Genome-wide Identification and Functional Analysis of RNAi Gene Families in Papaya (Carica papaya L.)

Abstract

RNA silencing plays a direct functional role in gene expression and defense against pathogens during various developmental stages in plants. This silencing process is regulated by the RNA interference (RNAi) pathway, which relies on double-stranded RNA (dsRNA) generated from small RNAs (sRNAs). Three important protein families regulate the functions of sRNAs: Dicer-like (DCLs), Argonautes (AGOs), and RNA-dependent RNA polymerases (RDRs). These major components have not been identified and characterized in papaya. In this study, we identified the RNAi gene families and comprehensively characterized their regulatory functions through a bioinformatics approach in papaya. The papaya genome contains 3 CaDCL, 8 CaAGO and 4 CaRDR genes, which comprise diverse functional regulatory elements compared to their corresponding Arabidopsis AtDCL, AtAGO, and AtRDR genes, indicating their pivotal roles in the RNA silencing mechanism. Phylogenetic tree and multiple sequence analyses reveal that each CaDCL, CaAGO and CaRDR protein clusters and aligns with the corresponding RNAi genes in Arabidopsis. The results from conserved domain, motif, and gene structure analyses indicate a higher level of similarity within the same gene family with some exceptions. The chromosomal and sub-cellular localization of the predicted proteins shows a well distributed pattern. Furthermore, the gene ontology (GO) analysis demonstrates that the candidate RNAi genes are associated with the RNAi silencing mechanism and related pathways. The network and sub-network analyses reveal interactions between various transcription factors (TFs), such as ERF, Dof, MIKC_MADS, NAC, and BBR-BPC families, and the identified RNAi gene families. Additionally, an analysis of cis-acting regulatory elements uncovers light-responsive (LR), stress-responsive (SR), hormone-responsive (HR), and other activities (OT) functions in the candidate CaDCL, CaAGO, and CaRDR genes. Tissue-specific expression patterns of papaya RNAi genes showed differential expression in various tissues. RNA-seq analysis revealed five candidates CpDCL1, CpAGO1, CpAGO5, CpAGO10a, and CpRDR6 that demonstrated potential in response to anthracnose-resistant in papaya. Over all, our findings provide useful information for the genetic improvement of papaya cultivars in breeding programs to overcome stress responses and offer a better understanding of these gene families in papaya.