20.500.12556/RUL-134657 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. Verticillium nonalfalfae RNA interference microRNA-like RNAs fungal pathogen plant-pathogen interactions Humulus lupulus Verticillium nonalfalfae verticilijska uvelost glivična okužba interferenca RNA interakcija rastlina-patogen true false true Angleški jezik Slovenski jezik Članek v reviji 2022-01-24 11:31:54 2022-01-24 11:31:59 2022-09-13 03:56:22 0000-00-00 00:00:00 2022 0 0 17 str. iss. 2, art. 900 Vol. 23 2022 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577.2 1422-0067 10.3390/ijms23020900 94636035 ijms-23-00900-v2.pdf ijms-23-00900-v2.pdf 1 4758A1524FCFB510A629F8844558ABB4 1035db4e28c9de1df5e26c1245bfd4630b05660c0dd66e6dafe84581157b1dbf 5c5c3f27-e57a-11ec-8aca-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=157077 https://www.mdpi.com/1422-0067/23/2/900 1 aa23c2e3-7d00-11ec-abdb-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=153201 Biotehniška fakulteta 0 0 0