Broad RNA Interference−Mediated Antiviral Immunity and Virus-Specific Inducible Responses in Drosophila

This information is current as Cordula Kemp, Stefanie Mueller, Akira Goto, Vincent of October 2, 2021. Barbier, Simona Paro, François Bonnay, Catherine Dostert, Laurent Troxler, Charles Hetru, Carine Meignin, Sébastien Pfeffer, Jules A. Hoffmann and Jean-Luc Imler J Immunol 2013; 190:650-658; Prepublished online 19 December 2012; doi: 10.4049/jimmunol.1102486 Downloaded from http://www.jimmunol.org/content/190/2/650

Supplementary http://www.jimmunol.org/content/suppl/2012/12/19/jimmunol.110248 http://www.jimmunol.org/ Material 6.DC1 References This article cites 57 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/190/2/650.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Broad RNA Interference–Mediated Antiviral Immunity and Virus-Specific Inducible Responses in Drosophila

Cordula Kemp,*,1 Stefanie Mueller,*,1,2 Akira Goto,* Vincent Barbier,* Simona Paro,* Franc¸ois Bonnay,* Catherine Dostert,* Laurent Troxler,* Charles Hetru,* Carine Meignin,* Se´bastien Pfeffer,† Jules A. Hoffmann,* and Jean-Luc Imler*,‡

The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity and has led to some important discoveries about the sensing and signaling of microbial infections. The response of Drosophila to virus infections remains poorly characterized and appears to involve two facets. On the one hand, RNA interference involves the recognition and processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible response controlled by the evolutionarily conserved JAK-STAT pathway contributes to the antiviral host defense. To clarify the

contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the Downloaded from resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by Drosophila C virus and cricket paralysis virus, two members of the Dicistroviridae family, which contrasts with the susceptibility of Dcr-2 mutant flies to many viruses, including the DNA virus invertebrate iridescent virus 6. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by Drosophila C virus or by two unrelated RNA viruses, Flock House virus and

Sindbis virus. Overall, our data reveal that RNA interference is an efficient antiviral mechanism, operating against a large http://www.jimmunol.org/ range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK-STAT pathway appears to be virus specific. The Journal of Immunology, 2013, 190: 650–658.

iruses represent an important class of pathogens, causing resistance have been described. In mammals, viral infection is first serious concern for human health, as well as important detected by pattern recognition receptors of the Toll- and RIG-I– V economic losses in crops and animals. Because they like families that sense the viral nucleic acid and trigger the in- replicate inside cells, and rely for the most part on host cell mo- duction of IFNs and other cytokines (1). These factors activate the production of antiviral molecules, such as protein kinase R

lecular machineries for their replication, viruses pose specific by guest on October 2, 2021 challenges to the immune system. Two major strategies of antiviral or oligo-29,59-adenylate synthetase, that contain the infection and contribute to the activation of the adaptive immune response (2). In plants, viral nucleic acids are recognized by enzymes of the *CNRS-UPR9022, Institut de Biologie Mole´culaire et Cellulaire, 67084 Strasbourg Dicer family, which produce small interfering RNAs (siRNAs) of † Cedex, France; CNRS-UPR9002, Institut de Biologie Mole´culaire et Cellulaire; 21–24 nucleotides. These siRNAs are then loaded onto molecules 67084 Strasbourg Cedex, France; and ‡Faculte´ des Sciences de la Vie, Universite´ de Strasbourg; 67083 Strasbourg Cedex, France of the Argonaute (AGO) family and will guide them toward RNAs 1C.K. and S.M. contributed equally to this work. with complementary sequences; targeted RNAs are then either 2Current address: Bernhard-Nocht-Institut for Tropical Medicine, Molecular Ento- sliced by AGO, or their translation is inhibited. This RNA in- mology, Hamburg, Germany terference (RNAi) mechanism provides efficient and sequence- Received for publication August 29, 2011. Accepted for publication November 5, specific protection against viral infections (3). 2012. RNAi also plays an important role in the control of viral in- This work was supported by the National Institutes of Health (PO1 AI070167), the fections in insects, as shown by the production of virus-derived Agence Nationale de la Recherche (ANR-09-MIEN-006-01), the Balzan Foundation siRNAs in infected flies, and the increased susceptibility to viral (to J.A.H.), the European Research Council (ERC Starting Grant ncRNAVIR 260767 to S.P.), the Investissement d’Avenir Program Laboratoire d’Excellence (NetRNA infection of Drosophila mutants for the genes Dcr-2 and AGO2 ANR-10-LABX-36), and the Centre National de la Recherche Scientifique. (3–6). In addition, several reports indicate that an inducible re- The sequences presented in this article have been submitted to the Gene Expression sponse also contributes to the control of viral infections (7–15). Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE31542 We previously showed that infection with Drosophila C virus and to the National Center for Biotechnology Information Small Read Archive (http://www.ncbi.nlm.nih.gov/sra) under accession number GSE41007. (DCV), a member of the Dicistroviridae family, leads to induction Address correspondence and reprint requests to Prof. Jean-Luc Imler, IBMC-CNRS/ of some 130 genes (11). Analysis of the regulation of one of these Universite´ de Strasbourg, Rue Rene Descartes, Strasbourg, Alsace 67084, France. genes, vir-1, revealed the presence of functionally important E-mail address: [email protected] binding sites for the transcription factor STAT in its promoter. The The online version of this article contains supplemental material. induction of vir-1, as well as several other DCV-induced genes, Abbreviations used in this article: AGO, Argonaute; CrPV, cricket paralysis virus; was found to be dependent on the gene hopscotch (hop), which Dcr-2, Dicer-2; DCV, Drosophila C virus; dpi, day postinfection; DXV, Drosophila X virus; FHV, Flock House virus; IIV-6, invertebrate iridescent virus type 6; Imd, encodes the only JAK kinase in Drosophila.Furthermore,hop immune deficiency; MEKK1, MEK kinase 1; RNAi, RNA interference; SINV, Sind- mutant flies succumb more rapidly than do wild-type controls, bis virus; siRNA, small interfering RNA; TotM, Turandot M; Upd, unpaired; VSV, with a higher viral load, to DCV infection (11). The Toll and vesicular stomatitis virus. immune deficiency (Imd) pathways, initially characterized for Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 their role in the control of bacterial and fungal infections, were www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102486 The Journal of Immunology 651 also thought to play a part in the control of viral infections. cells. DCV, CrPV, FHV, and IIV-6 were titrated on Kc167 (DCV, CrPV, Whereas the Toll pathway was associated with resistance to the and FHV) or S2 (IIV-6) cells by the Reed–Muench method to calculate Drosophila X virus (DXV) (15), the Imd pathway was implicated 50% tissue culture–infective dose and converted to PFU with a conversion factor of 0.7. in the control of Sindbis virus (SINV) (7) and cricket paralysis virus (CrPV) (9). RNA analysis Altogether, the data in the present literature point to the in- Total RNA from infected flies was isolated using TRI Reagent RT bro- volvement of both RNAi and an inducible expression of effector moanisole solution (MRC), according to the manufacturer’s instructions. molecules to counter viral infections in insects (5, 16). However, Total RNA, 1 mg, was reverse transcribed using iScript cDNA Synthesis whereas RNAi was shown to contribute to resistance to several Kit (Bio-Rad). The reverse transcription was run in the T3000 Thermo- cycler (Biometra), with the following PCR program: step 1: 65˚C for 5 RNA viruses (with either single-stranded genomes of both polar- min, step 2: 4˚C for 5 min, step 3: 25˚C for 10 min, step 4: 42˚C for 60 ities or double-stranded genomes), most studies on the inducible min, and step 5: 70˚C for 15 min. A total of 100 ng cDNA was used for response have so far focused on a single virus. As a result, the quantitative real-time PCR, using the iQ Custom SYBR Green Supermix global significance of the inducible response for the control of Kit (Bio-Rad). The PCR was performed using the CFX384 Real-Time System (Bio-Rad) with the following program: step 1: 95˚C for 3 min, viral infections remains poorly understood. In particular, it is step 2: 95˚C for 10 s, step 3: 55˚C for 30 s, repeated 39 times from step 2. unclear at present if the JAK-STAT pathway is involved in a Primers used for qPCR were as follows: RpL32 (forward 59-GACGCTTC- general antiviral response, providing broad antiviral immunity, or AAGGGACAGTATCTG-39; reverse 59-AAACGCGGTTCTGCATGAG-39), if it acts specifically on a critical step in the replication cycle of a vir-1 (forward 59-GATCCCAATTTTCCCATCAA-39; reverse 59-GATTAC- 9 9 specific virus or virus family. To address this important question, AGCTGGGTGCACAA-3 ), drosomycin (forward 5 -CGTGAGAACCTT- TTCCAATATGATG-39; reverse 59-TCCCAGGACCACCAGCAT-39), and we have compared the resistance of a mutant for the JAK-STAT diptericin (forward 59-GCTGCGCAATCGCTTCTACT-39; reverse 59- Downloaded from pathway to infection by seven RNA or DNA viruses. We find that TGGTGGAGTGGGCTTCATG-39). Turandot M (TotM), upd, upd2, and hop mutant flies are more susceptible than wild-type controls to upd3 expression levels were quantified using the Brilliant II QRT-PCR infections by the Dicistroviridae DCV and CrPV, but exhibit either Core Reagent Kit, 1-step (Stratagene). The reaction took place in a total volume of 20 ml, using the Taqman Gene Expression Assay [TotM no or a weak phenotype for other viruses, suggesting that the (Dm02362087 s1), upd (os) (Dm01843792_g1), upd2 (Dm01844134 g1), JAK-STAT pathway–dependent inducible response is virus spe- upd3 (custom-designed upd3exon2-ANY), and RpL32 (Dm02151827 g1), cific. Genome-wide transcript profiling shows that infection by all from Applied Biosystems]. We used the 7500 Fast Real-Time PCR two other RNA viruses, Flock House virus (FHV; Nodaviridae) System (Applied Biosystems) with following PCR program: step 1: 45˚C for http://www.jimmunol.org/ and SINV (Alphaviridae), leads to upregulation of $400 genes, 30 min, step 2: 95˚C for 10 min, step 3: 95˚C for 15 s, step 4: 60˚C for 1 min, repeated 39 times from step 3. In all cases, gene expression was which only partially overlap with those induced by DCV. Overall, normalized to the ribosomal protein gene RpL32. our data indicate that the siRNA pathway exerts broad antiviral For IIV-6, the expression of the annotated genes 206R, 224L, 244L, and activity and affects both RNA and DNA viruses, with virus- 261R was assessed by strand-specific RT-PCR. We used SuperScript III specific inducible responses contributing to the control of viral Reverse Transcriptase specifically adapted for gene-specific priming and followed the manufacturer’s protocol (Invitrogen). Briefly, primer pairs infections in Drosophila. were designed to amplify regions of the IIV-6 genome exhibiting or not exhibiting a high density of small RNA reads. Total RNA,1 mg, extracted from infected S2 cells was reverse transcribed with 2 pmol of either for- Materials and Methods by guest on October 2, 2021 ward (F) or reverse (R) primer and 200 U of SuperScript III Reverse Fly strain culture and infection Transcriptase. The reaction was then incubated for 1 h at 55˚C. Then 1 ml Oregon-R (OR)andyw were used as wild-type control flies. The hopM38/msv1, of the resulting cDNA was used to perform 25 cycles of PCR, using Taq Dcr-2L811fsX, and Dcr-2R416X mutant flies were previously described (17– DNA polymerase (Invitrogen) and both F and R primers. The primer pairs 9 9 19). A genomic rescue of the Dcr-2 gene was established with the Fosmid were as follows: 206R (forward: 5 -AAGGAAAGTGGCGAGTACGA-3 , 9 9 9 FlyFos017074 (transgeneome.mpi-cbg.de) inserted at the landing site reverse 5 -AACAAACCCGTTTTCTTCCA-3 ); 224L (forward: 5 -CCACC- 9 9 9 attP40 (2L), and the transgenic chromosome was recombined with the ATCACATTGACCTTG-3 ,reverse:5-ATAAGCGAACCCGAAATCA-3 ); 9 9 9 deficiency Df(2R)BSC45, which uncovers the Dcr-2 locus. For the rescue 244L (forward: 5 -TGGAAAAGAGTGGTCCCATTT-3 ,reverse:5-TGT- 9 9 experiments, Dcr-2 mutants were crossed with the deficiency Df(2R) ACCTCCCGGAAGATTT-3 ); 261R (forward: 5 -CAGCCCCATCCGAAT- 9 9 9 BSC45 or the Df(2R)BSC45–Dcr-2 rescue line. Flies were fed on standard TACTA-3 ,reverse:5-CTGCAACTGCAGAAATTTGA-3 ). The PCR bands cornmeal–agar medium at 25˚C. All fly lines were tested for Wolbachia were sequenced to verify their viral origin. infection and cured whenever necessary. Viral stocks were prepared in 10 Statistical analysis mM Tris-HCl, pH 7.5, with the exception of vesicular stomatitis virus (VSV), which was used directly from Vero cell culture supernatant [VSV An unpaired two-tailed Student t test was used for statistical analysis of 4 3 109 PFU/ml; DCV 5 3 1010 PFU/ml; CrPV 1 3 109 PFU/ml; FHV data with GraphPad Prism (GraphPad Software). The p values , 0.05 were 5.5 3 109 PFU/ml; DXV 4.4 3 107 PFU/ml, invertebrate iridescent virus considered statistically significant. Survival curves were plotted and ana- type 6 (IIV-6) 4.4 3 1011 PFU/ml; and SINV 5 3 108 PFU/ml]. Infections lyzed by log-rank analysis (Kaplan–Meier method) using GraphPad Prism were performed with 4- to 6-d-old adult flies by intrathoracic injection (GraphPad Software). (Nanoject II apparatus; Drummond Scientific) with viral particles, indi- cated in the figure legends. Injection of the same volume (4.6 nL) of 10 DNA microarray analysis mM Tris-HCl, pH 7.5, was used as a control. For bacterial infection, flies For each sample, Tris-injected, DCV-infected (11), and FHV- and SINV- wereprickedwithathinneedlepreviouslydippedinaconcentrated infected, three biologically independent samples comprising 45 male Escherichia coli Micrococcus luteus overnight culture of and in Luria– Oregon-R flies were used. RNA extraction, biotinylation, and hybridization Bertani medium. Infected flies were then incubated at room temperature, to Affymetrix Drosophila GeneChip microarrays (Affymetrix) were per- hopM38/msv1 or at 29˚C in the case of and the corresponding control flies, formed as described (20). The Affymetrix Microarray Suite 5.0 (Affy- and monitored daily for survival, or frozen for RNA isolation and virus metrix) or Excel (Microsoft) with a combination of built-in functions and titration at the indicated time points. custom formulae was used for data analysis. Raw data were sorted with the Cell culture and virus titration “absent-marginal-present flags” generated by the Microarray Suite func- tions. Although an absent flag might indicate that no mRNA of a particular Vero R cells were grown in DMEM (Invitrogen) supplemented with 10% type was present in a sample, marginal flags and absent flags may indicate FCS (Biowest), penicillin/streptomycin (Invitrogen), nonessential amino problems with the hybridization; therefore, only data points marked as acid mix (Invitrogen), 10 mM pyruvate (Life Technologies), and 200 mM L- present in at least one replicate were retained. The remaining data mass glutamine (Invitrogen). Kc167 and S2 cells were grown in Schneider’s for each microarray was then normalized to itself, making 1 the median medium (Biowest) supplemented with 10% FCS, GlutaMAX (Invitrogen), of all the measurements. A gene was considered induced if present in at and penicillin/streptomycin (1003 mix, 10 mg/ml/10,000 U; Invitrogen). least one replicate, with a virus/Tris ratio higher than 2 for at least one of VSV and SINV were titrated from infected flies by plaque assay on Vero R the time points. Classification of gene functions was analyzed by David 652 VIRUS-SPECIFIC INDUCIBLE RESPONSE IN DROSOPHILA

