Characterization of regulatory genes Plhffp and Plpif1 involved in conidiation regulation in Purpureocillium lavendulum.

Purpureocillium lavendulum is an important biocontrol agent against plant-parasitic nematodes, primarily infecting them with conidia. However, research on the regulatory genes and pathways involved in its conidiation is still limited. In this study, we employed Agrobacterium tumefaciens-mediated genetic transformation to generate 4,870 random T-DNA insertion mutants of P. lavendulum. Among these mutants, 131 strains exhibited abnormal conidiation, and further in-depth investigations were conducted on two strains (designated as #5-197 and #5-119) that showed significantly reduced conidiation. Through whole-genome re-sequencing and genome walking, we identified the T-DNA insertion sites in these strains and determined the corresponding genes affected by the insertions, namely Plhffp and Plpif1. Both genes were knocked out through homologous recombination, and phenotypic analysis revealed a significant difference in conidiation between the knockout strains and the wild-type strain (ku80). Upon complementation of the ΔPlpif1 strain with the corresponding wildtype allele, conidiation was restored to a level comparable to ku80, providing further evidence of the involvement of this gene in conidiation regulation in P. lavendulum. The knockout of Plhffp or Plpif1 reduced the antioxidant capacity of P. lavendulum, and the absence of Plhffp also resulted in decreased resistance to SDS, suggesting that this gene may be involved in the integrity of the cell wall. RT-qPCR showed that knockout of Plhffp or Plpif1 altered expression levels of several known genes associated with conidiation. Additionally, the analysis of nematode infection assays with Caenorhabditis elegans indicated that the knockout of Plhffp and Plpif1 indirectly reduced the pathogenicity of P. lavendulum towards the nematodes. The results demonstrate that Agrobacterium tumefaciens - mediated T-DNA insertion mutagenesis, gene knockout, and complementation can be highly effective for identifying functionally important genes in P. lavendulum.

Mycolicibacterium arseniciresistens sp. nov., isolated from lead-zinc mine tailing, and reclassification of two Mycobacterium species as Mycolicibacterium palauense comb. nov. and Mycolicibacterium grossiae comb. nov.

A Gram-positive, acid-fast, aerobic, rapidly growing and non-motile strain was isolated from lead-zinc mine tailing sampled in Lanping, Yunnan province, Southwest China. 16S rRNA gene sequence analysis showed that the most closely related species of strain KC 300T was Mycolicibacterium litorale CGMCC 4.5724T (98.47 %). Additionally, phylogenomic and specific conserved signature indel analysis revealed that strain KC 300T should be a member of genus Mycolicibacterium, and Mycobacterium palauense CECT 8779T and Mycobacterium grossiae DSM 104744T should also members of genus Mycolicibacterium. The genome size of strain KC 300T was 6.2 Mb with an in silico DNA G+C content of 69.2 mol%. Chemotaxonomic characteristics of strain KC 300T were also consistent with the genus Mycolicibacterium. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values, as well as phenotypic, physiological and biochemical characteristics, support that strain KC 300T represents a new species within the genus Mycolicibacterium, for which the name Mycolicibacterium arseniciresistens sp. nov. is proposed, with the type strain KC 300T (=CGMCC 1.19494T=JCM 35915T). In addition, we reclassified Mycobacterium palauense and Mycobacterium grossiae as Mycolicibacterium palauense comb. nov. and Mycolicibacterium grossiae comb. nov., respectively.

Secondary metabolites from the Actinomadura sp. and their cytotoxic activity.

Actinomadura sp., which is usually found in muddy habitats, produces various secondary metabolites with biological activities. In this study, five new compounds named formosensin A (1), formosensin B (2), oxanthroquinone-3-O-α-d-mannose (8), oxanthromicin A (9), and oxanthromicin B (10) were isolated from the culture of Actinomadura sp. together with five known compounds (3-7). Their structures were elucidated by extensive spectroscopic methods including NMR and MS. In particular, the absolute configurations of compounds 1 and 2 were determined using computational methods. Moreover, compounds 1-2 and 8-10 were screened for cytotoxic activity using a panel of human tumor cell lines. Compound 9 induced significant cytotoxicity in five human tumor cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW480) with IC50 values of 8.7, 17.5, 15.0, 17.8, and 14.6 µM, respectively. These findings suggested that compound 9 could provide therapeutic benefits in the treatment of tumor-related diseases.

