AbstractDuring the immune response there is a significant increase in the number of protrusions of the chloroplast stroma known as stromules. These stromules were reported over 100 years ago; however, their function has remained elusive. Recently, it was shown that chloroplast stromules are induced during the plant innate immune response and serve as positive regulators of the immune response. Toward this, we wanted to identify genetic components required for stromule formation. Using a candidate approach, we found a kinesin containing a calponin homology domain, Kinesin required for Inducing Stromules 1 (KIS1) which induces constitutive stromule formation when overexpressed and is required for N-TNL mediated stromule formation and immunity to Tobacco mosaic virus (TMV). Further, we used pathogen effectors as a probe to identify proteins which are required for stromule formation and subsequent perinuclear chloroplast clustering during the N-mediated immune response. Using TurboID-based proximity labeling we identified four proteins which interact with the Type 3 secretion system (T3SS) effector SH9 and are required for immunity to TMV. Toward this, we showed that one SH9 interactor, Sh9 interactor 1 is required for perinuclear chloroplast clustering during the N-mediated immune response. Our results with KIS1 represent the first known component required for stromule formation. Further, our initial characterization of Sh9 interactor 1 suggests that perinuclear chloroplast clustering is required for immunity to TMV and is specifically inhibited by the effector Sh9.