K.M., Werner, T., Stoven, S., Hultmark, D., Anderson, K.V., 2002. Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 296, 359-362. Costa, A., Jan, E., Sarnow, P., Schneider, D., 2009. The Imd pathway is involved in antiviral immune responses in Drosophila. PLoS One 4, e7436. Dushay, M.S., Asling, B., Hultmark, D., 1996. Origins of immunity: Relish, a compound Rel-like gene in the antibacterial defense of Drosophila. Proc. Natl. Acad. Sci. USA 93, 10343-10347. 19

ACCEPTED MANUSCRIPT Feng, N., Wang, D., Wen, R., Li, F., 2014. Functional analysis on immune deficiency (IMD) homolog gene in Chinese shrimp Fenneropenaeus chinensis. Mol. Biol. Rep. 41, 1437-1444. Ganesan, S., Aggarwal, K., Paquette, N., Silverman, N., 2011. NF-κB/Rel Proteins and the Humoral Immune Responses of Drosophila melanogaster. Curr. Top.

PT

Microbiol. Immunol. 349, 25-60. Georgel, P., Naitza, S., Kappler, C., Ferrandon, D., Zachary, D., Swimmer, C.,

RI

Kopczynski, C., Duyk, G., Reichhart, J.M., Hoffmann, J.A., 2001. Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial

SC

defense and can promote apoptosis. Dev. Cell 1, 503-514.

Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J.A., Ferrandon,

NU

D., Royet, J., 2002. The Drosophila immune response against Gram-negative bacteria

MA

is mediated by a peptidoglycan recognition protein. Nature 416, 640-644. Gross, I., Georgel, P., Kappler, C., Reichhart, J.M., Hoffmann, J.A., 1996. Drosophila immunity: a comparative analysis of the Rel proteins dorsal and Dif in the induction

D

of the genes encoding diptericin and cecropin. Nucleic Acids Res. 24, 1238-1245.

PT E

Guan, R., Mariuzza, R.A., 2007. Peptidoglycan recognition proteins of the innate immune system. Trends Microbiol. 15, 127-134.

CE

Hedengren, M., Borge, K., Hultmark, D., 2000. Expression and evolution of the Drosophila attacin/diptericin gene family. Biochem. Biophys. Res. Commun. 279,

AC

574-581.

Helenius, I.T., Krupinski, T., Turnbull, D.W., Gruenbaum, Y., Silverman, N., Johnson, E.A., Sporn, P.H., Sznajder, J.I., Beitel, G.J., 2009. Elevated CO2 suppresses specific Drosophila innate immune responses and resistance to bacterial infection. Proc. Natl. Acad. Sci. USA 106, 18710-18715. Ip, Y.T., Reach, M., Engström, Y., Kadalayil, L., Cai, H., González-Crespo, S., Tatei, K., Levine, M., 1993. Dif, a dorsal-related gene that mediates an immune response in Drosophila. Cell 75, 753-763. 20

ACCEPTED MANUSCRIPT Kaneko, T., Goldman, W.E., Mellroth, P., Steiner, H., Fukase, K., Kusumoto, S., Harley, W., Fox, A., Golenbock, D., Silverman, N., 2004. Monomeric and polymeric gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway. Immunity 20, 637-649. Kaneko, T., Silverman, N., 2005. Bacterial recognition and signalling by the

PT

Drosophila IMD pathway. Cell. Microbiol. 7, 461-469. Kaneko, T., Yano, T., Aggarwal, K., Lim, J.H., Ueda, K., Oshima, Y., Peach, C.,

RI

Erturk-Hasdemir, D., Goldman, W.E., Oh, B.H., Kurata, S., Silverman, N., 2006. PGRP-LC and PGRP-LE have essential yet distinct functions in the Drosophila

SC

immune response to monomeric DAP-type peptidoglycan. Nat. Immunol. 7, 715-723. Koyama, H., Kato, D., Minakuchi, C., Tanaka, T., Yokoi, K., Miura, K., 2015.

