A highly conserved single-stranded DNA-binding protein (SSB) triggers a HR in tobacco.

<p>(A) HR induction by the SSB_Xoc protein of <i>X. oryzae</i> pv. <i>oryzicola</i>. <i>A. tumefaciens</i> GV3101 (OD₆₀₀ = 0.5) containing <i>hpa1</i>, <i>ssb_X</i> and <i>bax</i> genes in the PVX vector pgR107 was inoculated into <i>N. benthamiana</i> tobacco leaves with a needleless syringe. Hpa1 and Bax were used as positive controls, and <i>A. tumefaciens</i> containing the empty vector PVX was used as a negative control. (B) Concentration of SSB_Xoc required for HR induction in tobacco cv. Xanthi. Purified SSB_Xoc was diluted in PBS buffer and inoculated into tobacco with needleless syringes. Hpa1, which functions as a harpin of <i>X. oryzae</i> pv. <i>oryzicola</i>, was used as a positive control and EVP as a negative control. (C) HR assays in tobacco inoculated with SSB_X homologues obtained from various bacterial species. SSB proteins were overproduced in <i>E. coli</i>, purified (see Methods), and diluted in PBS buffer to different concentrations from 0.01 to 50 µM. A typical image of HRs on tobacco leaves caused by the proteins at 1 µM was taken in this report. Numbers represent sections of leaves inoculated with the following: 1, EVP; 2, SSB_Xoc from <i>X. oryzae</i> pv. <i>oryzicola</i> RS105; 3, SSB_Xoo from <i>X. oryzae</i> pv. <i>oryzae</i> PXO99^A; 4, SSB_Xac from <i>X. axonopodis</i> pv. <i>citri</i> 306; 5, SSB_Xcv from <i>X. campestris</i> pv. <i>vesicatoria</i> 85-10; 6, SSB_Xcc from <i>X. campestris</i> pv. <i>campestris</i> 8004; 7, SSB_Pf from <i>P. fluorescens</i> Pf-5; 8, Hpa1_Xoc, from <i>X. oryzae</i> pv. <i>oryzicola</i> RS105; 9, SSB_Ea from <i>E. amylovora</i> 0065; 10, SSB_Ec from <i>E. coli</i> BL21 (DES); 11, SSB_Rs from <i>R. solanacearum</i> ZJ2731; and 12, SSB_Pst from <i>P. syringae pv. tomato</i> DC3000. (D) Assays for SSBx and Hpa1-induced HR in response to various metabolic inhibitors. Tobacco plants were infiltrated with SSB_X (1 µM) or Hpa1 (0.5 µM), which was heat-treated or incubated (see methods) with one of the following: 1 µM LaCl₃, 0.71 µM actinomycin D, 0.1 µM cycloheximide or protease K (0.5 U/ml). Leaf panels: 1, Hpa1; 2, heat-treated Hpa1; 3, protease K-treated Hpa1; 4, Hpa1 plus 1 µM LaCl₃; 5, Hpa1 plus 0.71 µM actinomycin D; 6, Hpa1 plus 0.1 µM cycloheximide; 7, SSB_X; 8, heat-treated SSB_Xoc; 9, protease K-treated SSB_Xoc; 10, SSB_Xoc plus LaCl₃; 11, SSB_Xoc plus 0.1 µM actinomycin D; 12, SSB_Xoc plus cycloheximide; 13, distilled water; and 14, EVP. Leaves in panels A to D were photographed 24–48 h after infiltration. (E) Analysis of DNA laddering in SSB_Xoc-treated tobacco leaves. Total genomic DNA was isolated from tobacco leaves 3, 6, 12, 24, 36 and 48 hpi with Hpa1 (0.5 µM), SSB_Xoc (1 µM) and EVP. DNA laddering was evaluated in 2% agarose gels. (F) Northern blot analysis in tobacco inoculated with SSB_X, Hpa1, or EVP. The marker genes, <i>HIN1</i>, <i>HSR203J</i>, <i>PR1a</i> and <i>PR1b</i>, were chosen as the targets. Total RNAs were extracted from tobacco leaves infiltrated with SSB_Xoc (1 µM), Hpa1 (0.5 µM), or PBS buffer. Aliquots (10 µg each) of the extracted RNAs were separated in 1% agarose gels, transferred onto membranes, and analyzed by northern blotting. Blots were hybridized with digoxigenin-labeled <i>HIN1</i>, <i>HSR203J</i>, <i>PR1a</i> and <i>PR1b</i> cDNA. The experiment was conducted twice with similar results.