Discovery of microRNA-like small RNAs in pathogenic plant fungus Verticillium nonalfalfae using high-throughput sequencing and qPCR and RLM-RACE validation

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

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.

• Language: English
• Keywords: Verticillium nonalfalfae, RNA interference, microRNA-like RNAs, fungal pathogen, plant-pathogen interactions
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2022
• Number of pages: 17 str.
• Numbering: Vol. 23, iss. 2, art. 900
• PID: 20.500.12556/RUL-134657
• UDC: 577.2
• ISSN on article: 1422-0067
• DOI: 10.3390/ijms23020900
• COBISS.SI-ID: 94636035
• Publication date in RUL: 24.01.2022

Record is a part of a journal

• Title: International journal of molecular sciences
• Shortened title: Int. j. mol. sci.
• Publisher: MDPI
• ISSN: 1422-0067
• COBISS.SI-ID: 2779162

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
• Licensing start date: 14.01.2022

Secondary language

• Language: Slovenian
• Keywords: Humulus lupulus, Verticillium nonalfalfae, verticilijska uvelost, glivična okužba, interferenca RNA, interakcija rastlina-patogen

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: 34110

• Funder: ARRS - Slovenian Research Agency
• Project number: P4-0077
• Name: Kmetijske rastline - genetika in sodobne tehnologije