Bioinformatics Resources 6.7 (21). The data set for FHV and SINV was which is characteristic of the accumulation of iridescent viral submitted to the Gene Expression Omnibus database (http://www.ncbi. particles, than in wild-type controls (Fig. 1A). Dcr-22/2 flies were nlm.nih.gov/geo/) with the accession number GSE31542. significantly more susceptible to IIV-6 infection than were the 2 2 Assembly, sequencing, and analysis of small RNA libraries corresponding wild-type (Fig. 1B). A fraction of Dcr-2 / flies injected with buffer also died in the course of the experiment, The small RNA library of S2 cells and whole flies was constructed as described (22) and sequenced by the Illumina 2G Analyzer. Reads were confirming the increased sensitivity to stress associated with then aligned to a reference consisting of the IIV-6 genome from the Na- mutations of the siRNA pathway (31). The decreased survival tional Center for Biotechnology Information (accession code NC_003038) time correlated with a 20-fold increased viral load in Dcr-2 mutant using the Bowtie program with standard parameters in genome assembly. flies at 10 d postinfection (dpi) (Fig. 1C). Similar results were Reads aligning to the IIV-6 genome with a maximum of one mismatch were retained and analyzed using in-house Perl scripts and Excel. Sequences were obtained when a different null allele of Dcr-2 was used, and the submitted to the National Center for Biotechnology Information Small Read IIV-6 susceptibility phenotype was rescued by a wild-type geno- 2 2 2 2 Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?) under the acces- mic Dcr-2 transgene (Fig. 1D). The r2d2 / and AGO2 / null sion number GSE41007. mutant flies also exhibited increased sensitivity to IIV-6 (Fig. 1E). AGO22/2 flies contained more viral DNA than did wild-type Results controls, confirming that this gene participates in the control of RNAi provides broad antiviral protection in Drosophila infection (Fig. 1F). Several independent studies, including our own, have established We next sequenced small RNA libraries prepared from IIV-6– that RNAi, and more precisely the siRNA pathway, serves as an infected S2 cells or adult flies. We observed several hundreds of efficient host defense against RNA viruses. These include viruses thousands of reads matching the IIV-6 genome in both infected S2 with a single-stranded genome of both (+) and (2) polarity and cells and wild-type flies, but not in control noninfected S2 cells Downloaded from dsRNA viruses (23–30), and we confirmed that flies mutant for (Supplemental Table I). The large majority of these reads had a Dcr-2 died more rapidly than wild-type controls when they were size of 21 nucleotides, which is characteristic for processing by infected with DCV, CrPV, FHV, SINV, VSV (Rhabdoviridae), and the RNase Dicer-2 (Dcr-2). This peak was absent from the library DXV (Birnaviridae) (data not shown). Next, we addressed the prepared from infected Dcr-22/2 mutant flies (Fig. 2A). These question whether the siRNA pathway also participated in the data indicate that Dcr-2 generates 21-nucleotide IIV-6–derived control of a DNA virus infection, and infected wild-type and RNAi siRNAs in infected flies, and raise the question of the nature of the http://www.jimmunol.org/ mutant flies with IIV-6 (Iridoviridae). Infection of Dcr-2 mutant substrate used by Dcr-2 in the context of this infection. As pre- flies led to a more rapid and intense appearance of blue color, viously reported for RNA viruses, the number of reads matching by guest on October 2, 2021

FIGURE 1. Dcr-2 is involved in host defense against the DNA virus IIV-6. (A) Upon injection of IIV-6 (5000 PFU) in wild-type (yw) and Dcr-2R416X mutant flies, typical blue paracrystalline structures appeared earlier in the abdomen (arrowhead) of the mutant flies. Representative individuals 10 dpi are shown. (B) Groups of 20 wild-type (yw)orDcr-2R416X mutant flies were injected with IIV-6 or Tris, and survival was monitored daily. The difference between the wild-type and Dcr-2 mutant flies is statistically significant. (C) Viral titer in groups of five wild-type (yw)orDcr-2R416X mutant flies was monitored 10 dpi. (D) Rescue of the hemizygous Dcr-2L811fsX for the IIV-6 susceptibility phenotype by a transposon expressing a wild-type Dcr-2 transgene. Dcr-2L811fsX hemizygous flies (Dcr-2L811fsX/Df) are significantly more susceptible than Dcr-2L811fsX hemizygous flies complemented by a wild- type Dcr-2 transgene (Dcr-2L811fsX/Df rescue). Df is Df(2R)BSC45, a deficiency that fully uncovers the Dcr-2 locus. All control and genomic rescued flies are in CantonS background. (E) Survival rate of wild-type (yw), R2D21, and AGO2414 mutant flies upon IIV-6 or Tris injection. (F) IIV-6 DNA load was determined by quantitative PCR in four groups of six flies of the indicated genotype at 10 dpi. For all panels, the data represent the mean and SD of at least three independent experiments, and the difference between controls and mutant flies is statistically significant. *p , 0.05, ***p , 0.001. All experiments are performed at 22˚C (A, C, F) or 25˚C (B, D, E). The Journal of Immunology 653 Downloaded from http://www.jimmunol.org/

FIGURE 2. Virus-derived siRNAs in S2 cells and Drosophila adult flies infected by the DNA virus IIV-6. RNA was extracted 5 dpi from S2 cells infected by IIV-6 (MOI 0.01) and adult wild-type (yw) or mutant (Dcr-2R416X) flies injected with IIV-6 (5000 PFU per fly). (A) Size distribution of the small RNAs matching the viral genome in S2 cells and adult flies of the indicated genotype. (B and C) Distribution of the 21-nucleotide siRNAs from the S2 cell (B) and yw adult fly (C) libraries along the IIV-6 genome. Each IIV-6–derived small RNA is represented by the position of its first nucleotide. The IIV-6–derived by guest on October 2, 2021 small RNAs matching the upper and lower strand of the DNA genome are respectively shown above (positive reads number) and below (negative reads number) the horizontal axis, which represents the 212482bp genome. In (B), the number of reads for four peaks going off-scale is indicated next to them, in italics. (D) Strand-specific RT-PCR with primers corresponding to the annotated viral genes 206R, 224L, 244L, and 261R. The experiment was performed in the presence (+) or absence (2) of RT. NI, Noninfected; F and R, forward and reverse strand primer used for reverse transcription. each strand of the viral genome was very similar (Supplemental mutant flies die more rapidly than do wild-type controls following Table I). However, unlike RNA viruses, the virus-derived siRNAs DCV infection, and contain ∼10-fold more virus (Fig. 3A). By were not uniformly distributed along the viral genome. Rather, contrast, we did not observe significant differences in survival several hotspots were observed, revealing that specific regions of between wild-type and hopM38/msv1 mutant flies upon infection the viral genome generate the siRNAs (Fig. 2B, 2C). These peaks with the alphavirus SINV (Fig. 4A), and the viral titers 2 dpi were do not correlate with the intensity of transcription of the viral not significantly different in wild-type and hopM38/msv1 mutant genome, and some highly transcribed regions are located in areas flies (data not shown), indicating that the JAK-STAT pathway does not generating significant levels of siRNAs (32). The strong not contribute to resistance to this virus. The hopM38/msv1 mutant symmetry of the peaks observed in S2 cells and wild-type flies flies, as well as wild-type flies, also resisted infections by the suggests that these regions are transcribed on both strands and rhabdovirus VSV and by the nodavirus FHV (Fig. 4B, 4C). A generate dsRNA. Indeed, we could detect bidirectional transcrip- slight reduction in survival was observed in the case of the dsRNA tion in the areas of the viral genome covered by the peaks (Fig. virus DXV (Birnaviridae) and the DNA virus IIV-6 (Fig. 4D, 4E). 2D). By contrast, transcription of only one strand of the DNA However, the difference between wild-type and hopM38/msv1 mutant genome was detected for the locus 261R, which is located in flies was only statistically significant in the case of DXV infection. a region that does not produce significant amounts of siRNAs. Furthermore, we did not observe statistically significant differences Overall, these results indicate that the siRNA pathway in Dro- in the DXV and IIV-6 viral titers in wild-type and hopM38/msv1 sophila can also protect against a DNA virus infection. mutant flies in the format of our assays (data not shown). Overall, our data indicate that the JAK-STAT pathway is critical for The JAK kinase Hopscotch does not confer broad antiviral host defense against DCV, but plays a minor role for DXV and IIV-6 immunity and is essentially dispensable in the case of FHV, SINV, and VSV. We To test the contribution of the JAK-STAT pathway in antiviral therefore tested CrPV, another member of the Dicistroviridae family immunity in Drosophila, we injected loss-of-function mutants of known to infect Drosophila. We observed a decrease in survival the JAK kinase Hopscotch (hopM38/msv1) with different ssRNA, and a significant increase in viral titers in CrPV-infected hopM38/msv1 dsRNA, and DNA viruses. As previously described, hopM38/msv1 mutant flies compared with wild-type flies (Fig. 3B). In conclusion, 654 VIRUS-SPECIFIC INDUCIBLE RESPONSE IN DROSOPHILA Downloaded from

FIGURE 3. The JAK kinase Hopscotch is involved in host defense against DCVand CrPV. (A and B) Groups of 20 wild-type (OR) or hopscotch (hopM38/msv1) mutant flies were injected with DCV (500 PFU) (A)orCrPV(5PFU)(B), and survival was monitored daily. The experiment was repeated three times, and data represent the mean and SD. In the right panels, viral titer was determined in groups of five flies 2 dpi for DCV (A) and 1 dpi for CrPV (B). The data http://www.jimmunol.org/ represent the mean and SD of three independent experiments, and the difference between wild-type and hop mutant flies is statistically significant. *p , 0.05, **p , 0.01, ***p , 0.001. (C) DCV and CrPV infection triggers induction of the genes upd2 and upd3, which encode cytokines activating the JAK/STAT pathway. Flies were infected with DCV or CrPV, and expression of upd, upd2, and upd3 was monitored in groups of 10 flies at the indicated time points by Taqman quantitative PCR. The results of at least two independent experiments are shown.

our data indicate that the JAK-STAT pathway in Drosophila confers expression in flies infected by FHV (2 and 3 dpi) or SINV (4 and 8 protection against some viruses—in particular, the Dicistroviridae— dpi), and compared the data with those obtained for DCV infection but does not provide broad antiviral immunity. (1 and 2 dpi). The time points for this analysis were chosen to take by guest on October 2, 2021 into account the different kinetics of replication and colonization Inducible gene expression in FHV- and SINV-infected flies of Drosophila by the different viruses (11, 24). For each virus, we The above results raised the question of whether an inducible observed a large overlap between the genes induced at the first and response contributes to host defense against viruses other than second time points. We then pursued our analysis, focusing on the DCV and CrPV. We therefore conducted a genome-wide micro- genes induced either at the first or at the second time point. The array analysis using Affymetrix DNA microarrays to monitor gene microarray data revealed that 487 and 201 genes were induced or

FIGURE 4. Susceptibility of flies mutant for the JAK kinase Hopscotch to infection by SINV, VSV, FHV, DXV, and IIV-6. Groups of 20 wild-type (OR) or hop mutant flies were injected with SINV (A), VSV (B), FHV (C), DXV (D), or IIV-6 (E), and survival was monitored. For VSV and SINV, the Tris buffer control injection is also shown, because hop mutant flies exhibited decreased survival at 29˚C after day 16 upon both buffer and virus injection. Kaplan–Meier analysis of the results of at least two independent experiments reveal a statistically significant difference in survival between wild-type and hop mutant flies only in the case of DXV. *p , 0.05. The Journal of Immunology 655 Downloaded from

