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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 Findings  We 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-1 KO  ,  dcl-2 KO  ,  dcl DKO   and  qde-2 KO   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/Significance  Our 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.

Identification of MicroRNA-Like RNAs in Mycelial and Yeast Phases of the Thermal Dimorphic Fungus Penicillium marneffei