Identification by subtractive suppression hybridization of bacteria-induced genes expressed in Manduca sexta fat body

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Abstract

Insect immune processes are mediated by programs of differential gene expression. To understand the molecular regulation of the immune response in the tobacco hornworm, Manduca sexta, the relevant subset of differentially expressed genes of interest must be identified, cloned and studied in detail. In this study, suppression subtractive hybridization, a PCR-based method for cDNA subtraction was performed to identify mRNAs from fat body of immunized larvae that are not present (or present at a low level) in control larvae. A subtracted cDNA library enriched in immune-inducible genes was constructed. Northern blot analysis of a sample of clones from our subtracted library indicated that >90% of the clones randomly selected from the subtracted library are immune inducible. Sequence analysis of 238 expressed sequence tags (ESTs) revealed that 120 ESTs, representing 54 distinct genes or gene families, had sequences identical or similar to previously characterized genes, some of which have been confirmed to be involved in innate immunity. These ESTs were categorized into seven groups, including pattern recognition proteins, serine proteinases and their inhibitors, and antimicrobial proteins. 112 ESTs, about 47.5% of the library, showed no significant similarity to any known genes. The sequences identified in this M. sexta library reflect our knowledge of insect immune strategies and may facilitate better understanding of insect immune responses.

Introduction

The humoral immune reactions of insects include activation of proteolytic pathways and the rapid and transient synthesis of immune-related polypeptides (Hultmark, 1993, Hoffmann, 1995, Meister et al., 1997, Gillespie et al., 1997, Lehrer and Ganz, 1999). Most of the characterized immune genes induced by microbial infection encode antibacterial or antifungal peptides, which are synthesized predominantly in fat body and released into hemolymph. Hemocytes and epithelial layers of the integument and the gut also participate in the synthesis of these molecules. These inducible genes are either not expressed or are constitutively expressed at a low level prior to infection. After immune challenge, their expression is stimulated within one to a few hours and reaches a maximum by 12–48 h later (Engström, 1998, Hoffmann, 1995). Extensive studies on how these differentially expressed antimicrobial genes are regulated at the transcriptional level by rel domain proteins have been carried out in Drosophila melanogaster (Hoffmann, 1995, Engström, 1998, Engström, 1999, Imler and Hoffmann, 2000).

Seven distinct antimicrobial peptides identified in Drosophila so far include an antifungal peptide called drosomycin, and six antibacterial peptides: cecropin, attacin, defensin, drosocin, diptericin and metchnikowin (Meister et al., 1997). In addition, analysis of the Drosophila genome indicates the presence of more than 30 genes for antimicrobial peptides (Khush and Lemaitre, 2000). The expression pattern of the antimicrobial peptide genes in Drosophila is not identical for different types of microbial infection, suggesting that different signaling pathways regulate immune gene expression in a distinct manner appropriate for a specific microbial challenge (Engström, 1998, Engström, 1999). The triggering of different pathways by various types of infection may result in the activation and nuclear translocalization of different Rel proteins, transcription factors whose hetero- and homo-dimeric combinations could generate a wide range of binding specificity and therefore facilitate the activation of different groups of antimicrobial genes (Engström, 1999).

In addition to the antimicrobial peptide genes, infection elicits expression of other types of genes in insects (Khush and Lemaitre, 2000). These include pattern recognition proteins, which bind to molecules associated with microorganisms and initiate specific immune responses (Yu et al., 2002), proteinases and proteinase inhibitors, which are involved in pathways such as prophenoloxidase activation and Toll signaling (Jiang and Kanost, 2000), nitric oxide synthase (Luckhart et al., 1998, Dimopoulos et al., 1998), and transferrin (Yoshiga et al., 1999).

