Duni Chand

The single functional blast resistance gene Pi54 activates a complex defence mechanism in rice

The Pi54 gene (Pi-k(h)) confers a high degree of resistance to diverse strains of the fungus Magnaporthe oryzae. In order to understand the genome-wide co-expression of genes in the transgenic rice plant Taipei 309 (TP) containing the Pi54 gene, microarray analysis was performed at 72 h post-inoculation of the M. oryzae strain PLP-1. A total of 1154 differentially expressing genes were identified in TP-Pi54 plants. Of these, 587 were up-regulated, whereas 567 genes were found to be down-regulated. 107 genes were found that were exclusively up-regulated and 58 genes that were down- regulated in the case of TP-Pi54. Various defence response genes, such as callose, laccase, PAL, and peroxidase, and genes related to transcription factors like NAC6, Dof zinc finger, MAD box, bZIP, and WRKY were found to be up-regulated in the transgenic line. The enzymatic activities of six plant defence response enzymes, such as peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, β-glucosidase, β-1,3-glucanase, and chitinase, were found to be significantly high in TP-Pi54 at different stages of inoculation by M. oryzae. The total phenol content also increased significantly in resistant transgenic plants after pathogen inoculation. This study suggests the activation of defence response and transcription factor-related genes and a higher expression of key enzymes involved in the defence response pathway in the rice line TP-Pi54, thus leading to incompatible host-pathogen interaction.

Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae

Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo–devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion–deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pinon/syn was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes.

An improved bioprocess for synthesis of acetohydroxamic acid using DTT (dithiothreitol) treated resting cells of Bacillus sp. APB-6.

Acyltransferase activity of amidase from Bacillus sp. APB-6 was enhanced (24 U) by multiple feedings of N-methylacetamide (70 mM) into the production medium. Hyperinduced whole resting cells of Bacillus sp. APB-6 corresponding to 4 g/L (dry cell weight), when treated with 10mM DTT (dithiothreitol) resulted in 93% molar conversion of acetamide (300 mM) to acetohydroxamic acid in presence of hydroxylamine-HCl (800 mM) after 30 min at 45°C in a 1L reaction mixture. After lyophilization, a 62 g powder containing 34% (wtwt(-1)) acetohydroxamic acid was recovered. This is the first report where DTT has been used to enhance acyltransfer reaction and such high molar conversion (%) of amide to hydroxamates was recorded at 1L scale.

Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature.

Rice blast resistant gene, Pi54 cloned from rice line, Tetep, is effective against diverse isolates of Magnaporthe oryzae. In this study, we prospected the allelic variants of the dominant blast resistance gene from a set of 92 rice lines to determine the nucleotide diversity, pattern of its molecular evolution, phylogenetic relationships and evolutionary dynamics, and to develop allele specific markers. High quality sequences were generated for homologs of Pi54 gene. Using comparative sequence analysis, InDels of variable sizes in all the alleles were observed. Profiling of the selected sites of SNP (Single Nucleotide Polymorphism) and amino acids (N sites ≥ 10) exhibited constant frequency distribution of mutational and substitutional sites between the resistance and susceptible rice lines, respectively. A total of 50 new haplotypes based on the nucleotide polymorphism was also identified. A unique haplotype (H_3) was found to be linked to all the resistant alleles isolated from indica rice lines. Unique leucine zipper and tyrosine sulfation sites were identified in the predicted Pi54 proteins. Selection signals were observed in entire coding sequence of resistance alleles, as compared to LRR domains for susceptible alleles. This is a maiden report of extensive variability of Pi54 alleles in different landraces and cultivated varieties, possibly, attributing broad-spectrum resistance to Magnaporthe oryzae. The sequence variation in two consensus region: 163 and 144 bp were used for the development of allele specific DNA markers. Validated markers can be used for the selection and identification of better allele(s) and their introgression in commercial rice cultivars employing marker assisted selection.

