Transcriptome Analysis of Capsicum Chlorosis Virus-Induced Hypersensitive Resistance Response in Bell Capsicum

Abstract

Background: Capsicum chlorosis virus (CaCV) is an emerging pathogen of capsicum, tomato and peanut crops in Australia and South-East Asia. Commercial capsicum cultivars with CaCV resistance are not yet available, but CaCV resistance identified in Capsicum chinense is being introgressed into commercial Bell capsicum. However, our knowledge of the molecular mechanisms leading to the resistance response to CaCV infection is limited. Therefore, transcriptome and expression profiling data provide an important resource to better understand CaCV resistance mechanisms.

Methodology/principal findings: We assembled capsicum transcriptomes and analysed gene expression using Illumina HiSeq platform combined with a tag-based digital gene expression system. Total RNA extracted from CaCV/mock inoculated CaCV resistant (R) and susceptible (S) capsicum at the time point when R line showed a strong hypersensitive response to CaCV infection was used in transcriptome assembly. Gene expression profiles of R and S capsicum in CaCV- and buffer-inoculated conditions were compared. None of the genes were differentially expressed (DE) between R and S cultivars when mock-inoculated, while 2484 genes were DE when inoculated with CaCV. Functional classification revealed that the most highly up-regulated DE genes in R capsicum included pathogenesis-related genes, cell death-associated genes, genes associated with hormone-mediated signalling pathways and genes encoding enzymes involved in synthesis of defense-related secondary metabolites. We selected 15 genes to confirm DE expression levels by real-time quantitative PCR.

Conclusion/significance: DE transcript profiling data provided comprehensive gene expression information to gain an understanding of the underlying CaCV resistance mechanisms. Further, we identified candidate CaCV resistance genes in the CaCV-resistant C. annuum x C. chinense breeding line. This knowledge will be useful in future for fine mapping of the CaCV resistance locus and potential genetic engineering of resistance into CaCV-susceptible crops.

Fig 1. Symptoms on inoculated leaves of C. annuum following CaCV infection 4 days after mechanical inoculation.

CaCV-resistant line showing coalescing necrotic lesions (A) and susceptible line without visible symptoms (B).

Fig 2. Gene ontology categories of differentially expressed genes in C. annuum in response to CaCV infection.

The number and percentage of up- or down-regulated genes in CaCV resistant line classified into three major domains were plotted using WEGO tool. Significant relationship (p< 0.05) between up- and down-regulated genes indicated with ‘*’.

Fig 3. Validation of RNA-Seq gene expression by real-time quantitative PCR (qPCR).

FPKM (fragments per kilobase of exon per million fragments mapped) values obtained by RNA-Seq analysis and relative expression levels obtained by qPCR for 15 selected genes in CaCV resistant (R) and susceptible (S) Capsicum annuum plants inoculated with capsicum chlorosis virus are shown. Error bars represent the standard error for three biological replicates. Putative resistance genes are consecutively numbered 1–12; PR, pathogenesis-related.