FIGURE 5. Microarray analysis of gene induction following infection by DCV, FHV, or SINV. (A) Venn diagram showing the number of upregulated genes (by a factor of at least 2) following infection by the three viruses. The total number of genes regulated by each virus is indicated in parentheses.(B) http://www.jimmunol.org/ FHV and SINV induce members of the same gene families, but FHV triggers a stronger response. The numbers of genes belonging to seven gene ontology functional categories induced by both FHV and SINV or by FHV only are shown. (C) Expression of vir-1 and TotM by quantitative PCR normalized for the expression of the housekeeping gene RpL32. Groups of 10 wild-type (OR) flies were injected with Tris buffer or the viruses DCV, CrPV, FHV, SINV, VSV, DXV, or IIV-6 or pricked with a needle dipped in a concentrated pellet of the Gram-positive bacterium M. luteus and the Gram-negative bacterium E. coli. RNA was extracted at 6 h, 1 d, 2 d, 3 d, and 4 d after challenge. The data represent the mean and SEs of at least two independent experiments. The p values were calculated for each time point individually versus the Tris-injected control. *p , 0.05, **p , 0.01, ***p , 0.001. upregulated by a factor of at least 2 upon infection by FHV and FHV are also induced by SINV (Fig. 5A). It is intriguing, though, by guest on October 2, 2021 SINV, respectively. When analyzed with the same criteria, 166 that many of the genes induced solely by FHV, but not by SINV, genes were induced by DCV (Fig. 5A, Supplemental Table II). are members of the same gene families as the genes coinduced by The data of this transcriptomic analysis call for two comments. both FHV and SINV. This peculiarity underlines the basic simi- First, we note that 42 genes were induced by all three viruses larities between the transcriptional response to the two viruses. In (Fig. 5A). We compared this set of genes with microarray studies addition, several genes associated with cell death are induced by performed on flies infected by fungi and bacteria (both extra- and FHV, but not SINV, which may reflect the higher virulence of intracellular) to identify a potential signature specific for viral FHV (Fig. 5B, Supplemental Tables II, III). Only 22% and 16% of infections (Supplemental Table III). We observed that a number of the genes induced by SINV and FHV, respectively, are also in- genes, such as Frost, are upregulated similarly by all types of duced by DCV, indicating that DCV, on one hand, and FHV and infections, suggesting that they are induced by the stress of the SINV, on the other hand, trigger different inducible responses infection, rather than by recognition of specific characteristics (Fig. 5A). We did not detect in our microarrays expression of the of the infecting microorganism. Of interest, other genes, such as genes encoding the unpaired (Upd) cytokines, which activate the Vago, Obp99b, Mal-B1, Nmda1, CG8147, CG1572, l(2)gd1, JAK-STAT pathway in Drosophila. However, quantitative RT-PCR CG14906, CG10911, and Tsp42EI, appear to be induced only in analysis revealed that upd2 and upd3, but not upd, are induced or response to viral infections, and may represent the core of an upregulated following DCV and CrPV infection (Fig. 3C). inducible antiviral gene expression program. The case of Obp99b is particularly striking, as this gene is strongly upregulated by Virus-specific pattern of gene induction FHV, SINV, and DCV, but inhibited following other types of in- To further characterize the transcriptional response triggered by fection. Clearly, the regulation and function of this molecule different viruses, wild-type flies were injected with DCV, CrPV, deserves further investigation. The genes CG4680, Eip75B, Sp7, FHV, SINV, VSV, DXV, and IIV-6, and gene induction was and CG10916 are induced both by the viruses and by the intra- measured at 6 h postinfection and 1, 2, 3, and 4 dpi. Gene ex- cellular bacterium Listeria (33), suggesting that they may partic- pression was monitored by quantitative RT-PCR, which provides ipate in the defense against intracellular intruders (Supplemental a more accurate quantification of gene expression than does hy- Table III). bridization to short oligonucleotide probes on microarrays (34). We A second comment is that the majority of upregulated genes are monitored expression of the DCV-induced gene vir-1 (11) and of induced by only one or two of the viruses, revealing virus-specific TotM, which, according to the microarrays, is induced by FHV and responses. Of interest, 84% of the genes upregulated by SINV SINV infection. We confirmed the induction of vir-1 by DCV and are also induced by FHV, pointing to a strong similarity between FHV (11) and detected a milder but significant induction of this the responses to the two viruses. FHV induced a higher number of gene by CrPV infection. By contrast, no induction of vir-1 by genes than did Sindbis virus, and only 34% of the genes induced by SINV, VSV, DXV, and IIV-6 was observed (Fig. 5C). For TotM, 656 VIRUS-SPECIFIC INDUCIBLE RESPONSE IN DROSOPHILA we confirmed the induction by FHV at different time points. In cells, as demonstrated by the critical role played by the dsRNA addition, we observed that TotM expression was significantly in- receptor TLR3 in the sensing of herpesvirus infection in mammals duced by DCV at late time points of infection (4 dpi). We note that (39, 40). Our data are consistent with a model whereby dsRNA induction of TotM by SINV, VSV, and DXV was 10–20 times generated from convergent transcription of the IIV-6 genome is stronger than the induction by FHV (Fig. 5C). The DNA virus IIV- processed by Dcr-2 and triggers RNAi. Thus, we conclude that 6 did not induce TotM at any measured time point. Interestingly, RNAi provides an efficient and highly specific RNA-based de- we observed different profiles for vir-1 and TotM induction after fense against many types of viruses in Drosophila and probably viral challenge. Overall, the viruses that kill wild-type flies rapidly other insects. This conclusion parallels the situation described (within 10 d), such as DCV, CrPV, and FHV, were potent inducers in plants. The vertebrates, which largely rely on the induction of of vir-1, whereas less pathogenic viruses, such as SINV, VSV, and IFNs to counter viral infections, appear to be the exception among DXV, did not induce vir-1. The opposite trend was observed for multicellular organisms (1). Of interest, however, the DExD/H TotM, which was most potently induced by SINV, VSV, and DXV. box helicase domains found in Dcr enzymes and RIG-I–like The different pattern of induction of vir-1 and TotM suggests that receptors, which sense the presence of viral RNAs in cells infected the two genes may be regulated differently, even though both were by RNA and DNA viruses, are phylogenetically related (10). This previously shown to be regulated by the JAK-STAT pathway (11, finding suggests that an essential domain of a core molecule from 17). Indeed, the MAP3K MEK kinase 1 (MEKK1) and the Imd the ancestral antiviral response, RNA silencing, was at some point pathways are also known to contribute to the induction of TotM recruited to sense viral RNAs in vertebrates and to subsequently induction in some contexts (17, 35). activate a signaling pathway leading to production of IFNs. Some antimicrobial peptide genes were also upregulated ac- cording to the microarrays, suggesting an overlap between antiviral Virus-specific induced gene expression in Drosophila Downloaded from immunity and antibacterial–antifungal defenses. We observed an Microarrays are powerful tools to monitor the global transcriptome enrichment for genes regulated by the Toll pathway [e.g., the of infected cells and compare the response to different infections. cytokine Spaetzle (Spz) and the antifungal peptides Drosomycine Despite its limitations for accurate measurements of the magnitude (Drs) and Metchnikowine (Mtk)] in the DCV-specific set of genes of expression changes, this technology provides useful information (Supplemental Table II). We also noted an enrichment of Imd on changes in gene expression (34). In this article, using whole- pathway–regulated genes, such as the antibacterial peptides Attacin- genome Affymetrix microarrays to analyze the transcriptome of http://www.jimmunol.org/ A and -C, Diptericin-B, and the transcription factor Relish, in flies infected by DCV, FHV, or SINV, we report the existence of virus- the genes upregulated by both DCV and FHV. However, when ex- specific responses to infection. These results are in keeping with pression of diptericin and drosomycin—two markers of activation a previous study pointing to autophagy as an antiviral defense of the Imd and Toll pathways, respectively—was monitored by mechanism against VSV, but not DCV, infection (14). The three quantitative RT-PCR, none of the viruses triggered an induction viruses we used belong to different families and present different comparable to that of bacterial and fungal infections, although the characteristics that make them valuable for the current study. For wounding associated with the injection procedure clearly led to example, 1) DCV and FHV replicate rapidly and kill Drosophila some expression of the genes (Supplemental Fig. 1). upon injection, whereas SINV does not at the dose used (11, 24); 2) by guest on October 2, 2021 DCV is a natural pathogen of Drosophila, whereas FHV and SINV Discussion have not been found in wild Drosophila populations (41); 3) FHV We have investigated the involvement of RNAi and the evolu- and DCV possess, respectively, a strong and moderate viral sup- tionarily conserved JAK-STAT signaling pathway in the resistance pressor of RNAi, whereas SINV presumably does not (28, 42, 43). to a panel of seven viruses representing several important families, The three viruses also have different tissue tropism and may be including the arboviruses SINV and VSV. Our data provide a associated with tissue-specific modifications in the physiology of contrasting picture: on the one hand, a broad antiviral immunity the infected host. For example, FHV was recently shown to be a based on RNAi contributing to the defense against both RNA and cardiotropic virus, affected by potassium channels regulating heart DNA viruses, and on the other hand, a virus-specific transcriptional function (44), whereas DCV infection causes intestinal obstruction response involving the JAK-STAT pathway but playing a critical (S. Chtarbanova and J.-L. Imler, manuscript in preparation). role only in the case of Dicistroviridae infection. Comparison of the transcriptomes of the flies infected by the three viruses revealed more similarities between FHV and SINV RNAi protects against a DNA virus infection than between each of these and DCV. This may reflect the co- The present study extends work from several groups, including our evolution of DCV with its host, and the fact that this virus may have own, showing that flies mutant for the siRNA pathway are more learned to ward off the antiviral arsenal of its host. Indeed, DCV sensitive than wild-type flies to a large panel of RNA viruses, and induces fewer genes than does FHV, even though the two viruses reveals that Dcr-2 is also required for the control of the DNA virus replicate with similar kinetics and lead to the rapid death of the IIV-6. We note, however, that the increase of viral titer in siRNA flies. The genes induced by FHV and SINV encode chaperonins pathway–mutant flies is not as strong as in the case of some RNA (Tcp or Hsp), glutathione transferases, cytochrome P450s, stress viruses [e.g., VSV (25)]. This finding could reflect either the ex- markers (Tot family), thioester-containing proteins, and cyto- pression of a viral suppressor of RNAi by IIV-6 or the fact that skeletal regulators, suggesting an involvement of oxidative stress only a portion of the viral genome is targeted by siRNAs. Indeed, and phagocytosis in the response to these viruses. The two viruses this virus encodes an RNaseIII enzyme, which could cleave also upregulate the gene egghead (egh), which encodes a molecule siRNA duplexes, as previously reported in plants infected by the involved in the uptake of dsRNA and antiviral immunity (27). sweet potato chlorotic stunt virus (36). The involvement of Dcr- Despite the large overlap between the genes upregulated by FHV mediated immune responses against DNA virus infections was and SINV, the former induce a more intense transcriptional re- previously noted in plants, in which secondary structures in the sponse than the latter. This observation may reflect the more ag- transcribed viral RNAs, or dsRNAs formed from overlapping bi- gressive replication of FHV in Drosophila. Indeed, the genes directional transcripts, can be processed into siRNAs (37, 38). specifically induced by FHV include not only additional members Production of dsRNA from DNA viruses also occurs in animal of the families mentioned above (Hsp, Tcp, Gst, cytP450, thioester- The Journal of Immunology 657 containing proteins), supporting the idea of a more intense response, as flies mutant for either RNAi or the inducible JAK-STAT but also genes associated with cell death. In addition, FHV upre- pathway succumb to infection 2–3 d before the controls, with an gulates several molecules previously connected to innate immunity ∼10-fold increase in viral titer. in Drosophila, such as Hel89B (45), POSH (46), or MEKK1 (35), Interestingly, even though hop mutant flies appear to be spe- or molecules that may downmodulate the strong response to virus cifically sensitive to Dicistroviridae, other viruses activate the infection (e.g., the genes CG9311 and Pez, encoding tyrosine JAK-STAT pathway. Indeed, we observed a slight increase in the phosphatases). Finally, we note that FHV induced eight genes lethality of hop mutant flies postinfection with DXV and IIV-6. encoding factors with RNA binding domains, including four In Aedes mosquitoes, the JAK/STAT pathway was also shown to DExD/H box helicases, which may participate in the sensing and activate a defense against Dengue, a member of the Flaviviridae neutralization of viral nucleic acids. This specificity may reflect family (54). We also note that the JAK-STAT pathway–regulated a response of the host to counter the effect of the strong suppressor gene vir-1 (11) is induced by DCV and CrPV, but also FHV, even of RNAi B2, a dsRNA-binding protein (47). though hop mutant flies resist FHV infection much as do wild-type An intriguing aspect of the transcriptome of virus-infected flies flies. One hypothesis to explain this apparent paradox is that some is the upregulation of genes regulated by the Toll and Imd pathways. genes may be induced in a JAK-STAT–independent manner in the We observed an enrichment of Toll pathway target genes induced context of viral infections. For example, the gene TotM, which is in flies infected by DCV, but not FHV or SINV, suggesting that induced by several viruses normally resisted by hop mutant flies, DCV infection triggers this pathway. Among the genes induced by can be induced by the MEKK1 pathway, in addition to the JAK- DCV, but not by the two other viruses, we also note the presence of STAT pathway (35). Indeed, we observed that TotM remains fully Ect4, which encodes a TIR domain cytoplasmic molecule. The induced by FHV and SINV in hop mutant flies (C. Dostert and mammalian ortholog of this gene, SARM, was proposed to par- J.-L. Imler, unpublished observations). However, this hypothesis Downloaded from ticipate as a negative regulator of TLR signaling in some antiviral cannot account for the induction of vir-1 by FHV, because it is defenses (48). Two other genes regulated by DCV and possibly strongly reduced in hop mutant flies (C. Dostert and J.-L. Imler, establishing a connection between RNA silencing and the inducible unpublished observations). This finding suggests that some aspects response are worth mentioning: headcase was identified in a screen of the JAK-STAT–induced response may be redundant of other as a regulator of the siRNA pathway (49), whereas CG9925 en- defenses for FHV, but not for DCV. The fact that FHV triggers codes a protein with a Tudor domain, a characteristic of several a stronger transcriptional response than does DCV (Fig. 5) is http://www.jimmunol.org/ components of the Piwi-interacting RNA pathway (50). consistent with this hypothesis. Unlike the Toll-regulated genes, several genes regulated by Imd A key question pertains to the nature of the receptor detecting were induced in flies infected by DCV or FHV, although not by Dicistroviridae infection and triggering the JAK-STAT–dependent SINV. The Toll and Imd pathways play a well-characterized role inducible response. Our data point to the induction of a specific in the regulation of bacterial and fungal infections, through the subset of genes, including the JAK-STAT–regulated gene vir-1 regulation of genes encoding antimicrobial peptides. These genes (11), by fast-killing viruses such as DCV and CrPV, but also FHV, are also upregulated by viral infection, although not significantly, which replicate rapidly to high titers upon injection in flies. Of compared with buffer injection. This low level of induction most note, vir-1 induction is not affected in flies expressing the dsRNA- by guest on October 2, 2021 likely explains our inability to detect antimicrobial peptides in binding protein B2, or in Dcr-2 mutant flies, indicating that this the hemolymph of DCV-infected flies (51). Although not formally gene is not induced following sensing of dsRNA (10). This finding establishing that the Toll and Imd pathways participate in the suggests that sensing tissue damage and/or cell death could con- antiviral response, these results certainly do not rule out such a tribute to this inducible response, a hypothesis corroborated by the role (7, 9, 15). Alternatively, induction of the antimicrobial genes association of the JAK-STAT pathway with the cellular response may involve the transcription factor FOXO, a known regulator of to a variety of stresses (17, 55–57). stress resistance, and may occur independently of the Toll and Imd In conclusion, our data confirm that, beyond RNAi, an inducible pathways (52). Whatever the mechanism of induction, the bio- response contributes to the control of some viral infections in logical significance of this weak induction of molecules normally Drosophila. However, this response is complex, and great care active in the micromolar range is unclear. One possibility is that should be exercised before generalizing the results obtained with the Drosophila antimicrobial peptides carry additional functions one single virus species. This unexpected complexity probably that do not require high-level expression. For example, some reflects the intricate association of viruses with their host cells in mammalian b-defensins play a dual role in innate immunity and, different tissues, their different strategies of replication or protein in addition to their antibacterial properties, interact with chemo- expression, or their acquisition of suppressors of host defense. kine receptors with affinities in the nanomolar range, thus medi- ating chemoattraction of phagocytic cells (53). Acknowledgments Dicistroviridae-specific contribution of the JAK-STAT pathway We thank Estelle Santiago and Miriam Yamba for excellent technical assis- to antiviral immunity tance, Phil Irving for help with the microarray experiments, Anette An unexpected finding reported in this article is that hop mutant Schneeman (The Scripps Research Institute, La Jolla, CA) for providing flies have a clear phenotype for DCV and CrPV, but not for the DXV and CrPV, Trevor Williams (Veracruz, Mexico) for providing IIV-6, and Ste´phanie Blandin and Dominique Ferrandon for critical reading of other viruses tested. This observation indicates that the JAK-STAT the manuscript and helpful suggestions. The microarray analysis and the pathway, in addition to RNAi, participates in host defense against deep sequencing were performed at the Plateforme Biopuces et Se´quen- members of the Dicistroviridae family. DCV infection leads to c¸age, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, Stras- induction of the genes encoding the cytokines Upd2 and Upd3, bourg, France. which may subsequently activate the JAK-STAT pathway in non- infected cells, triggering an antiviral program of gene expression. Altogether, our results highlight that the contribution of the in- Disclosures ducible response to the control of DCV is similar to that of RNAi, The authors have no financial conflicts of interest. 658 VIRUS-SPECIFIC INDUCIBLE RESPONSE IN DROSOPHILA

