The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8S- ITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4?-acetoxy-12,13- epoxy-?(9)-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 ?g mL(-1). Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 ?g mL(-1). However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 ?g mL(-1)). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin.
The proneural genes are fundamental regulators of neuronal development in all metazoans. A critical role of the fly proneural factor Atonal (Ato(Dm)) is to induce photoreceptor neuron formation in Drosophila, whereas its murine homolog, Atonal7(Mm) (aka Ath5) is essential for the development of the ganglion cells of the vertebrate eye. Here, we identify the Bombyx mori ato homolog (ato(Bm) ). In a pattern strikingly reminiscent of ato(Dm), the ato(Bm) mRNA is expressed as a stripe in the silkworm eye disc. Its DNA-binding and protein-protein interaction domain is highly homologous to the Ato(Dm) bHLH. Targeted expression of Ato(Bm) in the endogenous ato(Dm) pattern rescues the eyeless phenotype of the fly ato(1) mutant and its ectopic expression induces similar gain-of-function phenotypes as Ato(Dm). Rescue experiments with chimeric proteins show that the non-bHLH portion of Ato(Bm) (N-region) can effectively substitute for the corresponding region of the fly transcription factor, even though no apparent conservation can be found at the amino acid level. On the contrary, the highly similar bHLH domain of Ato(Bm) cannot similarly substitute for the corresponding region of Ato(Dm). Thus, the bHLH(Bm) domain requires the Ato(Bm) N-region to function effectively, whereas the bHLH(Dm) domain can operate well with either N-region. These findings suggest a role for the non-bHLH portion of Ato proteins in modulating the function of the bHLH domain in eye neurogenesis and implicate specific aa residues of the bHLH in this process.
Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation.
VP37 protein of Broad bean wilt virus 2 (BBWV-2) is a multifunctional protein that binds single-strand nucleic acids, interacts with viral coat protein (CP) and potentiates the virus cell-to-cell movement in its host plant. In this study, tubule-like structures filled with virus-like particles were observed by Electron Microscopy in plasmodesmata in walls of Chenopodium quinoa leaf cells infected with BBWV-2. Immunogold labeling using VP37 protein specific antibody demonstrates that the VP37 is a component of the tubular structures. When VP37 was fused with the green fluorescent protein (VP37-GFP) and expressed in BY-2 protoplasts or in insect Tn cells, green fluorescent tubules of various lengths were produced, protruding from the surface of the expressing cells. These findings suggest that the movement of BBWV-2 between cells is mediated by the tubular structures that contain the VP37 protein, and the VP37 protein itself is capable of inducing these tubule-like structures in cells. Our results also suggest that the plant and insect cell factors involved in the tubule formation have conserved features.
Kinesins are microtubule-based motors involved in various intracellular transports. Neurons, flagellated cells, and pigment cells have been traditionally used as model systems to study the cellular functions of kinesins. Here, we report silkworm posterior silkgland (PSG), specialized cells with an extensive endomembrane system for intracellular transport and efficient secretion of fibroin, as a novel model for kinesin study. To investigate kinesin-driven intracellular transport in PSG cells, we cloned five silkworm kinesin-like proteins (KLPs), BmKinesin-1, BmKinesin-6, BmKinesin-7, BmKinesin-13, and BmKinesin-14A. We determined their expression patterns by relative real-time PCR and western blotting. Immunofluorescence microscopy verified their colocalization with microtubules. By combining pull-down assays, LC-MS/MS, and western blotting analysis, we identified many potential cargoes of BmKinesin-1 in PSG, including fibroin-containing granules and exuperantia-associated ribonucleoprotein (RNP) complexes. Moreover, BmKinesin-13 overexpression disrupted the microtubule network in BmN cells, which is consistent with a role of Kinesin-13 in regulating microtubule dynamics in other organisms. On the basis of these results, we concluded that PSG might have advantages in elucidating mechanisms of intracellular transport in secretory tissues and could serve as a potential model for kinesin studies.
To develop a method based on immunoreactions for detection of Ectropis obliqua Nucleopolyhedrovirus (EoNPV), the polyhedra of the virus were purified and used to immunize the mouse BALB/c. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. A hybridoma cell line which can stably secrete the monoclonal antibody against EoNPV was achieved by using indirect ELISA screening and cloning methods, and was named as 7D3. Meanwhile, the polyhedrin gene was cloned from EoNPV and expressed in E. coli. Western blotting analysis showed that the monoclonal antibody prepared from 7D3 could specifically react with the recombinant polyhedrin. An indirect ELISA method based on this monoclonal antibody for detecting EoNPV in infected tea looper was developed.
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