Jiao Pan

Identification and Functional Demonstration of miRNAs in the Fungus Cryptococcus neoformans

microRNAs (miRNAs), endogenous posttranscriptional repressors by base-pairing of their cognate mRNAs in plants and animals, have mostly been thought lost in the kingdom of fungi. Here, we report the identification of miRNAs from the fungus Cryptococcus neoformans. With bioinformatics and Northern blotting approaches, we found that these miRNAs and their hairpin precursors were present in this fungus. The size of miR1 and miR2 is 22 nt and 18 nt, respectively. The precursors are about ∼70 nt in length that is close to mammalian pre-miRNAs. Characteristic features of miRNAs are also found in miR1/2. We demonstrated that the identified miRNAs, miR1 and miR2, caused transgene silencing via the canonical RNAi pathway. Bioinformantics analysis helps to reveal a number of identical sequences of the miR1/2 in transposable elements (TEs) and pseudogenes, prompting us to think that fungal miRNAs might be involved in the regulation of the activity of transposons and the expression of pseudogenes. This study identified functional miRNAs in C. neoformans, and sheds light on the diversity and evolutionary origin of eukaryotic miRNAs.

A new taxol-producing fungus ( Pestalotiopsis malicola ) and evidence for taxol as a transient product in the culture

Fungal production of the anti-tumor taxol is an effective way of making this drug in industries. We reported here a new taxol-producing fungus, NK101, from plant debris in the soil. Based on the culture characteristics, conidia structure and molecular evidence, NK101 was classified as Pestalotiopsis malicola . Taxol was verified in both the culture and the mycelium in a high level (186 µg/L). The time course of yield suggests that taxol was present as a transient product in the fungus. This work may show the diversity of using fungi to produce taxol.

A copper-responsive factor gene CUF1 is required for copper induction of laccase in Cryptococcus neoformans.

The multicopper laccase is a major virulence factor in Cryptococcus neoformans. Its expression regulation is complex. We presented molecular evidence to show that laccase expression was induced by high concentrations of exogenous copper. Melanin production and laccase enzymatic activity increased dramatically in response to the addition of copper to the media. Reverse transcription-PCR amplification of the laccase gene LAC1 mRNA revealed that the induction occurred at the transcriptional level, which required the copper-responsive factor-encoding gene CUF1. Disruption of CUF1 demolished the activation of LAC1 transcription by copper, whereas the reconstituted strain restored the phenotypic defects. Furthermore, copper induction was shown to be independent of derepression by glucose starvation, a well-established activation factor for laccase expression. These results demonstrate a role of the copper-responsive factor gene CUF1 in the expression of laccase in C. neoformans.

A PKS gene, pks-1, is involved in chaetoglobosin biosynthesis, pigmentation and sporulation in Chaetomium globosum

Chaetomium globosum is one of the most common fungi in nature. It is best known for producing chaetoglobosins; however, the molecular basis of chaetoglobosin biosynthesis is poorly understood in this fungus. In this study, we utilized RNA interference (RNAi) to characterize a polyketide synthase gene, pks-1, in C. globosum that is involved in the production of chaetoglobosin A. When pks-1 was knocked down by RNAi, the production of chaetoglobosin A dramatically decreased. Knock-down mutants also displayed a pigment-deficient phenotype. These results suggest that the two polyketides, melanin and chaetoglobosin, are likely to share common biosynthetic steps. Most importantly, we found that pks-1 also plays a critical role in sporulation. The silenced mutants of pks-1 lost the ability to produce spores. We propose that polyketides may modulate cellular development via an unidentified action. We also suggest that C. globosum pks-1 is unique because of its triple role in melanin formation, chaetoglobosin biosynthesis and sporulation. This work may shed light on chaetoglobosin biosynthesis and indicates a relationship between secondary metabolism and fungal morphogenesis.

A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.

Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This ‘suicide’ CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans.

Regulation of copper homeostasis by Cuf1 associates with its subcellular localization in the pathogenic yeast Cryptococcus neoformans H99

Here, we present further characterization of cryptococcal CUF1 in copper homeostasis. We demonstrated that CUF1 was involved both in copper acquisition and in copper detoxification in response to copper variation. This was verified by direct measurement of the quantity of intracellular copper with flame atomic absorption spectrometry (FAAS) and molecular evidence. In copper-limited growth, the mutant cuf1Δ exhibited copper deficiency, growth defect on glycerol and sensitivity to hydrogen peroxide and methionine. A novel function of cryptococcal CUF1 is revealed in copper detoxification when copper is in excess. The mutant cuf1Δ showed severe hypersensitivity to exogenous copper, while a high level of copper was accumulated shown by FAAS, suggesting that CUF1 may be required in copper export events. On cloning of cDNA, it was found that Cuf1 distinguishably harbors functional elements that are found in Ace1 and Mac1 of Saccharomyces cerevisiae. The regulation of copper homeostasis by Cuf1 is realized by its subcellular localization. Epifluorescence microscopy observed that, upon copper depletion, Cuf1 was localized exclusively to the nucleus as an activator for CTR4 transcription, while it was located to the cell periphery in the presence of exogenous copper. This work reveals a unique copper regulator and may provide insights into the copper metabolism in fungi.

