Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae.

Urban Kunej,Sebastjan Radišek,Jernej Jakše,Nataša Štajner

doi:10.3390/ijms22084224

Abstract

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

Highlights

Post-transcriptional gene silencing (PTSG), or RNA interference (RNAi), is considered an evolutionarily conserved mechanism since it has been found across all main eukaryotic lineages and is involved in many biological processes [1]
The RNAi mechanism is referred to as a conserved gene silencing process mediated by small RNAs and RNAi components
Obtaining expression profiles of RNAi core components in susceptible and resistant hop cultivars would enable us to know which RNAi pathways play an important role during Verticillium wilt pathogenesis and which physiological processes are affected by the RNAi mediated response to fungal infection

Summary

Introduction

Post-transcriptional gene silencing (PTSG), or RNA interference (RNAi), is considered an evolutionarily conserved mechanism since it has been found across all main eukaryotic lineages and is involved in many biological processes [1]. Small non-coding RNAs (sRNAs), namely small interfering RNAs (siRNAs) or microRNAs (miRNAs), have the central role in dictating the RNAi process and are classified based on their biogenesis, precursor structure and mode of action. While miRNAs are derived from single-stranded precursors with a hairpin or stem-loop structure, siRNAs are produced from longer double-stranded RNAs (dsRNAs) [2]. Despite their differences, the mode of action of both classes of sRNAs converges in utilization of the RNAi machinery, i.e., proteins that guide gene silencing, and play an important role in the defense mechanism of plants against various biotic and abiotic stresses [3]. All DCLs consist of a DExD/H-box helicase domain, a centrally positioned PAZ domain

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Conclusion