Genome-wide identification and characterization of phased small interfering RNA genes in response to Botrytis cinerea infection in Solanum lycopersicum

Phased small interfering RNAs (phasiRNAs) are encoded by a novel class of genes known as phasiRNA producing (PHAS) genes. These genes play important regulatory roles by targeting protein coding transcripts in plant species. In this study, 91 regions were identified as potential PHAS loci in tomato, with additional evidence that seven of them can be triggered by five miRNAs. Among the identified loci, 51 were located in genic regions, and the remaining 40 were located in intergenic regions. The transient overexpression of PHAS15 and PHAS26 demonstrated that phasiRNAs predicted by PhaseTank were indeed generated from their respective PHAS loci. Using sRNA-seq data from B. cinerea-infected tomato leaves, we identified 50 B. cinerea-responsive phasiRNAs with increased abundance and five with decreased abundance. Moreover, 164 targets of these differentially expressed phasiRNAs were predicted, and 94 of them were confirmed experimentally using degradome data. Gene ontology analysis of the targets revealed an enrichment of genes with functions related to defense responses and signaling regulation. These results suggest that a large number of endogenous siRNAs, such as phasiRNAs, have not yet been identified in tomato and underscore the urgent need to systematically identify and functionally analyze siRNAs in tomato.