IQD1 Function in Defense Responses

The main objective of the proposed research was to study IQD1's mechanism of action and elucidate its role in plant protection. Preliminary experiments suggest that IQD1 binds CaM in a Ca²⁺-dependent manner and functions in general defense responses. We propose to identify proteins and genes that interact with IQD1, which may provide some clues to its mechanism of action. We also plan to dissect IQD1's integration in defense pathways and to study and modulate its binding affinity to CaM in order to enhance crop resistance. Our specific objectives were: (1) Analysis of IQD1's CaM-binding properties; (2) Identification of IQD1 targets;(3) Dissection of IQD1 integration into defense signaling pathways. Analysis of IQD1's CaM-binding properties defined four potential classes of sequences that should affect CaM binding: one is predicted to raise the affinity for Ca²⁺-dependent interaction but have no effect on Ca²⁺-independent binding; a second is predicted to act like the first mutation but eliminate Ca²⁺-independent binding; a third has no predicted effect on Ca²⁺-dependent binding but eliminates Ca²⁺-independent binding; and the fourth is predicted to eliminate or greatly reduce both Ca²⁺-dependent and Ca²⁺-independent binding. Following yeast two hybrid analysis we found that IQD1 interact with AtSR1 (Arabidopsis thalianaSIGNALRESPONSIVE1), a calcium/calmodulin-binding transcription factor, which has been shown to play an important role in biotic and abiotic stresses. We tested IQD1 interaction with both N-terminal or C-terminal half of SR1. These studies have uncovered that only the N-terminal half of the SR1 interacts with the IQD1. Since IQD1 has an important role in herbivory, its interaction with SR1 suggests that it might also be involved in plant responses to insect herbivory. Since AtSR1, like IQD1, is a calmodulin-binding protein and the mutant showed increased sensitivity to a herbivore, we analyzed WT, Atsr1 and the complemented line for the levels of GS to determine if the increased susceptibility of Atsr1 plants to T. ni feeding is associated with altered GS content. In general, Atsr1 showed a significant reduction in both aliphatic and aromatic GS levels as compared to WT. In order to study IQD1's molecular basis integration into hormone-signaling pathways we tested the epistatic relationships between IQD1 and hormone-signaling mutants. For that purpose we construct double mutants between IQD1ᴼXᴾ and mutants defective in plant-hormone signaling and GS accumulation. Epitasis with SA mutant NahG and npr1-1 and JA mutant jar1-1 suggested IQD1 function is dependent on both JA and SA as indicated by B. cinerea infection assays. We also verified the glucosinolate content in the crosses siblings and found that aliphatic GSL content is reduced in the double transgenic plants NahG:IQD1ᴼXᴾ as compare to parental lines while the aliphatic GSL content in the npr1-1:IQD1ᴼXᴾ and jar1-1: IQD1ᴼXᴾ double mutants was intimidated to the parental lines. This suggests that GSL content dependency on SA is downstream to IQD1. As a whole, this project should contribute to the development of new defense strategies that will improve crop protection and reduce yield losses and the amount of pesticides required; these will genuinely benefit farmers, consumers and the environment.