Chryseobacterium luquanense sp. nov., a casein-hydrolysing bacterium from the Jiaozi Mountain in Yunnan, PR China.

Strain KC 927T was isolated during an investigation of the soil bacteria diversity on Jiaozi Mountain, central Yunnan, Southwest China. The strain was Gram-stain-negative, rod-shaped, non-motile, oxidase-negative, catalase-positive and aerobic. Results of 16S rRNA gene alignment and phylogenetic analysis indicated that strain KC 927T was a member of the genus Chryseobacterium and closely related to Chryseobacterium caseinilyticum GCR10T (98.4%), Chryseobacterium piscicola DSM 21068T (98.3 %) and 'Chryseobacterium formosus' CCTCC AB 2015118T (97.9 %). With a genome size of 4 348 708 bp, strain KC 927T had 33.5 mol% DNA G+C content and contained 4012 protein-coding genes and 77 RNA genes. The average nucleotide identity and digital DNA-DNA hybridization values between strain KC 927T and C. caseinilyticum GCR10T, C. piscicola DSM 21068T and 'C. formosus' CCTCC AB 2015118T were 80.1, 79.6 and 90.7 %, and 25.5, 23.6 and 42.0 %, respectively. The main polar lipid of strain KC 927T was phosphatidylethanolamine and the respiratory quinone was MK-6. The major fatty acids (≥10 %) were iso-C15 : 0, iso-C17 : 1 ω9c and iso-C17 : 0 3-OH. Evidence from phenotypic, phylogenetic and chemotaxonomic analyses support that strain KC 927T represents a new species of the genus Chryseobacterium, for which the name Chryseobacterium luquanense sp. nov. is proposed. The type strain is KC 927T (=CGMCC 1.18760T=JCM 35707T).

Chromosome-level Dinobdella ferox genome provided a molecular model for its specific parasitism.

BACKGROUND: Dinobdella ferox is the most frequently reported leech species parasitizing the mammalian nasal cavity. However, the molecular mechanism of this special parasitic behavior has remained largely unknown. METHODS: PacBio long-read sequencing, next-generation sequencing (NGS), and Hi-C sequencing were employed in this study to generate a novel genome of D. ferox, which was annotated with strong certainty using bioinformatics methods. The phylogenetic and genomic alterations of D. ferox were then studied extensively alongside the genomes of other closely related species. The obligatory parasitism mechanism of D. ferox was investigated using RNA-seq and proteomics data. RESULTS: PacBio long-read sequencing and NGS yielded an assembly of 228 Mb and contig N50 of 2.16 Mb. Along Hi-C sequencing, 96% of the sequences were anchored to nine linkage groups and a high-quality chromosome-level genome was generated. The completed genome included 19,242 protein-coding genes. For elucidating the molecular mechanism of nasal parasitism, transcriptome data were acquired from the digestive tract and front/rear ends of D. ferox. Examining secretory proteins in D. ferox saliva helped to identify intimate connections between these proteins and membrane proteins in nasal epithelial cells. These interacting proteins played important roles in extracellular matrix (ECM)-receptor interaction, tight junction, focal adhesion, and adherens junction. The interaction between D. ferox and mammalian nasal epithelial cells included three major steps of pattern recognition, mucin connection and breakdown, and repair of ECM. The remodeling of ECM between epithelial cells of the nasal mucosa and epithelial cells of D. ferox may produce a stable adhesion environment for parasitism. CONCLUSIONS: Our study represents the first-ever attempt to propose a molecular model for specific parasitism. This molecular model may serve as a practical reference for parasitism models of other species and a theoretical foundation for a molecular process of parasitism.

