Reprogramming of root cells during nitrogen-fixing symbiosis entails dynamic polysome association of coding and non-coding RNAs

GMUCT 2.0 data were analyzed to globally identify 5’ ends differentially accumulated in SKI3 RNAi roots as compared with GUS RNAi, as well as those that change in abundance in response to rhizobia SUPERKILLER3 (*SKI3),* a member of SKI complex that guides RNAs to the 3 to 5' exoribonuclease of the exosome complex. In the absence of a functional SKI complex to thread transcripts to the exosome, the bulk of the mRNAs that accumulate in the cytoplasm are diverted to the 5 -to-3 XRN4 pathway for degradation. We explored the function of *SKI3* during root nodule symbiosis using RNA interference to knockdown *SKI3* in *M. truncatula* hairy roots. To evaluate whether silencing of *SKI3* in *M. truncatula* roots altered RNA decay in response to rhizobia, we performed genome-wide mapping of uncapped and cleaved transcripts (GMUCT 2.0) on *GUS* RNA and *SKI3*RNAi roots. This method identifies products of miRNA or siRNA guided-endonucleolytic cleavage as well as decapped mRNAs by selecting for molecules with a free 5 monophosphate (5 P) (Willmann et al., 2014). GMUCT 2.0 data were analyzed to globally identify 5' ends differentially accumulated in *SKI3* RNAi roots as compared with *GUS* RNAi, as well as those that change in abundance in response to rhizobia. GMUCT 2.0 libraries were prepared from RNA samples isolated from GUS RNAi or SKI3 RNAi roots inoculated with water (mock) or S. meliloti for 48 hours. Three biological replicates were conducted.