Identification and study of additional insect genes that are differentially expressed after immune challenge will lead to a more complete understanding of the breadth and regulation of insect immune responses. A conventional method for discovering immunity genes has been to purify a protein based on characters such as binding, enzymatic, or antimicrobial activity, obtain amino acid sequence information and then initiate DNA cloning. During the last decade, isolation of multiple differentially expressed genes without knowing their sequences or protein characters has been accomplished by several techniques, including subtractive cDNA hybridization and PCR-based mRNA differential display. Differential display has been used successfully for the discovery of immune-related genes in two lepidopteran species, Trichoplusia ni (Kang et al., 1996) and Hyphantria cunea (Shin et al., 1998).

The recently developed suppression subtractive hybridization (SSH) method (Diatchenko et al., 1996, Diatchenko et al., 1999) is based on suppression PCR, which selectively amplifies differentially expressed cDNA fragments and simultaneously suppresses amplification of common cDNAs. SSH overcomes bias for highly abundant mRNA by incorporating a normalization step to equalize the numbers of individual cDNA species, utilizing second order kinetics during subtraction. SSH has been used to identify differentially expressed genes in response to infection in rice (Xiong et al., 2001) and in rainbow trout (Bayne et al., 2001). In insects, SSH was adopted to identify immune-inducible genes in the mosquito, Anopheles gambiae (Oduol et al., 2000) and tsetse fly, Glossina morsitans morsitans (Hao et al., 2001). In this paper, we describe the use of the SSH technique to identify immune response genes in a lepidopteran insect, Manduca sexta.

Two-dimensional polyacrylamide gel electrophoresis, comparing M. sexta plasma proteins from bacteria-injected larvae with control larvae, detected 22 bacteria-induced proteins (Hurlbert et al., 1985), but only a few of these have been subsequently well characterized. The immune-inducible genes known in M. sexta encode antimicrobial peptides and proteins: cecropins (Dickinson et al., 1988), attacin (Kanost et al., 1990), and lysozyme (Mulnix and Dunn, 1994); pattern recognition proteins: hemolin (Ladendorff and Kanost, 1991) and two immulectins (Yu et al., 1999, Yu and Kanost, 2000); serine proteinases: prophenoloxidase activating proteinase (Jiang et al., 1998) and proteinases of unknown function (Jiang et al., 1999, Finnerty and Granados, 1997, Finnerty et al., 1999); and a serine proteinase inhibitor from the serpin family, serpin-2 (Gan et al., 2001). In the present study, use of SSH to construct a M. sexta larval fat body cDNA library enriched in differentially expressed genes related to immune response has resulted in identification of these previously identified genes and a large number of additional bacteria-induced genes with potential functions in innate immunity.

Section snippets

Insects

M. sexta eggs were originally obtained from Carolina Biological Supply. Larvae were reared as described by Dunn and Drake (1983).

Preparation of total RNA and poly (A)+ RNA

Fat body dissected from day 2, fifth instar larvae at 24 h after injection with either 10 μl filter-sterilized saline (0.85% NaCl) or 100 μg Micrococcus lysodeikticus (Sigma) in 10 μl sterile saline was frozen immediately in liquid nitrogen and stored at −80 °C. Total RNA was isolated from fat body using a RAPID Total RNA Isolation Kit (Eppendorf-5 Prime, Inc.). Poly

Construction of subtractive cDNA library by using SSH

SSH was performed to identify M. sexta genes that are expressed in fat body in response to bacterial invasion. Two mRNA populations were prepared. One mRNA population, isolated from fat body of fifth instar larvae immunized with M. lysodeikticus, contained differentially expressed genes and was used as the tester RNA. The other population of mRNAs, isolated from control larvae injected with saline, provided a driver whose transcripts, common to both populations, should be eliminated during the

Acknowledgements

We thank Maureen Gorman for helpful comments on the manuscript and for confirming the induced expression of newly identified moricin and cecropin genes. This work was supported by National Institutes of Health Grants GM41247. This is contribution 02-442-J from the Kansas Agricultural Experiment Station.

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