Ethanol mediates the interaction between Caenorhabditis elegans and the nematophagous fungus Purpureocillium lavendulum.

Accurately recognizing pathogens by the host is vital for initiating appropriate immune response against infecting microorganisms. Caenorhabditis elegans has no known receptor to recognize pathogen-associated molecular pattern. However, recent studies showed that nematodes have a strong specificity for transcriptomes infected by different pathogens, indicating that they can identify different pathogenic microorganisms. However, the mechanism(s) for such specificity remains largely unknown. In this study, we showed that the nematophagous fungus Purpureocillium lavendulum can infect the intestinal tract of the nematode C. elegans and the infection led to the accumulation of reactive oxygen species (ROS) in the infected intestinal tract, which suppressed fungal growth. Co-transcriptional analysis revealed that fungal genes related to anaerobic respiration and ethanol production were up-regulated during infection. Meanwhile, the ethanol dehydrogenase Sodh-1 in C. elegans was also up-regulated. Together, these results suggested that the infecting fungi encounter hypoxia stress in the nematode gut and that ethanol may play a role in the host-pathogen interaction. Ethanol production in vitro during fungal cultivation in hypoxia conditions was confirmed by gas chromatography-mass spectrometry. Direct treatment of C. elegans with ethanol elevated the sodh-1 expression and ROS accumulation while repressing a series of immunity genes that were also repressed during fungal infection. Mutation of sodh-1 in C. elegans blocked ROS accumulation and increased the nematode's susceptibility to fungal infection. Our study revealed a new recognition and antifungal mechanism in C. elegans. The novel mechanism of ethanol-mediated interaction between the fungus and nematode provides new insights into fungal pathogenesis and for developing alternative biocontrol of pathogenic nematodes by nematophagous fungi. IMPORTANCE Nematodes are among the most abundant animals on our planet. Many of them are parasites in animals and plants and cause human and animal health problems as well as agricultural losses. Studying the interaction of nematodes and their microbial pathogens is of great importance for the biocontrol of animal and plant parasitic nematodes. In this study, we found that the model nematode Caenorhabditis elegans can recognize its fungal pathogen, the nematophagous fungus Purpureocillium lavendulum, through fungal-produced ethanol. Then the nematode elevated the reactive oxygen species production in the gut to inhibit fungal growth in an ethanol dehydrogenase-dependent manner. With this mechanism, novel biocontrol strategies may be developed targeting the ethanol receptor or metabolic pathway of nematodes. Meanwhile, as a volatile organic compound, ethanol should be taken seriously as a vector molecule in the microbial-host interaction in nature.

Characterization of the complete mitochondrial genome of the nematode-trapping fungus Drechslerella dactyloides.

The complete mitochondrial genome of Drechslerella dactyloides was characterized in this study. This mitogenome is a closed circular molecule of 246860 bp in length with a GC content of 26.16%, including 87 predicted protein-coding genes, 29 transfer RNA genes, and two rRNA gens. Phylogenetic analyses based on concatenated amino acid sequences at 14 conserved mitochondrial protein-coding genes showed that D. dactyloides was closely related to Dactylellina haptotyla.

Streptothiazolidine B, a new cytotoxic alkaloid produced by Streptomyces violaceoruber.

A new cytotoxic alkaloid, named streptothiazolidine B (1), together with three known compounds (2-4), were isolated from Streptomyces violaceoruber. The structure of the undescribed compound was established using 1D and 2D NMR, and HRESIMS. Streptothiazolidine B was isolated and identified as an amide alkaloid with a unique thiazolidine side chain and its absolute configuration was determined by a combination of NOESY experiment and ECD analysis. Streptothiazolidine B exhibited significant cytotoxic activities against two human tumor cell lines, Li-7 and A2780, with IC50 values of 7.8, and 9.1 µM. Meanwhile, compound 4 showed obvious cytotoxic activities against four human tumor cell lines, THP-1, HT29, Li-7 and A2780, with IC50 values ranging from 3.1 to 10.2 µM.

