PUCHI delimits the spatial domain of nodule organogenesis associated with auxin patterning and NIN-dependent transcription in Lotus japonicus.

Root nodule organogenesis requires the activation of symbiotic developmental programs within a spatially restricted region of the root. Although many regulators of nodule initiation have been identified, the mechanisms that delimit the domain of organogenic cell proliferation remain poorly understood. In this study, we show that the AP2/ERF transcription factors PUCHI1 and PUCHI2 function in spatially restricting cortical cell division during nodulation in Lotus japonicus. The loss of PUCHI function increased infection thread formation and nodule primordium initiation; however, it did not increase the number of mature nodules. Instead, puchi1 and puchi2 mutants frequently formed clustered nodules accompanied by ectopic cortical cell divisions surrounding developing primordia. Constitutive activation of CCaMK induced broadened spontaneous proliferative structures in the mutant background even in the absence of rhizobia, indicating that this phenotype was not simply a consequence of enhanced infection. PUCHI1 expression was induced during early symbiotic signalling downstream of the NODULE INCEPTION (NIN)-associated transcriptional network; both PUCHI genes were preferentially expressed in the basal region of developing primordia, corresponding to sites of ectopic proliferation in the mutant. Transcriptome and reporter analyses suggested that PUCHI regulated auxin-response patterning, potentially via STY1-, YUCCA11-, TAR2-, and Forked1-like-associated pathways. Additionally, PUCHI1 promoted NIN expression via the cytokinin-responsive CE region of the distal promoter independently of LHK1-mediated cytokinin perception. These findings indicate that PUCHI genes define the spatial domain of organogenic cell proliferation during nodulation; they coordinate auxin-related patterning with the NIN transcriptional module to ensure localised nodule formation.