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- Title
ChIP-Seq Analysis Protocol for Identification of PsIPD3 and PsNIN Transcription Factors Binding Sites in Pisum sativum genome.
- Authors
Dolgikh, A. V.; Kantsurova, E. S.; Dolgikh, E. A.
- Abstract
Legumes have the unique ability to form symbiosis with nitrogen-fixing bacteria of the genus Rhizobium by forming special symbiotic organs called nodules. The process of symbiosis initiation and development between legumes and rhizobia is tightly controlled by a complex network of transcription factors. Control of root nodule development and infection thread formation involves the recognition of rhizobial signal molecules through specific receptors, which trigger signal transduction cascades. IPD3 and NIN transcription factors begin to act early in this process. The IPD3 transcription factor has been previously shown to regulate infection processes associated with rhizobial invasion and activate several downstream components, including NIN transcription factor. NIN, in turn, plays an important role in the control of infection process as well as in nodule organogenesis. Nevertheless, their targets in developing nodules of legumes such as Pisum sativum and Medicago truncatula remain unclear. In our study, we used chromatin immunoprecipitation combined with the next-generation sequencing (ChIP-Seq) method to identify targets of PsIPD3 and PsNIN in the Pisum sativum genome. To do this, we generated transgenic plants carrying the p35S::PsIPD3~3XFLAG::t35S and p35S::PsNIN~3XFLAG::t35S constructs using the Agrobacterium rhizogenes-mediated transformation approach. Immunoprecipitation of chromatin bound to PsIPD3~3XFLAG and PsNIN~3XFLAG proteins was performed using anti-FLAG antibodies, and libraries generated from bound chromatin were sequenced on the Illumina NovaSeq 6000 platform. Here, the methodical approaches for preparation of IPD3-targets-enriched and NIN-targets-enriched samples in pea plants using ChiP-Seq are discussed.
- Subjects
ROOT-tubercles; TRANSCRIPTION factors; BINDING sites; MEDICAGO; MEDICAGO truncatula; GENOMES; NITROGEN-fixing bacteria; NUCLEOTIDE sequencing
- Publication
Russian Journal of Plant Physiology, 2023, Vol 70, Issue 9, p1
- ISSN
1021-4437
- Publication type
Article
- DOI
10.1134/S1021443723603403