Salicylic Acid (SA) is an important plant hormone which acts as a therapeutic agent in the plant in response to biotic and abiotic stress. It plays a significant role in growth and development. SABP2, a methyl salicylate esterase is a key player in SA mediated defense signaling. It catalyzes the conversion of mobile methyl salicylate to salicylic acid. During infection, accumulation of salicylic acid in the distal organ in response to the primary infection elsewhere primes the plant to defend against subsequent infection by the mechanism known as Systemic Acquired Resistance (SAR). SIP68, one of the interacting proteins of SABP2 is a glucosyltransferase protein. Glucosyltransferase protein catalyzes the formation of the glycosidic bond by transferring glucose molecule from donor to acceptor molecules. Plant glucosyltransferase is widely distributed in nature playing the dual role of activating and inactivating enzymes. They are also associated with changing the protein stability and solubility of compounds. Since SABP2 has a role in SA mediated defense signaling and glucosyltransferase proteins are associated with physiological function thus, there is a possibility of SIP68 associated with the major or supportive role in either or both functions. The purified recombinant SIP68 protein was tested for glucosyltransferase activity using radioactive method. The purified SIP68 glucosylates various artificially available flavonoid compounds with highest activity detected with Kaempferol (flavonol) followed by quercetin but negligible activity with SA. HPLC based glucosyltransferase assay further verified SIP68 as a flavonoid UDP-glucosyltransferase, not SA glucosyltransferase. Our interest is to further characterize SIP68 and assess its role in plant defense mechanism. Knowing its expression pattern inside plant cell will help us to assess its activity pattern inside the cell. For this enhanced Green Fluorescent Protein (eGFP) tagged SIP68 was transiently expressed inside the plant cell. Confocal microscopy imaging suggests SIP68 likely to be localized in the cytoplasm which will be further confirmed by subcellular fractionation. To assess the role of SIP68 in plant defense mechanism transgenic line expressing altered SIP68 gene was generated using CRISPR Cas9 technique. Verified transgenic line challenged under different biotic and abiotic stress will help us to understand the role of SIP68 in plant defense mechanism. Our research will help us to understand defense mechanism in tobacco model system enabling us to use the knowledge to develop the resistant varieties of crops that are capable of withstanding the adverse condition of pathogenic as well environmental challenges.

Flavonoids are chemically modified by glucosylation, hydroxylation, methylation, etc. During glucosylation, the sugar moiety from UDP-sugar is transferred to aglycone flavonoid substrates by glucosyltransferases (GTs). Grapefruit contains 5 different glucosyltransferases that demonstrate differences in not only substrate but also position specificity. Previous research obtained 3 putative 5’ grapefruit GT clones using SMART RACE RT-PCR with a degenerate gene specific primer based on a highly conserved sequence area in the Plant Secondary Product Glucosyltransferase box. The objective of this research was to use clone specific primers to obtain 3’ clones of the 3 previously mentioned 5’ clones as well as verify putative GT candidacy based on sequence data. Two of the 3 putative GT candidates were designated non-GTs following 3’end sequencing. During pursuit of sequence for the remaining 5’ clone, 1 full-length clone and 1 partial putative GT clone were obtained. To verify GT status, the clones must undergo expression/biochemical characterization.