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Screening for Novel Mutants involved in Abscisic Acid Signaling and Effector Triggered Immunity Signaling, and CRISPR/Cas9 Mediated Double Mutant Generation of Tandem Genes VICTR and VICTL1 in Arabidopsis thaliana

  • Author(s): Garin, Vince Anthony
  • Advisor(s): Schroeder, Julian
  • et al.

Abscisic acid (ABA) is an important phytohormone whose major functions include regulation of seed germination, stomatal movement, and other plant responses to biotic and abiotic stress. Recent research has revealed that ABA signaling exhibits cross-talk with effector triggered immunity, a pathway in pathogen defense signaling. However, the exact mechanisms of this crosstalk remain unknown. Previously, our lab discovered that a new small molecule DFPM ([5-(3,4-dichlorophenyl)furan-2-yl]- piperidine-1-ylmethanethione) induces ETI-linked signaling and inhibits ABA signaling, providing us with a method to screen for genes involved in the crosstalk between ETI signaling and ABA signaling. Using this screening method, the novel mutant rda3 (resistant to dfpm induced aba inhibition 3) was isolated here. rda3 exhibits resistance to DFPM induced ABA signaling inhibition in the leaves of Arabidopsis thaliana. Its genetic locus is yet to be determined. In addition, the lab previously identified another gene involved in DFPM/ABA crosstalk called VICTR, (VARATION IN COMPOUND TRIGGERED ROOT), which codes for a Nucleotide Binding – Leucine Rich Repeat (NB-LRR) protein involved in early ETI signaling. It has a highly homologous tandem gene VICTL1, and since NB-LRR genes are often functionally redundant, generating a victr victl1 double mutant will allow us to more accurately characterize both genes. The close proximity of VICTR and VICTL1 makes a higher order mutant by cross-pollination inefficient, so we used CRISPR/Cas9 to mutate VICTR in victl1 mutant plants. Although we isolated plants harboring victr-like phenotypes, eliminating the CRISPR/Cas9 construct from the mutant plants and creating stable mutations remain a challenge.

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