NU

Peptidoglycan recognition protein genes and their roles in the innate immune pathways of the red flour beetle, Tribolium castaneum. J. Invertebr. Pathol. 132, 86-

MA

100.

Kurata, S., 2014. Peptidoglycan recognition proteins in Drosophila immunity.

D

Developmental and Comparative Immunology 42, 36-41.

PT E

Lan, J.F., Zhou, J., Zhang, X.W., Wang, Z.H., Zhao, X.F., Ren, Q., Wang, J.X., 2013. Characterization of an immune deficiency homolog (IMD) in shrimp (Fenneropenaeus chinensis) and crayfish (Procambarus clarkii). Developmental and

CE

Comparative Immunology 41, 608-617.

AC

Lemaitre, B., Kromer-Metzger, E., Michaut, L., Nicolas, E., Meister, M., Georgel, P., Reichhart, J.M., Hoffmann, J.A., 1995. A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense. Proc. Natl. Acad. Sci. USA 92, 9465-9469. Leulier, F., Parquet, C., Pili-Floury, S., Ryu, J.-H., Caroff, M., Lee, W.-J., MenginLecreulx, D., Lemaitre, B., 2003. The Drosophila immune system detects bacteria through specific peptidoglycan recognition. Nat. Immunol. 4, 478-484.

21

ACCEPTED MANUSCRIPT Meister, S., Agianian, B., Turlure, F., Relogio, A., Morlais, I., Kafatos, F.C., Christophides, G.K., 2009. Anopheles gambiae PGRPLC-mediated defense against bacteria modulates infections with malaria parasites. PLoS Path. 5, e1000542. Mellroth, P., Karlsson, J., Hakansson, J., Schultz, N., Goldman, W.E., Steiner, H., 2005. Ligand-induced dimerization of Drosophila peptidoglycan recognition proteins

PT

in vitro. Proc. Natl. Acad. Sci. USA 102, 6455-6460. Morisato, D., Anderson, K.V., 1994. The spatzle gene encodes a component of the

RI

extracellular signaling pathway establishing the dorsal-ventral pattern of the

SC

Drosophila embryo. Cell 76, 677-688.

Myllymäki, H., Valanne, S., Rämet, M., 2014. The Drosophila imd signaling

NU

pathway. J. Immunol. 192, 3455-3462.

Paquette, N., Broemer, M., Aggarwal, K., Chen, L., Husson, M., Erturk-Hasdemir, D.,

MA

Reichhart, J.M., Meier, P., Silverman, N., 2010. Caspase-mediated cleavage, IAP binding, and ubiquitination: linking three mechanisms crucial for Drosophila NF-

D

kappaB signaling. Mol. Cell 37, 172-182.

Ramet, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B., Ezekowitz, R.A., 2002.

PT E

Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416, 644-648.

CE

Rao, X.J., Yu, X.Q., 2010. Lipoteichoic acid and lipopolysaccharide can activate antimicrobial peptide expression in the tobacco hornworm Manduca sexta. Dev.

AC

Comp. Immunol. 34, 1119-1128. Reichhart, J.M., Georgel, P., Meister, M., Lemaitre, B., Kappler, C., Hoffmann, J.A., 1993. Expression and nuclear translocation of the rel/NF-kappa B-related morphogen dorsal during the immune response of Drosophila. Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie. 316, 1218-1224. Romeo, Y., Lemaitre, B., 2008. Drosophila immunity: methods for monitoring the activity of Toll and Imd signaling pathways. Methods Mol. Biol. 415, 379-394.

22

ACCEPTED MANUSCRIPT Royet, J., Gupta, D., Dziarski, R., 2011. Peptidoglycan recognition proteins: modulators of the microbiome and inflammation. Nat. Rev. Immunol. 11, 837-851. Samakovlis, C., Asling, B., Boman, H.G., Gateff, E., Hultmark, D., 1992. In vitro induction of cecropin genes-an immune response in a Drosophila blood cell line. Biochem. Biophys. Res. Commun. 188, 1169-1175.