Streptomyces plumbidurans sp. nov., a Pb2+-tolerant actinomycete.

A novel actinobacterium, designated KC 17012T, was isolated from lead zinc tailings collected from Lanping, Yunnan, PR China. Comparative 16S rRNA gene sequencing showed that KC 17012T belonged to the genus Streptomyces and was most closely related to the type strains of Streptomyces neyagawaensis (98.34%), Streptomyces panaciradicis (98.34%) and Streptomyces heilongjiangensis (98.27%). Phylogenetic tree analysis revealed strain KC 17012T formed a distinct clade. The genome size was 8.64 Mbp with a DNA G+C content of 70.8%. Digital DNA-DNA hybridization and average nucleotide identity values between the genome sequence of strain KC 17012T and those of S. neyagawaensis JCM 4796T (25.3 and 81.5 %) and S. panaciradicis NBRC 109811T (30.1 and 85.7 %) were below the thresholds of 70 and 96% for prokaryotic conspecific assignation. The strain formed long straight aerial hyphae which generated regular short rod spores with spiny surfaces. Growth occurred at 10-45 °C, pH 6-8 and with 0-9 % NaCl (w/v). Strain KC 17012T contained ll-diaminopimelic acid and the major whole-cell hydrolysates included glucose, mannose and ribose. The menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The major cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, one unidentified lipid and one unidentified phospholipid. On the basis of the results of a polyphasic taxonomic study, it is concluded that KC 17012T represents a novel species of the genus Streptomyces, for which the name Streptomyces plumbidurans sp. nov., is proposed. The type strain is KC 17012T (CGMCC 4.7704T=JCM 35204T).

Siccirubricoccus soli sp. nov., a novel bacterial species isolated from Jiaozi Mountain soil.

An isolate of Gram-stain-negative and strictly aerobic bacterium, designated KC 17139T, was isolated from Jiaozi Mountain sample in Yunnan, China. Cells were non-motile cocci to oval, catalase-positive and oxidase-positive. Growth occurred at 0-7% NaCl (w/v; optimum, 0%), pH 6.0-8.0 (optimum, pH 7.0) and 15-45 °C (optimum, 28-37 °C). The polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC) and four unidentified aminolipids (UALs). Strain KC 17139T contained summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c), C18:1 2OH and C16:0 as major cellular fatty acids (> 5%) and ubiquinone-10 as the sole isoprenoid quinone. The 16S rRNA gene sequence analysis indicated that strain KC 17139T shared highest similarities with Siccirubricoccus phaeus 1-3T (96.7%) and Siccirubricoccus deserti SYSU D8009T (95.0%). Strain KC 17139T clustered with the two Siccirubricoccus type strains, but formed a separate branch in both 16S rRNA gene and genome-scale phylogenetic dendrograms. The genomic DNA G + C content of strain KC 17139T was 71.2%. Genomic comparisons between strain KC 17139T and its close relatives showed the highest digital DNA-DNA hybridisation to S. phaeus (35.5%), highest average nucleotide identity to S. phaeus (88.2%), indicating that strain KC 17139T represents a novel species. On the basis of results of phenotypic, chemotaxonomic and molecular analysis, we report a new bacterium strain KC 17139T belonged to genus Siccirubricoccus, for which the name Siccirubricoccus soli sp. nov. is proposed. The type strain is KC 17139T (= CGMCC 1.18756T = JCM 35132T).

Insights into gut microbiota communities of Poecilobdella manillensis, a prevalent Asian medicinal leech.

