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Query: UNIPROT:P06889 (Mol)
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In order to compare transcription profiles in cultivars of Malus domestica that are differentially sensitive to apple scab (Venturia inaequalis), two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) method. Subtraction hybridization was performed between cDNAs from uninfected young leaves of the resistant cultivar Remo and the susceptible Elstar. In total, 480 EST clones were obtained: 218 (ELSTAR) clones represent transcripts that are preferentially expressed in Elstar, while the other 262 (REMO) are derived from RNAs that are more highly expressed in Remo. The putative functions of about 50% of the cloned sequences could be identified by sequencing and subsequent homology searches in databases or by dot-blot hybridization to known targets. In the resistant cv. Remo the levels of transcripts encoding a number of proteins related to plant defense (such as beta-1,3-glucanase, ribonuclease-like PR10, cysteine protease inhibitor, endochitinase, ferrochelatase, and ADP-ribosylation factor) or detoxification of reactive oxygen species (such as superoxide dismutase) were highly up-regulated relative to the amounts present in cv. Elstar. Most surprising was the large number of clones derived from mRNAs for metallothioneins of type 3 (91 out of 262) found in the REMO population. The corresponding transcripts were only present in small amounts in young uninfected leaves of the cv. Elstar, but were up-regulated in the susceptible cultivar after inoculation with V. inaequalis. These results indicate that constitutively high-level expression of PR proteins may protect cv. Remo from infection by different plant pathogens.
Mol Genet Genomics 2005 Jun
PMID:Characterization by suppression subtractive hybridization of transcripts that are differentially expressed in leaves of apple scab-resistant and susceptible cultivars of Malus domestica. 1581 49

Cell separation in Schizosaccharomyces pombe is achieved by the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ringlike structure that surrounds the septum. The requirements for the correct localization of both hydrolases as a ring were analyzed using green fluorescent protein fusion proteins. Targeting to the septum required a functional exocyst, because both proteins failed to localize correctly in sec8-1 or exo70delta mutants, suggesting that Agn1 and Eng1 might be two of the cargo proteins present in the vesicles that accumulate in exocyst mutants. Septins and Mid2 were also required for correct formation of a ring. In their absence, Eng1 and Agn1 were found in a disk-like structure that spanned the septum, rather than in a ring. Even though septin and mid2delta mutants have a cell separation defect, the septum and the distribution of linear beta-1,3-glucans were normal in these cells, suggesting that mislocalization of Eng1 and Agn1 might be the reason underlying the failure to separate efficiently. Thus, one of the functions of the septin ring would be to act as a positional marker for the localization of hydrolytic proteins to the medial region.
Mol Biol Cell 2005 Oct
PMID:Role of septins and the exocyst complex in the function of hydrolytic enzymes responsible for fission yeast cell separation. 1607 82

Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and beta-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported.
Mol Genet Genomics 2006 Feb
PMID:Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1. 1634 85

The retaining endo-1,3-beta-D-glucanase (LV) with molecular mass of 36 kDa was purified to homogeneity from the crystalline styles of scallop Mizuhopecten yessoensis. The purified enzyme catalyzed hydrolysis of laminaran as endo-enzyme forming glucose, laminaribiose and higher oligosaccharides as products (Km approximately 600 microg/mL). The 1,3-beta-D-glucanase effectively catalyzed transglycosylation reaction that is typical of endo-enzymes too. Optima of pH and temperature were at 4.5 and 45 degrees C, respectively. cDNA encoding the endo-1,3-beta-D-glucanase was cloned by PCR-based methods. It contained an open reading frame that encoded 339-amino acids protein. The predicted endo-1,3-beta-D-glucanase amino acid sequence included a characteristic domain of the glycosyl hydrolases family 16 and revealed closest homology with 1,3-beta-D-glucanases from bivalve Pseudocardium sachalinensis, sea urchin Strongylocentrotus purpuratus and invertebrates lipopolysaccharide and beta-1,3-glucan-binding proteins. The fold of the LV was more closely related to kappa-carrageenase, agarase and 1,3;1,4-beta-D-glucanase from glycosyl hydrolases family 16. Homology model of the endo-1,3-beta-D-glucanase from M. yessoensis was obtained with MOE on the base of the crystal structure of kappa-carrageenase from P. carrageonovora as template. Putative three-dimensional structures of the LV complexes with substrate laminarihexaose or glucanase inhibitor halistanol sulfate showed that the binding sites of the halistanol sulfate and laminarihexaose are located in the enzyme catalytic site and overlapped.
Comp Biochem Physiol B Biochem Mol Biol 2006 Apr
PMID:Purification, cDNA cloning and homology modeling of endo-1,3-beta-D-glucanase from scallop Mizuhopecten yessoensis. 1647 36

