Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation.

Taja Jeseničnik,Urban Kunej,Nataša Štajner,Jernej Jakše,Katarina Košmelj,Ajay Kumar Mishra,Sebastjan Radišek

doi:10.3390/ijms23020900

Abstract

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

Highlights

Introduction iationsThe soil fungus Verticillium nonalfalfae (V. nonalfalfae) is a causal agent of vascular wilt diseases in many important agriculture crops, trees and other woody plants [1,2].Taxonomically, V. nonalfalfae belongs to a group of 10 species that form the anamorphic genus Verticillium sensu stricto in the class Sordariomycetes in the phylum Ascomycota
For each of the eight sequenced samples (Rec_XSM mycelia, Rec_CD mycelia, Rec_conidia, Rec_resting mycelia, T2_XSM mycelia, T2_CD mycelia, T2_conidia and T2_resting mycelia), approximately 10 million reads were obtained via high-throughput sequencing (Table 1)
To gain insight into the potential effects of MicroRNA-like RNAs (milRNAs) on the RNA interference (RNAi)-mediated growth, development and pathogenicity of V. nonalfalfae, we investigated the expression patterns of all nine validated milRNAs in two specific tissues, conidia and mycelia grown in xylem-simulating media (XSM), of two different isolates

Summary

Introduction

The soil fungus Verticillium nonalfalfae (V. nonalfalfae) is a causal agent of vascular wilt diseases in many important agriculture crops, trees and other woody plants [1,2]. V. nonalfalfae belongs to a group of 10 species that form the anamorphic genus Verticillium sensu stricto in the class Sordariomycetes in the phylum Ascomycota (www.mycobank.org, accessed on 21 September 2021). Penetration hyphae infect host roots, and the infection progresses with the colonization of xylem vessels and results in the wilting of the affected parts of plants or, in the case of severe disease forms, dieback of the entire plant [3]. Hop (Humulus lupulus L.) is one of the most susceptible hosts to V. nonalfalfae, as the fungus causes outbreaks in many hop growing regions of Europe and worldwide [4,5].

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