PT

Schneider, D.S., Jin, Y., Morisato, D., Anderson, K.V., 1994. A processed form of the Spätzle protein defines dorsal-ventral polarity in the Drosophila embryo.

RI

Development 120, 1243-1250.

SC

Stenbak, C.R., Ryu, J.H., Leulier, F., Pili-Floury, S., Parquet, C., Herve, M., Chaput, C., Boneca, I.G., Lee, W.J., Lemaitre, B., Mengin-Lecreulx, D., 2004. Peptidoglycan

pathway. J. Immunol. 173, 7339-7348.

NU

molecular requirements allowing detection by the Drosophila immune deficiency

MA

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725-

D

2729.

Tanaka, H., Ishibashi, J., Fujita, K., Nakajima, Y., Sagisaka, A., Tomimoto, K.,

PT E

Suzuki, N., Yoshiyama, M., Kaneko, Y., Iwasaki, T., Sunagawa, T., Yamaji, K., Asaoka, A., Mita, K., Yamakawa, M., 2008. A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem. Mol.

CE

Biol. 38, 1087-1110.

AC

Tanaka, H., Sagisaka, A., 2016. Involvement of Peptidoglycan Recognition Protein L6 in Activation of Immune Deficiency Pathway in the Immune Responsive Silkworm Cells. Arch. Insect Biochem. Physiol. 92, 143-156. Tanji, T., Hu, X., Weber, A.N.R., Ip, Y.T., 2007. Toll and IMD pathways synergistically activate an innate immune response in Drosophila melanogaster. Mol. Cell. Biol. 27, 4578-4588. Valanne, S., Wang, J.H., Ramet, M., 2011. The Drosophila Toll signaling pathway. J. Immunol. 186, 649-656. 23

ACCEPTED MANUSCRIPT Vollmer, W., Blanot, D., de Pedro, M.A., 2008. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149-167. Wang, L., Song, J., Bao, X.Y., Chen, P., Yi, H.S., Pan, M.H., Lu, C., 2016. BmDredd is an initiator caspase and participates in Emodin-induced apoptosis in the silkworm, Bombyx mori. Gene 591, 362-368.

PT

Wang, S., Beerntsen, B.T., 2015. Functional implications of the peptidoglycan recognition proteins in the immunity of the yellow fever mosquito, Aedes aegypti.

RI

Insect Mol. Biol. 24, 293-310.

SC

Werner, T., Liu, G., Kang, D., Ekengren, S., Steiner, H., Hultmark, D., 2000. A family of peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster.

NU

Proc. Natl. Acad. Sci. USA 97, 13772-13777.

Xia, Q., Zhou, Z., Lu, C., Cheng, D., Dai, F., Li, B., Zhao, P., Zha, X., Cheng, T.,

MA

Chai, C., Pan, G., Xu, J., Liu, C., Lin, Y., Qian, J., Hou, Y., Wu, Z., Li, G., Pan, M., Li, C., Shen, Y., Lan, X., Yuan, L., Li, T., Xu, H., Yang, G., Wan, Y., Zhu, Y., Yu, M., Shen, W., Wu, D., Xiang, Z., Yu, J., Wang, J., Li, R., Shi, J., Li, H., Li, G., Su, J.,

D

Wang, X., Li, G., Zhang, Z., Wu, Q., Li, J., Zhang, Q., Wei, N., Xu, J., Sun, H., Dong,

PT E

L., Liu, D., Zhao, S., Zhao, X., Meng, Q., Lan, F., Huang, X., Li, Y., Fang, L., Li, C., Li, D., Sun, Y., Zhang, Z., Yang, Z., Huang, Y., Xi, Y., Qi, Q., He, D., Huang, H., Zhang, X., Wang, Z., Li, W., Cao, Y., Yu, Y., Yu, H., Li, J., Ye, J., Chen, H., Zhou,

CE

Y., Liu, B., Wang, J., Ye, J., Ji, H., Li, S., Ni, P., Zhang, J., Zhang, Y., Zheng, H., Mao, B., Wang, W., Ye, C., Li, S., Wang, J., Wong, G.K., Yang, H., Biology