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Leibbrandt, Rde. M. Simoes, S. Gruber, U. Puc, et al. 2009. sophila requires systemic RNA interference spread. Nature 458: 346–350. Genome-wide RNAi screen identifies genes involved in intestinal pathogenic 28.vanRij,R.P.,M.C.Saleh,B.Berry,C.Foo,A.Houk,C.Antoniewski,and bacterial infection. Science 325: 340–343. R. Andino. 2006. The RNA silencing endonuclease Argonaute 2 mediates 57. Jiang, H., P. H. Patel, A. Kohlmaier, M. O. Grenley, D. G. McEwen, and specific antiviral immunity in Drosophila melanogaster. Genes Dev. 20: 2985– B. A. Edgar. 2009. Cytokine/Jak/Stat signaling mediates regeneration and ho- 2995. meostasis in the Drosophila midgut. Cell 137: 1343–1355. &'('      

       

                           

                           

      !"#$% Supplemental Table II: List of genes induced by DCV, FHV and SINV

DCV specific (82 genes) Probe number Gene name Synomymus DCV * Putative function Key words 143500_at rho 12.5 signal transduction 153603_at CG10927 4.9 Unknown Cytidine/deoxycytidylate deaminase zinc-binding region 151144_at CG13553 4.2 152245_at CG13335 3.6 151473_i_at syt synaptotagmin p65 3.4 enzyme 143125_at dl dorsal 3.3 DNA binding NF-kappa-B/Rel/dorsal 150552_at CG9996 3.1 Unknown Glycosyl transferase family 8 143770_at Mtk Metchnikowin 3.0 Antifungal peptide activity

141691_at spz spatzle 3.0 Toll binding

141444_at CG2471 3.0 actin binding Leucine-rich repeat 141366_at CG10764 serine protease 3.0 serine-type endopeptidase activity Peptidase trypsin-like serine and cysteine proteases 154443_at CG1667 2.9 unknown 151661_s_at Nrt Neurotactin 2.8 cell adhesion Esterase/lipase/thioesterase

141718_at D12 2.8 transcription factor YEATS family

147495_at CG10081 2.8 Unknown Peptidase M28

143869_at vri vrille 2.8 RNA polymerase II transcription factor activity Basic-leucine zipper (bZIP) transcription factor

151648_at CalpB Calpain-B 2.7 calpain activity Peptidase C2 calpain 152979_at CG31531 2.7

152405_at CG6014 2.7 Unknown C-type lectin

147951_at CG13905 2.7

154983_at CG15634 2.7 AICARFT/IMPCHase bienzyme, Methylglyoxal synthase-like 153761_at CG11089 2.6 Unknown domain

141974_at CG4573 2.6 tyrosine--tRNA ligase; enzyme

153004_at CG8603 2.6 motor

153432_at Thor Thor 2.6 Eukaryotic initiation factor 4E binding Eukaryotic translation initiation factor 4E binding

142200_at CG32477 2.5

151361_at RhoGAP18B 2.5 GTPase activator activity Rho GTPase activation protein

148533_at ninjurin A ninjurin 2.5 cell adhesion 142429_at CG14089 2.5 Collagen triple helix repeat 142097_at Myosin 31DF myosin I 2.5 Actin binding Myosin head (motor domain), IQ calmodulin-binding region

154415_at CG3279 2.5 v-SNARE activity Vesicle transport v-SNARE

Fibronectin type III, Immunoglobulin-like, Peptidase M14 143665_at fra frazzled 2.5 netrin receptor activity carboxypeptidase A

153174_at CG9925 2.5 Maternal tudor protein, Zn-finger MYND type

151557_i_at CG32737 2.5 144197_at rdgB 2.5 phosphatidylinositol transporter activity Phosphatidylinositol transfer protein 147151_at CG18279 2.4 155065_at hdc headcase 2.3 signal transduction 151217_at U snoRNA host gene 1 2.3 151871_at CG30494 2.3 ligand binding or carrier

Zn-finger C2H2 type, Cytochrome c heme-binding site, 144505_at CG32767 2.3 Nucleic acid binding Immunoglobulin/MHC

152889_at dei delilah 2.3 transcription factor activity Basic helix-loop-helix dimerization domain bHLH Eukaryotic protein of unknown function DUF292, Pancreatic 153798_at CG10103 2.3 transcription factor ribonuclease General substrate transporter, Sugar transporter 154436_at rtet tetracycline resistance 2.3 sugar porter activity superfamily 147575_at CG18413 2.3 1-phosphatidylinositol-45- 1-phosphatidylinositol-45-biphosphate 142250_at norpA 2.3 biphosphate phosphodiesterase phosphodiesterase ; EC:3.1.4.11 153304_at bip1 2.2

145526_at CG4213 2.2 motor

148109_at CG12014 2.2 iduronate-2-sulfatase activity Sulfatase 153934_at CG12190 2.2 transcription factor Zn-finger Ran-binding 153814_at CG17612 2.2 nucleic acid binding Zn-finger C2H2 type

143293_at pbl pebble 2.2 guanyl-nucleotide exchange factor activity Rhodopsin-like GPCR superfamily

Dynein heavy chain, Peptidase eukaryotic cysteine 143726_at Dhc93AB Dynein heavy chain at 93AB 2.2 Dynein_heavy;microtubule motor activity peptidase active site, AAA ATPase 155084_at CG17260 2.2 nucleic acid binding Zn-finger RING

148881_at CG13059 2.2

154573_at CG8149 2.2 SAP;DNA binding;4 DNA-binding SAP 146703_at CG30158 2.2 GTP binding Ras GTPase superfamily

153520_at 5-HT7 Serotonin receptor 7 2.2 G-protein coupled serotonin receptor activity Rhodopsin-like GPCR superfamily

154930_at CG3165 3'-5' exonuclease 2.1 enzyme

154682_at Krn Keren 2.1 EGF receptor binding EGF-like domain

146238_at CG32955 2.1 kinesin;motor activity 143507_at mys integrin-beta-subunit 2.1 cell adhesion receptor 143605_at Drs Drosomycin 2.1 antifungal peptide activity Gamma thionin, Knottin

151022_at CG33188 2.1 protein binding Zn-finger AN1-like

147490_at Rgk1 GTP binding protein-like 2.1 RAS small monomeric GTPase activity Ras GTPase superfamily

148775_at CG10006 2.1 Zip;metal ion transporter activity Zinc transporter ZIP 152824_at Ect4 2.1 Sterile alpha motif SAM, ARM repeat fold, TIR Thrombospondin type I, von Willebrand factor type C, 153034_at CG32183 2.1 growth factor activity Cystine knot C-terminal

144380_at CG14770 2.1

Homeodomain-like, ARID (AT-rich interaction domain) 151685_r_at CG32813 2.1 transcription factor protein

148681_at CG12520 2.1

154808_at CG4953 2.1 Protein of unknown function DUF974, Actinoxanthin-like

Peptidase S1 chymotrypsin, Apoptosis regulator Bcl-2 151834_at CG32130 2.0 BAG;protein binding protein BAG 141962_at CG5787 2.0 transcription factor; DNA binding Sodium/calcium exchanger membrane region, K+- 151600_at Nckx30C sodium/calcium exchanger 2.0 calcium potassium:sodium antiporter activity dependent Na+/Ca+ exchanger related-protein 148381_r_at CG13674 2.0

141379_at Ag5r Antigen 5-related 2.0 Allergen V5/Tpx-1 related

148130_at ida imaginal discs arrested 2.0 TPR-like

143472_at Ten-m Tenascin major 2.0 cell adhesion EGF-like domain 143221_at Gliotactin gliotactin 2.0 serine esterase 150044_at CG18213 2.0 Zn-finger MYND type

146411_at CG15145 2.0

154066_at wus wurst 2.0 chaperone Heat shock protein DnaJ N-terminal FHV specific (279 genes)

Probe number Gene name Synomymus FHV * Putative function Key words

143470_at LysX Lysozyme X 6.9 lysozyme activity, cell death Glycoside hydrolase family 22

IPBinding-protein-dependent transport systems inner 141566_at CG10805 6.0 membrane component, Aldo/keto reductase, HEAT 152381_at Pcaf histone acetyltransferase 5.3 histone acetyltransferase activity Bromodomain, GCN5-related N-acetyltransferase

152230_at CG6778 4.5 glycine--tRNA ligase

155116_at POSH Plenty of SH3s 4.3 receptor signaling complex scaffold activity Zn-finger RING, Neutrophil cytosol factor 2

Leucine-rich repeat, TIR, Interleukin-1 receptor type I/Toll 152907_at 18w 18 wheeler 4.2 transmembrane receptor activity precursor

152730_at CG5205 4.2 RNA helicase activity Sec63 domain, DEAD/DEAH box helicase, AAA ATPase

149122_at CG5571 4.0 Rhodopsin-like GPCR superfamily GTP binding, hydrolase activity acting on acid Protein synthesis factor GTP-binding, Elongation factor Tu 144194_at Dgp-1 3.8 anhydrides in phosphorus-containing domain 2 anhydrides 147210_at CG12868 3.8

Tyrosine specific protein phosphatase, Rhodopsin-like 142986_at CG9311 protein phosphatase-like 3.7 protein-tyrosine-phosphatase activity GPCR superfamily

143828_at Apc APC-like 3.6 beta-catenin binding, microtubule binding Armadillo, APC cysteine-rich

145304_at psh persephone 3.6 serine-type endopeptidase activity Peptidase S1 chymotrypsin

154332_at CG12876 ALix 3.6 signal transducer activity, cell death BRO1, Rho GTPase activation protein

cytidine deaminase activity, 146014_at CG8353 cytidine deaminase-like 3.6 Cytidine/deoxycytidylate deaminase zinc-binding region dCMP_cyt_deam;zinc ion binding importin-alpha export receptor activity, cell 143953_at Cas CAS/CSE1 segregation protein 3.6 CAS/CSE C-terminal, Importin-beta N-terminal death, phagocytosis retinoid binding/fatty acid binding lipid binding, Vitellogenin_N;lipid transporter von Willebrand factor type D, Lipid transport protein N- 153828_at CG15828 3.5 protein-like glycoprotein activity terminal, Protein of unknown function DUF1081 peroxisomal carnitine O- 141776_at CG12428 3.4 carnitine O-octanoyltransferase activity Acyltransferase ChoActase/COT/CPT octanoyltransferase protein-tyrosine-phosphatase activity, Tyrosine specific protein phosphatase and dual specificity 145889_at Pez protein phosphatase-like 3.4 cytoskeletal protein binding protein phosphatase 149757_at GstD3 Glutathione S transferase D3 3.4 glutathione transferase activity Glutathione S-transferase

143031_at CG7627 ATP-binding cassette transporter 3.4 ATP-binding cassette (ABC) transporter activity ABC transporter, AAA ATPase

145025_at CG12177 purine nucleosidase 3.3 enzyme Inosine/uridine-preferring nucleoside hydrolase

149173_at CG7632 3.3 enzyme Annexin, Esterase/lipase/thioesterase

150261_at CG4335 3.3 gamma-butyrobetaine dioxygenase activity Gamma-butyrobetaine hydroxylase

receptor signaling protein serine/threonine 141756_at Mekk1 heat shock construct of Inoue 3.3 kinase activity, MAP kinase kinase kinase Serine/threonine protein kinase activity

149754_at GstD10 Glutathione S transferase D10 3.3 glutathione transferase activity Glutathione S-transferase

152949_at Cyp6d4 cytochrome P450 CYP6D4 3.2 cytochrome P450 activity Cytochrome P450, E-class P450 group I

152798_at Cyp12c1 cytochrome P450 CYP12C1 3.2 cytochrome P450 activity Cytochrome P450, E-class P450 group I

ATP dependent RNA helicase activity, DEAD/DEAH box helicase, ATP-dependent helicase DEAD- 153844_at Rs1 3.2 DEAD;ATP binding box

143384_at trx trithorax 3.2 DNA binding Zn-finger-like PHD finger, FY-rich domain SET-related region