AIMS: Medicinal leeches (Annelida: Hirudinea) are fresh water ectoparasitic species which have been applied as traditional therapy. However, gut microbiota could bring high risks of opportunistic infections after leeching and arouses great interests. Here, gut bacterial and fungal communities of an Asian prevalent leech Poecilobdella manillensis were characterized and analysed through culture-independent sequencing. METHODS AND RESULTS: With high coverage in 18 samples (>0.999), a more complicated community was apparent after comparing with previous leech studies. A total of 779/939 OTUs of bacteria and fungi were detected from leech guts. The bacterial community was dominated by the phylum Bacteroidetes and Synergistetes. Genera Mucinivorans and Fretibacterium accounted mostly at the genus level, and genus Aeromonas showed an extremely low abundance (2.02%) on average. The fungal community was dominated by the phylum Ascomycota and Basidiomycota. At the genus level, the dominant OTUs included Mortierella, Geminibasidium and Fusarium. The analysis of core taxa included those above dominant genera and some low-abundance genera (>1%). The functional annotation of the bacterial community showed a close correlation with metabolism (34.8 ± 0.6%). Some fungal species were predicted as opportunistic human pathogens including Fusarium and Chaetomiaceae. CONCLUSIONS: The present study provides fundamental rationales for further studies of such issues as bacteria-fungi-host interactions, host fitness, potential pathogens, and infecting risks after leeching. It shall facilitate in-depth explorations on the safe utilization of leech therapy. SIGNIFICANCE AND IMPACT OF STUDY: Present paper is the first-ever exploration on microbiota of a prevalent Asian medicinal leech based on culture-independent technical. And it is also the first report of gut fungi community of medicinal leech. The diversity and composition of bacteria in P. manillensis was far different from that of the European leech. The main components and core OTUs indicate a particular gut environment of medicinal leech. Unknown bacterial and fungal species were also recovered from leech gut.

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus.

Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.

Cryo-EM structure of mouse TRPML2 in lipid nanodiscs.

In mammalians, transient receptor potential mucolipin ion channels (TRPMLs) exhibit variable permeability to cations such as Ca2+, Fe2+, Zn2+, and Na+ and can be activated by the phosphoinositide PI(3,5)P2 in the endolysosomal system. Loss or dysfunction of TRPMLs has been implicated in lysosomal storage disorders, infectious diseases, and metabolic diseases. TRPML2 has recently been identified as a mechanosensitive and hypotonicity-sensitive channel in endolysosomal organelles, which distinguishes it from TRPML1 and TRPML3. However, the molecular and gating mechanism of TRPML2 remains elusive. Here, we present the cryo-EM structure of the full-length mouse TRPML2 in lipid nanodiscs at 3.14 Å resolution. The TRPML2 homotetramer structure at pH 7.4 in the apo state reveals an inactive conformation and some unique features of the extracytosolic/luminal domain and voltage sensor-like domain that have implications for the ion-conducting pathway. This structure enables new comparisons between the different subgroups of TRPML channels with available structures and provides structural insights into the conservation and diversity of TRPML channels. These comparisons have broad implications for understanding a variety of molecular mechanisms of TRPMLs in different pH conditions, including with and without bound agonists and antagonists.

Characterization of the complete mitochondrial genome of the nematophagous fungus Purpureocillium lavendulum.

The complete mitochondrial genome of Purpureocillium lavendulum was characterized in this study. This mitogenome is a closed circular molecule of 23,567 bp in length with a GC content of 28.46%, including 15 protein-coding genes, 25 transfer RNA genes, 2 ribosomal RNA genes. Phylogenetic analyses based on sequences at the 14 concatenated mitochondrial protein-coding genes showed that P. lavendulum was closely related to Hirsutella minnesotensis.

Class IV Lasso Peptides Synergistically Induce Proliferation of Cancer Cells and Sensitize Them to Doxorubicin.

Heterologous expression of a biosynthesis gene cluster from Amycolatopsis sp. resulted in the discovery of two unique class IV lasso peptides, felipeptins A1 and A2. A mixture of felipeptins stimulated proliferation of cancer cells, while having no such effect on the normal cells. Detailed investigation revealed, that pre-treatment of cancer cells with a mixture of felipeptins resulted in downregulation of the tumor suppressor Rb, making the cancer cells to proliferate faster. Pre-treatment with felipeptins made cancer cells considerably more sensitive to the anticancer agent doxorubicin and re-sensitized doxorubicin-resistant cells to this drug. Structural characterization and binding experiments showed an interaction between felipeptins resulting in complex formation, which explains their synergistic effect. This discovery may open an alternative avenue in cancer treatment, helping to eliminate quiescent cells that often lead to cancer relapse.

