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Two coiled-coil regions of Xanthomonas oryzae pv. oryzae harpin differ in oligomerization and hypersensitive response induction

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Abstract

Hpa1Xoo (harpin) is a type III secreted protein of the rice blight bacterial pathogen Xanthomonas oryzae pv. oryzae that elicits a hypersensitive response (HR) in nonhost tobacco. Hpa1Xoo is predicted to contain two potential coiled-coil (CC) regions, one at the N-terminus with a high probability of formation, and one at the C-terminus with a lower probability of formation. We constructed several CC-equivalent peptides by a chemosynthetic method, and investigated the structure–function of the predicted Hpa1Xoo CC regions, using biophysical and biochemical approaches. Both peptides elicited an HR in tobacco. Mutant versions of the N- and C-terminal peptides that were predicted to disrupt or favor CC formation were generated. The resulting altered HR activity and oligomerization indicated that the N-terminal CC region is essential for eliciting HR, but the C-terminus is not. The results also indicate that a 14-residue fragment (LDQLLCQLISALLQ) within the N-terminal CC region is a minimal and independent functional element for HR-induction in tobacco leaves. We propose that HR-induction requires a specific oligomerization of the CC regions of Hpa1Xoo.

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Acknowledgments

This work was supported by grants from the National Key Basic Research Plan of China (2003CB114204 and 2006CB101902), the National Key project of China (2004BA901A36), and the Key Project of Science and Technology of Jiangsu (BE-2005-604).

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Authors and Affiliations

  1. Department of Plant Pathology, Nanjing Agricultural University, Weigang 1, Nanjing, 210095, People’s Republic of China

    Zhaolin Ji, Congfeng Song & Jinsheng Wang

  2. Rice Research Institute, Anhui Academy of Agricultural Sciences, Nongke South Road 40, Hefei, 230031, People’s Republic of China

    Xuzhong Lu

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  1. Zhaolin Ji
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  2. Congfeng Song
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  3. Xuzhong Lu
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  4. Jinsheng Wang
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Corresponding author

Correspondence to Jinsheng Wang.

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726_2010_643_MOESM1_ESM.tif

Supplemental Fig. S1. HR induced by gradients of N14 and C21-1 on tobacco leaves. a, HR 5 d after N14 infiltration. 1, HarpinXoo (positive control); 2, H2O (negative control); 3, N14 with 7.0 μM; 4, 13.9 μM; 5, 27.8 μM; 6, 37.1 μM; 7, 55.6μM; 8, 111.3 μM. The concentration of 27.8 μM elicited weak HR on tobacco leaves. b, HR 24 hours after C21-1 infiltration. 1, C21-1 with 10.7 μM; 2, 21.3 μM; 3, 42.6 μM; 4, H2O (negative control); 5, HarpinXoo (positive control). Weak HR was seen at a minimum C21-1 concentration of 21.3 μM (TIFF 6.28 mb)

726_2010_643_MOESM2_ESM.tif

Supplemental Fig. S2. Tobacco leaf with HR 24 h after infiltration of high concentration of aqueous WC14, N14-L1S and C21-2. 1, H2O (negative control); 2, WC14 (520.3 μM); 3, N14-L1S (568.4 μM); 4, C21-2 (412.5 μM); 5, HarpinXoo (positive control) (TIFF 1917 kb)

726_2010_643_MOESM3_ESM.tif

Supplemental Fig. S3. Structural analysis of synthetic peptides N14, N14-L1S, C21-1, C21-2, and WC14 by CD, SEC and AUC. A, N14 and mutant N14-L1S from the N-terminal α-helical region of Hpa1Xoo. B,C21-1 and mutant C21-2 from the C-terminal α-helical region of Hpa1Xoo. C, peptide WC14. CDspectra were recorded at 20°C from 190 to 260 nm. A Superdex-30 HiLoad 16/60 prepgrade columnwas used in SEC for the peptides, details are given in “Materials and methods”. Data from AUC ofWC14 were analyzed by the continuous distribution (c(s)) method using the SedFit program. Aqueous solutions of peptides N14 (55.6 μM), N14-L1S (56.8 μM), C21-1 (42.6 μM), C21-2 (41.3μM), and WC14 (26.0 μM) were used for analysis (TIFF 1484 kb)

