Priya Prabhakar

The effect of mutation on proteins of blast resistance gene was analysed by detecting and comparing the natural variations (SNPs and InDels) present in 30 alleles of Pi-ta gene in Oryza spp. O. sativa indica. Three-dimensional structure of Pi-ta proteins were predicted through I-TASSER software. 3D refine web server (http://sysbio.rnet.missouri.edu/3Drefine/) was used to bring the initial model closer to the native structure. The stereo chemical quality of the model was checked using the ‘Protein Quality Assessment’ parameter option of Discovery Studio. Quantitative structural evaluations were performed to find out the stability and disease response of Pi-ta alleles upon mutation. Average number of nucleotide difference between two sequences was found lower in Oryza indica in comparison with the nucleotide difference surveyed from O. rufipogon. InDel diversity per site (Πi =0.00061) was found lower than nucleotide diversity per site (Π =0.00255) in the selected accessions of Pi-ta gene. More number of synonymous substitutions were observed in non-coding region. Susceptible indica cultivar C101A51 showed maximum number of InDels events in the gene. Most of the changes in secondary structure elements were observed in NBS-LRR region. More number of coils were found in LRR region in comparison to helix. The predicted in silico protein structure of the Pi-ta alleles was observed to be affected by mutation i.e. SNPs and InDels. Quantitative assessments of these structures showed differences in value of ASA, SFE and number of H-bonds. Normal mode analysis of modelled proteins showed differences in region of maximum atomic displacements between different alleles of Pi-ta gene. A total of 101 accessions of AvrPita proteins were found in NCBI from which 12 dominant effectors Avr-pita proteins and 8 recessive Avr-pita proteins were selected from 19 isolates of M.oryzae strain. All these sequences were not 100% similar. Value of SFE and number of H-bonds were less in recessive Avr proteins in comparison to dominant effector proteins. An approximate degree of flexibility for proteins was determined by secondary structure elements present in the structure. BAK40873.1 and BAK40874.1 contain more number of coils as compared to other structures which can lead to more degree of flexibility than others. CCD21951.1 contains least number of coils than others, so it is most rigid structure. In normal mode analysis, region of maximum atomic displacements was found different in recessive and dominant effector proteins. In another experiment, direct interaction between proteins of Pi-ta alleles and their corresponding Avr-pita was observed by two methods. First method is interaction in gene-for-gene manner and second is in silico mutagenesis and interaction of polymorphic sites in the LRR region of R proteins. Maximum BE was observed in complex of BAF91347.1 and BAK40873.1. It also contained maximum number of hydrogen-bonds. Least BE was observed between docking complexes of Pita proteins from susceptible Oryza cultivars and recessive Avr proteins. Mutation analysis of the polymorphic sites in the LRR region showed that mutability of these sites is different. The mean of the binding free energy change distribution over 19 mutations was found negative for 8 out of 11 sites. A very less correlation was found between mutations that increase stability and mutations that favour complex formation.