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

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.