EPIGENETIC AND EPITRANSCRIPTOMIC MARKS AFFECTING RNA TRANSCRIPTION, STABILITY, AND TRANSLATABILITY DURING THE DEVELOPMENT OF LATERAL ROOT ORGANS IN LEGUMES
Marcas epigenéticas y epitranscriptómicas que afectan la transcripción, estabilidad y traducibilidad de RNAs durante el desarrollo de órganos laterales de raíces de leguminosas
María Eugenia Zanetti (PI), Flavio Blanco (CoPI), Mauricio Reynoso (CoPI), Soledad Traubenik (Postdoc), Cristina Kirolinko (PhD student), Milagros Ferrari (PhD student), Jesica Rivero (undergraduate student) Marianela Cueva (undergraduate student), Milagros Yacullo (undergraduate student) and Camila Coronel (undergraduate student)
Legume plants form two types of post-embryonic organs in their roots: lateral roots and nodules. Lateral roots serve for soil anchor, water, and nutrient acquisition, whereas nodules are the result of a symbiotic interaction with soil bacteria that allow legumes to overcome nitrogen deficiencies in the soil. Both types of organs have a direct impact on plant growth and development and constitute important agricultural traits. Our research is focused on the characterization of the transcriptional and translational reprograming of root cells during the formation of both types of lateral root organs, as well as changes in mRNA stability mediated by long and small non-coding RNAs during these two developmental processes. Our current aims are to understand the dynamics of epigenetic marks- DNA methylation and histone modifications- that affect chromatin accessibility at transcriptionally active loci during lateral root and nodule organogenesis. In addition, we aim to elucidate how epitranscriptomic marks -particularly N-6 Adenosine methylation (m6A)- affect RNA stability and translatability during the formation of both lateral organs. We use genetic and biochemical approaches combined with high throughput sequencing technologies to elucidate the function of proteins that are crucial for these cellular processes. These results will contribute to a better understanding of the multitier mechanisms that governs the reprogramming of gene expression during the formation of lateral roots and nitrogen fixing nodules. From a biotechnological perspective, this information can be translated into the development of novel strategies for improvement of crop productivity in agriculture.

MULTI-LEVEL REGULATORY MECHANISMS GOVERNING STRAIN SPECIFICITY IN NITROGEN-FIXING SYMBIOSIS
Circuitos regulatorios que controlan la expresión génica en tipos celulares específicos durante la selectividad de cepa en la interacción simbiótica fijadora de nitrógeno
Flavio Blanco (PI), María Eugenia Zanetti (CoPI), Mauricio Reynoso (CoPI), Claudio Rivero (PhD student), Carla Roda (PhD student), Jennifer Artunián (PhD student), Andrés Eylenstein (PhD student), Lourdes Nassarov (undergraduate student) and Marina Cretton (undergraduate student)
During the legume-rhizobia interaction, root cells undergo a genetic reprogramming that results in the execution of two independent genetic programs that are spatially and temporarily coordinated: infection and the formation of a post-embryonic organ, the nodule, where nitrogen fixation takes place. Biological Nitrogen fixation has been used in agriculture by introducing legumes in crop rotation together with selected strains of rhizobia optimized for nitrogen fixation. To understand genetic changes during this biological process, cell-specific responses, and the multiple layers of gene regulation, from transcriptional to translational level, should be analyzed and integrated. We aim to dissect such responses in epidermal and cortical cells, which are involved in infection and nodule organogenesis, respectively. Genetic reprogramming is evaluated at transcriptional, post-transcriptional and translational levels, including changes in chromatin remodelling, DNA methylation, mRNA dynamics and its translational status. We apply INTACT (A) to purify cell-specific nuclei, ATAC (B), a technique used to characterize dynamic changes in the chromatin state, bisulfite sequencing to assess the dynamics of DNA methylation, RNA-seq, small RNA-seq for transcriptomics and TRAP (C) to study the translational level. Using this information, we have identified regulatory events associated with the efficiency of the symbiotic interaction, but also those that are common across the different accession x strain interactions. Currently, we are functionally characterizing several genes involved in the strain-specific response. These results could help to develop breeding programs that optimize symbiotic outcomes in crop species by improving the compatibility between both symbiotic partners. In the long term, this will contribute to improve agricultural productivity and sustainability by reducing the use of nitrogen fertilizers.

CONSERVED REGULATORY LANDSCAPE OF ROOT LEGUME PLASTICITY
Control conservado de la plasticidad de las raíces de leguminosas
Mauricio Reynoso (PI), María Eugenia Zanetti (CoPI), Flavio Blanco (CoPI), Agustina Tati (undergraduate student) and Camila Bori (undergraduate student)
Increased frequency of extreme climatic events such as floods and droughts affect plant development on both natural and agronomical environments. Plants have the unique capacity of adapting their development to cope with biotic and abiotic stresses derived from these changing conditions. Under this context, the global plant biology community has progressed toward understanding strategies required for plant resilience and aims to increase crop productivity while reducing a negative environmental impact.
Legumes establish a symbiotic association with rhizobia bacteria capable of fixing atmospheric nitrogen. The interaction leads to the cellular reprogramming of root cells to form post-embryonic organs called nodules, which provide a low oxygen compartment required for nitrogen fixation. Previous studies have found that conserved regulatory networks to flooding across species include a low oxygen signature response. We aim to characterize regulatory mechanisms that could involve transcription factors (TFs) conserved in low oxygen response with focus on their role in cellular reprogramming for lateral root development under floods and nodule development during legume-rhizobia interaction. Evidence supports that most cis-regulatory elements are allocated in chromatin accessible regions present in different cell-populations. We use of a combination of INTACT and ATAC-seq on cells required for lateral root and nodule development that experiment low oxygen conditions to dissect regulatory changes exerted by these TFs. Conserved TFs showing a role in the cellular reprograming are being further evaluated using chromatin immunopurification. This new information will be integrated in the context of already described pathways to provide a better understanding of the key biological processes required for plant root plasticity