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In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. We identified two groups of exonucleases from the Herpes Virus and the bacteriophage T7 families that conferred an up to 38-fold increase in HDR frequencies when fused to Cas9/Cas12a in a Tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrate stable and heritable knock-ins of in wheat in 1% of the primary transformants. Our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses. These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence. Here, we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana (Bs) and Fusarium graminearum (Fg) at the metabolite level. We identify hordedanes, a previously undescribed set of labdane-related diterpenoids with antimicrobial properties, as critical players in these interactions. Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 600-kb gene cluster. Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes, including one of the most functionally decorated products 19-b-hydroxy-hordetrienoic acid (19-OH-HTA). Barley mutants in the diterpene synthase genes of this cluster are unable to produce hordedanes but, unexpectedly, show reduced Bs colonization. By contrast, colonization by Fusarium graminearum, another fungal pathogen of barley and wheat, is 4-fold higher in the mutants completely lacking hordedanes. Accordingly, 19-OH-HTA enhances both germination and growth of Bs, whereas it inhibits other pathogenic fungi, including Fg. Analysis of microscopy and transcriptomics data suggest that hordedanes delay the necrotrophic phase of Bs. Taken together, these results show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization.

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.

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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