Characterization and application of small RNAs and RNA silencing mechanisms in fungi

Although extensively cataloged and functionally diverse in plants and animals, the role and targets of small RNAs remain mostly uncharacterized in filamentous fungi. To date, much of the knowledge of small RNAs in filamentous fungi has been derived from studies of a limited group of fungi, most notably in Neurospora crassa. While most of the recently discovered classes of small RNAs appear to be unique to fungi some are commonly found in eukaryotes. It is noteworthy that the RNA silencing protein machinery involved in small RNA biogenesis has also diverged greatly, particularly within filamentous fungi, and may explain the diversity of small RNA classes. In this review, we summarize important classes of eukaryotic small RNAs and provide a current analysis of the RNA silencing machinery based on available fungal genome sequences. Finally, we discuss opportunities for exploiting knowledge of small RNAs and RNA silencing for practical application such as engineering plants resistant to fungal pathogens.     

Characterization and application of small RNAs and RNA silencing mechanisms in fungi

Although extensively cataloged and functionally diverse in plants and animals, the role and targets of small RNAs remain mostly uncharacterized in filamentous fungi. To date, much of the knowledge of small RNAs in filamentous fungi has been derived from studies of a limited group of fungi, most notably in Neurospora crassa. While most of the recently discovered classes of small RNAs appear to be unique to fungi some are commonly found in eukaryotes. It is noteworthy that the RNA silencing protein machinery involved in small RNA biogenesis has also diverged greatly, particularly within filamentous fungi, and may explain the diversity of small RNA classes. In this review, we summarize important classes of eukaryotic small RNAs and provide a current analysis of the RNA silencing machinery based on available fungal genome sequences. Finally, we discuss opportunities for exploiting knowledge of small RNAs and RNA silencing for practical application such as engineering plants resistant to fungal pathogens.     

Identification of microRNA-Like RNAs in the Filamentous Fungus Trichoderma reesei by Solexa Sequencing

microRNAs (miRNAs) are non-coding small RNAs (sRNAs) capable of negatively regulating gene expression. Recently, microRNA-like small RNAs (milRNAs) were discovered in several filamentous fungi but not yet in Trichoderma reesei, an industrial filamentous fungus that can secrete abundant hydrolases. To explore the presence of milRNA in T. reesei and evaluate their expression under induction of cellulose, two T. reesei sRNA libraries of cellulose induction (IN) and non-induction (CON) were generated and sequenced using Solexa sequencing technology. A total of 726 and 631 sRNAs were obtained from the IN and CON samples, respectively. Global expression analysis showed an extensively differential expression of sRNAs in T. reesei under the two conditions. Thirteen predicted milRNAs were identified in T. reesei based on the short hairpin structure analysis. The milRNA profiles obtained in deep sequencing were further validated by RT-qPCR assay. Computational analysis predicted a number of potential targets relating to many processes including regulation of enzyme expression. The presence and differential expression of T. reesei milRNAs imply that milRNA might play a role in T. reesei growth and cellulase induction. This work lays foundation for further functional study of fungal milRNAs and their industrial application.     

milRNApredictor: Genome-free prediction of fungi milRNAs by incorporating k-mer scheme and distance-dependent pair potential

MicroRNA-like small RNAs (milRNAs) with length of 21–22 nucleotides are a type of small non-coding RNAs that are firstly found in Neurospora crassa in 2010. Identifying milRNAs of species without genomic information is a difficult problem. Here, knowledge-based energy features are developed to identify milRNAs by tactfully incorporating k-mer scheme and distance-dependent pair potential. Compared with k-mer scheme, features developed here can alleviate the inherent curse of dimensionality in k-scheme once k becomes large. In addition, milRNApredictor built on novel features performs comparably to k-mer scheme, and achieves sensitivity of 74.21%, and specificity of 75.72% based on 10-fold cross-validation. Furthermore, for novel miRNA prediction, there exists high overlap of results from milRNApredictor and state-of-the-art mirnovo. However, milRNApredictor is simpler to use with reduced requirements of input data and dependencies. Taken together, milRNApredictor can be used to de novo identify fungi milRNAs and other very short small RNAs of non-model organisms.     

The lifestyle transition of Arthrobotrys oligospora is mediated by microRNA-like RNAs

The lifestyle transition of fungi, defined as switching from taking organic material as nutrients to pathogens, is a fundamental phenomenon in nature. However, the mechanisms of such transition remain largely unknown. Here we show microRNA-like RNAs(milRNAs) play a key role in fungal lifestyle transition for the first time. We identified milRNAs by small RNA sequencing in Arthrobotrys oligospora, a known nematode-trapping fungus. Among them, 7 highly expressed milRNAs were confirmed by northern-blot analysis. Knocking out two milRNAs significantly decreased A. oligospora's ability to switch lifestyles. We further identified that two of these milRNAs were associated with argonaute protein QDE-2 by RNA-immunoprecipitation(RIP) analysis. Three of the predicted target genes of milRNAs were found in immunoprecipitation(IP)products of QDE-2. Disruption of argonaute gene qde-2 also led to serious defects in lifestyle transition. Interestingly, knocking out individual milRNAs or qde-2 lead to diverse responses under different conditions, and qde-2 itself may be targeted by the milRNAs. Collectively, it indicates the lifestyle transition of fungi is mediated by milRNAs through RNA interference(RNAi)machinery, revealing the wide existence of miRNAs in fungi kingdom and providing new insights into understanding the adaptation of fungi from scavengers to predators and the mechanisms underlying fungal infections.     

