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. 2022 Jan 26;96(2):e0171521.
doi: 10.1128/JVI.01715-21. Epub 2021 Nov 10.

Alternative Splicing Landscape of Small Brown Planthopper and Different Response of JNK2 Isoforms to Rice Stripe Virus Infection

Lu Tong #  1   2 , Xiaofang Chen #  1   2 , Wei Wang #  1 , Yan Xiao  1   2 , Jinting Yu  1   2 , Hong Lu  1 , Feng Cui  1   2
Affiliations

Alternative Splicing Landscape of Small Brown Planthopper and Different Response of JNK2 Isoforms to Rice Stripe Virus Infection

Lu Tong et al. J Virol. .

Abstract

Alternative splicing (AS) is a frequent posttranscriptional regulatory event occurring in response to various endogenous and exogenous stimuli in most eukaryotic organisms. However, little is known about the effects of insect-transmitted viruses on AS events in insect vectors. The present study used third-generation sequencing technology and RNA sequencing (RNA-Seq) to evaluate the AS response in the small brown planthopper Laodelphax striatellus to rice stripe virus (RSV). The full-length transcriptome of L. striatellus was obtained using single-molecule real-time sequencing technology (SMRT). Posttranscriptional regulatory events, including AS, alternative polyadenylation, and fusion transcripts, were analyzed. A total of 28,175 nonredundant transcript isoforms included 24,950 transcripts assigned to 8,500 annotated genes of L. striatellus, and 5,000 of these genes (58.8%) had AS events. RNA-Seq of the gut samples of insects infected by RSV for 8 d identified 3,458 differentially expressed transcripts (DETs); 2,185 of these DETs were transcribed from 1,568 genes that had AS events, indicating that 31.4% of alternatively spliced genes responded to RSV infection of the gut. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, experienced exon skipping, resulting in three transcript isoforms. These three isoforms differentially responded to RSV infection during development and in various organs. Injection of double-stranded RNAs targeting all or two isoforms indicated that three or at least two JNK2 isoforms facilitated RSV accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors. IMPORTANCE Alternative splicing (AS) is a regulatory mechanism that occurs after gene transcription. AS events can enrich protein diversity to promote the reactions of the organisms to various endogenous and exogenous stimulations. It is not known how insect vectors exploit AS events to cope with transmitted viruses. The present study used third-generation sequencing technology to obtain the profile of AS events in the small brown planthopper Laodelphax striatellus, which is an efficient vector for rice stripe virus (RSV). The results indicated that 31.4% of alternatively spliced genes responded to RSV infection in the gut of planthoppers. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, produced three transcript isoforms by AS. These three isoforms showed different responses to RSV infection, and at least two isoforms facilitated viral accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors.