Functional Characterization of Core Regulatory Genes Involved in Sporulation of the Nematophagous Fungus Purpureocillium lavendulum.

The nematophagous fungus Purpureocillium lavendulum is a natural enemy of plant-parasitic nematodes, which cause severe economic losses in agriculture worldwide. The production of asexual spores (conidia) in P. lavendulum is crucial for its biocontrol activity against nematodes. In this study, we characterized the core regulatory genes involved in conidiation of P. lavendulum at the molecular level. The central regulatory pathway is composed of three genes, P. lavendulumbrlA (PlbrlA), PlabaA, and PlwetA, which regulate the early, middle, and late stages of asexual development, respectively. The deletion of PlbrlA completely inhibited conidiation, with only conidiophore stalks produced. PlAbaA determines the differentiation of conidia from phialides. The deletion of PlwetA affected many phenotypes related to conidial maturation, including abscission of conidia from conidium strings, thickening of the cell wall layers, vacuole generation inside the cytoplasm, production of trehalose, tolerance to heat shock, etc. Comparative analyses showed that the upstream regulators of the core regulatory pathway of conidiation, especially the "fluffy" genes, were different from those in Aspergillus Besides their roles in conidiation, the central regulators also influence the production of secondary metabolites, such as the leucinostatins, in P. lavendulum Our study revealed a set of essential genes controlling conidiation in P. lavendulum and provided a framework for further molecular genetic studies on fungus-nematode interactions and for the biocontrol of plant-parasitic nematodes.IMPORTANCE Plant-parasitic nematodes cause serious damage to crops throughout the world. Purpureocillium lavendulum is a nematophagous fungus which is a natural enemy of nematodes and a potential biocontrol agent against plant-parasitic nematodes. The conidia play an important role during infection of nematodes. In this study, we identified and characterized genes involved in regulating asexual development of P. lavendulum We found that these genes not only regulate conidiation but also influence secondary-metabolite production. This work provides a basis for future studies of fungus-nematode interactions and nematode biocontrol.

Characterization of cold stress responses in different rapeseed ecotypes based on metabolomics and transcriptomics analyses.

The winter oilseed ecotype is more tolerant to low temperature than the spring ecotype. Transcriptome and metabolome analyses of leaf samples of five spring Brassica napus L. (B. napus) ecotype lines and five winter B. napus ecotype lines treated at 4 °C and 28 °C were performed. A total of 25,460 differentially expressed genes (DEGs) of the spring oilseed ecotype and 28,512 DEGs of the winter oilseed ecotype were identified after cold stress; there were 41 differentially expressed metabolites (DEMs) in the spring and 47 in the winter oilseed ecotypes. Moreover, more than 46.2% DEGs were commonly detected in both ecotypes, and the extent of the changes were much more pronounced in the winter than spring ecotype. By contrast, only six DEMs were detected in both the spring and winter oilseed ecotypes. Eighty-one DEMs mainly belonged to primary metabolites, including amino acids, organic acids and sugars. The large number of specific genes and metabolites emphasizes the complex regulatory mechanisms involved in the cold stress response in oilseed rape. Furthermore, these data suggest that lipid, ABA, secondary metabolism, signal transduction and transcription factors may play distinct roles in the spring and winter ecotypes in response to cold stress. Differences in gene expression and metabolite levels after cold stress treatment may have contributed to the cold tolerance of the different oilseed ecotypes.

Genome-wide exploration and characterization of miR172/euAP2 genes in Brassica napus L. for likely role in flower organ development.

