Meng-Meng Liu

Transcriptome analysis of genes involved in defence response in Polyporus umbellatus with Armillaria mellea infection.

Polyporus umbellatus, a species symbiotic with Armillaria mellea and it also exhibits substantial defence response to Armillaria mellea infection. There are no genomics resources databases for understanding the molecular mechanism underlying the infection stress of P. umbellatus. Therefore, we performed a large-scale transcriptome sequencing of this fungus with A. mellea infection using Illumina sequencing technology. The assembly of the clean reads resulted in 120,576 transcripts, including 38,444 unigenes. Additionally, we performed a gene expression profiling analysis upon infection treatment. The results indicated significant differences in the gene expression profiles between the control and the infection group. In total, 10933 genes were identified between the two groups. Based on the differentially expressed genes, a Gene Ontology annotation analysis showed many defence-relevant categories. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes pathway analysis uncovered some important pathways. Furthermore, the expression patterns of 13 putative genes that are involved in defence response resulting from quantitative real-time PCR were consistent with their transcript abundance changes as identified by RNA-seq. The sequenced genes covered a considerable proportion of the P. umbellatus transcriptome, and the expression results may be useful to strengthen the knowledge on the defence response of this fungus defend against Armillaria mellea invasion.

Oxalic acid and sclerotial differentiation of Polyporus umbellatus

The present investigation aimed to uncover the effects of exogenous oxalic acid during the sclerotial formation of Polyporus umbellatus, with an emphasis on determining the content of the endogenic oxalic acid in the fungus. To this end, the oxalic acid content of the vegetative mycelia, sclerotia, culture mediums and sclerotial exudate were measured using High Performance Liquid Chromatography (HPLC). Furthermore, the lipid peroxidation was estimated by detecting thiobarbituric bituric acid reactive substances (TBARS). The results showed that the exogenous oxalic acid caused a delay in sclerotial differentiation (of up to 9 or more days), suppressed the sclerotial biomass and decreased the lipid peroxidation significantly in a concentration-dependent manner. Oxalic acid was found at very low levels in the mycelia and the maltose medium, whereas it was found at high levels in the mycelia and sucrose medium. After sclerotial differentiation, oxalic acid accumulated at high levels in both the sclerotia and the sclerotial exudate. Oxalic acid was therefore found to inhibit P. umbellatus sclerotial formation.

Transcriptomic analyses reveal clathrin-mediated endocytosis involved in symbiotic seed germination of Gastrodia elata

BackgroundGastrodia elata is a well-known medicinal orchid. In nature, the germination rate of G. elata is extremely poor, because there is no endosperm within the mature seed. It is crucial for G. elata to obtain nutrition from mycorrhizal fungi (Mycena) at the early-stage of germination. After germination, the seed gives rise to a protocorm. However, there are no “omic” studies on understanding the interaction between Gastrodia and Mycena. Here, we used transcriptomic approaches to explore changes in seed germination of G. elata.ResultsBased on RNA-Seq, a total of ~221 million clean reads were assembled denovo into 139,756 unigenes, including 42,140 unigenes that were annotated in public databases. Meanwhile, 1750 unigenes were identified as differentially expressed genes. Most of these differentially expressed genes were putatively involved in energy metabolism, plant defense, molecular signaling, and secondary metabolism. Additionally, numerous genes involved in clathrin-mediated endocytosis were identified from our data. Most of these genes (e.g., clathrin, adaptor protein, dynamin, HSC70) were basally expressed in seeds and highly expressed in protocorms.ConclusionsOur data suggested that clathrin-mediated endocytosis could play important roles in symbiotic seed germination of G. elata with Mycena infections.

ESTs Analysis of Putative Genes Engaged in Polyporus umbellatus Sclerotial Development

Polyporus umbellatus is one of the most widely used and precious medicinal fungi and the underground sclerotia are known to be with great medicinal value. However, the molecular mechanisms involved in sclerotial development are poorly understood. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of Polyporus umbellatus to identify genes expressing differently between mycelium and sclerotia. In this library, a total of 1202 clones were sequenced, assembled into 222 contigs and 524 singletons which were further searched against the NCBI nonredundant (NR) protein database (E-value cutoff, 10−5). Based on sequence similarity with known proteins, 378 sequences between mycelium and sclerotial were identified and classified into different functional categories through Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs). We have finally identified a majority of differentially expressed genes (constituting 5.6% of the present library) between the two different periods. An expression level of 32 selected expressed sequence tags (ESTs) generated from the above SSH cDNA library was studied through RT-PCR. This study provides the first global overview of genes putatively involved in Polyporus umbellatus sclerotial development and provides a preliminary basis for further functional research in terms of regulated gene expression in sclerotial production.

