MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21-nt in length which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, miRNAs and their target genes remain largely unknown in hot pepper (Capsicum annuum), one of the most important crops cultivated worldwide. We here employed high-throughput small-RNA and degradome sequencing to comprehensively identify small-RNAs and their targets in pepper. From these, we identified several novel targets of miRNAs, including the major de novo methylation enzyme involved in RNA-directed DNA methylation in plants. Furthermore, we identified several highly abundant 22-nt miRNA families that target conserved domains in NB-LRRs and trigger the production of phased secondary siRNAs. We showed that transient co-expression of can-miR482 with Rpi-blb1, one of the potato NB-LRRs, resulted in the attenuation of the hypersenstive responses in Nicotiana benthamiana, suggesting that interaction between miR-482 family and disease resistance proteins is likely to serve as a conserved trigger for defense mechanism in Solanaceae. This work provides the first reliable draft of the pepper small RNA transcriptome that offers an expanded picture of miRNAs in relation to NB-LRR regulation, providing a basis for understanding the functional roles of miRNAs in disease resistance pepper.

MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21-nt in length which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, miRNAs and their target genes remain largely unknown in hot pepper (Capsicum annuum), one of the most important crops cultivated worldwide. We here employed high-throughput sequencing to comprehensively identify small RNAs and their targets in pepper. From these, we identified several novel targets of miRNAs, including the major de novo methylation enzyme involved in RNA-directed DNA methylation in plants. Furthermore, we identified several highly abundant 22-nt miRNA families that target conserved domains in NB-LRRs. We showed that transient co-expression of the miRNA with NB-LRRs, resulted in the attenuation of the hypersenstive responses in Nicotiana benthamiana, suggesting that interaction between miRNA family and disease resistance proteins is likely to serve as a conserved trigger for defense mechanism in Solanaceae. This work provides the first reliable draft of the small RNA transcriptome in pepper that offers an expanded picture of miRNAs in relation to NB-LRR regulation, providing a basis for understanding the functional roles of miRNAs in disease resistance.