The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus.

Bacterial accommodation inside living plant cells is restricted to the nitrogen-fixing root nodule symbiosis. In many legumes, bacterial uptake is mediated via tubular structures called infection threads (ITs). To identify plant genes required for successful symbiotic infection, we screened an ethyl methanesulfonate mutagenized population of Lotus japonicus for mutants defective in IT formation and cloned the responsible gene, ERN1, encoding an AP2/ERF transcription factor. We performed phenotypic analysis of two independent L. japonicus mutant alleles and investigated the regulation of ERN1 via transactivation and DNA-protein interaction assays. In ern1 mutant roots, nodule primordia formed, but most remained uninfected and bacterial entry via ITs into the root epidermis was abolished. Infected cortical nodule cells contained bacteroids, but transcellular ITs were rarely observed. A subset exhibited localized cell wall degradation and loss of cell integrity associated with bacteroid spread into neighbouring cells and the apoplast. Functional promoter studies revealed that CYCLOPS binds in a sequence-specific manner to a motif within the ERN1 promoter and in combination with CCaMK positively regulates ERN1 transcription. We conclude that the activation of ERN1 by CCaMK/CYCLOPS complex is an important step controlling IT-mediated bacterial progression into plant cells.

A CCaMK-CYCLOPS-DELLA Complex Activates Transcription of RAM1 to Regulate Arbuscule Branching.

Intracellular arbuscular mycorrhiza symbiosis between plants and glomeromycotan fungi leads to formation of highly branched fungal arbuscules that release mineral nutrients to the plant host. Their development is regulated in plants by a mechanistically unresolved interplay between symbiosis, nutrient, and hormone (gibberellin) signaling. Using a positional cloning strategy and a retrotransposon insertion line, we identify two novel alleles of Lotus japonicus REDUCED ARBUSCULAR MYCORRHIZA1 (RAM1) encoding a GRAS protein. We confirm that RAM1 is a central regulator of arbuscule development: arbuscule branching is arrested in L. japonicus ram1 mutants, and ectopic expression of RAM1 activates genes critical for arbuscule development in the absence of fungal symbionts. Epistasis analysis places RAM1 downstream of CCaMK, CYCLOPS, and DELLA because ectopic expression of RAM1 restores arbuscule formation in cyclops mutants and in the presence of suppressive gibberellin. The corresponding proteins form a complex that activates RAM1 expression via binding of CYCLOPS to a cis element in the RAM1 promoter. We thus reveal a transcriptional cascade in arbuscule development that employs the promoter of RAM1 as integrator of symbiotic (transmitted via CCaMK and CYCLOPS) and hormonal (gibberellin) signals.

CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development.

Nuclear calcium oscillations are a hallmark of symbiotically stimulated plant root cells. Activation of the central nuclear decoder, calcium- and calmodulin-dependent kinase (CCaMK), triggers the entire symbiotic program including root nodule organogenesis, but the mechanism of signal transduction by CCaMK was unknown. We show that CYCLOPS, a direct phosphorylation substrate of CCaMK, is a DNA-binding transcriptional activator. Two phosphorylated serine residues within the N-terminal negative regulatory domain of CYCLOPS are necessary for its activity. CYCLOPS binds DNA in a sequence-specific and phosphorylation-dependent manner and transactivates the NODULE INCEPTION (NIN) gene. A phosphomimetic version of CYCLOPS was sufficient to trigger root nodule organogenesis in the absence of rhizobia and CCaMK. CYCLOPS thus induces a transcriptional activation cascade, in which NIN and a heterotrimeric NF-Y complex act in hierarchical succession to initiate symbiotic root nodule development.