Yan Li

The genome of Pleurotus eryngii provides insights into the mechanisms of wood decay

Pleurotus eryngii (DC.) Quél. is widely used for bioconverting lignocellulosic byproducts into biofuel and value added products. Sequencing and annotating the genome of a monokaryon strain P. eryngii 183 allows us to gain a better understanding of carbohydrate-active enzymes (CAZymes) and oxidoreductases for degradation of lignocellulose in white-rot fungi. The genomic data provides insights into genomic basis of degradation mechanisms of lignin and cellulose and may pave new avenues for lignocellulose bioconversion.

The complete mitochondrial genome of the Basidiomycete edible fungus Pleurotus eryngii

Abstract The complete mitochondrial genome of the edible fungus Pleurotus eryngii (oyster mushroom) was determined using Illumina sequencing. This mitogenome is a circular molecule of 72,650 bp in length with a GC content of 26.28%. Gene prediction showed that 40 putative protein-coding genes, the small ribosomal RNA subunits (rns), and 23 tRNAs were located on the same strand. The mitogenome of P. eryngii has a similar structure to that of P. ostreatus in both gene content and gene order. The mitogenome information of P. eryngii should contribute to our understanding of the diversity and evolution of Pleurotaceae and Agaricales.

The complete mitochondrial genome of the widely cultivated edible fungus Lentinula edodes

Abstract The complete mitochondrial genome of the widely cultivated edible fungus Lentinula edodes was determined using the next-generation sequencing technology. The circular molecule is 116,819 bp in length with a GC content of 30.75%. Conserved genes including 13 putative protein-coding genes and 24 tRNAs were located on the same strand. We detected 14 introns invading 4 genes, including cob, cox1, nad1, and nad5. The phylogenetic analysis confirmed that L. edodes was a number of Agaricales. This mitochondrial genome may open new avenues for understanding the phylogeny and evolution of Omphalotaceae and Agaricales.

Evaluation of the ribosomal DNA internal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing

The nuclear ribosomal DNA internal transcribed spacer (ITS) has been widely used to assess the fungal composition in different environments by deep sequencing. To evaluate the ITS in the analysis of fungal diversity, comparisons of the clustering and taxonomy generated by sequencing with different portions of the whole fragment were conducted in this study. For a total of 83,120 full-length ITS sequences obtained from the UNITE database, it was found that, on average, ITS1 varied more than ITS2 within the kingdom Fungi; this variation included length and GC content variations and polymorphisms, with some polymorphisms specific to particular fungal groups. The taxonomic accuracy for ITS was higher than that for ITS1 or ITS2. The commonly used operational taxonomic unit (OTU) for evaluating fungal diversity and richness assigned several species to a single OTU even with clustering at 99.00% sequence similarity. The clustering and taxonomic capacities did not differ between ITS1 and ITS2. However, the OTU commonality between ITS1 and ITS2 was very low. To test this observation further, 219,741 pyrosequencing reads, including 39,840 full-length ITS sequences, were obtained from 10 soil samples and were clustered into OTUs. The pyrosequencing results agreed with the results of the in silico analysis. ITS1 might overestimate the fungal diversity and richness. Analyses using ITS, ITS1 and ITS2 yielded several different taxa, and the taxonomic preferences for ITS and ITS2 were similar. The results demonstrated that ITS2 alone might be a more suitable marker for revealing the operational taxonomic richness and taxonomy specifics of fungal communities when the full-length ITS is not available.

Three complete mitochondrial genomes of straw-rotting edible fungus Volvariella volvacea using next generation sequencing

Abstract The straw-rotting edible fungus Volvariella volvacea is a widely cultivated edible fungus across China and Southeast Asian countries. Three complete mitochondrial genomes of V. volvacea from China, Thailand, and India were determined using the next-generation sequencing technology. The genome sizes of the three strains (China, Thailand, and India) were 62,541 bp, 64,531 bp, and 65,668 bp with GC contents of 38.46%, 38.56%, and 38.52%, respectively. All the genomes encoded 14 conserved protein-coding genes, the small ribosomal RNA subunits (rns), large ribosomal RNA subunits (rnl), and 23 tRNAs were located on the same strand. In the putative protein-coding genes, four introns were distributed in cox1 in the genomes of V23-1 and V8. 5 introns (four introns invaded into cox1and one intron invaded into cob) were detected in Tai8. The phylogenetic analysis confirmed that V. volvacea was a number of Agaricales. This mitochondrial genome may open new avenues for understanding the phylogeny and evolution of Pluteaceae and Agaricales.

