A Laser Dissection-RNAseq Analysis Highlights the Activation of Cytokinin Pathways by Nod Factors in the Medicago truncatula Root Epidermis

Abstract

Nod factors (NFs) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobiumlegume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified NF (4 and 24 h of treatment) in the root epidermis of the model legume Medicago truncatula. Tissue-specific transcriptome analysis was achieved by laser-capture microdissection coupled to high-depth RNA sequencing. The expression of 17,191 genes was detected in the epidermis, among which 1,070 were found to be regulated by NF addition, including previously characterized NF-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at 4 h, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, including perception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism, and defense, with numerous NF-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (CK) pathways was indicated, based on the induction of CK metabolic and signaling genes, including the CRE1 receptor essential to promote nodulation. These transcriptional activations were independently validated using promoter:b-glucuronidase fusions with the MtCRE1 CK receptor gene and a CK response reporter (TWO COMPONENT SIGNALING SENSOR NEW). A CK pretreatment reduced the NF induction of the EARLY NODULIN11 (ENOD11) symbiotic marker, while a CK-degrading enzyme (CYTOKININ OXIDASE/DEHYDROGENASE3) ectopically expressed in the root epidermis led to increased NF induction of ENOD11 and nodulation. Therefore, CK may play both positive and negative roles in M. truncatula nodulation.

Cytokinin Biosynthesis Promotes Cortical Cell Responses during Nodule Development

“…In L. japonicus, TCS readout and Lhk1 promoter activity occur first in the cortex before extending to epidermal cells (Held et al, 2014). In M. truncatula, contrasting results have been reported from TCSn and TCS reporter studies following NF application (van Zeijl et al, 2015;Jardinaud et al, 2016). TCSn activity was detected in the epidermis and supported by expression analysis of laser-capture microdissected cells, while TCS activity was found in cortical cells in response to NF application.…”

“…In L. japonicus, TCS activity occurred first in the dividing cells of the developing nodule and later in the epidermis (Held et al, 2014). Epidermal cytokinin responses to NF also have been reported in M. truncatula hairy roots using the more recently developed TCS new (TCSn) reporter (Zürcher et al, 2013;Jardinaud et al, 2016). It is likely that both cytokinin accumulation and receptor availability modulate these cytokinin response domains.…”

“…Several additional M. truncatula biosynthetic genes responding to NF treatment also were identified in the Ipt, Cyp735a, and Log families (van Zeijl et al, 2015). Cytokinin metabolism genes also were identified in laser-dissected epidermis following NF treatment (Jardinaud et al, 2016). Although LjIpt3 was suggested to contribute to nodule organogenesis in an RNA interference study (Chen et al, 2014), mutant analysis indicated that LjIpt3 in fact plays a shoot-dependent negative regulatory role in nodulation (Sasaki et al, 2014).…”

Laser Capture Microdissection Protocol for Xylem Tissues of Woody Plants

“…Good results in terms of RNA quality and RNA sequencing have been observed also after incubation of plant material in polyethylene glycol (PEG)-ethanol mixture and subsequent inclusion in PEG-modified Steedman’s wax and polyethylene naphtalate (PEN)-coated slides for LCM (Roux et al, 2014). This protocol was developed further and 100% PEG was used as an embedding medium with sections mounted on PEN slides (Jardinaud et al, 2016). Much thicker longitudal root sections (35 μm) of barrel medic have been prepared for LCM using embedding in tissue-freezing medium and collected mycorrhizal material was used for gene expression profiling (Gaude et al, 2012).…”

Medicago truncatula: Genetic and Genomic Resources

“…Another M. truncatula gene expression database that will include data from M. truncatula RNA‐seq experiments is under development (Patrick Zhao, Noble Research Institute; personal communication). Results from specific RNA‐seq experiments are already available at https://iant.toulouse.inra.fr/symbimics/ (Jardinaud et al., ) and at http://pages.discovery.wisc.edu/∼sroy/medicago_symbiosis_transcriptome/index.html (Larrainzar et al., ).…”