Publications - Stress and Develop Biology

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.

The HD-ZIP class I transcription factor, HvHOX1 (Homeobox 1) or VRS1 (Vulgare Row-type Spike 1 or Six-rowed Spike 1), regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic function of HvHOX1 and HvHOX2 during spikelet development is still fragmentary. Here, we show that compared to HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of these genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley.

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.

In nature plants are constantly challenged by simultaneous abiotic and biotic stresses, and under conflicting stress scenarios prioritization of stress responses is required for plant survival. Calcium-dependent protein kinase CPK5 is a central hub in local and distal immune signaling, required upstream of hormone salicylic acid (SA)-dependent systemic acquired resistance (SAR). Here we show that CPK5 signaling-dependent immune responses are effectively blocked and pathogen resistance is reverted either upon treatment of plants with abscisic acid (ABA) or in genetic mutant backgrounds lacking PP2C phosphatase activities including abi1-2. Consistently, enhanced immune responses occur upon co-expression of CPK5 kinase with active variants of ABI1 phosphatase ABI1G180S and ABI1G181A. Biochemical studies and mass spectrometry-based phosphosite analysis reveal a direct ABI1 phosphatase-catalyzed de-phosphorylation of CPK5 at T98, a CPK5 auto-phosphorylation site. CPK5T98A, mimicking continuous de-phosphorylation through ABI1, correlates with an increase in kinase activity and CPK5 function in ROS production. CPK5T98D, mimicking a CPK5 auto-phosphorylated status under ABA-induced phosphatase inhibition, leads to inactivated CPK5 causative to an immediate stop of immune responses.Our work reveals an elegant mechanism for plant stress prioritization, where the ABA-dependent phosphatase ABI1, negative regulator of abiotic responses, functions as positive regulator of biotic stress responses, stabilizing CPK5-dependent immune responses in the absence of ABA. This mechanism allows continuous immune signaling during pathogen survey in environmentally non-challenging conditions. Under severe abiotic stress, immune signaling is discontinued via a direct biochemical intersection through a phosphatase/kinase pair recruiting two key regulatory enzymes of these antagonistic signaling pathways.

The calmodulin-binding transcription activator 3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced genes in plants. Post-transcriptional or -translational mechanisms have been proposed to control CAMTA3’s role in the crosstalk between immune and chilling responses. Here, we show that treatment with the bacterial flg22 elicitor, but not cold stress, induces a phospho-mobility shift of CAMTA3 proteins. Correspondingly, CAMTA3 is directly phosphorylated by two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, which triggers CAMTA3 nuclear export and destabilization. SR1IP1, a substrate E3 ubiquitin ligase adaptor required for pathogen-induced CAMTA3 degradation, is shown here to be likely plasma-membrane-localized and therefore cannot physically interact with the nuclear CAMTA3. Despite the flg22-inducible re-localization of CAMTA3 to the cytoplasm, we failed to detect CAMTA3-SR1IP1 complexes. Hence, the role of SR1IP1 for CAMTA3 degradation needs to be re-evaluated. Surprisingly, flg22 elicitation can still induce nuclear export and phospho-mobility shift of a phospho-null CAMTA3 that cannot be phosphorylated by MAPKs, suggesting the participation of additional flg22-responsive kinase(s). A constitutively-active calcium-dependent protein kinase, CPK5, can stimulate a phospho-mobility shift in CAMTA3 similar to that induced by flg22. Although CPK5 can interact with CAMTA3, it did not directly phosphorylate CAMTA3, suggesting the requirement of a still unidentified downstream kinase or additional components. Overall, at least two flg22-responsive kinase pathways target CAMTA3 to induce degradation that presumably serves to remove CAMTA3 from target promoters and de-repress expression of defence genes.