141436_at CG3505 3.2 serine-type endopeptidase activity Peptidase trypsin-like serine and cysteine proteases

152481_at CG9951 3.2 motor Protein of unknown function DUF812

141381_at CG30069 3.2 RNA_pol_Rpb1_R;DNA binding Short-chain dehydrogenase/reductase SDR

Pol III transcription termination factor activity, RNA-binding region RNP-1 (RNA recognition motif), RNA- 143668_at La La autoantigen-like 3.2 RNA binding, 5S rRNA primary transcript binding protein Lupus Lal binding

153930_at CG5224 3.1 glutathione transferase activity Glutathione S-transferase

143374_at T-cp1 Tcp1-like 3.1 chaperone activity, phagocytosis Chaperonin Cpn60/TCP-1, GroEL-like chaperone ATPase

153051_at CG4841 3.1

143696_at CG4038 3.0 Gar1 protein RNA-binding region

141466_at CG15099 3.0 protein kinase Dopey N-terminal

154265_at CG8258 T-complex protein 1 theta-subunit 3.0 chaperonin ATPase activity Chaperonin Cpn60/TCP-1, GroEL-like chaperone ATPase Cyclin-like F-box, Zn-finger CXXC type, Cytochrome c heme- 151745_at CG11033 3.0 binding site, Leucine-rich repeat cysteine-containing subtype, Transcription factor jumonji jmjC

152411_at CG10286 3.0 ARM repeat fold

DEAD/DEAH box helicase, Heavy metal 146573_at CG9253 3.0 ATP dependent RNA helicase activity transport/detoxification protein

152638_at CG3363 2.9 DNA binding

154861_at asf1 anti-silencing factor 1 2.9 cell cycle regulator Anti-silencing protein ASF1-like, p53-like transcription factor

SNF2 related domain, DEAD/DEAH box helicase, ARM repeat 152420_at Hel89B Helicase 89B 2.9 ATP dependent DNA helicase activity fold UBA/THIF-type NAD/FAD binding fold, Ubiquitin-activating 152946_at Uba1 Ubiquitin activating enzyme 1 2.9 ubiquitin activating enzyme activity enzyme repeat 151728_at CG18596 2.9 SpoU_methylase;RNA binding tRNA/rRNA methyltransferase (SpoU), ARM repeat fold damaged DNA binding, CPSF_A;nucleic acid CPSF A subunit C-terminal, Immunoglobulin/major 147940_at CG13900 spliceosomal protein-like 2.9 binding histocompatibility complex actin binding, structural constituent of 154742_at Arp66B Actin-related protein 66B 2.8 Actin/actin-like, phagocytosis cytoskeleton Aminoacyl-tRNA synthetase class Ia, Valyl-tRNA synthetase 141603_at Aats-val Valyl-tRNA synthetase 2.8 glutamate-tRNA ligase activity class Ia

Zn-finger MYND type, Programmed cell death protein 2 C- 147841_at Zfrp8 Zinc finger protein RP-8 2.8 DNA binding terminal, 4Fe-4S ferredoxin iron-sulfur binding domain peripheral-type benzodiazepine 151838_at CG2789 2.8 benzodiazepine receptor activity TspO/MBR-related protein receptor

154028_at CG9200 2.8 myb_DNA-binding;DNA binding Myb DNA-binding domain, Homeodomain-like

154859_at CG17904 2.8 nucleotide binding

Alpha-2-macroglobulin, Terpenoid cylases/protein 152023_at TepIII Thiolester containing protein III 2.8 endopeptidase inhibitor activity, phagocytosis prenyltransferase alpha-alpha toroid

146827_at CG8738 2.8 serine-type endopeptidase activity Peptidase trypsin-like serine and cysteine proteases

150418_at sav salvador 2.8 enzyme, cell death WW/Rsp5/WWP domain

153299_at CG12292 2.8 Protein of unknown function DUF803

152623_at Est2 Esterase-2 2.7 carboxylesterase activity Carboxylesterase type B, Esterase/lipase/thioesterase

154156_at CG5033 2.7 transcription factor activity G-protein beta WD-40 repeat aldehyde reductase activity, trans-12- 148662_at CG10638 aldehyde reductase-like 2.7 Aldo/keto reductase dihydrobenzene-12-diol dehydrogenase activity

154452_at c11.1 2.7 ARM repeat fold

carnitine-acylcarnitine carrier carrier activity, acyl carnitine transporter Mitochondrial substrate carrier, Mitochondrial carrier 154670_at CG3476 2.7 protein activity protein, Adenine nucleotide translocator 1

transcription factor activity, ligand-dependent Zn-finger C4-type steroid receptor, Vitamin D receptor, 143144_at eg eagle 2.7 nuclear receptor activity Steroid nuclear receptor ligand-binding

154451_at CG2691 2.7 ARM repeat fold

143782_at Cyp4p1 Cytochrome P450-4p1 2.7 cytochrome P450 activity Cytochrome P450, E-class P450 group I

tRNA synthetase class II (D K and N), OB-fold nucleic acid 153336_at Aats-asp Aspartyl-tRNA synthetase 2.7 aspartate-tRNA ligase activity binding domain, Aspartyl-tRNA synthetase archea/euk type CUB, EGF-like domain, Aspartic acid and asparagine 141481_at CG32702 2.7 metalloendopeptidase activity hydroxylation site

type-I transmembrane ER-resident protein kinase activity, eukaryotic elongation 149333_at PEK 2.7 Serine/threonine protein kinase, Bacterial quinoprotein serine/threonine kinase-like factor-2 kinase activity

antibacterial peptide activity, wide-spectrum Alpha-2-macroglobulin, Serpin, Terpenoid cylases/protein 145970_at TepII Thiolester containing protein II 2.7 protease inhibitor activity, phagocytosis prenyltransferase alpha-alpha toroid endothelin-converting enzyme activity, 142141_at CG4723 2.7 Peptidase M13 neprilysin Peptidase_M13_N;metallopeptidase activity

141207_at CG4453 nucleoporin 2.7 endopeptidase

protein carrier activity, RAN protein binding, 151799_at msk moleskin 2.6 Importin-beta N-terminal, ARM repeat fold protein transporter activity 144700_at CG15347 2.6 Protein of unknown function DUF753

146788_at CG11210 2.6 Protein of unknown function DUF221

149968_at CG5399 2.6

154083_at Smg5 2.6 Nucleotide binding protein PINc

sphingomyelin phosphodiesterase activator Beige/BEACH domain, G-protein beta WD-40 repeat, 152479_at CG6734 2.6 activity Immunoglobulin/MHC 142720_at CG11660 2.6 protein kinase activity Protein of unknown function RIO1, Tyrosine protein kinase

142550_at l(3)mbt lethal (3) malignant brain tumor 2.6 transcription factor activity Sterile alpha motif (SAM)/Pointed domain, Mbt repeat

145098_at Lsd-2 Lipid storage droplet-2 2.6 Perilipin

RAB-protein geranylgeranyltransferase activity, 154565_at CG12007 2.6 Protein prenyltransferase PPTA;protein prenyltransferase activity 145074_at CG5321 2.6 gamma-butyrobetaine dioxygenase activity Gamma-butyrobetaine hydroxylase Interferon-related protein conserved region, Ankyrin, ARM 145681_at CG31694 interferon-like 2.6 signal transduction repeat fold

153088_at pst pastrel 2.6

antibacterial peptide activity, endopeptidase 146503_at TepIV Thiolester containing protein IV 2.6 Alpha-2-macroglobulin, HPr serine phosphorylation site inhibitor activity

154692_at CG18240 2.6 Cytochrome P450, E-class P450 group I

GTP binding, GTP_EFTU;translation elongation Protein synthesis factor GTP-binding, Elongation factor Tu 141564_at CG2017 2.6 factor activity domain 2 latrotoxin receptor activity, Gal_Lectin;sugar D-galactoside/L-rhamnose binding SUEL lectin domain, G- 146823_at Cirl latrophilin receptor-like 2.6 binding protein coupled receptor family 2 (secretin-like) glucuronosyltransferase activity, 147690_at Ugt58Fa 2.6 UDPGT;transferase activity transferring hexosyl UDP-glucoronosyl/UDP-glucosyl transferase groups Pre-mRNA processing ribonucleoprotein binding region, 153330_at Nop56 2.6 Nucleic acid-binding OB-fold

154194_at CG8939 2.5 rRNA methyltransferase activity Ribosomal RNA methyltransferase RrmJ/FtsJ

Pancreatic trypsin inhibitor (Kunitz), Immunoglobulin-like, 142182_s_at Ppn Papilin 2.5 Thrombospondin type I, EGF-like domain

electron carrier activity, fer2;electron 143690_at Fdxh Ferredoxin 2.5 Ferredoxin, Adrenodoxin, Cytochrome c heme-binding site transporter activity

[acyl-carrier protein] S-acetyltransferase Zinc-containing alcohol dehydrogenase superfamily, 151887_at v(2)k05816 2.5 activity, enoyl-[acyl-carrier protein] reductase Thioesterase, Phosphopantetheine-binding domain, Acyl (NADPH B-specific) activity transferase domain

154537_at CG31738 2.5 cell adhesion Fibronectin type III, Ribosomal protein S2

144074_at CG4025 2.5

148524_at CG14152 2.5 149907_r_at CG8066 cystatin-like 2.5 cysteine protease inhibitor activity Sarcocystatin, Cysteine protease inhibitor 154829_at CG7728 2.5 Protein of unknown function DUF663 oxidoreductase activity acting on CH-OH group Short-chain dehydrogenase/reductase SDR, Glucose/ribitol 149345_at CG12171 alcohol dehydrogenase-like 2.5 of donors, adh_short;oxidoreductase activity dehydrogenase 152727_at CG7224 2.5 153358_at Nmd3 Nonsense-mediated mRNA 3 2.5 RNA binding, protein binding NMD3 family, Cytochrome c heme-binding site

153152_at CG11920 2.5 Brix domain

alpha-tocopherol transfer protein- 154616_at CG31633 2.5 ligand binding or carrier Cyclin-like F-box, Four-helical cytokine like 154916_at CG7417 2.5 transcription factor activity Ubiquitin system component Cue, Zn-finger Ran-binding

145612_at CG7291 2.5 signal transduction E1 protein and Def2/Der2 allergen

141658_at CG5853 ATP-binding cassette transporter 2.5 ATP-binding cassette (ABC) transporter activity Phagocytosis, Phosphopantetheine attachment site

142836_at CG32041 heat shock protein 22 kD 2.5 heat shock protein

146844_at CG30349 2.5 G-protein beta WD-40 repeat

143606_at Cyp18a1 Cytochrome P450-18a1 2.5 cytochrome P450 activity Cytochrome P450, E-class P450 group I guanine deaminase activity, 155054_at CG18143 2.5 Amidohydrolase, Calcium-binding EF-hand Amidohydro_1;hydrolase activity Zn-finger C2H2 type, Zinc-finger protein of unknown 152398_at CG10631 2.5 nucleic acid binding function DM3 154928_at wdn wings down 2.4 RNA polymerase II transcription factor activity Zn-finger C2H2 type

145309_at CG6335 histidine--tRNA ligase 2.4 enzyme

tRNA synthetases class-II (G H P and S), Aminoacyl-transfer 142167_at CG17259 serine--tRNA ligase-like 2.4 serine-tRNA ligase activity RNA synthetase class II 145230_at CG5010 2.4 CHCH transcription factor activity, myb_DNA- 142244_at CG9305 2.4 Myb DNA-binding domain binding;DNA binding 153052_at CG10420 2.4 ARM repeat fold

154647_at CG8877 2.4 pre-mRNA splicing factor activity Peptidase M67 Mov34

glucose transporter activity, General substrate transporter, Sugar transporter 143040_at CG4797 sugar transporter-like 2.4 sugar_tr;transporter activity superfamily

142263_at CG32836 2.4 ligand binding or carrier

helicase activity, DNA dependent ATPase SNF2 related domain, DNA topoisomerase II, ATP-dependent 151511_r_at dom domino 2.4 activity, general RNA polymerase II DNA ligase transcription factor activity

153862_at l(3)07882 2.4 Nop14-like protein

RAB-protein geranylgeranyltransferase activity, 153458_at Rep Rab escort protein 2.4 Rab GTPase activator Rab escort protein activity

G-protein beta WD-40 repeat, Lissencephaly type-1-like 141309_at CG7611 2.4 chaperone homology motif

polypeptide N- polypeptide N-acetylgalactosaminyltransferase 152222_at CG4445 2.4 Glycosyl transferase family 2, Ricin B lectin domain acetylgalactosaminyltransferase activity

146718_at Tsp42Er Tetraspanin 42Er 2.4 CD9/CD37/CD63 antigen, Tetraspanin protein serine/threonine phosphatase activity, Serine/threonine-specific protein phosphatase and bis(5- 143976_at Pp4-19C Protein phosphatase 19C 2.4 CTD phosphatase activity, nucleosyl)-tetraphosphatase Metallophos;hydrolase activity Carboxylesterase type B, TONB Box N terminus, 154994_at clt cricklet 2.3 carboxylesterase activity Esterase/lipase/thioesterase

145265_at CG6762 2.3 ParB-like nuclease

154620_at CG6181 2.3 motor G-protein beta WD-40 repeat

151373_at CG32795 2.3

147699_at CG4250 2.3 EGF-like domain

152100_at CG31705 2.3 DNA binding

153963_at CG16971 2.3

153320_at Smg6 2.3 RNA binding Nucleotide binding protein PINc, TPR-like

RAB small monomeric GTPase activity, GTP Ras GTPase superfamily, ARF/SAR superfamily, Sigma-54 152357_at Rab-RP4 Rab-related protein 4 2.3 binding, ras;small monomeric GTPase activity factor interaction domain ATP dependent RNA helicase activity, KH, DEAD/DEAH box helicase, ATP-dependent helicase 154657_at CG7878 2.3 KH;nucleic acid binding DEAD-box

143627_at cora coracle 2.3 cytoskeletal protein binding Band 4.1, Ezrin/radixin/moesin ERM, FERM

Lethal(2) giant larvae protein, Regulator of chromosome 153565_at l(2)gl lethal (2) giant larvae 2.3 myosin II binding, cell death condensation RCC1 taste receptor activity, G-protein coupled 144536_at Tre1 heat shock construct of Ishimoto 2.3 Rhodopsin-like GPCR superfamily receptor activity unknown ligand, cell death DNA binding, RNA binding, rrm;nucleic acid 154081_at mod modulo 2.3 RNA-binding region RNP-1 (RNA recognition motif) binding

