Research ArticleCROP SCIENCE

The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

See allHide authors and affiliations

Science Advances  24 Jul 2015:
Vol. 1, no. 6, e1500245
DOI: 10.1126/sciadv.1500245
  • Fig. 1 raxX, a small ORF located upstream of the raxSTAB operon, is required for activation of XA21-mediated immunity.

    (A) raxST, raxA, and raxB are encoded in a single operon. A 1.0-kb region upstream of raxST and a 1.7-kb region downstream of raxB were deleted in PXO99Δ1.0Sp and PXO99Δ1.7Sp, respectively. The raxX ORF is located ~0.4 kb upstream of raxST and in the opposite orientation. (B) TP309 (open bars) or XA21-TP309 (black bars) were inoculated by clipping with scissors dipped in Xoo suspensions. Bars indicate the mean lesion length ± SE measured 14 days after inoculation (n ≥ 14). The “*” indicates statistically significant difference from PXO99 using Dunnett’s test (α = 0.01). No statistical differences in lesion length were observed on TP309 inoculated with the different strains. The experiment was repeated at least three times with similar results. (C) XA21-TP309 rice leaves display water-soaked lesions 2 weeks after inoculation with the indicated strains. (D) Growth of PXO99 (□), PXO99ΔraxX (△), and PXO99ΔraxX(praxX) (○) in rice leaves inoculated as in (B). In planta bacterial growth analysis was carried out as described (22). Bacterial quantification was determined as the number of colony-forming units (CFU) extracted per inoculated leaf. For the final data point, “*” indicates statistically significant difference from PXO99 using Dunnett’s test (α = 0.01, n = 4). The experiment was repeated twice with similar results.

  • Fig. 2 RaxST sulfates RaxX on tyrosine 41.

    (A) The predicted PAPS binding residue R35 of RaxST and Y41 of RaxX are required for activation of XA21-mediated immunity. TP309 (open bars) and XA21-TP309 (black bars) were inoculated with the indicated Xoo strains, and lesion lengths were measured 14 days later as described in Fig. 1B. Bars indicate the mean lesion length ± SE (n ≥ 14). The “*” indicates statistically significant difference from PXO99 using Dunnett’s test (α = 0.01). (B) Ultraviolet photodissociation (UVPD) mass spectrum of a tyrosine-sulfated peptide (HVGGGDsYPPPGANPK, 2–, m/z 770) from trypsin digestion of in vitro sulfated RaxX39. After incubation with Escherichia coli–expressed and purified His-RaxST, RaxX39 was digested with trypsin and analyzed by LC-UVPD-MS/MS in the negative nanoelectrospray mode to generate a, c, x, y, and z product ions, which are defined in the fragmentation key in the upper left corner of the spectrum. SO3 is retained on all product ions, allowing the sulfate modification to be localized to Y41. Neutral losses of SO3 from the precursor ion and charge reduced radical precursor ion are denoted as (M-2H-SO3)2− and (M-2H-SO3)1−•, respectively, in the spectrum. The ion labeled “m7” refers to the neutral loss of the sulfotyrosine side chain without additional fragmentation of the peptide backbone. Examination of extracted ion chromatograms of the sulfated and nonsulfated peptides suggests that the sulfated peptide is 100× lower in abundance than the nonsulfated peptide (see fig. S9). (C and D) RaxX-His proteins purified from PXO99(praxX-His) and PXO99ΔraxST(praxX-His) were analyzed by selected reaction monitoring-MS (SRM-MS) (fig. S8). Total peak areas (arbitrary units) were quantitated for sulfated (C) and nonsulfated (D) tryptic RaxX peptides covering Y41.

  • Fig. 3 Sulfated RaxX triggers XA21-mediated defense responses.

