Phosphoprotein analysis of soybean root hairs in response to Bradyrhizobium japonicum inoculation
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The nitrogen fixing symbiosis in soybean (Glycine max) is initiated by colonization of root hairs by the symbiotic bacterium Bradyrhizobium japonicum. Specificity in this symbiosis is determined very early during the initial stages of host-symbiont recognition which involve specific receptor kinases and subsequent kinase cascades. We characterized the phosphoproteome of isolated root hair cells colonized by B. japonicum and quantify the phosphorylation changes using isobaric tag 8-plex ITRAQ coupled to LC-MS/MS analysis. 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated in response to B. japonicum inoculation. By using in silico proteolytic digestion, we showed that the palaeopolyploid soybean genome has less unique peptides making it a challenging organism for quantitative proteomic analysis. Trypsin was shown to be the best choice for digestion of soybean proteins to maximize the number of unique peptides. To screen for the protein kinases that can phosphorylate the 240 phosphoproteins above, a synthetic peptide cocktail derived from 273 phosphopeptides significantly regulated by B. japonicum inoculation was developed. This peptide cocktail was shown to work well in a solution-phase, label-free peptide kinase assay in conjunction with MS/MS analysis. Using a RNAi-mediated gene silencing approach, GmRIN4a and GmRIN4b, the two RPM1-interacting proteins whose phosphorylation status increased upon B. japonicum inoculation, were shown to play an important role in the rhizobium-legume symbiosis. Efforts are now underway to investigate the function of GmRIN4 in more detailed.
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