Positive selection pressure on rice blast resistance allele Piz-t makes it divergent in Indian land races

Abstract Allelic variants of the broad-spectrum blast resistance gene, Piz-t, have been analyzed in 48 rice lines selected after phenotyping across three blast hot-spot regions of India. Single Nucleotide Polymorhisms in the form of transitions were more frequent than the transversions in the alleles. On the basis of nucleotide polymorphism, 46 haplotypes have been identified, with major haplotypes forming three main haplogroups. The Piz-t alleles formed mostly region-specific clusters. Resistant and susceptible Piz-t alleles were grouped into separate sub-clusters. The value of Tajimas D was negative indicating positive selection on Piz-t locus. Sequence variations were more abundant in the leucine rich repeats (LRR) region than in the NB-ARC (nucleotide-binding adaptor shared by APAF-1,R proteins, and CED-4) region, indicating that the LRR region has played a more important role in the evolution of this allele. The detailed molecular analysis of the Piz-t locus provide insights to high degree of inter-and intra-specific relationship among the Indian land races of rice which will help in the selection of better alleles for future rice breeding programs.

Thermostable Fe/Mn superoxide dismutase from Bacillus licheniformis SPB-13 from thermal springs of Himalayan region: Purification, characterization and antioxidative potential

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that scavenges free radicals and increases the longevity. In this study, a thermostable superoxide dismutase (SOD) from Bacillus licheniformis SPB-13, from Himalayan region was purified to homogeneity using ion exchange chromatography (DEAE-Sepharose). The SDS and native PAGE analysis showed that SOD is composed of two subunits of 32 kDa each and total molecular mass of the enzyme was estimated as 68 kDa. The specific activity of enzyme was 3965.51 U/mg and was purified to 16.17 folds. The SOD showed maximum activity with 60 mM Tris-HCl buffer at pH 8.0 for 2 min of incubation. Enzyme along with FeCl3 as metal ion remained active till 70 °C. After reaction variables optimization, enzyme activity increased from 3965.51 to 4015.72 U/mg. Kinetic analysis of SOD showed km of 1.4 mM of NADH and Vmax of 10000 U/mg of protein. Turnover number (kcat) and catalytic efficiency (kcat/Km) were found to be 11,333 s-1 and 7092.2 s-1·mM-1 NADH. The activation energy (Ea) was calculated as 2.67 kJ·mol-1. After typing, it was found to be a member of Fe/Mn SOD family with IC50 value of 25 μg/ml, prevented the cell death at a concentration of 30 μg/ml and it increased the cell viability by 30%.

pUC-IVT, a modified pUC19 based in vitro transcription vector

The popular cloning vector, pUC19, was modified into an in vitro transcription system, pUC-IVT, by incorporation of T7 promoter sequence and appropriate restriction sites for cloning of gene of interest and generation of transcript. Strategy was also devised to eliminate incorporation of undesirable sequences at both 5′ and 3′ ends of the transcript for its use in many sensitive downstream applications. All the major restriction sites, except SphI and PstI present in the MCS of pUC19, were retained in pUC-IVT for facile cloning. Any DNA fragment having 5′ blunt end, and cohesive 3′ end, generated by restriction enzymes present in MCS of pUC19, except SphI and PstI, can be directionally cloned into pUC-IVT backbone generated by restricting with StuI and the same restriction enzyme used for 3′-end generation of target DNA fragment. EcoRI digested modified gene coding for archaebacterial (Synechocystis sp.) precursor tRNA-glutamine (ptRNAGln), harbouring recognition site for a type IIS restriction enzyme, FokI, was ligated onto the StuI and EcoRI digested pUC-IVT to generate pUC-IVT::ptRNAGln. In vitro run-off transcription was performed using FokI digested linearized pUC-IVT::ptRNAGln plasmid DNA as template to get ptRNAGln transcript. Reconstituted Escherichia coli ribonuclease P (RNase P) efficiently cleaved 5′ leader sequence of ptRNAGln to generate mature tRNAGln.