726_2010_643_MOESM4_ESM.tif

Supplemental Fig. S4. Tobacco leaf with HR 5 d after gradient infiltration of aqueous Hpa1Xoo-N21. 1, HarpinXoo(positive control); 2, Hpa1Xoo-N21 aqueous solution with 21.1 μM; 3, 10.6 μM; 4, 4.2 μM ; 5, 2.8μM; 6, 2.1 μM; 7, H2O (negative control) (TIFF 3133 kb)

726_2010_643_MOESM5_ESM.tif

Supplemental Fig. S5. Structure analysis of synthetic peptides Hpa1Xoo-N21. a, CD spectrum of Hpa1Xoo-N21 at20°C. b, molecular weight analytical profile of Hpa1Xoo-N21 fitted by AUC data using thecontinuous distribution (c(s)) method using the SedFit program. c, SEC elution profile ofHpa1Xoo-N21 using a Superdex-30 HiLoad 16/60 prepgrade column (TIFF 715 kb)

726_2010_643_MOESM6_ESM.tif

Supplemental Fig. S6. CD analysis of 21-residue peptides from the N-terminal α-helix of Hpa1 from Xanthomonas.a, CD spectra of peptides Hpa1Xoo-N21 (thick and black line), Hpa1Xoc-N21 (thin and black line),HpaGXag-N21 (thick and gray line), XopAXcv-N21 (thin and gray line), and Hpa1Xcc-N21 (dottedline). b, thermal unfolding curve of peptide Hpa1Xoc-N21. Circles show the melting curve, and theblack dashed line gives its fitted line. c and d, melting curves and the corresponding first-orderderivatives of peptides HpaGXag-N21 and Hpa1Xoo-N21. Triangles, melting curve of HpaGXag-N21;squares, melting curve of Hpa1Xoo-N21; black dashed line, fitted line; black or gray line, firstderivative. Peptides in aqueous solutions were monitored by the [θ]222, continuously from 20 to100°C at a scan rate of 1°C/min. Peptides were used at 42.3 μM for Hpa1Xoo-N21, 36.7 μM forHpa1Xoc-N21, 33.3 μM for HpaGXag-N21, 37.4 μM for Hpa1Xcc-N21, and 36.4 μM for XopAXcv-N21 (TIFF 1622 kb)

726_2010_643_MOESM7_ESM.tif

Supplemental Fig. S7. Circular dichroism spectrum of peptide N14-L1S in 50% TFE. N14-L1S was at 63.16 μM. N14-L1S in 50% TFE showed 35.8% α-helix, 27.9% β-sheet, no turn and 36.2% random coil. (TIFF 332 kb)

726_2010_643_MOESM8_ESM.tif

Supplemental Fig. S8. Tobacco leaf HR induced by N14-L1S in 2%-4% TFE. 1, H2O (negative control); 2,N14-L1S (15.16 μM) in 2% TFE; 3, N14-L1S (18.95 μM) in 2.5% TFE; 4, N14-L1S (25.26 μM) in3.3% TFE; 5, N14-L1S (30.31 μM) in 4% TFE; 6, HarpinXoo (positive control); 7, 2% TFE; 8, 2.5%TFE; 9, 3.3% TFE; 10, 4% TFE. TFE at 2% did not induce tobacco cell death. Peptide N14-LIS in2% TFE elicited HR in tobacco leaves. Photograph taken 5 d after infiltration (TIFF 4250 kb)

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Ji, Z., Song, C., Lu, X. et al. Two coiled-coil regions of Xanthomonas oryzae pv. oryzae harpin differ in oligomerization and hypersensitive response induction. Amino Acids 40, 381–392 (2011). https://doi.org/10.1007/s00726-010-0643-y

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