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Reactive oxygen species (ROS) are important messengers in eukaryotic organisms and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase (RLK)-dependent signaling networks. Here we show that the cysteine-rich RLK CRK2 kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RBOHD in Arabidopsis. Functional CRK2 is required for the full elicitor-induced ROS burst and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment and substitution of S703 with alanine reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating MAMP-triggered ROS production.One-sentence summaryCRK2 associates with and activates RBOHD to trigger MAMP-induced ROS production and reveals a novel regulatory mechanism for plant NADPH oxidases through phosphorylation of the C-terminus.

Cyclic Nucleotide Gated Ion Channels (CNGCs) are non-selective cation channels that are involved in regulating responses to both biotic and abiotic stresses in plants. CNGC2 has been implicated in plant immunity and Ca 2+ signaling through the study of the autoimmune phenotypes exhibited by the null mutant, defense, no death1 (dnd1) . However, dnd1 also shows additional phenotypes that are unique among autoimmune mutants. This suggests that CNGC2 plays multiple biological roles beyond pathogen defense. In this study, we cloned the gene that encodes the first suppressor of dnd1 (cngc2), REPRESSOR OF DEFENSE, NO DEATH1 (RDD1) , which encodes an auxin biosynthesis gene, YUCCA6 (YUC6) . We found that dnd1 (cngc2) is defective in auxin-mediated root growth inhibition and gravitropic responses in roots. Consistently with these auxin resistance phenotypes, we found dnd1 shows a dampened response to exogenous auxin compared to wildtype plants using the auxin inducible DR5::GUS and DII:VENUS reporter systems. Finally, auxin-induced Ca 2+ influx was examined using the Förster resonance energy transfer (FRET)-based, genetically encoded Ca 2+ indicator yellow cameleon (YC)-Nano65. We captured severe defects in auxin-induced Ca 2+ increase in dnd1 . These defects were rescued by the rdd1 mutation. Our findings highlight the unexpected involvement of CNGC2 in auxin-induced Ca 2+ signaling and calls into question the current interpretation of dnd1 phenotypes in defense signaling.

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.