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Science 326 (5954): 850-853

Copyright © 2009 by the American Association for the Advancement of Science

A Type I–Secreted, Sulfated Peptide Triggers XA21-Mediated Innate Immunity

Sang-Won Lee*, Sang-Wook Han*, Malinee Sririyanum, Chang-Jin Park, Young-Su Seo, and Pamela C. Ronald{dagger}

Department of Plant Pathology, University of California, Davis, CA 95616, USA.


Figure 1
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  		Fig. 1. Isolation of Ax21. (A) RP-HPLC elution profile of peptides secreted from Xoo strain PXO99 (carrying Ax21 activity). Peptide-enriched samples from the PXO99 supernatant were separated on a reversed-phase C18 column (1 x 250 mm, flow rate 0.05 ml/min) with a 10 to 90% acetonitrile gradient containing 0.1% trifluoroacetic acid. A280, absorbance at 280 nm. (B) Lesion length measurements of XA21 rice leaves pretreated with RP-HPLC fractions followed by inoculation with PXO99{Delta}raxST. Lesion lengths were measured 12 days after PXO99{Delta}raxST inoculation. Each value is the mean ± SD from nine inoculated leaves. (C) Deduced amino acid sequence of Ax21. The two peptides (boxed) identified from the biologically active fraction were sequenced using LC-MS/MS. Predicted sulfated tyrosines Y22 and Y144 are underlined. The dashed box indicates one of the peptide used in the Ax21 bioassay shown in Fig. 3. (D) Mass (LTQ) spectrum of the axY22 peptide corresponding to the N-terminal region [first box in (C)] of Ax21. The spectrum corresponding to the peptide derived from the C-terminal region [second box in (C)] of Ax21 is shown in fig. S1.

 

Figure 2
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  		Fig. 2. A mutation in ax21 abolishes Ax21 activity. (A) Lesion lengths of rice leaves measured 12 days after inoculation with Xoo strains PXO99, PXO99{Delta}raxST, or PXO99{Delta}ax21. Suspensions of each strain [108 colony-forming units (CFU)/ml] were scissor-inoculated onto rice leaves (TP309-XA21, resistant to PXO99; TP309, susceptible to PXO99). Images are representative of five independent experiments. (B) Growth of PXO99, PXO99{Delta}raxST, and PXO99{Delta}ax21 populations in inoculated rice leaves. Bacteria were extracted from the leaves at 0, 3, 6, 9, and 12 days after inoculation, plated on selective media after serial dilution, and colonies counted after a 3-day incubation at 28°C. Each value is the mean ± SD from nine inoculated leaves.

 

Figure 3
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  		Fig. 3. The axYS22 peptide is sufficient to trigger XA21-mediated immunity. (A) Synthetic peptides, including three corresponding to the N-terminal region of AX21 (axYS22, axY22, and axY22A), three corresponding to the central region (axYS144, axY144, and axY144A), and one corresponding to the C-terminal region (axM178), were tested for activity. Abbreviations for amino acid residues: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. (B) Five hours after peptide pretreatment, leaves were inoculated with PXO99{Delta}raxST and the lesions measured 12 days later. Each value is the mean ± SD from six leaves. (C) Growth of PXO99{Delta}raxST populations over time. TP309-XA21 leaves were pretreated with PXO99 supernatant (PXO99sup), water, or the synthetic peptides (axYS22 and axY22, 100 µM each). Bacterial cells were extracted from the leaves at 0, 5, 10, and 15 days after inoculation, plated on selective media after serial dilution, and colonies counted after a 3-day incubation at 28°C. Each value is the mean ± SD from eight inoculated leaves.

 

Figure 4
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  		Fig. 4. XA21 is required for axYS22 binding. HA-tagged axYS22 cross-links to a 140-kD polypeptide that is immunoprecipitated by an antibody to Myc (Myc-XA21). (A) Before immunoprecipitation, the loading of equal amounts of protein (50 µg) from Kitaake and Myc-XA21 leaf extracts was confirmed using an antibody to actin (input). (B) Leaf extracts were incubated with 1 mM HA-axYS22 in the presence (+, 5 mM; ++, 10 mM) or absence (–) of the competitors axYS22 lacking the HA tag or flg22ave. After binding, cross-linking was initiated by the addition of sulfo-Ethylene Glycol bis (Succinimidyl Succinate). Duplicate protein gels were analyzed after separation by SDS-PAGE using antibodies to Myc (top) and to HA (bottom). Myc-XA21 and a proteolytic cleavage product of Myc-XA21 were detected at 140 and 110 kD, respectively, as reported previously (32). Arrows indicate the XA21 and Ax21-XA21 complexes.

 

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