    (A) Amino acid sequence of RaxX from PXO99. RaxX derivative peptides of varying lengths were tested for their ability to activate XA21-dependent signaling. Sulfated peptides shown in black trigger XA21-mediated defense responses, whereas nonsulfated peptides and the sulfated peptide RaxX_18 shown in red do not (figs. S13 to S21). “EC50 value on Ubi::XA21” refers to the EC50 values determined by monitoring total ROS production over 3 hours after application of the RaxX protein and peptide derivatives (fig. S20) at different concentrations on Kitaake rice expressing XA21 under the control of the ubiquitin promoter (Ubi::XA21) (n = 6). (B) ROS production in leaves of Kitaake and Ubi::XA21 rice plants treated with H2O (mock), RaxX21-Y, or RaxX21-sY (250 nM) (n = 6). RLU, relative light units. (C) Ethylene production in leaves of Kitaake and Ubi::XA21 rice plants after 4 hours of treatment with H2O (mock), RaxX21-Y, or RaxX21-sY (1 μM). The “*” indicates statistically significant difference from mock treatment using Dunnett’s test (α = 0.01, n = 3). (D) Temporal changes in defense marker gene (Os04g10010, PR10b, and Os12g36830) expression in leaves of Kitaake and Ubi::XA21 rice plants treated with H2O (mock), RaxX21-Y, or RaxX21-sY (500 nM, n = 3). All data points depict means ± SE. These experiments were repeated at least three times with similar results.

  • Fig. 4 Comparative genomics and mutational analyses identify key amino acids required for RaxX activity.

    (A) Alignment of amino acid sequences of RaxX from PXO99 and IXO685. The region corresponding to RaxX21 is boxed. (B and C) RaxX from the field strain IXO685 does not trigger XA21-mediated immune response. (B) TP309 (open bars) and XA21-TP309 (black bars) were inoculated with the indicated Xoo strains, and lesion lengths were measured 14 days later as described in Fig. 1B. Bars indicate the mean lesion length ± SE (n ≥ 14). (C) ROS production in leaves of Kitaake (open bars) and Ubi::XA21 (black bars) rice plants treated with the indicated peptides (250 nM) or mock treatment. Bars depict mean RLU over 3 hours ± SE (n = 6). (D and E) RaxX point mutation analysis reveals that P44 and P48 are required for activation of XA21-mediated immune response. (D) TP309 (open bars) and XA21-TP309 (black bars) were inoculated with the indicated Xoo strains, and lesions were measured 14 days later as described in Fig. 1B. Bars indicate the mean lesion length ± SE (n ≥ 14). (E) ROS production in leaves of Kitaake (open bars) and Ubi::XA21 (black bars) rice plants treated with the indicated peptides (250 nM) or mock treatment. Bars depict average RLU ± SE (n = 6). For (B) to (D), the “*” indicates statistically significant difference from mock treatments using Dunnett’s test (α = 0.01). These experiments were repeated at least three times with similar results.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/6/e1500245/DC1

    Fig. S1. Sequence alignment of 15 raxX alleles.

    Fig. S2. raxX is required for activation of XA21-mediated immunity in the Ubi::XA21 Kitaake (Ubi::XA21) genetic background.

    Fig. S3. Alignment of the PAPS binding motif of RaxST from PXO99, TPST1 from human, and TPST2 from human.

    Fig. S4. RaxX Y41 is required for activation of XA21-mediated immunity.

    Fig. S5. RaxX Y41F does not trigger the XA21-mediated immune response.

    Fig. S6. Purification of the full-length RaxST protein carrying an N-terminal His-tag.

    Fig. S7. Expanded MS1 spectrum of HVGGGDsYPPPGANPK from trypsin digestion of in vitro sulfated RaxX39.

    Fig. S8. Extracted ion chromatograms (XIC) showing the difference in ion abundances for the sulfated and nonsulfated peptide HVGGGDYPPPGANPK (2− charge state).

    Fig. S9. Positive UVPD mass spectrum of HVGGGDsYPPPGANPK from trypsin digestion of in vitro sulfated RaxX39.

    Fig. S10. RaxX-His proteins were purified from PXO99(praxX-His) and PXO99ΔraxST(praxX-His).

    Fig. S11. SRM-MS analysis of the tryptic RaxX peptide HVGGGDYPPPGANPK from PXO99(praxX-His) and PXO99ΔraxST(praxX-His).

    Fig. S12. Expression and sulfation of RaxX60-Y and RaxX60-sY.

    Fig. S13. Heterologously expressed, full-length RaxX60-sY activates XA21-mediated defense gene expression.

    Fig. S14. Chemically synthesized RaxX39-sY activates XA21-mediated defense gene expression.

    Fig. S15. Digestion of RaxX39-sY by the four site-specific proteases GluC, trypsin, ArgC, and ApsN.

    Fig. S16. A 21–amino acid sulfated peptide derived from RaxX is sufficient to activate XA21-mediated defense gene expression and the production of ROS.

    Fig. S17. Chemically synthesized RaxX21-sY is sufficient to activate XA21-mediated defense gene expression.