To gain a better understanding on the function of the potato Solanum tuberosum Multiprotein Bridging Factor 1 protein (StMBF1) its interaction with the TATA box binding protein (TBP) was demonstrated. In addition we reported that StMBF1 rescues the yeast mbf1 mutant phenotype, indicating its role as a plant co-activator. These data reinforce the hypothesis that MBF1 function is also conserved among non closely related plant species. In addition, measurement of StMBF1 protein level by Western blot using anti-StMBF1 antibodies indicated that the protein level increased upon H(2)O(2) and heat shock treatments. However, the potato beta-1,3-glucanase protein level was not changed under the same experimental conditions. These data indicate that StMBF1 participates in the cell stress response against oxidative stress allowing us to suggest that MBF1 genes from different plant groups may share similar functions.
J Biochem Mol Biol 2006 Jul 31
PMID:The potato transcriptional co-activator StMBF1 is up-regulated in response to oxidative stress and interacts with the TATA-box binding protein. 1688 77

The modes of action of the antagonistic yeast Pichia anomala (strain K) have been studied; however, thus far, there has been no clear demonstration of the involvement of exo-beta-1,3-glucanase in determining the level of protection against Botrytis cinerea afforded by this biocontrol agent on apple. In the present study, the exo-beta-1,3-glucanase-encoding genes PAEXG1 and PAEXG2, previously sequenced from the strain K genome, were separately and sequentially disrupted. Transfer of the URA3-Blaster technique to strain K, allowing multiple use of URA3 marker gene, first was validated by efficient inactivation of the PaTRP1 gene and recovery of a double auxotrophic strain (uracil and tryptophan). The PAEXG1 and PAEXG2 genes then were inactivated separately and sequentially with the unique URA3 marker gene. The resulting mutant strains showed a significantly reduced efficiency of biocontrol of B. cinerea when applied to wounded apple fruit, the calculated protection level dropping from 71% (parental strain) to 8% (mutated strain) under some experimental conditions. This suggests that exo-beta-1,3-glucanases play a role in the biological control of B. cinerea on apple. Furthermore, biological control experiments carried out in this study underline the complexity of the host-antagonist-pathogen interaction. Two experimental parameters (yeast inoculum concentration and physiological stage of the fruit) were found to influence dramatically the protection level. Results also suggest that, under some conditions, the contribution of exo-beta-1,3-glucanase to biological control may be masked by other modes of action, such as competition.
Mol Plant Microbe Interact 2007 Apr
PMID:Separate and combined disruptions of two exo-beta-1,3-glucanase genes decrease the efficiency of Pichia anomala (strain K) biocontrol against Botrytis cinerea on apple. 1742 7

Mitogen-activated protein kinases (MAPKs) constitute one of the most critical signaling components in plants. A typical example is wound-induced protein kinase (WIPK), which functions during pathogen responses in tobacco plants (Nicotiana tabacum). Searching for direct down-stream components, we previously isolated a novel transcription factor, which was activated upon phosphorylation by WIPK and designated as N. tabacum WIPK-interacting factor (NtWIF). Overexpression of NtWIF in tobacco plants enhanced the hypersensitive response (HR) upon tobacco mosaic virus infection and cryptogein treatment, while its silencing by RNAi suppressed such HR. NtWIF contains a specific motif similar to the B3 DNA binding domain, which recognizes the core TGTCTC motif called the auxin-responsive element (ARE). Using synthetic ARE sequences, NtWIF was also shown to recognize the ARE motifs and to transactivate the Luciferase (Luc)-reporter gene driven by such AREs in tobacco BY2 cultured cells. Subsequent microarray screening of NtWIF overexpressing tobacco identified 49 stress-responsive genes, and in silico analyses of available promoter regions of these genes revealed beta-1,3-glucanase, ACS2, P-450, and WIPK itself to contain the ARE core motif consisted of either TGTCTC or TGTCCT. Gel shift assay showed NtWIF to efficiently bind to both sequences. Assays with 1.5-kb PR-Q and 1.2 kb WIPK promoter regions, each fused to the Luc-reporter gene, indicated NtWIF to exhibit a clear transactivation activity, which was increased up to 3-fold upon phosphorylation by WIPK. These results revealed that NtWIF directly regulates multiple stress-responsive genes containing the ARE motif in their promoters, thereby partly filling up the last step of the MAPK cascade.
Plant Mol Biol 2007 Dec
PMID:Transactivation of wound-responsive genes containing the core sequence of the auxin-responsive element by a wound-induced protein kinase-activated transcription factor in tobacco plants. 1792 10