Octicosapeptide/Phox/Bem1p, Zn-finger ZZ type, Peptidase 152056_at ref(2)P refractory to sigma P 2.3 eukaryotic cysteine peptidase, Ubiquitin-associated domain

153126_at CG11523 2.3

SNF;neurotransmitter:sodium symporter 145975_at CG13795 2.3 Sodium:neurotransmitter symporter activity

152368_at CG8913 2.3 peroxidase activity Animal haem peroxidase, Haem peroxidase

142736_s_at Hmgs HMG Coenzyme A synthase 2.3 hydroxymethylglutaryl-CoA synthase activity Hydroxymethylglutaryl-coenzyme A synthase

154193_at Set 2.3 cyclin binding, NAP;DNA binding Nucleosome assembly protein (NAP)

149558_at CG16777 2.3

galactose-specific C-type lectin- 146490_at CG9978 2.3 lectin_c;sugar binding C-type lectin like 147547_at CG18067 2.3 3'5'-cyclic nucleotide phosphodiesterase

Protein of unknown function DUF139, EGF-like domain, 142395_at CG31839 nimrodB2 2.3 cell adhesion Growth factor receptor 151642_at stich1 sticky ch1 2.3 Basic helix-loop-helix dimerization domain bHLH 141751_at east enhanced adult sensory threshold 2.3 Peptidase M14 carboxypeptidase A

144771_at CG9691 2.3

150295_at CG7044 2.3 ARM repeat fold

uridine phosphorylase activity, Purine and other phosphorylases family 1, Uridine 145684_at CG17224 2.3 PNP_UDP_1;catalytic activity phosphorylase eukaryotic

152444_at ken ken and barbie 2.3 transcription factor activity, phagocytosis Zn-finger C2H2 type, BTB/POZ domain, Immunoglobulin-like

methylenetetrahydrofolate methylenetetrahydrofolate reductase (NADPH) 148573_at CG7560 2.3 Methylenetetrahydrofolate reductase reductase activity 151946_at Os9 Olfactory-specific 9 2.3 pheromone binding

152518_at CG8231 T-complex protein 1 zeta-subunit 2.3 chaperonin ATPase activity Chaperonin Cpn60/TCP-1, GroEL-like chaperone ATPase

translation initiation factor activity, MIF4G;RNA eIF4-gamma/eIF5/eIF2-epsilon, Initiation factor eIF-4 gamma 152430_at l(2)01424 NAT1 2.3 binding, cell death middle, ARM repeat fold

152280_at CG2467 2.3 Endoglin/CD105 antigen

Serine palmitoyltransferase 147131_at Spt-I 2.2 serine C-palmitoyltransferase activity subunit I

149759_at GstD5 Glutathione S transferase D5 2.2 glutathione transferase activity Glutathione S-transferase

152817_at CG1648 2.2

specific RNA polymerase II transcription factor 153515_at Dr Drop 2.2 Homeobox, Homeodomain-like activity

144950_at CG32654 glutamate receptor-like 2.2 enzyme

149937_at CG6912 2.2

translation release factor activity, Elongation factor Tu, Translation factor, Protein synthesis 142364_at HBS1 translation initiation factor 3-like 2.2 GTP_EFTU;translation elongation factor activity factor GTP-binding

Pheromone-binding protein- pheromone binding, phenylalkylamine binding, Pheromone/general odorant binding protein PBP/GOBP, 143646_at Pbprp1 2.2 related protein 1 PBP_GOBP;odorant binding Insect pheromone/odorant binding protein PhBP CBF/Mak21 family, Peptidase eukaryotic cysteine peptidase, 154719_at CG1234 2.2 ARM repeat fold 146710_at Tsp42Ef Tetraspanin 42Ef 2.2 CD9/CD37/CD63 antigen, Tetraspanin

144142_at Kaz1 2.2 serine protease inhibitor

154940_at CG6876 2.2 pre-mRNA splicing factor activity Pre-mRNA processing ribonucleoprotein binding region

146453_at CG31753 2.2 nucleic acid binding

BRK, SNF2 related domain, Helicase, Chromo, DEAD/DEAH 141644_at kis kismet 2.2 DNA helicase activity box helicase 147097_at CG30044 2.2 serine-type endopeptidase activity, 143699_at Ser6 Serine protease 6 2.2 Peptidase trypsin-like serine and cysteine proteases trypsin;trypsin activity

152874_at CG30015 2.2 motor

144115_at cib ciboulot 2.2 actin binding Thymosin beta-4

144890_at CG15220 replication factor A 14 kD subunit 2.2 DNA binding Nucleic acid-binding OB-fold

149419_at CG1105 2.2 Arrestin

146699_at Spn4 Serine protease inhibitor 4 2.2 serine-type endopeptidase inhibitor activity Serpin, Endoplasmic reticulum targeting sequence

142349_at yellow-f yellow-f 2.2 intramolecular isomerase activity Major royal jelly protein

149057_at CG9295 cuticle protein 2.2 structural constituent of cuticle (sensu Insecta) Insect cuticle protein

154058_at CG5295 2.2 Patatin;catalytic activity Patatin

142674_at CG8282 sorting nexin-like 2.2 Phox-like

154433_at CG7338 2.2 Protein of unknown function DUF663 Filamin/ABP280 repeat, Calponin-like actin-binding, Actin- 151902_at jbug jitterbug 2.2 actin binding binding actinin-type Ribosomal_L7Ae;structural constituent of Ribosomal protein L7Ae/L30e/S12e/Gadd45, High mobility 142579_at NHP2 2.2 ribosome group-like nuclear protein

141491_at CG8465 2.2 enzyme inhibitor Ankyrin, Ribosomal protein L9 N-terminal-like

C2 domain, Pleckstrin-like, Ras GTPase-activating protein, 143456_at Gap1 GTPase-activating protein 1 2.2 Ras GTPase activator activity Rho GTPase activation protein 154505_at CG9107 2.2 rrm;nucleic acid binding RNA-binding region RNP-1 (RNA recognition motif)

154039_at CG11738 2.2 RNA binding KH

141457_at Mtor Megator 2.2 motor

141622_at CG5841 ankyrin-like 2.1 apoptosis inhibitor Ankyrin, Zn-finger ZZ type, Mib_herc2, Zn-finger RING glycine dehydrogenase (decarboxylating) 149624_at CG3999 glycine dehydrogenase p protein 2.1 Glycine cleavage system P-protein activity cystathionine beta-synthase activity, Cysteine synthase/cystathionine beta-synthase P- 154013_at CG1753 cystathionine beta-synthase-like 2.1 PALP;lyase activity phosphate-binding site, Cystathionine beta-synthase Aminotransferase class I and II, Aminolevulinic acid 141461_at Alas Aminolevulinate synthase 2.1 5-aminolevulinate synthase activity synthase endonuclease G activity, Endonuclease;nucleic 154735_at CG8862 2.1 DNA/RNA non-specific endonuclease acid binding 155000_at CG7806 ATP-binding cassette transporter 2.1 ion channel

149590_at CG8516 2.1

150379_at CG6937 RNA binding protein-like 2.1 RNA binding, rrm;nucleic acid binding RNA-binding region RNP-1 (RNA recognition motif)

cathepsin L activity, KRAB;nucleic acid binding, Peptidase C1A papain, KRAB box, Peptidase eukaryotic 141762_at Cp1 Cysteine proteinase-1 2.1 Cell death cysteine peptidase active site

151904_at CG31012 2.1 SH3/SH2 adaptor protein activity SH3, Neutrophil cytosol factor 2

155134_at MICAL-like alpha-actinin-like 2.1 actin binding Zn-binding protein LIM, Calponin-like actin-binding

AAA ATPase VAT, Cell division protein 48 CDC48 domain 2, 143738_at Nsf2 NEM-sensitive fusion protein 2 2.1 cdc48_N;ATP binding Aspartate decarboxylase-like fold 143000_at CG5525 2.1 chaperonin ATPase activity Chaperonin Cpn60/TCP-1, GroEL-like chaperone ATPase

144243_at Spn6 Serine protease inhibitor 6 2.1 serine-type endopeptidase inhibitor activity Serpin SNF4/AMP-activated protein protein serine/threonine kinase activity, 154116_at SNF4A 2.1 CBS domain kinase gamma subunit SNF1A/AMP-activated protein kinase activity

149138_at CG4858 2.1 nucleotide binding Mrp family

estradiol 17 beta-dehydrogenase- steroid dehydrogenase activity, Short-chain dehydrogenase/reductase SDR, Glucose/ribitol 146386_at CG13284 2.1 like adh_short;oxidoreductase activity dehydrogenase, phagocytosis 145647_at Cyp309a1 cytochrome P450 CYP309A1 2.1 cytochrome P450 activity Cytochrome P450, E-class P450 group I

147354_at CG15917 2.1

152511_at Ela Elastin-like 2.1 Collagen triple helix repeat

142566_at CG2926 2.1 PHD;DNA binding Zn-finger RING, Zn-finger-like PHD finger

152581_at Spn5 Serine protease inhibitor 5 2.1 serine-type endopeptidase inhibitor activity Serpin

152470_at CG10189 2.1

152019_at to takeout 2.1 Odorant binding protein 155057_at CG10107 2.1 cysteine-type peptidase activity Peptidase C48 SUMO/Sentrin/Ubl1 153909_at CG2972 2.1 Protein of unknown function DUF133 149260_s_at CG10712 2.1 DNA binding 145393_at CG32528 2.1 actin binding Calponin-like actin-binding 145547_r_at CG13947 2.1

152066_at BcDNA:GH06451 asparagine--tRNA ligase-like 2.1 enzyme

144015_at Smrter Smr 2.1 transcription co-repressor, phagocytosis ecdysone receptor co-repressor Rabex-5, guanyl-nucleotide guanyl-nucleotide exchange factor activity, zf- 141553_at CG9139 2.1 Zn-finger A20-like, Vacuolar sorting protein 9 exchange factor A20;DNA binding 149020_at CG6843 CBF1 interacting corepressor 2.1 transcription factor binding ubiquitin-specific protease activity, Peptidase C19 ubiquitin carboxyl-terminal hydrolase family 154133_at CG15817 2.1 UCH;cysteine-type endopeptidase activity 2 Laminin-type EGF-like domain, Galactose-binding like, 154252_at LanA Laminin A 2.1 structural protein Concanavalin A-like lectin/glucanase

145466_at CG32521 2.1

150514_at CG13618 2.1 Odorant binding protein

thiol-disulfide exchange intermediate activity, 141539_at Trx-2 thioredoxin-2 2.1 Thioredoxin type domain, Thioredoxin domain 2 thiored;electron transporter activity

152780_at CG7967 2.1 Protein of unknown function DUF605

147696_at CG13510 2.1

peptidoglycan recognition protein- N-acetylmuramoyl-L-alanine amidase family 2, Animal 142657_at PGRP-SB1 2.1 peptidoglycan recognition activity like peptidoglycan recognition protein PGRP tRNA binding, translation initiation factor 142531_at eIF-2 Eukaryotic initiation factor 2&bgr; 2.1 Translation initiation factor IF5 activity, GTP binding Saccharomyces cerevisiae UAS 144174_at inx2 2.1 innexin channel activity Innexin construct a of Stebbings

150746_at CG12883 2.1

143957_at CG11971 2.1 nucleic acid binding Zn-finger C2H2 type

143982_at Chd3 DEAH-box helicase 2.1 ATP dependent DNA helicase ; EC:3.6.1.3

154495_at CG6459 2.1 defense/immunity protein Mitochondrial glycoprotein

143058_f_at Act5C Actin 5C 2.1 structural constituent of cytoskeleton Actin/actin-like, phagocytosis

Deoxyribonuclease/rho motif-related TRAM, Radical SAM, 153079_at CG6550 2.1 MiaB-like tRNA modifying enzyme archaeal-type 144369_at CG11378 2.1 Protein of unknown function DUF1397

146611_at CG6448 2.1 receptor

ubiquitin-protein ligase activity, apoptosis Zn-finger RING, Baculovirus inhibitor of apoptosis protein 152631_at th thread 2.1 inhibitor activity repeat (BIR) Pheromone/general odorant binding protein PBP/GOBP, 150834_at Obp99c Odorant-binding protein 99c 2.1 PBP_GOBP;odorant binding Insect pheromone/odorant binding protein PhBP

154669_at CG5080 2.1 motor alpha-tocopherol transfer protein- Cellular retinaldehyde-binding)/triple function, alpha- 146898_at CG12926 2.1 tocopherol binding like tocopherol transport transcription factor activity, RNA polymerase II Paired amphipathic helix, H+-transporting two-sector 141716_at Sin3A 2.0 transcription factor activity ATPase

143784_at Fer2LCH Ferritin 2 light chain homologue 2.0 ferrous iron binding, ferritin;binding Ferritin/ribonucleotide reductase-like AAA ATPase central region, MIT, Viral coat and capsid 154205_at spas spastin 2.0 ATPase activity protein 152140_at CG8678 2.0 enzyme G-protein beta WD-40 repeat

146771_at CG1882 2.0 enzyme Alpha/beta hydrolase fold, Esterase/lipase/thioesterase

149251_at l(3)04053 2.0

147486_i_at CG18606 2.0

142778_at bcn92 2.0 Complex 1 LYR protein

high affinity inorganic phosphate:sodium 142212_at l(2)08717 sodium/phosphate cotransporter 2.0 General substrate transporter, Major facilitator superfamily symporter activity, sugar_tr;transporter activity [2Fe-2S]-binding, Ferredoxin, 2Fe-2S ferredoxin iron-sulfur 154786_at CG18522 2.0 fer2;electron transporter activity binding site 147364_at CG14478 2.0 C-5 cytosine-specific DNA methylase

150582_at CG13659 2.0 Protein of unknown function DUF227

serine-type endopeptidase activity, 148144_at CG14990 2.0 Peptidase trypsin-like serine and cysteine proteases trypsin;trypsin activity

154757_at CG14991 mitogen inducible protein-like 2.0 Pleckstrin-like /// IPR009065 // FERM 2OG-Fe(II) oxygenase superfamily, Prolyl 4-hydroxylase 154136_at CG31120 2.0 alpha subunit 146493_at CG9987 2.0 Protein of unknown function UPF0027