FoQDE2-dependent milRNA promotes Fusarium oxysporum f. sp. cubense virulence by silencing a glycosyl hydrolase coding gene expression

MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein-coding gene expression primarily found in plants and animals. Fungi produce microRNA-like RNAs (milRNAs) that are structurally similar to miRNAs and functionally important in various biological processes. The fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Banana Fusarium vascular wilt that threatens global banana production. It remains uncharacterized about the biosynthesis and functions of milRNAs in Foc. In this study, we investigated the biological function of milRNAs contributing to Foc pathogenesis. Within 24 hours post infecting the host, the Argonaute coding gene FoQDE2, and two Dicer coding genes FoDCL1 and FoDCL2, all of which are involved in milRNA biosynthesis, were significantly induced. FoQDE2 deletion mutant exhibited decreased virulence, suggesting the involvement of milRNA biosynthesis in the Foc pathogenesis. By small RNA sequencing, we identified 364 small RNA-producing loci in the Foc genome, 25 of which were significantly down-regulated in the FoQDE2 deletion mutant, from which milR-87 was verified as a FoQDE2-depedent milRNA based on qRT-PCR and Northern blot analysis. Compared to the wild-type, the deletion mutant of milR-87 was significantly reduced in virulence, while overexpression of milR-87 enhanced disease severity, confirming that milR-87 is crucial for Foc virulence in the infection process. We furthermore identified FOIG_15013 (a glycosyl hydrolase-coding gene) as the direct target of milR-87 based on the expression of FOIG_15013-GFP fusion protein. The FOIG_15013 deletion mutant displayed similar phenotypes as the overexpression of milR-87, with a dramatic increase in the growth, conidiation and virulence. Transient expression of FOIG_15013 in Nicotiana benthamiana leaves activates the host defense responses. Collectively, this study documents the involvement of milRNAs in the manifestation of the devastating fungal disease in banana, and demonstrates the importance of milRNAs in the pathogenesis and other biological processes. Further analyses of the biosynthesis and expression regulation of fungal milRNAs may offer a novel strategy to combat devastating fungal diseases.     

Free living amoebae could enhance Fusarium oxysporum growth

Free living amoebae and Fusarium oxysporum can be recovered in the same environment and may potentially interact. The presence of these protozoa could lead to an increased development of this filamentous fungus. To assess this potential risk, the interactions between two free living amoebae, Acanthamoeba castellanii and Hartmanella vermiformis, and F. oxysporum, which can be isolated from soil and water, were studied. After 48 hr of coincubation in tap water, culturable fungi were quantified. In addition, the interactions between the free living amoebae and the fungus were investigated using electron microscopy. We show that the presence of amoeba trophozoites increased the growth of F. oxysporum without fungal influence on amoebae viability. In the same way, incubation of the fungus with culture supernatants of the two amoebae induced fungal germination and increased fungal growth. The results of this study confirm that the presence of amoebae should be taken into consideration in the different environments where they may be in contact with Fusarium.     

综述: 表观遗传学研究的模式生物——粗糙脉孢菌

丝状真菌粗糙脉孢菌是一种作为遗传学研究的经典模式生物．通过对粗糙脉孢菌5S rRNA基因的组成和在染色体上分布的研究,揭示了丝状真菌中存在的一种基因组防御机制——重复序列诱导的DNA点突变(RIP)．通过对发生突变的5S rRNA假基因的研究还发现,粗糙脉孢菌中存在一种重要的表观遗传修饰——DNA甲基化,随后的深入研究使粗糙脉孢菌成为解析DNA甲基化机制的最重要模式生物之一．粗糙脉孢菌基因转化操作引起的营养生长阶段同源基因的沉默(quelling)是由RNAi途径调控的,同时该途径也是调控减数分裂过程中非配对DNA诱发的基因沉默(meiotic silencing)的关键．由于粗糙脉孢菌基因组简单,且存在与高等真核生物相同的DNA甲基化和多种组蛋白的修饰,使其成为今后深入研究组蛋白修饰与染色质重塑等表观遗传现象参与基因表达调控和基因组稳定性维持的重要模式生物之一．     

Cloning and sequence of the Ascobolus immersusS-adenosyl-l-methionine synthetase-encoding gene

The structural gene encoding S-adenosyl-l-methionine synthetase (SAM-S) in the fungus Ascobolus immersus has been cloned and sequenced. It contains a 1179-bp ORF, interrupted by three introns, encoding a 393-amino-acid protein (42 978 Da) that is 90% homologous to the SAM-S of the filamentous fungus Neurospora crassa, indicating that these fungi are closely related species     

Neurospora crassa,a Model System for Epigenetics Research

The filamentous fungus Neurospora crassa has provided a rich source of knowledge on epigenetic phenomena that would have been difficult or impossible to gain from other systems. Neurospora sports features found in higher eukaryotes but absent in both budding and fission yeast, including DNA methylation and H3K27 methylation, and also has distinct RNA interference (RNAi)-based silencing mechanisms operating in mitotic and meiotic cells. This has provided an unexpected wealth of information on gene silencing systems. One silencing mechanism, named repeat-induced point mutation (RIP), has both epigenetic and genetic aspects and provided the first example of a homology-based genome defense system. A second silencing mechanism, named quelling, is an RNAi-based mechanism that results in silencing of transgenes and their native homologs. A third, named meiotic silencing, is also RNAi-based but is distinct from quelling in its time of action, targets, and apparent purpose.