To better understand the nature of resistance of tomato to the whitefly (Bemisia tabaci, B biotype)-transmitted Tomato yellow leaf curl virus (TYLCV), whiteflies and TYLCV were considered as particular cases of biotic stresses and virus resistance as a particular case of successful response to these stresses. Two inbred tomato lines issued from the same breeding program that used Solanum habrochaites as a TYLCV resistance source, one susceptible and the other resistant, were used to compare the expression of key proteins involved at different stages of the plant response with stresses: mitogen-activated protein kinases (MAPKs), cellular heat shock proteins (HSPs, proteases), and pathogenesis-related (PR) proteins. The two biotic stresses-non-viruliferous whitefly feeding and virus infection with viruliferous insects--led to a slow decline in abundance of MAPKs, HSPs, and chloroplast protease FtsH (but not chloroplast protease ClpC), and induced the activities of the PR proteins, beta-1,3-glucanase, and peroxidase. This decline was less pronounced in virus-resistant than in virus-susceptible lines. Contrary to whitefly infestation and virus infection, inoculation with the fungus Sclerotinia sclerotiorum induced a rapid accumulation of the stress proteins studied, followed by a decline; the virus-susceptible and -resistant tomato lines behaved similarly in response to the fungus.
Mol Plant Microbe Interact 2007 Nov
PMID:Expression of stress-response proteins upon whitefly-mediated inoculation of Tomato yellow leaf curl virus in susceptible and resistant tomato plants. 1797 49

Cell separation in Schizosaccharomyces pombe is achieved through the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ring-like structure that surrounds the septum. Correct localization of these hydrolases requires the presence of both the septins and the exocyst. In this work, we show that the glucanase Eng1 contains a region at the C-terminus that acts as a carbohydrate-binding module (CBM) and that it is not present in other members of glycoside hydrolases family 81 (GH81). In vitro, the purified CBM has affinity for beta-1,3-glucan chains with a minimum degree of polymerization of 30 glucose units. Deletion of the CBM results in a protein that is largely defective in complementing the separation defect of eng1Delta mutants. This defect is due to a reduction in the catalytic activity against insoluble substrates and to a defect in targeting of Eng1 to the septum, as the truncated protein localizes to the lateral cell wall of the cell. Thus, the targeting of Eng1 to the primary septum requires not only trans-factors (septins and the exocyst complex) but also a cis-element localized to the C-terminus of the protein.
Mol Microbiol 2008 Jul
PMID:The Schizosaccharomyces pombe endo-1,3-beta-glucanase Eng1 contains a novel carbohydrate binding module required for septum localization. 1846 95

To achieve a high level of purity in the purification of recombinant proteins for therapeutic or analytical application, it is necessary to use several chromatographic steps. There is a range of techniques available including anion and cation exchange, which can be carried out at different pHs, hydrophobic interaction chromatography, gel filtration and affinity chromatography. In the case of a complex mixture of partially unknown proteins or a clarified cell extract, there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98%, 99.5% or 99.9%). This review shows how an initial 'proteomic' characterization of the complex mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a specific level of purity with a minimum number of steps. The chosen methodology was implemented in a computer- based Expert System. Two algorithms were developed, the first algorithm was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants and the second algorithm was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The application of the Expert System approach was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with a supernatant of Bacillus subtilis producing a recombinant beta-1,3-glucanase. Once the type of chromatography is chosen, optimization of the operating conditions is essential. Chromatographic elution curves for a three-protein mixture (alpha-lactoalbumin, ovalbumin and beta-lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. In order to optimize operating conditions, a cost function was built that included the effect of the different production stages, namely fermentation, purification and concentration. This cost function was also successfully used for the determination of the fraction of product to be collected (peak cutting) in chromatography. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters.
J Mol Recognit
PMID:Protein purification using chromatography: selection of type, modelling and optimization of operating conditions. 1854 92


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