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Calcium-dependent protein kinases (CPKs) can decode and translate intracellular calcium signals to induce plant immunity. Mutation of the exocyst subunit gene EXO70B1 causes autoimmunity that depends on CPK5 and the Toll/interleukin-1 receptor (TIR) domain resistance protein TIR-NBS2 (TN2), where direct interaction with TN2 stabilizes CPK5 kinase activity. However, how the CPK5–TN2 interaction initiates downstream immune responses remains unclear. Here, we show that, besides CPK5 activity, the physical interaction between CPK5 and functional TN2 triggers immune activation in exo70B1 and may represent reciprocal regulation between CPK5 and the TIR domain functions of TN2 in Arabidopsis (Arabidopsis thaliana). Moreover, we detected differential phosphorylation of the calmodulin-binding transcription activator 3 (CAMTA3) in the cpk5 background. CPK5 directly phosphorylates CAMTA3 at S964, contributing to its destabilization. The gain-of-function CAMTA3A855V variant that resists CPK5-induced degradation rescues immunity activated through CPK5 overexpression or exo70B1 mutation. Thus, CPK5-mediated immunity is executed through CAMTA3 repressor degradation via phosphorylation-induced and/or calmodulin-regulated processes. Conversely, autoimmunity in camta3 also partially requires functional CPK5. While the TIR domain activity of TN2 remains to be tested, our study uncovers a TN2–CPK5–CAMTA3 signaling module for exo70B1-mediated autoimmunity, highlighting the direct embedding of a calcium-sensing decoder element within resistance signalosomes.

Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here we developed a genetically encoded FRET (Förster resonance energy transfer)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.

In plant cells, plastids form elongated extensions called stromules, the regulation and purposes of which remain unclear. Here, we quantitatively explore how different stromule structures serve to enhance the ability of a plastid to interact with other organelles: increasing the effective space for interaction and biomolecular exchange between organelles. Interestingly, electron microscopy and confocal imaging showed that the cytoplasm in Arabidopsis thaliana and Nicotiana benthamiana epidermal cells is extremely thin (around 100 nm in regions without organelles), meaning that inter-organelle interactions effectively take place in 2D. We combine these imaging modalities with mathematical modeling and new in planta experiments to demonstrate how different stromule varieties (single or multiple, linear or branching) could be employed to optimize different aspects of inter-organelle interaction capacity in this 2D space. We found that stromule formation and branching provide a proportionally higher benefit to interaction capacity in 2D than in 3D. Additionally, this benefit depends on optimal plastid spacing. We hypothesize that cells can promote the formation of different stromule architectures in the quasi-2D cytoplasm to optimize their interaction interface to meet specific requirements. These results provide new insight into the mechanisms underlying the transition from low to high stromule numbers, the consequences for interaction with smaller organelles, how plastid access and plastid to nucleus signaling are balanced and the impact of plastid density on organelle interaction.

Secreted immune proteases Rcr3 (Required for Cladosporium resistance-3) and Pip1 (Phytophthora- inhibited protease-1) of tomato (Solanum lycopersicum) are both inhibited by Avr2 from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity. Despite substantial divergence in Rcr3 orthologs from eggplant (Solanum melongena) and tobacco (Nicotiana spp.), minimal alterations were sufficient to trigger Avr2/Cf-2-mediated immune signalling. By contrast, tomato Pip1 was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side of the protein next to the substrate-binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues, and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

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Tomato (Solanum lycopersicum) fruit shape is related to microtubule organization and the activity of microtubule-associated proteins (MAPs). However, insights into the mechanism of fruit shape formation from a cell biology perspective remain limited. Analysis of the tissue expression profiles of different microtubule regulators revealed that functionally distinct classes of MAPs, including members of the plant-specific MICROTUBULE-ASSOCIATED PROTEIN 70 (MAP70) and IQ67 DOMAIN (IQD, also named SUN in tomato) families, are differentially expressed during fruit development. SlMAP70-1–3 and SlIQD21a are highly expressed during fruit initiation, which relates to the dramatic microtubule pattern rearrangements throughout this developmental stage of tomato fruits. Transgenic tomato lines overexpressing SlMAP70-1 or SlIQD21a produced elongated fruits with reduced cell circularity and microtubule anisotropy, while their loss-of-function mutants showed the opposite phenotype, harboring flatter fruits. Fruits were further elongated in plants coexpressing both SlMAP70-1 and SlIQD21a. We demonstrated that SlMAP70s and SlIQD21a physically interact and that the elongated fruit phenotype is likely due to microtubule stabilization induced by the SlMAP70–SlIQD21a interaction. Together, our results identify SlMAP70 proteins and SlIQD21a as important regulators of fruit elongation and demonstrate that manipulating microtubule function during early fruit development provides an effective approach to alter fruit shape.

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