Tissue-specific expression of Arabidopsis NPR1 gene in rice for sheath blight resistance without compromising phenotypic cost.

Rice sheath blight disease, caused by the fungus Rhizoctonia solani, is considered the second most important disease of rice after blast. NPR1 (non expressor of PR1) is the central regulator of systemic acquired resistance (SAR) conferring broad spectrum resistance to various pathogens. Previous reports have indicated that constitutive expression of the Arabidopsis thaliana NPR1 (AtNPR1) gene results in disease resistance in rice but has a negative impact on growth and agronomic traits. Here, we report that green tissue-specific expression of AtNPR1 in rice confers resistance to the sheath blight pathogen, with no concomitant abnormalities in plant growth and yield parameters. Elevated levels of NPR1 activated the defence pathway in the transgenic plants by inducing expression of endogenous genes such as PR1b, RC24, and PR10A. Enhanced sheath blight resistance of the transgenic plants was evaluated using three different bioassay systems. A partially isolated toxin from R. solani was used in the bioassays to measure the resistance level. Studies of the phenotype and yield showed that the transgenic plants did not exhibit any kind of phenotypic imbalances. Our results demonstrate that green tissue-specific expression of AtNPR1 is an effective strategy for controlling the sheath blight pathogen. The present work in rice can be extended to other crop plants severely damaged by the pathogen.

Antibiotics, arsenate and H2O2 induce the promoter of Staphylococcus aureus cspC gene more strongly than cold.

Proteins expressed by the bacterial cold shock genes are highly conserved at sequence level and perform various biological functions in both the cold-stressed and normal cells. To study the effects of various agents on the cold shock genes of Staphylococcus aureus, we have cloned the upstream region of cspC from S. aureus Newman and found that the above region possesses appreciable promoter (P(c)) activity even at 37 degrees C. A reporter S. aureus strain CHANDA2, constructed by inserting the P(c)-lacZ transcriptional fusion into S. aureus RN4220 genome, was found to express very low level of beta-galactosidase after cold shock, indicating that low temperature induces P(c) very weakly. Interestingly, transcription from P(c ) was induced very strongly by several antibiotics, hydrogen peroxide and arsenate salt. Cold shock proteins expressed by S. aureus are highly identical at sequence level and bear single-strand nucleic acid binding motifs. A 16 nt downstream box and a 13 nt upstream box were identified at the downstream of initiation codon and at the upstream of ribosome binding site of csp transcripts. Their roles in S. aureus cold shock gene expression have been discussed elaborately.

Detection of antistaphylococcal and toxic compounds by biological assay systems developed with a reporter Staphylococcus aureus strain harboring a heat inducible promoter - lacZ transcriptional fusion.

Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cell-wall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region (P(g)) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the P(g)-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that P(g) in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced P(g) efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals.

Purification and characterization of repressor of temperate S. aureus phage phi11.

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O(L) and O(R), respectively. Equilibrium binding studies indicate that His-CI binds to O(R) with a little more strongly than O(L) and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O(L) and O(R) harbor a nearly identical inverted repeat and studies show that phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 CI even nearly resembles to that of lambda, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.