Keywords: JNK; RNA-Seq; SMRT; alternative splicing; full-length transcriptome; insect vector; plant virus; rice stripe virus; small brown planthopper.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Basic information about the sequencing of the transcriptome of the small brown planthopper by PacBio SMRT. (A) Classification of the reads of inserts. (B) Classification of 24,950 isoforms. (C) Functional annotation of 1,297 novel isoforms. (D) Species distributions of 1,297 novel isoforms.
FIG 2
FIG 2
Alternative splicing (AS) events. (A) Quantitative relationship of the isoforms and 8,500 annotated genes. (B) GO terms for 5% of the genes harboring the largest number of isoforms. (C) KEGG pathways for 5% of the genes harboring the largest number of isoforms. (D) Six types of AS models and quantitative distribution of 13,640 AS events.
FIG 3
FIG 3
Alternative polyadenylation (APA) and fusion transcripts. (A) Quantitative relationships of poly(A) sites and gene numbers. (B) Nucleotide composition of the 50 nucleotide regions upstream and downstream of all poly(A) sites. (C) Classification of 747 fusion transcripts. (D) GO terms for the fusion transcripts.
FIG 4
FIG 4
Differentially expressed transcripts (DETs) in the gut infected by RSV for 8 d. (A) Number of DETs. (B) Volcano plot of DETs. The Log2 (fold change) values were plotted against the -Log10 (P value). P < 0.05 was set as a threshold for DETs. (C) Quantitative relationships between DETs and their source genes.
FIG 5
FIG 5
Alternatively spliced transcripts of JNK2. (A) The model graph showing the alternative splicing patterns of JNK2. Exons are represented by boxes and introns are represented by lines. The translation start codon 'ATG’ and translation stop codon ‘TGA’ are indicated by arrows. Gray boxes correspond to skipped exons. Two conserved phosphorylation sites, Thr176 and Tyr178 in JNK2-1, are located at exon 5 and indicated by a triangle in each isoform. The locations of dsRNAs designed to knock down the expression of both JNK2-1 and JNK2-2 (dsJNK2-1&2-2) or both JNK2-1 and JNK2-3 (dsJNK2-1&2-3) or all three isoforms of JNK2 (dsJNK2) are indicated above JNK2-1. (B) Western blot assay of expressed recombinant proteins of three JNK2 isoforms with His tags in Escherichia coli cells. The recombinant proteins with expected molecular weights of 44.2 kDa for JNK2-1-His, 32.3 kDa for JNK2-2-His, and 23.4 kDa for JNK2-3-His were detected by an anti-His monoclonal antibody (arrows). The products of the pET28a vector were used as a negative control. (C) Phylogenetic analysis of proteins homologous to JNK from several insect species, human, and mouse using the maximum likelihood method (Jones-Taylor-Thornton model). Bootstrap analysis (1,000 replicates) was used to evaluate internal support of the tree topology. Bootstrap values higher than 60% are shown at the nodes. The sequences of Sogatella furcifera JNKs and Nilaparvata lugens JNKs were retrieved from the GigaScience database (http://dx.doi.org/10.5524/100255 and http://dx.doi.org/10.5524/100139). Other sequences were retrieved from GenBank.
FIG 6
FIG 6
Temporal and spatial expression of alternatively spliced transcripts of JNK2. The relative transcript levels of three JNK2 isoforms normalized to the levels of EF2 during development (A) and in various organs (B) of nonviruliferous planthoppers. Five nymph stages, female, and male adults were sampled. Different letters on the columns represent significant differences.
FIG 7
FIG 7
Responses of alternatively spliced transcripts of JNK2 to RSV infection at different developmental stages. Comparison of the relative transcript levels of JNK2-1 (A), JNK2-2 (B) and JNK2-3 (C) normalized to the levels of three reference genes at each developmental stage in viruliferous (V) and nonviruliferous (NV) planthoppers. *, P < 0.05. **, P < 0.01. ns, no significant difference.
FIG 8
FIG 8
Responses of alternatively spliced transcripts of JNK2 to RSV infection in various organs. Comparison of the relative transcript levels of JNK2-1 (A), JNK2-2 (B) and JNK2-3 (C) normalized to the levels of three reference genes in various organs in viruliferous (V) and nonviruliferous (NV) planthoppers. *, P < 0.05. **, P < 0.01. ns, no significant difference.
FIG 9
FIG 9
Effect of alternatively spliced transcripts of JNK2 on RSV accumulation. (A) The relative transcript levels of three JNK2 isoforms and relative RNA level of RSV NP normalized to the level of EF2 in the planthoppers that were injected with dsRNA targeting the common region of the three isoforms of JNK2 (dsJNK2) or dsRNA for GFP (dsGFP) and fed RSV-infected rice seedlings for 8 d. (B) and (C) are the same as (A) except that planthoppers were injected with dsRNA targeting both JNK2-1 and JNK2-3 (dsJNK2-1&2-3) (B) or with dsRNA targeting both JNK2-1 and JNK2-2 (dsJNK2-1&2-2) (C). **, P < 0.01. ns, no significant difference.
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