| Summary: | The circadian clock is an endogenous mechanism that provides a wide variety of organisms with the ability to anticipate daily environmental changes. It was shown that under cyclic and constant light growth conditions Arabidopsis exhibits rhythmicity in Botrytis cinerea resistance, with maximal resistance observed when leaves were inoculated at dawn. Crucially, this mechanism was confirmed to be under circadian clock regulation. To understand how the circadian clock was driving an effective defence response, genes that were more rapidly induced or repressed after inoculation at dawn compared to night were identified. This indicated a complex interaction between the circadian clock and the defence regulatory network. Phytohormone defence signalling, in particular, jasmonate (JA) and ethylene responses, was shown to contribute to the observed rhythmic variation in resistance. This was further confirmed by the identification of a JA signalling mutant (jaz6), which displayed no difference in resistance to B. cinerea following inoculation at dawn or night under cyclic or constant light conditions. Given the central role of JAZ6 in the circadian defence response against B. cinerea, it was likely transcription factors (TFs) bound by JAZ6 were potential links between the plant circadian clock and the defence response. Elucidating the TFs that interacted with JAZ6 revealed JAZ6 to be able to interact with a TF shown to be crucial to the B. cinerea defence response, EIN3. Moreover, JAZ6 was also able to interact with a central regulator of circadian clock, FHY3. Both TF interactors indicate JAZ6 is a linking protein between the circadian clock and the defence response against B. cinerea. To further understand how the circadian clock was mediating the plant defence response against B. cinerea genome-wide chromatin accessibility data was generated using ATAC-Seq. This aimed to enable the comparison of chromatin accessibility as well as TF binding in regions surrounding genes related to B. cinerea defences between the two time-points. This protocol was not optimized for plant tissue. Steps within in! laboratory-based ATAC-Seq protocol were therefore pinpointed for tissue specific optimization. Thus, a protocol specific to ATAC-Seq data analysis was proposed.
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