Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor

Abstract

BackgroundIn plants, the multistep phosphorelay signaling pathway mediates responses to environmental factors and plant hormones. This system is composed of three successive partners: hybrid Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). Among the third partners, B-type RR family members are the final output elements of the pathway; they act as transcription factors and clearly play a pivotal role in the early response to cytokinin in Arabidopsis. While interactions studies between partners belonging to the multistep phosphorelay system are mainly focused on protagonists involved in cytokinin or ethylene pathways, very few reports are available concerning partners of osmotic stress signaling pathway.ResultsIn Populus, we identified eight B-type RR proteins, RR12-16, 19, 21 and 22 in the Dorskamp genotype. To assess HPt/B-type RR interactions and consequently determine potential third partners in the osmosensing multistep phosphorelay system, we performed global yeast two-hybrid (Y2H) assays in combination with Bimolecular Fluorescence Complementation (BiFC) assays in plant cells. We found that all B-type RRs are able to interact with HPt predominant partners (HPt2, 7 and 9) of HK1, which is putatively involved in the osmosensing pathway. However, different profiles of interaction are observed depending on the studied HPt. HPt/RR interactions displayed a nuclear localization, while the nuclear and cytosolic localization of HPt and nuclear localization of RR proteins were validated. Although the nuclear localization of HPt/RR interaction was expected, this work constitutes the first evidence of such an interaction in plants. Furthermore, the pertinence of this partnership is reinforced by highlighting a co-expression of B-type RR transcripts and the other partners (HK1 and HPts) belonging to a potential osmosensing pathway.ConclusionBased on the interaction studies between identified B-type RR and HPt proteins, and the co-expression analysis of transcripts of these potential partners in poplar organs, our results favor the model that RR12, 13, 14, 16 and 19 are able to interact with the main partners of HK1, HPt2, 7 and 9, and this HPt/RR interaction occurs within the nucleus. On the whole, the five B-type RRs of interest could be third protagonists putatively involved in the osmosensing signaling pathway in Populus.

Insights into B-type RR members as signaling partners acting downstream of HPt partners of HK1 in the osmotic stress response in Populus

“…The different YFP N -HPt clones were identical to those used in Bertheau et al (2012). The desired combination of BiFC vectors was used to transiently transform Catharanthus roseus cells by particle bombardment according to Guirimand et al (2009).…”

“…The BD-HPt (HPt2, 7 and 9 fused to LexA DNA-binding domain) clones were constructed previously as described in Bertheau et al (2012). The transformations of yeast strain L40D were performed using the lithium acetate method from Gietz et al (1995).…”

“…The transformations of yeast strain L40D were performed using the lithium acetate method from Gietz et al (1995). Cell yeast suspensions (10 mL) of a dilution series of OD 600 from 0.2 to 0.002 were dropped onto selective medium (-LW) and interaction selective medium (-LWH) supplemented with 60 mM or 20 mM of 3AT for HPt2 or HPt7 and HPt9 respectively, and incubated for 4 days at 30 C. X-gal assays (Bertheau et al, 2012) were directly performed on grown drops resulted from the first growth assay. The b-galactosidase activity reflecting the HPt/RR18 interactions was then measured as previously described in Bertheau et al (2012) using three independent yeast clones, and values were reinstated with those obtained for the other HPt/B-type RR interactions (Bertheau et al, 2012).…”

“…Among the 11 poplar B-type RRs identified by an in silico analysis (Ramírez-Carvajal et al, 2008;Singh and Kumar, 2012), we have succeeded to isolate and study eight of them from Populus Dorskamp genotype (Bertheau et al, 2012). One of the three missing RRs, the poplar RR18 presents high sequence similarities with ARR18 considered as an actor in the osmosensing signaling pathway in Arabidopsis (Veerabagu et al, 2014).…”

“…Thus, by interaction studies coupled with transcriptional co-expression analysis, we discovered existence of a preferential interaction network among HK1 and three HPts (HPt2, 7 and 9) Bertheau et al, 2013) and among these HPts and five B-type RRs (RR12, 13, 14, 16 and 19) (Bertheau et al, 2012).…”