AC

Analysis, G., 2004. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science 306, 1937-1940. Yang, Z., Ji, L., Jie, Y., QingQing, C., ZhongYuan, S., JinMei, W., 2016. Peptidoglycan recognition protein L1 is involved in the Imd pathway in the silkworm, Bombyx mori. Acta Entomol. Sin. 59, 164-171. Yoshida, H., Kinoshita, K., Ashida, M., 1996. Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori. J. Biol. Chem. 271, 13854-13860. 24

ACCEPTED MANUSCRIPT Zhang, Z.T., Zhu, S.Y., 2009. Drosomycin, an essential component of antifungal defence in Drosophila. Insect Mol. Biol. 18, 549-556. Legends Figure 1. Sequence and gene information of BmPGRP-L1. (A) Nucleotide and deduced amino acid sequence of BmPGRP-L1. The cDNA sequence is shown above the deduced protein sequence. The transmembrane region is underlined. The PGRP

PT

domain is shaded grey. The potential N- and O-glycosylation sites are shaded black. (B) Genomic structure of BmPGRP-L1 gene was shown. Numbers indicated the positions

RI

on chromosome 1. The diagram was drawn to scale. The accession number and the

SC

genomic range was shown above the diagram.

Figure 2. Phylogenetic analysis and sequence alignment of BmPGRP-L1. (A) The

NU

amino acid sequences of 30 PGRPs were analyzed. The evolutionary history was inferred using the Neighbor-Joining method. Accession numbers are shown before taxa

MA

information in brackets. BmPGRP-L1 is shown in red. The percentages of bootstrap values are shown next to branches. (B) The alignment of PGRP domains from BmPGRP-L1 and homologs. Conserved residues were shaded black. Five conserved

D

residues for the amidase activity were shaded grey and denoted by a star on the top.

PT E

Figure 3. Sequence and phylogenetic tree of BmIMD. (A) Nucleotide and deduced amino acid sequence of BmIMD. The death domain is shaded grey. The potential Nand O-glycosylation sites are shaded black. The potential polyubiquitination sites are

CE

circled (K92, K132, K147 and K199). (B) The unrooted Neighbor-Joining phylogenetic tree of BmIMD and homologous proteins. The tree is drawn to scale, with branch

AC

lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. BmIMD and DmIMD are indicated by stars. (C) An alignment of 19 IMDs from different insect species. The conserved residues are shaded black. Red box of solid line: the IAP-binding motif (IBM); blue box of dashed line: the caspase cleavage site. Genbank accession numbers are: VaIMD, AFK75941; PcIMD, AFK75940; HgIMD, AFK75936; AlIMD, AFK75935; PmIMD, KPJ08677; PxIMD, KPI95000; SeIMD, AFK75934; HaIMD, XP_021192466; HsIMD, AFK75939; BmIMD, XP_004921531; 25

ACCEPTED MANUSCRIPT PxyIMD, XP_011567077; AtIMD, XP_013197162; NvIMD, XP_017783673; TcIMD, XP_008199405;

ScIMD,

XP_013107116;

DsIMD,

AAQ64725;

AgIMD,

XP_001688608; AdIMD, ETN65294; DmIMD, NP_573394. Figure 4. Recombinant protein purification and the expression of deletion constructs in S2 cells. (A) The extracellular region of BmPGRP-L1 was expressed in E. coli with a HisTag. The recombinant protein was analyzed by coomassie brilliant

PT

blue (CBB) staining or western blotting using either HisTag antibody or BmPGRP-L1 antibody. Noninduced: noninduced bacteria lysate; Induced: IPTG induced bacteria

RI

lysate; Soluble: soluble proteins from induced bacteria; Insoluble: insoluble proteins; rPGRP-L1(ecto): purified target protein; BSA: 1 μg BSA. The position of target protein

SC

was indicated by arrow heads. Recombinant BmActin3 was analyzed by the purified antibody. (B) Deletion constructs of BmPGRP-L1 and BmIMD were cloned into

NU

pAc5.1/V5-His B vector for expression in S2 cells. V5-6xHis tag and FLAG tag was fused to the C-terminus of BmIMD and BmPGRP-L1, respectively. The diagrams were

MA

drawn to scale. TM: transmembrane region. (C) S2 cells were transfected with fulllength or deletion constructs and analyzed by western blot with anti-6xHis (for BmIMD)

D

and anti-FLAG (for BmPGRP-L1) antibodies.

