Analysis of genes expression of Spodoptera exigua larvae upon AcMNPV infection

Abstract

Background: The impact of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection on host gene expression in Spodoptera exigua 4th instar larvae was investigated through the use of 454 sequencing-based RNA-seq of cDNA libraries developed from insects challenged with active AcMNPV or heat-inactivated AcMNPV.

Methodology/principal findings: By comparing the two cDNA libraries, we show that 201 host genes are significantly up-regulated and 234 genes are significantly down-regulated by active AcMNPV infection. Down-regulated host genes included genes encoding antimicrobial peptides, namely three gloverin isoforms and an attacin, indicating that the viral infection actively repressed the expression of a portion of the host immune gene repertoire. Another interesting group of down-regulated host genes included genes encoding two juvenile hormone binding proteins and a hexamerin, all of which are involved in juvenile hormone regulation. The expression of these genes was enhanced by the topical application of Juvenile Hormone III (JHIII) in the insects challenged with heat-inactivated AcMNPV. However, infection with the active virus strongly suppresses the expression of these three genes, regardless of the absence or presence of JHIII.

Conclusions/significance: Using RNA-seq, we have identified groups of immune-regulated and juvenile hormone-regulated genes that are suppressed by infection with active AcMNPV. This information and further studies on the regulation of host gene expression by AcMNPV will provide the tools needed to enhance the utility of the virus as an effective protein expression system and as an insecticide.

Figure 1. KEGG analysis of total (A), UP and DOWN (B) contigs.

(A) Functional groups defined by KEGG were classified as Carbohydrate metabolism, Lipid metabolism, Nucleotide metabolism, Amino acid metabolism, Biosynthesis of other secondary metabolites, Xenobiotics biodegradation and metabolism, Transcription, Translation, Folding Sorting and Degradation, Replication and Repair, Membrane Transport, Signal Transduction, and Cellular Processes. The immune response category was directly defined by comparing to Drosophila immune genes using BLASTN. (B) The contigs that showed significantly different expression (a binominal probability of <0.1) were classified as UP or DOWN, where UP indicates genes that are up-regulated by active AcMNPV infection compared to heat-inactivated AcMNPV challenge and DOWN indicates genes that are down-regulated by active AcMNPV infection compared to heat-inactivated AcMNPV challenge.

Figure 2. Graph of the numbers of I-reads and A-reads for each contig.

The number of I-reads and A-reads of each contig were graphed on an x,y plot. For convenience, contigs were plotted on two separate graphs: for contigs shown in the left panel, the number of I- or A-reads is smaller than 150; for contigs shown in the right panel, the number of I or A-reads is equal to or larger than 150. The linear trendline (with the intercept set as zero) and the slope are indicated by a line and an equation. UP and DOWN contigs are indicated as green circles and red boxes, respectively.

Figure 3. Validation of the RNA-seq results by quantitative real-time PCR (qPCR).

The expression profiles of 10 UP and 10 DOWN contigs (randomly selected) were analysed by qPCR to validate the RNA-seq results. The tested contigs are indicated in red in Tables S2 and S3.

Figure 4. Graph of the number of reads from contigs encoding ribosomal proteins.

The expression of four ribosomal protein genes, RpS20, RpSL12, RpL19 and RpS3A (indicated by green circles), which have been cited as examples of global down-regulation of host transcription in Sf9 cells , was not significantly altered in our experiments, clearly indicating that there is no global down-regulation of host transcripts.

Figure 5. Graph of the number of reads from contigs encoding host immune-related peptides/proteins (A) and serine proteases (B).

Three gloverin genes and one attacin gene are indicated by red boxes and a green triangle, respectively. The linear trendline (with the intercept set at zero) and the slope are indicated by a line and an equation.

Figure 6. The expression profiles of the GLV1 (A) and ATT1 (B) genes.

The relative expression levels of the GLV1 and ATT1 genes 6 h after each treatment were examined by qPCR. Mock, treatment with Sf9 growth medium; AcMNPV, culture medium with AcMNPV; Inactivated AcMNPV, culture medium with heat-inactivated AcMNPV.

Figure 7. The expression profiles of two JHBP genes and one hexamerin gene.

Relative expression levels of two JHBP genes and one hexamerin gene 12 h after each treatment were examined by qPCR. Mock, treatment with Sf9 growth medium; AcMNPV, culture medium with AcMNPV; Inactivated AcMNPV, culture medium with heat-inactivated AcMNPV; AcMNPVΔEGT, culture medium with AcMNPVΔEGT. JHIII was topically applied directly after each treatment.