Massively parallel pyrosequencing-based transcriptome analyses of small brown planthopper (Laodelphax striatellus), a vector insect transmitting rice stripe virus (RSV)

Abstract

Background: The small brown planthopper (Laodelphax striatellus) is an important agricultural pest that not only damages rice plants by sap-sucking, but also acts as a vector that transmits rice stripe virus (RSV), which can cause even more serious yield loss. Despite being a model organism for studying entomology, population biology, plant protection, molecular interactions among plants, viruses and insects, only a few genomic sequences are available for this species. To investigate its transcriptome and determine the differences between viruliferous and naïve L. striatellus, we employed 454-FLX high-throughput pyrosequencing to generate EST databases of this insect.

Results: We obtained 201,281 and 218,681 high-quality reads from viruliferous and naïve L. striatellus, respectively, with an average read length as 230 bp. These reads were assembled into contigs and two EST databases were generated. When all reads were combined, 16,885 contigs and 24,607 singletons (a total of 41,492 unigenes) were obtained, which represents a transcriptome of the insect. BlastX search against the NCBI-NR database revealed that only 6,873 (16.6%) of these unigenes have significant matches. Comparison of the distribution of GO classification among viruliferous, naïve, and combined EST databases indicated that these libraries are broadly representative of the L. striatellus transcriptomes. Functionally diverse transcripts from RSV, endosymbiotic bacteria Wolbachia and yeast-like symbiotes were identified, which reflects the possible lifestyles of these microbial symbionts that live in the cells of the host insect. Comparative genomic analysis revealed that L. striatellus encodes similar innate immunity regulatory systems as other insects, such as RNA interference, JAK/STAT and partial Imd cascades, which might be involved in defense against viral infection. In addition, we determined the differences in gene expression between vector and naïve samples, which generated a list of candidate genes that are potentially involved in the symbiosis of L. striatellus and RSV.

Conclusions: To our knowledge, the present study is the first description of a genomic project for L. striatellus. The identification of transcripts from RSV, Wolbachia, yeast-like symbiotes and genes abundantly expressed in viruliferous insect, provided a starting-point for investigating the molecular basis of symbiosis among these organisms.

Figure 1

BlastX similarity search of the combined L. striatellus EST library. (A) Distribution of matched sequences by BlastX search against the NCBI-NR database. Significant matched sequences were defined as e-value ≤ 10^-3. (B) Similarity of L. striatellus ESTs compared with putative proteomes of other arthropods. The cutoff e-value was set as ≤ 10^-3.

Figure 2

Comparison of the distribution of GO terms. The x-axis shows subgroups of molecular functions from GO, the Y-axis shows the percentage of the matched EST sequences. Distribution of GO terms of ESTs from viruliferous (vector), naïve, and combined samples are compared.

Figure 3

Abundances of transcripts of RSV ORFs. RdRP: RNA dependent RNA polymerase; NS2: function-unknown protein; NSvc2: putative glycoprotein; NS3: RNAi suppressor; NCP: nucleocapsid; SP: disease-specific protein; NSvc4: movement protein. The Y-axis shows the number of reads identified in the viruliferous EST library.

Figure 4

RT-PCR profiles of putative transcripts from viruliferous and naïve L. striatellus samples. V: viruliferous; N: naïve. The arrow next to the gel pictures indicates increased or decreased expression of a particular gene in viruliferous vs. naïve sample. At least two rounds of independent replication were used for each primer pair. The putative elongation factor gene (Contig2248) was used as a reference. The sequences of PCR primers used in this analysis are listed in Additional file 6.