Biosynthesis and secretion of the microbial sulfated peptide RaxX and binding to the rice XA21 immune receptor

Abstract

The rice immune receptor XA21 is activated by the sulfated microbial peptide required for activation of XA21-mediated immunity X (RaxX) produced by Xanthomonas oryzae pv. oryzae (Xoo). Mutational studies and targeted proteomics revealed that the RaxX precursor peptide (proRaxX) is processed and secreted by the protease/transporter RaxB, the function of which can be partially fulfilled by a noncognate peptidase-containing transporter component B (PctB). proRaxX 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 prokaryotic member of a previously unclassified and understudied group of eukaryotic tyrosine sulfated ribosomally synthesized, posttranslationally modified peptides (RiPPs). 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.

Keywords: ABC transporter; RIPP; immunogen; peptidase; sulfation.

Fig. 1.

Biosynthetic pathways of RiPPs and RaxX. (A) General RiPP biosynthetic pathway. The RiPP precursor (propeptide) and biosynthetic proteins are ribosomally synthesized. The core, which becomes the final RiPP product, is posttranslationally modified by enzyme(s) encoded in the same genomic region. Multiple posttranslational modifications can take place on a single propeptide. The N-terminal leader is enzymatically removed by a protease, and the modified core is exported by a transporter, releasing the mature bioactive RiPP. (B) RaxX biosynthetic pathway. proRaxX is ribosomally synthesized, and the core is sulfated by the sulfotransferase RaxST encoded upstream. We hypothesize that the PCAT RaxB removes the N-terminal leader and transports the sulfated mature RaxX peptide through the T1SS composed of RaxB, the periplasmic adaptor protein RaxA, and the genetically unlinked outer membrane protein RaxC.

Fig. 2.

Sulfated RaxX peptide binds XA21^ECD with high affinity. Quantification of binding between sY (red) and nY (blue) RaxX21 and sulfated PSY1 (gray) peptides with the XA21 ectodomain (XA21^ECD) by MST. Data points indicate the fraction of fluorescently labeled XA21 bound to the peptides during the assay (fraction bound [−]). The K_d and confidence intervals shown in brackets are indicated in nanomolar. SE bars are representative of at least two independent measurements performed with independent protein preparations.

Fig. 3.

Mature RaxX is detected in Xoo supernatants. (A) proRaxX sequence. Numbers above refer to the residue position relative to the predicted cleavage site; numbers below are relative to propeptide. Cleavage of proRaxX after Gly38, Gly39 (orange) would result in a leader with hydrophobic residues (yellow) at conserved locations typical of PCAT substrates and a core containing Tyr41 (green) sulfated by RaxST. Shown below this precursor are the synthetic RaxX peptide derivatives (RaxX21, RaxX16, RaxX13, and RaxX11) and the two tryptic peptide targets detected by SRM-MS. (B and C) SRM-MS chromatograms of nonprocessed proRaxX (B) and the predicted mature RaxX (C) tryptic peptides detected from ΔraxX(praxX-His) cell lysates or from PXO99 or ΔraxX supernatants. Lines correspond to the individual SRM transitions monitored. The legends indicate the detected peptide b- and y-series fragment ions.

Fig. 4.

Genetic map of candidate RaxX biosynthetic proteins. (A) raxX is encoded upstream of the raxSTAB operon, containing genes for the RaxST sulfotransferase and components of a T1SS: the RaxA periplasmic adaptor protein and the RaxB PCAT. (B) Genes for components of a second peptidase-containing transporter system are not genetically linked to raxX: pctA and pctB encoding a periplasmic adaptor protein and PCAT, respectively.

Fig. 5.

The ΔraxB ΔpctB double mutant does not secrete RaxX, but secretion can be restored with the addition of the praxSTAB plasmid. (A–C) Rice plants were inoculated by scissor clipping with PXO99-derived strains. (A) Bars represent the mean + SD of lesions (centimeters) measured 14 d postinoculation (dpi) of TP309 (blue dots) or XA21-TP309 (red dots) plants (n = 21–28). (B) Inoculated XA21-TP309 leaves 14 dpi. (Scale bar: 1 cm.) (C) Bacterial densities in planta. Data points represent the mean log cfu per 10-cm leaf section ± SE (n = 3). (D) SRM-MS was used to detect the presence of RaxX in Xoo supernatants. Bars represent the total peak area of the chromatograms shown in SI Appendix, Fig. S3B. Similar results were observed in five (A) or two (C) other experiments, except that ΔraxB formed lesions comparable with PXO99 in roughly one-half. **P < 0.01 compared with PXO99 using Dunnett’s test.

Fig. 6.

Mutation of the RaxB peptidase catalytic triad impairs proRaxX maturation and secretion. (A) Alignment of the peptidase C and H motifs of RaxB and PctB to select PCATs or ABC transporters with a CLD (listed in Materials and Methods). The Cys–His–Asp/Asn catalytic triad is highlighted in yellow, the oxyanion hole Gln is in green, and residues common in at least six of the transporters are in gray. (B–D) Rice plants were inoculated by scissor clipping with the indicated strains. (B) Bars represent the mean + SD of lesion measurements (centimeters) on TP309 (blue dots) or XA21-TP309 (red dots) plants 14 d postinoculation (dpi; n = 10–16). (C) Inoculated XA21-TP309 leaves 14 dpi. (Scale bar: 1 cm.) (D) Bacterial densities in planta. Data points represent the mean log cfu per 10-cm leaf section ± SE (n = 3). Performed at the same time as Fig. 5C. (E) SRM-MS chromatograms of RaxX tryptic peptide detected in supernatants from the ΔraxB ΔpctB double mutant-derived strains. Similar results were observed in five (B) or two (D) other independent experiments. **P < 0.01 compared with PXO99 using Dunnett’s test.

Fig. 7.

Mutation of the GG cleavage site compromises proRaxX maturation and secretion. (A) Sequence logo of the leader from all Xanthomonas proRaxX alleles. Conserved GG preceding the cleavage site is boxed in orange; hydrophobic residues in conserved positions typical of PCAT substrates are boxed in black. (B and C) Rice plants were inoculated by scissor clipping. (B) Bars represent the mean + SD of lesion measurements (centimeters) on TP309 (blue dots) or XA21-TP309 (red dots) plants 14 d postinoculation (dpi; n = 8–13). Similar results were observed in at least six independent experiments. (C) Bars represent the mean log cfu per leaf section ± SE measured 14 dpi (n = 4). (D) SRM-MS chromatograms of RaxX tryptic peptide detected in Xoo supernatants. (E) Quantification of the total peak area shown in D. (F) SRM-MS chromatograms of RaxX tryptic peptide detected in supernatants from the wild-type PXO99 and ΔraxST mutant strains. *P < 0.05 compared with PXO99 using Dunnett’s test; **P < 0.01 compared with PXO99 using Dunnett’s test.