The binding of native biglycan and decorin to pepsinextracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcore TM 2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K D ) of ϳ30 nM. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the ␣2(VI) chain probably plays a role in the interaction.Collagens are a large family of extracellular structural proteins made up of three ␣ chains that are intracellularly associated and folded into specific structures including characteristic triple helical domains (1). The major class, recognized as the fibril-forming collagens, contains molecules with one large uninterrupted triple helical domain (for review, see Refs. 1 and 2). Other members of the collagen family have one or more non-triple helical domains, which may constitute the major part of the protein. Most of these collagens do not form prominent lateral aggregates in a manner similar to that of the fibril-forming collagens. Instead, they form complex aggregates together with other matrix macromolecules. Collagen VI is one example, forming multimolecular filamentous beaded structures after secretion from the cell (for review, see Ref. 3). This collagen is composed of three different peptide chains (␣1(VI), ␣2(VI), and ␣3(VI)), which form the basic unit consisting of a relatively short triple helical domain flanked by two large multidomain globular regions (4). These are composed primarily of repeating units of von Willebrand type A domains (5). Collagen VI assembles intracellularly into antiparallel, overlapping dimers that then align and form tetramers (6). These structures are stabilized by disulfide bonds. Secreted tetramers assemble extracellularly in a characteristic end-to-end fashion into thin (3-10 nm) beaded filaments with a periodicity of about 100 nm (7-9). Further supramolecular assembly includes lateral associations of the beaded filaments into microfibrils (8, 9).Collagen VI is ubiquitous. It can be found intermingled with fibril-forming collagens and is often enriched in the pericellular matrix (for review, see Refs. 3 and 10). Decreased amounts of secreted collagen VI resulting from mutations in COL6A1 have been shown in Bethlem myopathy (11, 12), a dominantly inherited disorder characterized by progressive muscle weakness and wasting....
Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusarium graminearum. Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichia coli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.
Oat (Avena sativa) is susceptible to Fusarium head blight (FHB). The quality of oat grain is threatened by the accumulation of mycotoxins, particularly the trichothecene deoxynivalenol (DON), which also acts as a virulence factor for the main pathogen Fusarium graminearum. The plant can defend itself, e.g., by DON detoxification by UGT-glycosyltransferases (UTGs) and accumulation of PR-proteins, even though these mechanisms do not deliver effective levels of resistance. We studied the ability of the fungal biocontrol agent (BCA) Clonostachys rosea to reduce FHB and mycotoxin accumulation. Greenhouse trials showed that C. rosea-inoculation of oat spikelets at anthesis 3 days prior to F. graminearum inoculation reduced both the amount of Fusarium DNA (79%) and DON level (80%) in mature oat kernels substantially. DON applied to C. rosea-treated spikelets resulted in higher conversion of DON to DON-3-Glc than in mock treated plants. Moreover, there was a significant enhancement of expression of two oat UGT-glycosyltransferase genes in C. rosea-treated oat. In addition, C. rosea treatment activated expression of genes encoding four PR-proteins and a WRKY23-like transcription factor, suggesting that C. rosea may induce resistance in oat. Thus, C. rosea IK726 has strong potential to be used as a BCA against FHB in oat as it inhibits F. graminearum infection effectively, whilst detoxifying DON mycotoxin rapidly.
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