Figure 5. Tissue distribution of BmPGRP-L1 and BmIMD. (A) The mRNA levels of

PT E

BmPGRP-L1 and BmIMD in different tissues were analyzed by qPCR. Fb: fat body; Hc: hemocytes; Mg: midgut; Mt: Malpighian tube; Te: testis; Ov: ovary; Ep: epidermis. ANOVA was performed and letters denoted level of significance. (B) Total proteins

CE

were extracted from hemocytes, midgut and fat body for western blotting using PGRP-

AC

L1 antibody. The position of PGRP-L1 band was denoted by an arrow head. Figure 6. mRNA levels of BmPGRP-L1, BmIMD and antimicrobial peptide genes in the midgut. After oral feeding of bacteria, the mRNA levels of BmPGRP-L1, BmIMD, CecropinA and Gloverin1 were analyzed by qPCR. Asterisks indicate levels of significant differences (*: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001). Student’s t-tests were performed for pairwise comparisons between control (PBS) and E. coli or S. aureus for each time point. Figure 7. mRNA levels of BmPGRP-L1, BmIMD and antimicrobial peptide genes in the fat body. After injection of bacteria, the mRNA levels of BmPGRP-L1, BmIMD, 26

ACCEPTED MANUSCRIPT CecropinA and Gloverin1 were analyzed by qPCR. Asterisks indicate levels of significant differences (*: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001). Student’s t-tests were performed for pairwise comparisons between control (PBS) and E. coli or S. aureus for each time point. Figure 8. mRNA levels of BmPGRP-L1, BmIMD and some immune-related genes in 2nd instar larvae. After oral feeding of bacteria to the 2nd instar larvae, the mRNA

PT

levels of BmPGRP-L1, BmIMD, BmFADD, BmDREDD, BmLebocin3 and BmCecropinA in whole worms were analyzed by qPCR. ANOVA (analysis of variance)

RI

was performed and Dunnett test was selected for multiple comparisons between control (PBS) and E. coli or S. aureus for each time point. Asterisks indicate levels of

SC

significant differences (*: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001).

NU

Figure 9. mRNA levels of antimicrobial peptide genes after in vivo blocking of BmPGRP-L1. Rabbit BmPGRP-L1 antiserum was injected to block the PGRP domain before injecting E. coli or B. subtilis. mRNA levels of BmGloverin1, BmGloverin2,

MA

BmCecropinA and BmMoricin were analyzed by qPCR. ANOVA was performed and Dunnett test was selected for multiple comparisons. Asterisks indicate levels of

D

significant differences (*: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001).

PT E

Figure 10. Overexpression of BmPGRP-L1 and BmIMD in S2 cells. (A) S2 cells were treated with E. coli or S. aureus for 6h and induction of antimicrobial peptide genes was measured by qPCR. (B) BmPGRP-L1 and BmIMD constructs were

CE

overexpressed in S2 cells for 72h. Cells transfected by pAC5.1B were used as the negative control. The mRNA levels of Drosomycin, DiptericinA and DiptericinB were

AC

measured by qPCR. ANOVA (analysis of variance) was performed and Dunnett test was selected for multiple comparisons. Asterisks indicate levels of significant differences (*: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001). Figure 11. Binding of recombinant BmPGRP-L1 to Dap- and Lys-type peptidoglycans. E. coli-PGN and S. aureus-PGN were immobilized on plates. Different amounts of recombinant BmPGRP-L1 were added to bind. EGFP was used as the negative control. Data represent mean ± SD.

