Abstract
Background Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, miRNAs in fungi were less well studied and their potential roles in fungal dimorphism were largely unknown. Based on P. marneffei genome sequence, we hypothesize that miRNA-like RNAs (milRNAs) may be expressed in the dimorphic fungus.Methodology/Principal FindingsWe attempted to identify milRNAs in P. marneffei in both mycelial and yeast phase using high-throughput sequencing technology. Small RNAs were more abundantly expressed in mycelial than yeast phase. Sequence analysis revealed 24 potential milRNA candidates, including 17 candidates in mycelial and seven in yeast phase. Two genes, dcl-1 and dcl-2, encoding putative Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were identified in P. marneffei. Phylogenetic analysis showed that dcl-2 of P. marneffei was more closely related to the homologues in other thermal dimorphic pathogenic fungi than to Penicillium chrysogenum and Aspergillus spp., suggesting the co-evolution of dcl-2 among the thermal dimorphic fungi. Moreover, dcl-2 demonstrated higher mRNA expression levels in mycelial than yeast phase by 7 folds (P<0.001). Northern blot analysis confirmed the expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase. Using dcl-1KO, dcl-2KO, dclDKO and qde-2KO deletion mutants, we showed that the biogenesis of both milRNAs were dependent on dcl-2 but not dcl-1 or qde-2. The mRNA expression levels of three predicted targets of PM-milR-M1 were upregulated in knockdown strain PM-milR-M1 KD, supporting regulatory function of milRNAs.Conclusions/SignificanceOur findings provided the first evidence for differential expression of milRNAs in different growth phases of thermal dimorphic fungi and shed light on the evolution of fungal proteins involved in milRNA biogenesis and possible role of post-transcriptional control in governing thermal dimorphism.
Highlights
Penicillium marneffei is the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in Southeast Asia [1,2,3,4]
Respectively compared to wild type strain PM1 (P,0.05 by student t test). This is the first report of miRNA-like RNAs (milRNAs) in a human thermal dimorphic pathogenic fungus and their differential expression in mycelial and yeast phases
No plant or animallike miRNAs was found in Aspergillus species by computational analysis of six Aspergillus genomes (Aspergillus nidulans, Aspergillus oryzae, Aspergillus fumigatus, Aspergillus terreus, Aspergillus clavatus, and Neosartorya fischeri) [67]. It was uncertain whether fungi have microRNAs until the recent discovery of milRNAs in the filamentous fungi, N. crassa, S. sclerotiorum and M. anisopliae, as well as the human pathogenic yeast, C. neoformans [30,31,32,33]
Summary
Penicillium marneffei is the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in Southeast Asia [1,2,3,4]. It has been renamed as Talaromyces based on phylogenetic analyses [5]. Cases of imported P. marneffei infections have been reported from countries outside Southeast Asia [11,12]. Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in China and Southeast Asia. Based on P. marneffei genome sequence, we hypothesize that miRNA-like RNAs (milRNAs) may be expressed in the dimorphic fungus
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