The RNA interference (RNAi) plays a critical role in gene regulation in a variety of eukaryotic organisms. However, the role of RNAi remains largely unclear in plant pathogenic fungi. In this study, we explored the roles of core components of the RNAi pathway in Fusarium graminearum, the major causal agent of wheat head blight. Our results demonstrated that the hairpin RNA (hpRNA) can efficiently silence the expression level of target gene, and the argonaute protein FgAgo1 and dicer protein FgDicer2 are important in this silencing process. RNAi machinery was not involved in growth, abiotic stress and pathogenesis in F. graminearum under tested conditions. We firstly applied high-throughput sequencing technology to elucidate small RNA (17–40 nucleotides) (sRNA) transcriptome in F. graminearum, and found that a total of forty-nine micro-like-RNA (milRNA) candidates were identified in the wild-type and ∆FgDICER2, and twenty-four of them were FgDicer2-dependent. Fg-milRNA-4 negatively regulated expression of its target gene. Taken together, our results indicated that the hpRNA-induced gene silencing was a valuable genetic tool for exploring gene function in F. graminearum. FgAgo1 and FgDicer2 proteins played a critical role in the hpRNA mediated gene silencing process. In addition, FgDicer2 was involved in sRNA transcription and milRNA generation in this fungus.

No abstract

BackgroundMicroRNAs (miRNAs) represent a class of small ncRNAs that repress gene expression on the post-transcriptional level by the degradation or translation inhibition of target mRNA.MethodologyThree small RNA libraries from oyster haemocytes were sequenced on the Illumina platform to investigate the latent immunomodulation of miRNAs after bacteria challenge and heat stress. Totally, 10,498,663, 8,588,606 and 9,679,663 high-quality reads were obtained in the control, bacteria and bacteria+heat library, respectively, from which 199 oyster miRNAs including 71 known and 128 novel ones were identified. Among these miRNAs, 6 known and 23 novel ones were predicted to possess more than one precursor-coding region, and cgi-miR-10a, cgi-miR-184b, cgi-miR-100, cgi-miR-1984 and cgi-miR-67a were observed to be the most abundant miRNAs in the control library. The expression levels of 22 miRNAs in the bacteria library were significantly higher than those in the control library, while there were another 33 miRNAs whose expression levels were significantly lower than that in the control library. Meanwhile, the expression levels of 65 miRNAs in the bacteria+heat library changed significantly compared to those in the bacteria library. The target genes of these differentially expressed miRNAs were annotated, and they fell in immune and stress-related GO terms including antioxidant, cell killing, death, immune system process, and response to stimulus. Furthermore, there were 42 differentially expressed miRNAs detected in both control/bacteria and bacteria/bacteria+heat comparisons, among which 9 miRNAs displayed the identical pattern in the two comparisons, and the expression alterations of 8 miRNAs were confirmed using quantitative RT-PCR.ConclusionsThese results indicated collectively that immune challenge could induce the expression of immune-related miRNAs, which might modulate the immune response such as redox reaction, phagocytosis and apoptosis, and the expression of some immune-related miRNAs could be also regulated by heat stress to improve the environmental adaption of oyster.

No abstract

BackgroundAs one of the most popular expression systems, recombinant protein expression in Pichia pastoris relies on the AOX1 promoter (PAOX1) which is strongly induced by methanol. However, the toxic and inflammatory nature of methanol restricts its application, especially in edible and medical products. Therefore, constructing a novel methanol-free system becomes necessary. The kinases involved in PAOX1 activation or repression by different carbon sources may be promising targets.ResultsWe identified two kinase mutants: Δgut1 and Δdak, both of which showed strong alcohol oxidase activity under non-methanol carbon sources. Based on these two kinases, we constructed two methanol-free expression systems: Δgut1-HpGCY1-glycerol (PAOX1 induced by glycerol) and Δdak-DHA (PAOX1 induced by DHA). By comparing their GFP expression efficiencies, the latter one showed better potential. To further test the Δdak-DHA system, three more recombinant proteins were expressed as examples. We found that the expression ability of our novel methanol-free Δdak-DHA system was generally better than the constitutive GAP promoter, and reached 50–60 % of the traditional methanol induced system.ConclusionsWe successfully constructed a novel methanol-free expression system Δdak-DHA. This modified expression platform preserved the favorable regulatable nature of PAOX1, providing a potential alternative to the traditional system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0578-4) contains supplementary material, which is available to authorized users.

No abstract

No abstract

DM9 domain was first identified in Drosophila melanogaster, and it was subsequently found to integrate with or without other protein domains across a wide range of invertebrates and vertebrates. In the present study, a member of DM9 domain containing protein (DM9CP) family from marine invertebrate Crassostrea gigas (designated CgDM9CP-1), which was only composed of two DM9 domains, was taken as a protein model to study the biological functions of DM9 domain and its molecular determinants. CgDM9CP-1 was found to exhibit high binding specificity and avidity toward d-mannose residue. It served as a pattern recognition receptor (PRR) with a broad range of recognition spectrum to various pathogen-associated molecular patterns, including lipopolysaccharide, peptidylglycan, mannan, and β-1, 3-glucan in a d-mannose-dependent manner, as well as bacteria and fungi. In order to reveal the molecular mechanism underlying its pattern recognition activity, the crystal structures of wild-type and loss-of-function mutants were solved, and Asp22 and Lys43 were found to be the critical residues for ligand recognition. Moreover, CgDM9CP-1 protein was found to mainly distribute on the surface of C. gigas hemocytes, and it could be translocated into cytoplasm and colocalized with the engulfed microbes during hemocyte phagocytosis. The present result clearly indicated that CgDM9CP-1 was a PRR, and it provided an important clue for the better understanding of DM9CP function.