Regulation of virulence factors, carbon utilization and virulence by SNF1 in Cryptococcus neoformans JEC21 and divergent actions of SNF1 between cryptococcal strains.

We describe here the functions of a Snf1/AMPK homolog in the human pathogenic yeast Cryptococcus neoformans, strain JEC21. We found that JEC21 SNF1 is a key regulator for the biosynthesis of the major virulence factors, stress resistance and alternative carbon source utilization. Disruption of JEC21 SNF1 results in defects of laccase activity and capsule production, sensitivity to cation stress. Especially, we found that JEC21 SNF1 is essential for growth at elevated temperature and for thermotolerance. To our knowledge, a role for Snf1 proteins in thermotolerance has not been reported. Furthermore, we observed a functional divergence between JEC21 SNF1 and its equivalent from serotype A strain H99. A high temperature is needed for H99 SNF1 to function in stress response and carbon source preference, but not for the JEC21 SNF1. Our results confirmed a critical role of JEC21 SNF1 in regulation of stress response and virulence. Revelation of divergent actions of SNF1 may help to understand the evolution of cryptococcal pathogenesis and provides insights into the strain-associated biosynthesis of virulence factors.

Optimized integration of T-DNA in the taxol-producing fungus Pestalotiopsis malicola

We previously reported a taxol-producing fungus Pestalotiopsis malicola . There, we described the transformation of the fungus mediated by Agrobacterium tumefaciens. T-DNA carrying the selection marker was transferred into the fungus and randomly integrated into the genome as shown by Southern blotting. Approximately 66% of the transformants obtained a single copy of T-DNA. Conditions of transformation were optimized. The method can be used for taxol-related genes searching and manipulation in P. malicola . This is the first report of T-DNA-mediated transformation in taxol-producing fungi. Key words: T-DNA, random insertional mutagenesis, taxol, Pestalotiopsis .

Requirement of a Tsp2-Type Tetraspanin for Laccase Repression and Stress Resistance in the Basidiomycete Cryptococcus neoformans

ABSTRACT Fungal laccases have been widely used in industry. The expression of laccase often is repressible by the primary carbon source glucose in many fungi. The underlying basis is largely unclear. We demonstrate here that a gene, TSP2-1, was required for laccase repression by glucose in the basidiomycete Cryptococcus neoformans. TSP2-1 encodes a Tsp2-type tetraspanin. The disruption of TSP2-1 resulted in constant melanin formation and the expression of the laccase gene LAC1. This derepression phenotype was restorable by 10 mM exogenous cyclic AMP (cAMP). A capsule defect in the mutant tsp2-1Δ also was restored by cAMP. The results indicate an interaction of Tsp2-1 with the cAMP-dependent protein kinase A (PKA) pathway that has been shown to modulate laccase repression and capsule biosynthesis in this fungus. Other roles of TSP2-1, e.g., in maintaining cell membrane integrity and stress resistance, also were defined. This work reveals a Tsp2-1-dependent glucose repression in C. neoformans. The function of Tsp2-type tetraspanin Tsp2-1 is described for the first time.

A B-type histone acetyltransferase Hat1 regulates secondary metabolism, conidiation, and cell wall integrity in the taxol-producing fungus Pestalotiopsis microspora.

In filamentous fungi, many gene clusters for the biosynthesis of secondary metabolites often stay silent under laboratory culture conditions because of the absence of communication with its natural environment. Epigenetic processes have been demonstrated to be critical in the expression of the genes or gene clusters. Here, we report the identification of a B‐type histone acetyltransferase, Hat1, and demonstrate its significant roles in secondary metabolism, conidiation, and the cell wall integrity in the fungus Pestalotiopsis microspora. An hat1 deletion strain shows a dramatic decrease of SMs in this fungus, suggesting hat1 functions as a global regulator on secondary metabolism. Moreover, the mutant strain hat1Δ delays to produce conidia with significantly decreased number of conidia, while shows little effect on vegetative growth, suggesting that it plays a critical role in conidiation. The hypersensitivity of hat1Δ to Congo red demonstrates that disruption of hat1 impairs the integrity of cell wall. Overexpression of the wild‐type hat1 allele enhances conidiation by boosting the number of conidia. This is the first report on the role of a B‐type histone acetyltransferase in fungal secondary metabolism and cell wall integrity.