Filamin/ABP280 repeat, Calponin-like actin-binding, Actin- 141641_at cher cheerio 2.0 actin binding binding actinin-type

143020_at Talin talin 2.0 actin binding, cytoskeletal anchoring activity

Short-chain dehydrogenase/reductase SDR, Glucose/ribitol 155055_at CG31549 2.0 oxidoreductase activity dehydrogenase

145021_at ligase4 DNA ligase 2.0 DNA ligase (ATP) activity ATP-dependent DNA ligase, BRCT

151208_at CG15068 2.0

PDZ/DHR/GLGF domain, Phosphotyrosine interaction 154462_at X11L 2.0 enzyme domain, Aminoacyl-tRNA synthetase class I G-protein coupled receptor activity, 149874_at CG9918 2.0 Rhodopsin-like GPCR superfamily neuropeptide receptor activity 141652_at CG5290 2.0 TPR repeat , TPR-like

142203_at CG14788 2.0 GTP-binding protein HSR1-related, GTP-binding domain

G-protein coupled receptor activity unknown 152436_at CG12290 G-protein coupled receptor-like 2.0 Rhodopsin-like GPCR superfamily ligand, rhodopsin-like receptor activity SINV specific (29 genes)

Probe number Gene name Synomymus SDV * Putative function Key words

structural protein of ribosome; small-subunit 142031_at RpS6 ribosomal protein S6 8.3 cytosol ribosomal protein 151466_at CG14599 3.8 cyclin-dependent protein kinase regulator 150138_at CG31232 2.8 Cyclin activity oxidoreductase activity acting on CH-OH group Short-chain dehydrogenase/reductase SDR, Rhodopsin-like 144428_at CG3603 2.6 of donors, acetoacetyl-CoA reductase activity GPCR superfamily

150711_at CG17189 2.6 Odorant binding protein

149283_at CG12585 2.5

149489_at CG7459 copper transporter-like 2.4 copper ion transporter activity Ctr copper transporter

151137_r_at IM4 Immune induced protein 4 2.3 defense/immunity protein activity

General substrate transporter, Sugar transporter 147027_at CG30035 2.3 sugar transporter activity superfamily

141289_at CG9821 2.3

142089_at BG:DS00941.14 2.3

female-specific independent of 150335_at fit 2.3 transformer 154079_at BCL7-like 2.2 BCL7 N-terminal

peroxidase activity, non-selenium glutathione Alkyl hydroperoxide reductase/ Thiol specific antioxidant/ 146946_s_at Prx2540-2 Peroxiredoxin 2540 2.2 peroxidase activity Mal allergen

153378_at Cg25C Collagen type IV 2.2 structural protein Collagen helix repeat, Collagen triple helix repeat

149564_at CG9386 2.2

152347_at CG11841 serine protease 2.2 serine-type endopeptidase activity Peptidase trypsin-like serine and cysteine proteases

150052_at CG31267 2.2 serine-type endopeptidase activity Peptidase trypsin-like serine and cysteine proteases

144239_at la costa 2.2

148228_at CG10592 alkaline phosphatase-like 2.1 alkaline phosphatase activity Alkaline phosphatase

151358_at CG14189 2.1

150743_at CG31058 2.1 ligand binding or carrier

1-phosphatidylinositol-4- 1-phosphatidylinositol-4-phosphate 5-kinase 147724_at PIP5K59B 2.1 Phosphatidylinositol-4-phosphate 5-kinase phosphate kinase activity

147206_at L Lobe 2.1

153731_at Hsp26 Heat shock protein 26 2.0 heat shock protein activity Heat shock protein Hsp20, chaperone

145020_at CG12175 2.0 transcription factor

148888_at CG4893 2.0 P25-alpha

146673_at CG11211 C-type lectin-like 2.0 mannose binding, lectin_c;sugar binding C-type lectin

151945_at RPE65 2.0 beta-carotene 1515'-monooxygenase activity Retinal pigment epithelial membrane protein DCV & FHV specific (39 genes) Probe number Gene name Synomymus DCV * FHV * Putative function Key words 141374_at AttA Attacin-A 8.7 5.2 Gram-negative antibacterial peptide activity Attacin N and C-terminal region 143334_at scb scab 7.9 3.5 calcium-dependent cell adhesion molecule activity Integrins alpha chain 146991_at CG9080 Listericin 7.0 3.6 147421_at CG14499 5.7 6.7 147473_at DptB Diptericin B 5.0 3.7 antibacterial peptide activity Attacin C-terminal region 147685_at CG4269 4.9 2.0 147220_s_at AttB Attacin-B 4.7 4.9 antimicrobial peptide activity Attacin N and C-terminal region 147406_at CG10910 4.3 3.1 structural protein specific RNA polymerase II transcription factor 151822_at Rel Relish 3.5 4.1 NF-kappa-B/Rel/dorsal activity 147127_s_at CG13323 3.2 2.3 143126_at dm diminutive 3.2 2.8 DNA binding, transcription factor activity Basic helix-loop-helix dimerization domain bHLH 152356_at CG32412 3.1 3.5 glutaminyl-peptide cyclotransferase activity Peptidase M28 glutaminyl cyclase 152901_at CG10383 3.0 2.6 ARM repeat fold triacylglycerol lipase activity, lipase;catalytic 141233_at CG5966 triacylglycerol lipase 2.8 3.9 Esterase/lipase/thioesterase activity 153553_at CG18273 2.8 2.4 specific RNA polymerase II transcription factor 143186_at h hairy 2.7 3.0 Basic helix-loop-helix dimerization domain bHLH activity, specific transcriptional repressor activity 146364_at CG5953 2.7 2.1 141483_at CG17836 2.7 2.6 protein dimerization activity, AT_hook;DNA binding HMG-I and HMG-Y DNA-binding domain (A+T-hook) G protein linked receptor- 149715_at mthl5 2.7 2.1 G-protein coupled receptor activity G-protein coupled receptor family 2 (secretin-like) like helicase activity, general RNA polymerase II 153722_at Xpd Xeroderma pigmentosum D 2.7 2.0 ATP-dependent helicase DEAH-box, Helicase c2 transcription factor activity 141271_at CG1021 2.7 2.7 motor

147150_at Attacin-C attacin-like 2.7 4.9 defense/immunity protein

modulator of the activity of 153855_at mae 2.7 3.5 protein binding Ets Peptidase C19 ubiquitin carboxyl-terminal hydrolase family 147126_i_at CG32479 2.6 2.5 Ubiquitin-specific protease activity 2

148424_at CG13311 2.4 2.1 structural molecule activity Alkaline phosphatase

EGF-like domain, Laminin-type EGF-like domain, 151918_s_at drpr draper 2.3 2.1 cell adhesion molecule activity Immunoglobulin/MHC

154372_at tamo 2.3 2.4 nuclear localization sequence binding Zn-finger Ran-binding, Immunoglobulin/MHC

141948_at Replication Protein A 70 replication protein A 70 2.3 2.0 single-stranded DNA binding 149642_at CG31272 sugar transporter-like 2.2 2.1 transporter activity General substrate transporter 153631_at CG8675 2.2 2.9 protein-tyrosine-phosphatase activity, cytokine Fibronectin type III, H+-transporting two-sector ATPase 142932_at dome domeless 2.1 2.3 binding alpha/beta subunit

151326_at CG32666 2.1 2.5

152225_at kraken kraken 2.1 3.1 serine hydrolase activity Alpha/beta hydrolase, Esterase/lipase/thioesterase

150549_at CG11819 2.1 2.6 protein kinase C2 calcium/lipid-binding domain CaLB 141265_at SH3PX1 2.0 3.0 SH3, Phox-like 153278_at Cct5 T-complex Chaperonin 5 2.0 2.6 chaperonin ATPase activity Chaperonin Cpn60/TCP-1, GroEL-like chaperone ATPase 150583_at CG31436 2.0 2.2 Protein of unknown function DUF227 153688_at CG16718 2.0 2.3 Protein of unknown function DUF590 146100_at CG13117 2.0 3.9 DCV & SINV specific (3 genes)

Probe number Gene name Synomymus DCV * SDV * Putative function Key words

145171_at CG14948 dpr18 2.2 1.7 Immunoglobulin-like

149738_r_at CG14742 2.2 1.7

151211_at CG16844 Immune induced molecule 3 2.0 1.8 defense/immunity protein FHV & SINV specific (127 genes) Probe number Gene name Synomymus FHV * SDV * Putative function Key words 145820_at TotM Turandot M 47.4 42.4 Stress-inducible humoral factor Turandot cathepsin L activity, Peptidase_C1;cysteine-type 152626_at CG11459 cathepsin L-like (inactive) 17.0 12.4 Peptidase C1A papain peptidase activity

144790_at CG2909 9.1 4.6

152902_at CG10341 8.4 2.8 NAF1

141701_at CG2064 8.3 4.5 adh_short;oxidoreductase activity Short-chain dehydrogenase/reductase SDR

149723_at CG12224 8.1 5.8 oxidoreductase activity Aldo/keto reductase 151551_i_at CG30090 serine protease 8.0 6.1 trypsin;trypsin activity Peptidase trypsin-like serine and cysteine proteases Asparaginase/glutaminase, AMP-dependent synthetase 144460_at CG6428 asparaginase 7.8 3.3 Lysophospholipase activity and ligase 147195_at CG12505 7.3 5.1 Retrotransposon gag protein, Zn-finger CCHC type

145788_at CG3008 6.6 3.5 protein kinase activity Ribulose bisphosphate carboxylase small chain

152598_at GstE1 Glutathione S transferase E1 6.4 2.5 glutathione transferase activity Glutathione S-transferase 149123_at trbl tribbles 6.4 3.5 protein serine/threonine kinase activity Protein kinase 147139_at CG10799 6.3 4.0 Peptidase M neutral zinc metallopeptidases zinc-binding 142206_s_at CG14527 5.4 4.2 Peptidase_M13_N;metallopeptidase activity site ATP-binding cassette ABC_membrane;ATP-binding cassette (ABC) ABC transporter, Nitrogenase component 1 alpha and beta 153894_at CG11897 5.4 2.6 transporter transporter activity subunits 149222_at CG7130 5.2 2.4 heat shock protein activity Heat shock protein DnaJ 150398_at CG4725 5.1 3.2 Peptidase_M13_N;metallopeptidase activity Peptidase M13 neprilysin

144845_at CG15203 5.0 3.0

OB-fold nucleic acid binding domain, Replication factor-A 142545_at RpA-70 Replication Protein A 70 5.0 2.5 single-stranded DNA binding protein 1 Peptidase S1 chymotrypsin, Low density lipoprotein- 148446_at Tequila 5.0 4.8 serine-type endopeptidase activity, chitin binding receptor class A, Chitin binding Peritrophin-A L-lactate dehydrogenase activity, 143201_at ImpL3 Ecdysone-inducible gene L3 4.9 2.1 Lactate/malate dehydrogenase ldh;oxidoreductase activity

155131_at CG9424 4.8 2.3 Lamino-associated polypeptide 2/emerin

151210_at CG16836 4.6 3.8

151310_at CG8620 4.6 4.5

149467_at CG2791 4.5 2.4 enzyme

153695_at CG6769 4.5 2.5 transcription factor Zn-finger C2H2 type

147525_at CG16898 4.5 2.3 Protein of unknown function DUF227 T-complex protein 1 beta- Chaperonin Cpn60/TCP-1, ATP-dependent helicase DEAD- 142977_at CG7033 4.4 3.1 chaperone activity subunit box 149755_at GstD9 Glutathione S transferase D9 4.4 2.3 glutathione transferase activity Glutathione S-transferase 144521_at CG15784 4.4 2.4 Winged helix DNA-binding 142142_at CG4721 4.3 2.4 Peptidase_M13_N;metallopeptidase activity Peptidase M13 neprilysin ATP-binding cassette 154740_at CG31793 4.3 3.0 ATP-binding cassette (ABC) transporter activity ABC transporter, Ribonucleotide reductase R1 subunit transporter-like trans-12-dihydrobenzene-12-diol dehydrogenase 142258_at CG12766 4.0 2.2 Aldo/keto reductase activity 147433_at CG15067 4.0 3.3 Cytochrome c 142969_at Cys Cystatin-like 4.0 4.0 cysteine protease inhibitor activity Sarcocystatin, Cysteine protease inhibitor

141583_at Cyp6a20 cytochrome P450 CYP6A20 3.9 2.1 cytochrome P450 activity Cytochrome P450, E-class P450 group I

145956_at CG5958 retinoid binding protein-like 3.9 2.2 retinal binding Cellular retinaldehyde-binding)/triple function

150031_at CG17560 3.9 3.5 Male sterility protein

142737_at CG5059 3.9 2.4

147912_at CG13877 3.7 3.4 phosphoserine transaminase activity, 153150_at CG11899 3.7 2.4 Phosphoserine aminotransferase aminotran_5;transaminase activity Structure specific DNA secondary structure binding, single-stranded 141661_at Ssrp 3.6 2.1 HMG1/2 (high mobility group) box recognition protein RNA binding

141242_at CG15092 3.6 2.6

protein serine/threonine kinase activity, JUN kinase 148051_at msn misshapen 3.6 2.1 Serine/threonine protein kinase kinase kinase kinase activity 153629_at mre11 meiotic recombination 11 3.6 2.1 endonuclease activity, exonuclease activity Metallo-phosphoesterase, DNA repair exonuclease

143198_at Hsp83 Heat shock protein 83 3.5 2.1 chaperone activity, HATPase_c;ATP binding Heat shock protein Hsp90

155148_at CG10527 3.5 3.5 farnesoic acid O-methyltransferase activity Protein of unknown function DM9

148108_at CG12012 3.5 2.6

144724_at CG7267 3.5 2.6

leucine-tRNA ligase activity, tRNA-synt_1;tRNA Aminoacyl-tRNA synthetase class Ia, Haem peroxidase 151752_s_at CG33123 leucine--tRNA ligase 3.4 2.1 ligase activity plant/fungal/bacterial neprilysin-like Peptidase M neutral zinc metallopeptidases zinc-binding 145896_at CG9508 3.4 2.0 neprilysin activity metalloendopeptidase site

149340_at rev7 3.4 2.2 cell cycle regulator DNA-binding HORMA

chaperonine-containing T- 153982_at Cct 3.4 2.1 chaperonin ATPase activity Chaperonin Cpn60/TCP-1, GroEL-like chaperone ATPase complex gamma-subunit

