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Transcriptional reprogramming is critical for plant immunity. Several calmodulin (CaM)-binding protein 60 (CBP60) family transcription factors (TFs) in Arabidopsis (Arabidopsis thaliana), including CBP60g, systemic acquired resistance deficient 1 (SARD1), CBP60a, and CBP60b, are critical for and show distinct roles in immunity. However, there are additional CBP60 members whose function is unclear. We report here that Arabidopsis CBP60c-f, 4 uncharacterized CBP60 members, play redundant roles with CBP60b in the transcriptional regulation of immunity responses, whose pCBP60b-driven expression compensates the loss of CBP60b. By contrast, neither CBP60g nor SARD1 is interchangeable with CBP60b, suggesting clade-specific functionalization. We further show that the function of CBP60b clade TFs relies on DNA-binding domains (DBDs) and CaM-binding domains, suggesting that they are downstream components of calcium signaling. Importantly, we demonstrate that CBP60s encoded in earliest land plant lineage Physcomitrium patens and Selaginella moellendorffii are functionally homologous to Arabidopsis CBP60b, suggesting that the CBP60b clade contains the prototype TFs of the CBP60 family. Furthermore, tomato and cucumber CBP60b-like genes rescue the defects of Arabidopsis cbp60b and activate the expression of tomato and cucumber SALICYLIC ACID INDUCTION DEFICIIENT2 (SID2) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes, suggesting that immune response pathways centered on CBP60b are also evolutionarily conserved. Together, these findings suggest that CBP60b clade TFs are functionally conserved in evolution and positively mediate immunity., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Plants utilize a plethora of peptide signals to regulate their immune response. Peptide ligands and their cognate receptors involved in immune signaling share common motifs among many species of vascular plants. However, the origin and evolution of immune peptides is still poorly understood. Here, we searched for genes encoding small secreted peptides in the genomes of three bryophyte lineages-mosses, liverworts and hornworts-that occupy a critical position in the study of land plant evolution. We found that bryophytes shared common predicted small secreted peptides (SSPs) with vascular plants. The number of SSPs is higher in the genomes of mosses than in both the liverwort Marchantia polymorpha and the hornwort Anthoceros sp. The synthetic peptide elicitors-AtPEP and StPEP-specific for vascular plants, triggered ROS production in the protonema of the moss Physcomitrella patens, suggesting the possibility of recognizing peptide ligands from angiosperms by moss receptors. Mass spectrometry analysis of the moss Physcomitrella patens, both the wild type and the Δcerk mutant secretomes, revealed peptides that specifically responded to chitosan treatment, suggesting their role in immune signaling.

Background: Plant α-dioxygenases catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids leading to the formation of oxylipins. In flowering plants, two main groups of α-DOXs have been described. While the α-DOX1 isoforms are mainly involved in defense responses against microbial infection and herbivores, the α-DOX2 isoforms are mostly related to development. To gain insight into the roles played by these enzymes during land plant evolution, we performed biochemical, genetic and molecular analyses to examine the function of the single copy moss Physcomitrella patens α-DOX (Ppα-DOX) in development and defense against pathogens., Results: Recombinant Ppα-DOX protein catalyzed the conversion of fatty acids into 2-hydroperoxy derivatives with a substrate preference for α-linolenic, linoleic and palmitic acids. Ppα-DOX is expressed during development in tips of young protonemal filaments with maximum expression levels in mitotically active undifferentiated apical cells. In leafy gametophores, Ppα-DOX is expressed in auxin producing tissues, including rhizoid and axillary hairs. Ppα-DOX transcript levels and Ppα-DOX activity increased in moss tissues infected with Botrytis cinerea or treated with Pectobacterium carotovorum elicitors. In B. cinerea infected leaves, Ppα-DOX-GUS proteins accumulated in cells surrounding infected cells, suggesting a protective mechanism. Targeted disruption of Ppα-DOX did not cause a visible developmental alteration and did not compromise the defense response. However, overexpressing Ppα-DOX, or incubating wild-type tissues with Ppα-DOX-derived oxylipins, principally the aldehyde heptadecatrienal, resulted in smaller moss colonies with less protonemal tissues, due to a reduction of caulonemal filament growth and a reduction of chloronemal cell size compared with normal tissues. In addition, Ppα-DOX overexpression and treatments with Ppα-DOX-derived oxylipins reduced cellular damage caused by elicitors of P. carotovorum., Conclusions: Our study shows that the unique α-DOX of the primitive land plant P. patens, although apparently not crucial, participates both in development and in the defense response against pathogens, suggesting that α-DOXs from flowering plants could have originated by duplication and successive functional diversification after the divergence from bryophytes.

Production of apoplastic reactive oxygen species (ROS), or oxidative burst, is among the first responses of plants upon recognition of microorganisms. It requires peroxidase or NADPH oxidase (NOX) activity and factors maintaining cellular redox homeostasis. Here, PpTSPO1 involved in mitochondrial tetrapyrrole transport and abiotic (salt) stress tolerance was tested for its role in biotic stress in Physcomitrella patens, a nonvascular plant (moss). The fungal elicitor chitin caused an immediate oxidative burst in wild-type P. patens but not in the previously described ΔPrx34 mutants lacking the chitin-responsive secreted class III peroxidase (Prx34). Oxidative burst in P. patens was associated with induction of the oxidative stress-related genes AOX, LOX7, and NOX, and also PpTSPO1. The available ΔPpTSPO1 knockout mutants overexpressed AOX and LOX7 constitutively, produced 2.6-fold more ROS than wild-type P. patens, and exhibited increased sensitivity to a fungal necrotrophic pathogen and a saprophyte. These results indicate that Prx34, which is pivotal for antifungal resistance, catalyzes ROS production in P. patens, while PpTSPO1 controls redox homeostasis. The capacity of TSPO to bind harmful free heme and porphyrins and scavenge them through autophagy, as shown in Arabidopsis under abiotic stress, seems important to maintenance of the homeostasis required for efficient pathogen defense.