|BHATTA, BED - University Of Arkansas|
|PONNIAH, SATHISH - University Of Arkansas|
|MUTHUSAMY, MANOHARAN - University Of Arkansas|
Submitted to: Plant and Animal Genome
Publication Type: Abstract Only
Publication Acceptance Date: 1/13/2018
Publication Date: 1/15/2018
Citation: Bhatta, B.P., Ponniah, S.K., Jia, Y., Muthusamy, M. 2018. CRISPR/Cas9-Mediated Gene Editing in Rice (Oryza sativa L. japonica cv. Katy) for Stable Resistance against Blast Fungus (Magnaporthe oryzae). Plant and Animal Genome, San Diego, California, January 13-17, 2018.
Technical Abstract: Rice blast is a recurring and devastating disease in the USA and worldwide. In the USA, the blast-resistance (R) genes found in a tropical japonica cultivar, Katy, reduce blast damages from 1990 to present. The cultivar is still used as a principal donor of blast R genes in developing numerous elite US resistant cultivars. The objective of this research is to identify the role of R genes in blast resistance of Katy using CRISPR/Cas9 technology targeting a "nucleotide binding site and leucine rich repeat" containing gene in rice (LOC_Os12g18374) at the Pi-ta/Pita2/Ptr region. Katy with the Pi-ta/Pita2/Ptr gene cluster is resistant to rice blast isolates that contain the corresponding avirulence gene AVR-Pita except for a virulent strain IE-1k that does not contain AVR-Pita. Two vectors containing single guide RNA (S1) encoding Alanine protein as well as combination of three gRNAs (S2) encoding Asparagine, Serine and Alanine protein were used to induce double-strand breaks at the intended target sites in Katy. Using Agrobacterium-mediated transformation, a total of 24 independent events were generated in each of the S1 and S2 transformations. Genomic DNA from putative mutant T0 plants was extracted and analyzed via PCR using M13/Ubiquitin, Cas9, and hygromycin primers. Results indicated that 100% of the T0 plants were positive for presence of M13/Ubiquitin, Cas9 and hygromycin. The genomic DNA from T0 mutant plants was further sequenced using gene-specific primer derived from upstream of the target locus. A series of indels and substitutions were observed in and around the gRNA sequence. T1 plants will be grown to identify the null segregants, and scored after blast infection and progress will be reported.