Characterization of microRNA-like RNAs associated with sclerotial development in Sclerotinia sclerotiorum

Highlights

• Sclerotinia sclerotiorum is a necrotrophic pathogen causing great losses worldwide.

• milRNAs associated with sclerotial development were identified.

• Histone acetyltransferase regulation was potentially related to sclerotial development.

Abstract

Sclerotinia sclerotiorum is a model necrotrophic pathogen causing great economic losses worldwide. Sclerotia are dormant structures that play significant biological and ecological roles in the life and disease cycles of S. sclerotiorum and other species of sclerotia-forming fungi. microRNA-like RNAs (milRNAs) as non-coding small RNAs play regulatory roles in fungal development and pathogenicity. Therefore, milRNAs associated with sclerotial development in S. sclerotiorum were investigated in this study. A total of 275 milRNAs with induced expression during sclerotia development were identified, in which 51 were differentially expressed. The target genes of all milRNAs were predicted. The putative functions of the targets regulated by milRNAs were annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The expression levels of six selected milRNAs that coordinated with their corresponding targets were validated by qRT-PCR. Among these six milRNAs, Ssc-milR-240 was potentially associated with sclerotial development by epigenetic regulation of its target histone acetyltransferase. This study will facilitate the better understanding of the milRNA regulation associated with sclerotial development in S. sclerotiorum and even other sclerotia-forming fungi. This work will provide novel insights into the molecular regulations of fungal morphogenesis and the candidate targets of milRNAs used for the sustainable management of plant diseases caused by S. sclerotiorum.

Introduction

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen that infects more than 400 plant species (Boland and Hall, 1994). This fungus causes over 60 plant diseases, which result in significant losses in both agricultural and horticultural crops (Bolton et al., 2006). Sclerotinia sclerotiorum produces a durable structure called sclerotium from which conidiomata develop for spore dispersal. Sclerotium also facilitates fungi to survive under challenging conditions (Smith et al. 2015). Therefore, sclerotia have important roles in both the life and disease cycles of S. sclerotiorum and other sclerotia-forming fungi (Liang et al., 2018). Sclerotia are composed of massive hyphae that can resist adverse environmental conditions including UV rays, high temperature and drought (Erental et al., 2008). Sclerotial development has been distinguished into three characteristic stages: initiation state that involves mycelial aggregation and initial formation; development stage that involves increase in sclerotial volume with the exudation of droplets and maturation stage that involves shape delimitation and pigmentation (Willetts and Bullock, 1992, Erental et al., 2008).

Studies have been conducted in order to delineate the molecular processes of sclerotial development in S. sclerotiorum (Erental et al., 2008, Chen et al., 2018). For instance, complete genome sequence for S. sclerotiorum has been annotated, which since then served as an important resource for molecular investigations of fungal development and pathogenesis (Derbyshire et al., 2018). Proteome-based studies have also been carried out in S. sclerotiorum, which have led to the better understanding of pathogen virulence and fungal biology (e.g., sclerotial development) (Yajima and Kav, 2006, Liang et al., 2010). In addition, studies on the transcriptome levels have also identified genes that may be important for S. sclerotiorum pathogenicity (Seifbarghi et al., 2017).

Recently, considerable interest in the roles of small, non-coding RNA molecules has led to a number of investigations aimed at understanding their specific roles in the regulation of gene expression (Wang and Chekanova, 2016). Small RNAs are non-coding RNAs derived from double-stranded RNA or hairpin RNA with a length of 15–40 nucleotides (nts) (Weiberg et al., 2014), which are further classified as short interfering RNAs (siRNAs), microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs) (Torres-Martínez and Ruiz-Vázquez, 2017). Small RNAs are involved in the regulation of many biological processes including maintenance of genome integrity, response to stresses, and regulation of developmental processes (Kumar et al., 2018, Singh et al., 2018). Some small RNAs produced in the model fungus and other fungal species have been identified as microRNA-like RNAs (milRNAs) and share many common features with miRNAs of animals and plants (Lee et al., 2010). It has also been demonstrated that similar regulatory mechanisms, mediated by milRNAs exist in most eukaryotes, and play fundamental roles in the growth and development of filamentous fungi (Jin et al., 2019, Shao et al., 2020).

Mature milRNAs are generally 20–24 nts that originate from double-stranded RNA precursors with unique stem-loop structures (Axtell, 2013). milRNAs have the ability to regulate the translation of target genes by complementary binding with the open reading frame (ORF) and/or untranslated regions (UTR), or modulate gene expression by promoting the degradation of target mRNA sequences (Sunkar and Zhu, 2004). It has also been demonstrated that milRNAs affect fungal growth and development (Lau et al., 2013). For instance, during vegetative growth, milRNAs differentially modulated mycelial growth and conidiation of a model fungus Aspergillus flavus and an entopathogenic fungus Metarhizium anisopliae (Zhou et al., 2012a, Wang et al., 2019). Similarly, during fungal reproduction, milRNAs induce and play regulatory functions during the sexual development of fungal species, including a phytopathogenic fungus Fusarium oxysporum and a medicinal fungus Antrodia cinnamomea (Chen et al., 2014, Lin et al., 2015). Certain milRNAs also play regulatory roles in perithecium formation and fruiting body development during sexual development in the pathogenic F. graminearum and the edible Cordyceps militaris (Zeng et al., 2018, Shao et al., 2019). Additionally, milRNAs are also involved in the mycotoxin biosynthesis in F. oxysporum and A. flavus (Bai et al., 2015, Jiang et al., 2017) as well as in mediating fungal pathogenicity and virulence of the human pathogen Trichophyton rubrum and phytopathogen Verticillium dahlia (Wang et al., 2018, Jin et al., 2019). Some milRNAs are also known to be involved in impairing host immune-responses to achieve fungal infection on plants such as Arabidopsis, tomato and wheat (Weiberg et al., 2013, Wang et al., 2017).

In recent years, high-throughput sequencing technologies were used for comprehensive investigations of milRNA regulation in fungal species (Jiang et al., 2017, Shao et al., 2020). Although a few investigations of milRNAs in fungi have been reported, the potential roles of milRNAs are still poorly known in the development and differentiation of fungal species (Chen et al., 2014, Lau et al., 2013). In this study we profiled the milRNAs associated with sclerotial development and mediating other functions in the phytopathogen S. sclerotiorum. Our findings were discussed within the biological context of this important pathogen and opportunities to use this information to develop rational approaches to sustainably manage S. sclerotiorum.