A novel assay for screening inhibitors targeting HIV-1 integrase dimerization based on Ni-NTA magnetic agarose beads

Human immunodeficiency virus (HIV)-1 integrase (IN), which mediates integration of viral cDNA into the cellular chromosome, is a validated antiviral drug target. Three IN inhibitors, raltegravir, elvitegravir and dolutegravir, have been clinically approved since 2008. However, drug resistance have emerged in infected patients receiving treatment using these drugs which share the same mechanism of action and have a low genetic barrier for resistance. Therefore, there is an urgent need to develop drugs with novel mechanism. IN requires a precise and dynamic equilibrium between several oligomeric species for its activities. The modulation of the process which is termed as IN oligomerization, presents an interesting allosteric target for drug development. In this research, we developed a magnetic beads based approach to assay the IN dimerization. Then, using the assay we screened a library of 1000 Food and Drug Administration (FDA)-approved drugs for IN dimerization inhibitors and identified dexlansoprazole as a potential IN dimerization inhibitor. In conclusion, the assay presented here has been proven to be sensitive and specific for the detection of IN dimerization as well as for the identification of antiviral drugs targeting IN dimerization. Moreover, a FDA-approved proton-pump inhibitors, dexlansoprazole, was identified as a potential inhibitor for IN dimerization.

Diversity Analysis of Polyporus umbellatus in China Using Inter-simple Sequence Repeat (ISSR) Markers.

Polyporus (P.) umbellatus, an endangered medicinal fungus in China, is distributed throughout most areas of the country. Thirty-seven natural P. umbellatus samples collected from 12 provinces in China were subjected to the inter-simple sequence repeat (ISSR) assay to investigate the genetic diversity within and among the 11 natural populations. Nine ISSR primers selected from 100 primers produced 88 discernible DNA bands, with 46 being polymorphic. The frequency of polymorphism varied from 19.57 to 93.48% with an average of 61.26% across all populations. At the population level, the within-population variance was much greater (92.04%) than the between-population variance (7.96%) as revealed by analysis of molecular variance. Eleven P. umbellatus populations were grouped into two major clusters, and the clustering pattern displayed four groups using the unweighted pair-group method with an arithmetic mean dendrogram. Principal coordinate analysis further indicated that the genetic diversity of P. umbellatus strains was unevenly distributed and displayed a clustered distribution pattern instead. Within these clusters, subgrouping (Henan and Hubei) and cluster II (Jilin and Heilongjiang) related to the geographic distribution were evident. The present study provides the first global overview of P. umbellatus diversity analysis in China, which may open up new opportunities in comparative genetic research on this medicinal fungus in other countries.

Cloning and expression of three thaumatin-like protein genes from Polyporus umbellatus

Genes encoding thaumatin-like protein (TLPs) are frequently found in fungal genomes. However, information on TLP genes in Polyporus umbellatus is still limited. In this study, three TLP genes were cloned from P. umbellatus. The full-length coding sequence of PuTLP1, PuTLP2 and PuTLP3 were 768, 759 and 561 bp long, respectively, encoding for 256, 253 and 187 amino acids. Phylogenetic trees showed that P. umbellatus PuTLP1, PuTLP2 and PuTLP3 were clustered with sequences from Gloeophyllum trabeum, Trametes versicolor and Stereum hirsutum, respectively. The expression patterns of the three TLP genes were higher in P. umbellatus with Armillaria mellea infection than in the sclerotia without A. mellea. Furthermore, over-expression of three PuTLPs were carried out in Escherichia coli BL21 (DE3) strain, and high quality proteins were obtained using Ni-NTA resin that can be used for preparation of specific antibodies. These results suggest that PuTLP1, PuTLP2 and PuTLP3 in P. umbellatus may be involved in the defense response to A. mellea infections.

The complete chloroplast genome of Anoectochilus roxburghii.

Abstract We determined the complete chloroplast (cp) genome of Anoectochilus roxburghii, a well-known medicinal orchid. The total genome size was 156,252 bp in length, containing a pair of inverted repeats (IRs) of 26,591 bp, a large single copy (LSC) of 84,665 bp and a small single copy (SSC) of 18,405 bp. The overall GC content of the genome was 37.71%. The cp genome of A. roxburghii contained 87 protein-coding genes, 38 tRNA genes, and eight rRNA genes. Of these 18 genes, one or two contained introns. A maximum parsimony phylogenetic tree revealed that the cp genome of A. roxburghii was closely related to that of the orchid within the Orchidoideae subfamily.

Morphological and Enzymatical Characterization of the Infection Process of Pythium ultimum in Dendrobium officinale (Orchidaceae)

Abstract Observations of the ultrastructural morphology of leaf segments of D. officinale with or without P. ultimum infection were made using light microscopy, scanning electron microscopy and transmission electron microscopy. FITC labelling was used to mark cellulose before and after infection by the pathogenic fungus, and observations were performed with a confocal laser-scanning microscope. The results showed that after infection by P. ultimum, the mycelia pierced the leaf tissue of D. officinale through the stomata of the guard cells gradually and changed into structures resembling penetration pegs under microscopy and SEM observations. The appressorium was observed to adhere to the stoma. Furthermore, the fluorescence intensity of cellulose labelled with FITC was much lower in D. officinale leaves infected by P. ultimum than in healthy leaves. Soft rot disease in D. officinale infection with P. ultimum might be related to direct penetration into the cell walls of the plant and the cellulases secreted by the pathogen, which partly contributed to step-by-step degradation of the cell walls of the host plant.