Characterization and functional analysis of a Relish gene from the Asian corn borer, Ostrinia furnacalis (Guenée)

Abstract

Pathogen-induced host immune responses reduce the efficacy of pathogens used to control pests. However, compared to the well-deciphered immunity system of Drosophila melanogaster, the immunity system of agricultural pests is largely unconfirmed through functional analysis. Beginning to unveil mechanisms of transcription regulation of immune genes in the Asian corn borer, Ostrinia furnacalis, we cloned the complementary DNA (cDNA) of a transcription factor Relish by rapid amplification of cDNA ends. The 3164 bp cDNA, designated Of-Relish, encodes a 956-residue protein. Bioinformatic analysis showed that Of-Relish had a Rel homology domain, a predicted cleavage site between Q⁴⁰⁹ and L⁴¹⁰ , six ankyrin repeats, and a death domain. The response of Of-Relish expression to the Gram-negative bacteria Pseudomonas aeruginosa was sooner and stronger than to the Gram-positive Micrococcus luteus. The antimicrobial peptide genes Attacin and Gloverin had similar expression patterns in response to the infections. Knockdown of Of-Relish led to a decrease in Attacin and Gloverin messenger RNA levels, suggesting that Attacin and Gloverin were regulated by Of-Relish. Together, the results suggested that Of-Relish is a key component of the IMD pathway in O. furnacalis, involved in defense against P. aeruginosa through activation of Attacin and Gloverin.

Keywords: IMD pathway; Ostrinia furnacalis; Relish; antibacterial peptides; insect immunity.

Fig. 1.

Structural characteristics of O. furnacalis Relish. (A) Amino acid sequence deduced from the cDNA. (B) Schematic drawing of the Relish domain structure. DNA binding: N-terminal DNA binding domain of Rel homology domain (RHD); IPT: domain identified in Ig-like fold, Plexins, and Transcription factors; C-terminal dimerization domain of RHD; Arrowhead indicates the putative cleavage site; ANK: Ankyrin; DD: death domain.

Fig. 2.

Phylogenetic analysis of the Relish sequences from O. furnacalis and other species. The phylogenetic tree was constructed using MEGA-X by the Neighbor-Joining (NJ) method and tested by the bootstrap method with 1000 replications. The numbers at the tree branches are the Bootstrap values. The Relish of Penaeus vannamei is used as an outgroup. D. melanogaster (NP_996189.1), D. simulans (EDX13677.1), D. busckii (ALC47935.1), Glossina morsitans (ABF47913.1), Aedes aegypti (AAEL007624, VectorBase), Anopheles stephensi (ASTE010360, VectorBase), Anopheles gambiae (AGAP006747, VectorBase), O. furnacalis (AXF67445.1), Helicoverpa armigera (AEO51739.1), Spodoptera litura (AIA24468.1), Spodoptera frugiperda (KAF9806827.1), Penaeus vannamei (ABR14713.1).

Fig. 3.

mRNA level changes of Of-Relish and AMPs after bacterial infection. qPCR analysis of Relish (A), Attacin (B), CecropinA (C), Gloverin (D), Lebocin4 (E), and Moricin (F) in O. furnacalis larvae after bacterial infection. Pa: P. aeruginosa; Ml: M. luteus. The housekeeping gene RPL8 was used to calibrate the expression of each gene, and the PBS group at 0 h post infection was regarded as 1. All reactions were completed in triplicate using independently extracted RNA samples. The significant differences are represented by ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 for pairwise comparisons by Student’s t-tests.

Fig. 4.

RNAi suppression of Of-Relish transcription. (A) Confirmation of the specific product of dsRelish by 1% agarose gel lectrophoresis. (B) Examination of RNAi efficiency at 84, 96, 108, and 120 h post injection of dsRelish. Injection of dsGFP was used as a control, and the housekeeping gene RPL8 was used to calibrate the levels of Relish transcripts. All reactions were completed in triplicate using independently extracted RNA samples. The significant differences are represented by ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 for pairwise comparisons by Student’s t-tests.

Fig. 5.

Effects of RNAi of Of-Relish on expression of AMPs in O. furnacalis larvae. qPCR analysis of Attacin (A) and Gloverin (B) mRNA levels after Relish RNAi knockdown and bacterial infection. Pa: P. aeruginosa; Ml: M. luteus. The housekeeping gene RPL8 was used an internal control to normalize the expression of each gene, and the PBS group at 0 h post infection was set at 1.0. All reactions were completed in triplicate using independently extracted RNA samples. Significant differences in pairwise comparisons are represented by ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 by Student’s t-tests.