Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity

<p>Abstract</p> <p>Background</p> <p>Lysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor.</p> <p>Results</p> <p>Using highly purified repressor (CI) of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD) and a C-terminal domain (CTD) which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25°C. Both CTD and CI contain significant amount of α-helix at 30°C but unfold partly at 42°C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to <it>O</it>₆₄and <it>O</it>_{<it>L </it>}types of asymmetric operators of L1 with variable affinity at 25°C. Interestingly, repressor – operator interaction is affected drastically at 42°C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42°C.</p> <p>Conclusion</p> <p>Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of λ and related phages at functional level but at structural level they are nearly similar.</p

Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight

Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, PD54O–544. Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice

Rice oxalate oxidasegene driven by green tissue-specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue-specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence-related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue-specific manner for sheath blight resistance

A point mutation at the C-terminal half of the repressor of temperate mycobacteriophage L1 affects its binding to the operator DNA

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to 42° C. While 40-95% operator-binding activity was shown to be retained at 35 to 42° C in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to 38° C, although the latter showed only 10% less binding compared to that of the former at 32° C. The CIts391 showed almost no binding at 42° C. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and 42° C. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at 32° C. Interestingly, the repressoroperator complexes preformed at 0oC have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to 32° C after preincubation at 42 to 52° C. All these data suggest that the 131st proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end

Chymotrypsin and trypsin digested His-CI fragments were analyzed by Tris-Tricine SDS-16

<p><b>Copyright information:</b></p><p>Taken from "Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity"</p><p>http://www.virologyj.com/content/4/1/64</p><p>Virology Journal 2007;4():64-64.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1934351.</p><p></p>5% PAGE followed by silver staining. Molecular masses (in kDa) of marker proteins are shown at the left side of gel. 'Chy' and 'Try' indicate chymotrypsin and trypsin, respectively whereas, a – h indicate intact repressor, different digested fragments of repressor, respectively. N-terminal ends of fragments c and h were sequenced. Western blotting analysis of chymotrypsin/trypsin digested His-CI fragments from 2 and 30 mins incubations by a standard procedure as indicated in Materials and method. Summary of proteolysis. The putative domains of CI and its amino acid residues involved in formation of hinge, NTD and CTD are indicated

Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity-2

<p><b>Copyright information:</b></p><p>Taken from "Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity"</p><p>http://www.virologyj.com/content/4/1/64</p><p>Virology Journal 2007;4():64-64.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1934351.</p><p></p>as calibrated with BSA (66 kDa, I), ovalbumin (46 kDa, II), carbonic anhydrase (29 kDa, III), and lysozyme (14.4 kDa, IV). Molecular weights were plotted against /, where and denote elution volume and void volume respectively. Void volume of column was determined from elution of blue dextran. Glutaraldehyde (GCHO) cross-linking. Nearly 0.5 μM His-CI or CTD was cross-linked with 0.1% GCHO and samples were analyzed by SDS-10% PAGE. Protein bands were visualized by silver staining. Horizontal arrows denote dimeric His-CI and CTD species

Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity-3

<p><b>Copyright information:</b></p><p>Taken from "Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity"</p><p>http://www.virologyj.com/content/4/1/64</p><p>Virology Journal 2007;4():64-64.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1934351.</p><p></p>estimated from inset gel shift assay pictures) versus CI concentration (0.05 – 0.45 μM and 0.1 – 0.8 μM CI with and , respectively) are shown. Nearly 0.1 nM labeled operator was used in each reaction. Plot of %operator bound versus time shows the kinetics of CI dissociation from and operators in presences of excess cold operator. The amount of operator bound in the shifted complex of the zero time aliquot was considered as 100%. Plot of log Keq versus 1/T shows equilibrium binding of CI to operator at temperatures ranging from 25° – 42°C. All curves/lines are best-fit curves/lines