Efficient transformation of Pleurotus eryngii with a safe selective marker mutated from the Pesdi1 gene

We introduced a site-directed mutation in the sdi1 gene and used it as a selective marker for the polyethylene glycol-mediated transformation of Pleurotus eryngii monokaryon protoplasts. The transformants displayed obvious and stable resistance to the fungicide carboxin indicating that the mutant Pesdi1 gene is an efficient selective marker.

Establishment of uracil auxotrophic dikaryotic strains of Lentinula edodes by crossbreeding

The uracil auxotrophic monokaryotic strain 423-9 of Lentinula edodes was crossed with nine monokaryons (cro2-2-9, W66-1, xd2-3-2, QingKe 20A, 241-1-1, 9015-1, L66-2, 241-1-2, and Qing 23A) derived from wild type strains of L. edodes. Nine dikaryotic hybrids were established from these crosses. These hybrids were fruited and 496 single spore isolates were obtained. Among these single spore isolates, 166 were identified as monokaryons under a microscope. We screened these monokaryons on selective medium and obtained 19 uracil auxotrophic monokaryons. By using the Monkaryon-monkaryon crossing method among the uracil auxotrophic monokaryons, 56 uracil auxotrophic dikaryotic strains were established on selective medium. These dikaryotic strains were unable to grow on minimal medium without uracil and exhibited slow growth rates on PDA plates compared to the wild type strain. The uracil auxotrophic dikaryotic strains also showed more vigorous growth on sawdust cultivation medium containing uracil than that without uracil. The fruiting tests showed that they formed normal fruiting bodies on the sawdust medium containing uracil. The results show that the uracil auxotrophic dikaryotic strain of L. edodes could be produced by mating, and will provide a valuable resource for future genetic studies and for spawn protection and identification.

Comparison and Validation of Putative Pathogenicity-Related Genes Identified by T-DNA Insertional Mutagenesis and Microarray Expression Profiling in Magnaporthe oryzae

High-throughput technologies of functional genomics such as T-DNA insertional mutagenesis and microarray expression profiling have been employed to identify genes related to pathogenicity in Magnaporthe oryzae. However, validation of the functions of individual genes identified by these high-throughput approaches is laborious. In this study, we compared two published lists of genes putatively related to pathogenicity in M. oryzae identified by T-DNA insertional mutagenesis (comprising 1024 genes) and microarray expression profiling (comprising 236 genes), respectively, and then validated the functions of some overlapped genes between the two lists by knocking them out using the method of target gene replacement. Surprisingly, only 13 genes were overlapped between the two lists, and none of the four genes selected from the overlapped genes exhibited visible phenotypic changes on vegetative growth, asexual reproduction, and infection ability in their knockout mutants. Our results suggest that both of the lists might contain large proportions of unrelated genes to pathogenicity and therefore comparing the two gene lists is hardly helpful for the identification of genes that are more likely to be involved in pathogenicity as we initially expected.

Comparative Proteomic Analysis of Light-Induced Mycelial Brown Film Formation in Lentinula edodes.

Light-induced brown film (BF) formation by the vegetative mycelium of Lentinula edodes is important for ensuring the quantity and quality of this edible mushroom. Nevertheless, the molecular mechanism underlying this phenotype is still unclear. In this study, a comparative proteomic analysis of mycelial BF formation in L. edodes was performed. Seventy-three protein spots with at least a twofold difference in abundance on two-dimensional electrophoresis (2DE) maps were observed, and 52 of them were successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF/MS). These proteins were classified into the following functional categories: small molecule metabolic processes (39%), response to oxidative stress (5%), and organic substance catabolic processes (5%), followed by oxidation-reduction processes (3%), single-organism catabolic processes (3%), positive regulation of protein complex assembly (3%), and protein metabolic processes (3%). Interestingly, four of the proteins that were upregulated in response to light exposure were nucleoside diphosphate kinases. To our knowledge, this is the first proteomic analysis of the mechanism of BF formation in L. edodes. Our data will provide a foundation for future detailed investigations of the proteins linked to BF formation.