145620_at CG4259 3.4 2.3 serine-type endopeptidase activity Peptidase trypsin-like serine and cysteine proteases

152325_at Lsd-1 Lipid storage droplet-1 3.4 3.4 chaperone Perilipin 147353_at CG11400 3.4 3.1

154266_at Ada2S Transcriptional adapter 2S 3.3 2.1 histone acetyltransferase activity Myb DNA-binding domain, Homeodomain-like

triacylglycerol lipase activity, sterol esterase 146165_at CG6113 triacylglycerol lipase-like 3.3 2.9 Alpha/beta hydrolase, Esterase/lipase/thioesterase activity

152063_at CG4716 3.2 2.5

Adenosine deaminase- 148973_at Adgf-A 3.2 2.7 adenosine deaminase activity Adenosine/AMP deaminase related growth factor A

142599_at CG31151 3.2 2.2 BAH;DNA binding Bromo adjacent region

149964_at CG14872 3.2 2.6 Lipocalin-related protein and Bos/Can/Equ allergen

154938_at CG1583 3.1 2.8 phospholipase A2 activity Phospholipase A2

146000_at CG7219 serpin 3.1 2.5 serine-type endopeptidase inhibitor activity Serpin

150482_at CG13607 3.1 2.3

142893_at Rtnl1 3.0 2.7 Reticulon, Calcium-binding EF-hand

150713_at CG6066 3.0 2.5 Protein of unknown function DUF926

147319_at CG8317 3.0 2.1

143845_at Rab6 Rab-protein 6 2.9 2.4 RAB small monomeric GTPase activity Ras GTPase superfamily

147564_at CG16799 lysozyme 2.9 2.4 defense/immunity protein activity, lysozyme activity Glycoside hydrolase family 22

145648_at Cyp309a2 2.9 2.9 cytochrome P450 activity Cytochrome P450, E-class P450 group I

141815_at PHGPx 2.9 2.0 glutathione peroxidase activity Glutathione peroxidase

141688_at ImpL2 Ecdysone-inducible gene L2 2.9 2.1 cell adhesion molecule activity Immunoglobulin subtype

transmembrane receptor protein serine/threonine 153075_at CG1773 serine protease-like 2.9 2.3 Peptidase trypsin-like serine and cysteine proteases kinase receptor-associated protein activity

145608_at CG15358 C-type lectin-like 2.9 2.0 lectin_c;sugar binding C-type lectin

152320_at CG6495 2.9 2.1 receptor Low density lipoprotein-receptor class A

147337_at CG6429 2.8 2.4

151967_at CG6415 aminomethyltransferase 2.8 2.1 aminomethyltransferase activity Glycine cleavage T protein (aminomethyl transferase)

Retinoid- and fatty-acid structural molecule activity, Vitellogenin_N;lipid 151538_s_at RfaBp 2.8 3.0 von Willebrand factor type D, Lipid transport protein binding protein transporter activity

143197_at Hsp68 Heat shock protein 68 2.8 2.6 heat shock protein activity Heat shock protein Hsp70

146311_at CG9928 2.7 2.1

148547_at Mocs1 2.7 2.0

152385_at Aats-gln Glutaminyl-tRNA synthetase 2.7 2.3 glutamine-tRNA ligase activity, ATP binding Glutamyl-tRNA synthetase class Ic

150616_at CG31380 2.7 2.3 Protein of unknown function DUF227

146558_at CG14400 2.7 2.4

151575_at CR32218 2.6 2.0 motor

149884_at CG9616 2.6 3.1

sodium-dependent 150628_at CG8932 2.6 2.0 sodium dependent multivitamin transporter activity Na+/solute symporter multivitamin transporter-like

151468_s_at egh egghead 2.6 2.6 beta-14-mannosyltransferase activity Peptidylprolyl isomerase FKBP-type

calcium-independent calcium-independent cytosolic phospholipase A2 142906_at CG6718 2.6 2.1 Patatin, Ankyrin phospholipase A2 activity Ubiquitin-associated domain, UBA-like, Zn-finger C2H2 148369_at CG8209 2.5 2.1 type

147607_at Glycogenin Glycogenin 2.5 2.1 glycogenin glucosyltransferase activity Glycosyl transferase family 8

144701_at CG12116 2.5 2.0 sepiapterin reductase activity

152613_at CG11255 adenosine kinase 2.5 2.3 adenosine kinase activity Carbohydrate kinase PfkB, Adenosine kinase

151841_at CG8586 2.5 2.1 serine-type endopeptidase activity Peptidase S1 chymotrypsin

replication factor A 30 kD 153194_at CG9273 2.5 2.3 DNA binding, tRNA_anti;nucleic acid binding OB-fold nucleic acid binding, Winged helix DNA-binding subunit

146526_at CG10680 2.5 2.7

144590_at CG32918 2.5 2.2 hydrogen-exporting ATPase activity 146216_at l(2)06225 ATP synthase g-subunit 2.5 2.1 Mitochondrial ATP synthase gamma phosphorylative mechanism

142954_at CG5773 2.5 2.3

151839_at CG31764 vir-1 2.4 2.1

cathepsin B activity, Peptidase_C1;cysteine-type 153218_at CG3074 2.4 2.3 Peptidase C1A papain, Somatomedin B peptidase activity Longevity-assurance protein (LAG1), TRAM LAG1 and 154405_at CG11642 2.4 2.2 CLN8 homology actin binding, calcium-dependent phospholipid 153673_at AnnIX Annexin IX 2.4 2.1 Annexin type V binding 142414_at CG11315 2.4 2.3 E1 protein and Def2/Der2 allergen

152591_at CG11395 2.4 2.0 structural protein

146726_at nec necrotic 2.4 2.3 serine-type endopeptidase inhibitor activity Serpin

146136_at Hand 2.3 3.1 transcription factor activity Basic helix-loop-helix dimerization domain bHLH

142768_at CG7407 2.3 2.2

glass multimer reporter 153313_at Rac2 2.3 2.8 small monomeric GTPase activity Ras GTPase superfamily construct of Nolan

151980_at CG5150 alkaline phosphatase-like 2.3 2.4 alkaline phosphatase activity Alkaline phosphatase

149053_at CG9451 acid phosphatase 2.2 2.5 acid phosphatase activity Histidine acid phosphatase Peptidase S9 prolyl oligopeptidase, 145223_at CG9059 dipeptidyl-peptidase IV-like 2.2 2.2 dipeptidyl-peptidase IV activity Esterase/lipase/thioesterase phenylalanine 4-monooxygenase activity, Aromatic amino acid hydroxylase, Phenylalanine-4- 153434_at Hn Henna 2.2 2.2 ACT;amino acid binding hydroxylase tetrameric form

155042_at scrib scribbled 2.2 2.2 cell proliferation Leucine-rich repeat, PDZ/DHR/GLGF domain

147665_at CG3290 alkaline phosphatase 2.2 2.1 alkaline phosphatase activity Alkaline phosphatase

144147_at BM-40-SPARC SPARC/ostenectin-like 2.1 2.0 ligand binding or carrier Calcium-binding EF-hand, Osteonectin-like

151851_at CG6206 alpha-mannosidase 2.1 2.2 alpha-mannosidase activity Glycoside hydrolase family 38

153583_at Hsp23 Heat shock protein 23 2.1 2.3 heat shock protein activity Heat shock protein Hsp20

145890_at CG9498 2.1 2.0 Protein of unknown function DUF227

150030_at CG17562 2.1 2.2 enzyme Male sterility protein

147336_at CG6426 calcium binding protein-like 2.0 2.9 ligand binding or carrier Destabilase, Calcium-binding EF-hand

4-nitrophenylphosphatase- 144903_at CG10352 2.0 2.0 4-nitrophenylphosphatase activity Haloacid dehalogenase-like hydrolase like

153365_at CG15825 2.0 2.1

143917_at lush lush 2.0 2.4 odorant binding Insect pheromone/odorant binding protein PhBP

ATP-binding cassette 145598_at CG17646 2.0 2.5 ATP-binding cassette (ABC) transporter activity ABC transporte, AAA ATPase transporter

152488_at CG8343 2.0 2.0 mannose binding, lectin_c;sugar binding C-type lectin

* Fold induction compared to Tris injection control Supplemental Table III: List of the 42 genes up-regulated by DCV, FHV and SINV

Fold induction Probe number Gene name Synomymus DCV FHV SINV E. c M. l B. b L. m Putative function 153941_at Fst Frost 19.7 10.9 2.9 16.9 23.8 8.6 15.5 Respond to cold. Stress response. 153231_at CG8791 7.5 3.7 2.5 3.5 -4.8 1.0 4.5 High affinity inorganic phosphate:sodium symporter activity 146285_at CG31764 Vir-1 7.1 9.5 4.6 4.1 3.1 1.9 3.7 Unknown, response to virus 145307_at CG15043 6.9 7.3 2.1 3.6 4.7 3.9 2.3 Unknown 146814_at CG12780 GNBP-like 6.5 5.4 3.1 1.6 1.9 2.2 2.7 Gram-negative bacterial cell surface binding 144846_at CG2081 Vago 5.3 6.0 4.7 1.0 0.9 1.0 1.5 Unknown, antiviral activity 141626_at CG7635 Mec2 4.8 2.9 2.2 2.7 2.9 4.0 2.5 Unknown, Band 7 protein; Stomatin 141353_at CG6687 Serpin 88Eb 4.5 10.3 6.5 2.7 5.0 7.4 5.7 Serine protease inhibitor Autophagic cell death. Pheromone/general odorant binding 150837_at Obp99b Odorant-binding protein 99b 3.8 10.8 4.6 0.2 0.2 0.2 0.3 protein 152122_at CG10912 3.6 7.7 2.3 1.6 1.8 2.4 2.7 Protein of unknown function DUF725 144998_at CG15745 3.4 10.9 8.5 2.3 2.7 2.7 2.5 Unknown 152721_at Idgf1 Imaginal disc growth factor 1 3.4 3.2 2.8 2.3 2.0 1.8 2.1 Chitinase II; Glycoside hydrolase, family 18 153233_at CG6357 3.3 4.9 2.6 1.3 1.7 2.0 2.5 Cysteine-type endopeptidase activity. 144882_at PGRP-SA 3.2 3.9 2.9 3.6 3.6 2.6 7.5 Peptidoglycan recognition 147430_at CG15066 IM23 3.1 2.2 3.2 7.8 16.4 19.0 1.6 Unknown 146256_at CG14934 Mal-B1 (Maltase B1) 3.1 7.7 3.7 1.0 1.6 1.0 1.1 Alpha amylase; Glycoside hydrolase, 149782_at Hsp70Bc 3.1 5.5 5.4 0.7 8.2 2.0 4.8 Heat shock protein 70 149477_at CG11671 3.1 5.0 2.0 1.1 1.5 2.3 2.1 Unknown 152112_at CG13625 2.9 2.4 2.1 1.3 1.5 2.1 1.0 Unknown 152183_at CG4680 2.9 13.7 8.6 0.8 0.9 1.4 3.1 Unknown N-methyl-D-aspartate receptor-associated 142415_at Nmda1 2.7 4.9 2.9 1.8 1.7 1.6 1.7 N-methyl-D-aspartate selective glutamate receptor activity protein 143682_at Tig Tiggrin 2.6 3.9 3.4 1.2 1.8 2.0 1.7 Haem oxygenase-like, multi-helical. 149139_at CG4786 Rcd2 (Reduction in Cnn dots 2) 2.6 3.2 2.5 8.8 8.1 2.7 1.5 Unknown, von Willebrand factor, type C 141511_at dos daughter of sevenless 2.5 4.9 2.8 2.2 3.2 2.9 1.0 Regulation of Ras protein signal transduction NAD-dependent methylenetetrahydrofolate NAD(P)-binding domain; Tetrahydrofolate dehydrogenase/ 151767_at Nmdmc 2.5 5.4 5.1 2.8 3.6 0.8 1.3 dehydrogenase cyclohydrolase CHMP2B (Charged multivesicular body 154760_at CG4618 2.5 2.5 2.3 1.7 2.7 2.9 1.8 Unknown protein 2b) 146873_at Cyp4p3 2.5 2.1 2.0 3.4 4.8 3.6 2.0 Cytochrome P450, E-class, group I 154821_at CG8147 2.5 5.1 4.6 1.0 0.3 1.0 0.4 Alkaline phosphatase-like 153197_at Eip75B Ecdysone-induced protein 75B 2.5 3.3 2.1 1.3 1.3 1.8 2.7 Nuclear hormone receptor, 153527_at CG1572 2.4 2.1 2.0 1.1 1.2 1.1 0.8 Unknown, MARVEL-like domain 143005_at CG4199 2.4 5.8 4.5 2.3 2.5 1.8 1.4 FAD/NAD-linked reductase 150036_at CG14907 2.4 5.4 2.6 2.3 2.1 2.7 2.7 Unknown 154140_at CG4713 l(2)gd1 (lethal (2) giant discs 1) 2.4 3.3 2.3 0.8 1.2 1.6 1.2 C2 calcium-dependent membrane targeting 146708_at Tsp42Ed Tetraspanin 42Ed 2.4 4.0 2.5 1.5 1.4 2.0 2.2 Unknown 144068_at IM2 Immune induced molecule 2 2.4 2.6 2.7 2.7 4.1 3.5 1.4 Unknown 143802_at CG14906 2.3 4.4 2.8 1.3 1.3 1.7 0.9 Methyltransferase activity 147410_at CG10911 2.3 2.9 2.3 1.4 1.4 1.5 1.2 Protein of unknown function DUF725 141685_at CG3066 Sp7 (Serine protease 7) 2.2 4.1 3.0 1.9 2.0 1.8 4.1 Clip domain chymotrypsin-type peptidase 143401_at Uro Urate oxidase 2.2 2.2 2.1 3.8 5.0 5.5 2.0 Urate oxidase activity 152618_at Tsp42El Tetraspanin 42El 2.1 4.0 2.2 0.8 0.9 1.4 1.5 Unknown 148843_at CG13075 2.1 3.1 2.2 4.5 5.8 3.8 4.7 Chitin binding 155151_at CG10916 2.0 6.7 4.3 1.3 1.2 1.7 2.1 Zinc ion binding

Upregulation superior to 2-fold is shown in bold. Data for Escherichia coli (E.c), Micrococcus luteus (M.l) and Beauveria bassiana (B.b) are from Ref 19. Data for Listeria monocytogenes (L.m) are from Ref 30.