27

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 1

28

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 2

29

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 3

30

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 4

31

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 5

32

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 6

33

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 7

34

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 8

35

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 9

36

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 10

37

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 11

38

ACCEPTED MANUSCRIPT

Table 1. List of Primers

CE

PT E

D

MA

NU

SC

RI

PT

Sequence (5’3’) CCCCATATGACGATCATGGCTAACTTTTCCTCC CCCCTCGAGAGATACCATTCCGTAGGTAACTTCACAGTC CGGGGTACCGCCGCCACCATGGCCCCTAGACGGGGCGTAC TGCTCTAGATCACTTATCGTCGTCATCCTTGTAATCATCCCACTCGTGAGGAGCGGTACG CGGGGTACCGCCGCCACCATGGCCAAAGACAAAGATATACCGTTTTCGCGC TGCTCTAGATCACTTATCGTCGTCATCCTTGTAATCAGATACCATTCCGTAGGTAAC CGGGGTACCGCCGCCACCATGGCCACTTTAAAAACAAAGC TGCTCTAGACTAATGAGATTAGTCTTTACAATTTCCTCTTC CGGGGTACCGCCGCCACCATGGCCAAAACTGTTATGAAAGCTACCATAAAG TGCTCTAGACTGTTTTTTTCAGAGTACAAATCTGC AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT CTAATACGACTCACTATAGGGC GCTAAACGGATGTGATATATGAGTATGCTTT GAGTATGCTTTGATTATTTGTCTGTGAATGTGTG ACAGCCTCGACCAGTAAATG TGCATGTTCGTCATCTACCG GTAGTGAAGACATCGAGCAGG GTGAAATACAGCATGAAGGCG ATCCTGCGTCTGGACTTGGC TGATGGAGTTGTATGTGGTTTCG CCAAGAAAAAAAGAAAGACTATTTGAAAC TCAATCTTGATAATTACACATTCTCGAA TTCGACCTGACGCTTTAGATG TGTCATACGGCTCATCTTGC TCGTGCCAGAGGTTCATCCA CGTCGGTAACGGTTTCCCATAT CCAACAGCATTATCAAAGC GGAAATTACAGAACGCAGA ATGATTGGTGATCACCTCA CAAACTTACAGTTAGACATACTTCG TGTGGCAATGTCTCTGGTG GCTCTTAGACCTTTACCGACTG TCTAAACAGCATAAGGCATTTC TGCACTCGGTCACTCTGAG AAGGGACAGTATCTGATGCCCAACA CATGAGCAGGACCTCCAGCTCG GCTGCGCAATCGCTTCTACT TGGTGGAGTGGGCTTCATG CGTGAGAACCTTTTCCAATATGATG TCCCAGGACCACCAGCAT TATTCATTGGACTGGCTTGTG ACTGGCGACGCACTCTGT AGTGCTGGCAGAGCCTCATCG AAATTGGACCCGGTCTTGGTTG

AC

Primer BmPGRP-L1ecto-F BmPGRP-L1ecto-R BmPGRP-N1 BmPGRP-C2 BmPGRP-N2 BmPGRP-C1 BmIMD-N1 BmIMD-C2 BmIMD-N2 BmIMD-C1 AP UAP AUAP GSP NestedGSP BmIMD-RTF BmIMD-RTR BmPGRP-L1-RTF BmPGRP-L1-RTR BmActinA3F BmActinA3R BmFADD-F BmFADD-R BmDREDD-F BmDREDD-R BmLeb3-F BmLeb3-R BmGlo1-F BmGlo1-R BmGlo2-F BmGlo2-R BmMoricin-F BmMoricin-R BmCecA-F BmCecA-R DmRp49-F DmRp49-R DmDipA-F DmDipA-R DmDrs-F DmDrs-R DmDipB-F DmDipB-R DmMet-F DmMet-R

39

Application Cloning Cloning Cloning Cloning Cloning Cloning Cloning Cloning Cloning Cloning 3’ RACE 3’ RACE 3’ RACE 3’ RACE 3’ RACE qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR