In Silico Designing of Xa13 Locus Specific TALENs for Introducing Bacterial Blight Resistance in Rice

Bacterial blight disease in rice is caused by Xanthomonas oryzae pv. oryzae (XOO) through binding of type III secretion system effectors to susceptibility genes in host. PthXo1 effectors secreted by XOO binds to promoter region of sugar transporter gene Os8N3 thereby activating dominant allele of Xa13 susceptibility gene in rice. Introgression of recessive xa13 allele by molecular breeding programs have successfully imparted resistance against PthXo1 effector mediated disease in few rice cultivars. However, molecular breeding mediated introgression of disease resistance in hundreds of susceptible cultivars is a daunting task. Recent advancements in the field of genome editing technology by use of engineered nucleases ZFN, TALEN and CRISPR-Cas9 system allow fast and précised modifications in the genome. Therefore, the present study focus on designing of Xa13 locus specific TALENs for introducing bacterial blight resistance in Indica rice through TALEN mediated genome editing. Total three hundred thirtythree TALENs were designed against promoter region of Xa13. Out of these, One-hundred thirtynine pairs of TALENs that follow Streubel’s guidelines and having targets with >40% GC content were retained. Further, Screening of these selected TALENs on basis of distance of their putative cleavage site from PthXo1 effectors binding site in rice genome resulted into eleven TALEN pairs. These eleven TALEN pairs were further screened for their number of putative targets in host genome and on the TAL score. Finally only three pairs possess a unique target site and score below a cut-off of four. Best scoring TALEN pair was selected for designing of TALEN coding gene sequences codon-optimized for high level expression in rice. These TALEN coding genes can be used for introducing deletions in Xa13 promoter and impart resistance against bacterial blight disease in rice.