    Fig. S18. Chemically synthesized RaxX21-sY activates gene expression in Kitaake lines expressing XA21 from its native promoter (XA21:XA21).

    Fig. S19. Chemically synthesized RaxX21-sY activates the production of ROS in Kitaake lines expressing XA21 from its native promoter (XA21:XA21).

    Fig. S20. ROS production dose-response curves in Ubi::XA21 leaves treated with chemically synthesized RaxX sulfated peptides.

    Fig. S21. The tyrosine-sulfated peptide axY(S)22, derived from Ax21, does not trigger XA21-mediated immune responses in rice.

    Fig. S22. Expression and purification of different RaxX alleles.

    Fig. S23. Complementation of Xoo isolates IXO651, IXO685, and IXO1221 with praxX confers the ability to activate XA21-mediated immunity.

    Fig. S24. RaxX is similar to the Arabidopsis thaliana peptide signaling factors PSY1 and to rice PSY1 homologs.

    Table S1. Marker genes and primers used for qPCR.

    Table S2. Plasmids used in this study.

    Table S3. Bacterial strains used in this study.

    References (6267)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Sequence alignment of 15 raxX alleles.
    • Fig. S2. raxX is required for activation of XA21-mediated immunity in the Ubi::XA21 Kitaake (Ubi::XA21) genetic background.
    • Fig. S3. Alignment of the PAPS binding motif of RaxST from PXO99, TPST1 from human, and TPST2 from human.
    • Fig. S4. RaxX Y41 is required for activation of XA21-mediated immunity.
    • Fig. S5. RaxX Y41F does not trigger the XA21-mediated immune response.
    • Fig. S6. Purification of the full-length RaxST protein carrying an N-terminal His-tag.
    • Fig. S7. Expanded MS1 spectrum of HVGGGDsYPPPGANPK from trypsin digestion of in vitro sulfated RaxX39.
    • Fig. S8. Extracted ion chromatograms (XIC) showing the difference in ion abundances for the sulfated and nonsulfated peptide HVGGGDYPPPGANPK (2− charge state).
    • Fig. S9. Positive UVPD mass spectrum of HVGGGDsYPPPGANPK from trypsin digestion of in vitro sulfated RaxX39.
    • Fig. S10. RaxX-His proteins were purified from PXO99(praxX-His) and PXO99ΔraxST(praxX-His).
    • Fig. S11. SRM-MS analysis of the tryptic RaxX peptide HVGGGDYPPPGANPK from PXO99(praxX-His) and PXO99ΔraxST(praxX-His).
    • Fig. S12. Expression and sulfation of RaxX60-Y and RaxX60-sY.
    • Fig. S13. Heterologously expressed, full-length RaxX60-sY activates XA21-mediated defense gene expression.
    • Fig. S14. Chemically synthesized RaxX39-sY activates XA21-mediated defense gene expression.
    • Fig. S15. Digestion of RaxX39-sY by the four site-specific proteases GluC, trypsin, ArgC, and ApsN.
    • Fig. S16. A 21–amino acid sulfated peptide derived from RaxX is sufficient to activate XA21-mediated defense gene expression and the production of ROS.
    • Fig. S17. Chemically synthesized RaxX21-sY is sufficient to activate XA21-mediated defense gene expression.
    • Fig. S18. Chemically synthesized RaxX21-sY activates gene expression in Kitaake lines expressing XA21 from its native promoter (XA21:XA21).
    • Fig. S19. Chemically synthesized RaxX21-sY activates the production of ROS in Kitaake lines expressing XA21 from its native promoter (XA21:XA21).
    • Fig. S20. ROS production dose-response curves in Ubi::XA21 leaves treated with chemically synthesized RaxX sulfated peptides.
    • Fig. S21. The tyrosine-sulfated peptide axY(S)22, derived from Ax21, does not trigger XA21-mediated immune responses in rice.
    • Fig. S22. Expression and purification of different RaxX alleles.
    • Fig. S23. Complementation of Xoo isolates IXO651, IXO685, and IXO1221 with praxX confers the ability to activate XA21-mediated immunity.
    • Fig. S24. RaxX is similar to the Arabidopsis thaliana peptide signaling factors PSY1 and to rice PSY1 homologs.
    • Table S1. Marker genes and primers used for qPCR.
    • Table S2. Plasmids used in this study.
    • Table S3. Bacterial strains used in this study.
    • References (62–67)

    Download PDF

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article