BACKGROUND: APETALA2-like genes encode plant-specific transcription factors, some of which possess one microRNA172 (miR172) binding site. The miR172 and its target euAP2 genes are involved in the process of phase transformation and flower organ development in many plants. However, the roles of miR172 and its target AP2 genes remain largely unknown in Brassica napus (B. napus). RESULTS: In this study, 19 euAP2 and four miR172 genes were identified in the B. napus genome. A sequence analysis suggested that 17 euAP2 genes were targeted by Bna-miR172 in the 3' coding region. EuAP2s were classified into five major groups in B.napus. This classification was consistent with the exon-intron structure and motif organization. An analysis of the nonsynonymous and synonymous substitution rates revealed that the euAP2 genes had gone through purifying selection. Whole genome duplication (WGD) or segmental duplication events played a major role in the expansion of the euAP2 gene family. A cis-regulatory element (CRE) analysis suggested that the euAP2s were involved in the response to light, hormones, stress, and developmental processes including circadian control, endosperm and meristem expression. Expression analysis of the miR172-targeted euAP2s in nine different tissues showed diverse spatiotemporal expression patterns. Most euAP2 genes were highly expressed in the floral organs, suggesting their specific functions in flower development. BnaAP2-1, BnaAP2-5 and BnaTOE1-2 had higher expression levels in late-flowering material than early-flowering material based on RNA-seq and qRT-PCR, indicating that they may act as floral suppressors. CONCLUSIONS: Overall, analyses of the evolution, structure, tissue specificity and expression of the euAP2 genes were peformed in B.napus. Based on the RNA-seq and experimental data, euAP2 may be involved in flower development. Three euAP2 genes (BnaAP2-1, BnaAP2-5 and BnaTOE1-2) might be regarded as floral suppressors. The results of this study provide insights for further functional characterization of the miR172 /euAP2 module in B.napus.

An efficient gene disruption system for the nematophagous fungus Purpureocillium lavendulum.

The fungus Purpureocillium lavendulum (formally Paecilomyces lilacinus) is a natural enemy of insects and plant-parasitic nematodes, and has been used as an important bio-control agent against agricultural pests all over the world. In order to understand the genetic mechanisms governing its biocontrol efficiency and other biological processes, an effective gene disruption system is needed. Here we report the development of an efficient system which integrates selective markers that differ from Purpureocillium lilacinum, a one-step construction method for gene knockout plasmids, and a ku80 knockout strain for efficient homologous recombination. With this system, we effectively disrupted the transcription factors in the central regulation pathway of sporulation and a serine protease which were contributed to nematode infection, demonstrating this system as an efficient gene disrupting system for further characterization of genes involved in the development and pathogenesis of this fungus.

Actinorectispora metalli sp. nov., a novel actinomycete isolated from a mine and emended description of the genus Actinorectispora.

A novel actinomycete, designated strain KC 198T, was isolated from rare earth mine. The results of analysis of the 16S rRNA gene sequence indicated that KC 198T was most closely related to Actinorectisporaindica YIM 75728T (98.4 %). Aerial hyphae differentiated into long, straight chains of cylindrical spores. Growth was observed at 10-45 °C (optimum 28 °C), with 0-10 % (w/v) NaCl (optimum, in the absence of NaCl) and at pH 6.0-8.0 (optimum pH 7.0). KC 198T possessed MK-9(H4) as the predominant respiratory quinone and a minor amount of MK-10(H4). Polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. Three unidentified lipids were also detected. The main cellular fatty acids were iso-C16 : 0 (30.9 %), iso-C16 : 1H (22.9 %) and iso-C15 : 0 (14.8 %). The genomic DNA G+C content was 66.8 mol%. On the basis of the phenotypic and genotypic characteristics, we propose that strain KC 198T represents a novel species of the genus Actinorectispora. The name Actinorectispora metalli sp. nov. is, therefore, proposed for the novel species with the type strain KC 198T (=CCTCC AA 2015043T=KCTC 39718T). The description of the genus Actinorectispora has also been emended.