The rice immune receptor XA21 is activated by the sulfated microbial peptide RaxX (required for activation of XA21-mediated immunity X) produced by Xanthomonas oryzae pv. oryzae (Xoo). Mutational studies and targeted proteomics revealed that RaxX is processed and secreted by the protease/transporter RaxB, whose function can be partially fulfilled by a noncognate peptidase-containing transporter B (PctB). RaxX is cleaved at a Gly-Gly motif, yielding a mature peptide that retains the necessary elements for RaxX function as an immunogen and host peptide hormone mimic. These results indicate that RaxX is a founding member of a previously unclassified and understudied group of tyrosine sulfated RiPPs (ribosomally synthesized, post-translationally modified peptides). We further demonstrate that sulfated RaxX directly binds XA21 with high affinity. This work reveals a complete, previously uncharacterized biological process: bacterial RiPP biosynthesis, secretion, binding to a eukaryotic receptor and triggering of a robust host immune response.

Here, we report that synthetic RaxX peptide directly binds the extracellular domain (ECD) of the XA21 immune receptor (XA21ECD), an interaction enhanced by tyrosine sulfation of RaxX. We also demonstrate that RaxB is required for proteolytic processing and secretion of RaxX and identify a second PCAT PctB (peptidase-containing transporter component B) that can partially compensate for deletion of raxB. Our genetics and targeted proteomics data indicate that RaxX is ribosomally synthesized as a precursor peptide and cleaved downstream of a GG-motif in a RaxB-dependent manner. This proteolytic event releases the mature RaxX core peptide containing the sulfated Tyr-41 and the minimal 16-residue active region. These results indicate that RaxX is a tyrosine sulfated RiPP, a previously undescribed class of RiPPs that mediate intercellular interactions.