Unique characteristics of Xanthomonas oryzae pv. oryzae AvrXa21 and implications for plant innate immunity

Lee et al. 10.1073/pnas.0605508103.

Supporting Information

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Supporting Figure 4
Supporting Table 2
Supporting Table 3

Supporting Figure 4

Fig. 4. AvrXa21 activity upon protease and heat treatment. Cell-free supernatants from the wild-type Xoo strain were treated with proteinase K, trypsin, or heat (95°C for 15 min), and the activity of the treated supernatants was measured in the AvrXa21 bioassay. Cell-free supernatants (50 ml) were concentrated to 25 ml by using evaporation and then mixed with 25 ml of the appropriate buffered protease solution for a final protease concentration of 0.1 mg/ml. The reaction mixtures were incubated at 37°C for 2 h and used in the AvrXa21 bioassay with Xa21 rice plants. For heat denaturation, the cell-free supernatant was placed in 95°C water bath for 15 min, transferred to room temperature to cool, and then tested with the AvrXa21 bioassay. Protease and heat denaturation treatments were combined by first heat-treating the wild-type cell-free supernatant and then treating with proteases as described above. Untreated cell-free supernatants from wild-type (avrXa21⁺) and raxST¯ (raxST¯) strains were used as a controls. The data are plotted as means (bar) and SDs from >10 scored leaves of two repeats.

Table 3. Primer sets for this study

Gene	Primer	Primer sequence	Amplification region, length
raxST	raxST1-F	5′-ACCATTTCATTTCCGGGCT-3′	62 to 291st, 230 bp
	raxST1-R	5′-CAGCTCCTGGCGATTCTGG-3′	62 to 291st, 230 bp
	raxST2-F	5′-GGGCACGGTGTTCGACACCA-3′	292 to 520th, 229 bp
	raxST2-R	5′-CACGCCCCGCGGCGCGGTCA-3′	292 to 520th, 229 bp
	raxST3-F	5′-CTGCGCCAGGCGTTCTACGG-3′	601 to 838th, 238 bp
	raxST3-R	5′-GCGAAGGCGCGCTTTCCCAG-3′	601 to 838th, 238 bp
raxP	raxP1-F	5′-GACCCTGCCGCCGCTGTCCC-3′	3 to 286th, 284 bp
	raxP1-R	5′-CGCCATCAGGATTGATGTGC-3′	3 to 286th, 284 bp
	raxP2-F	5′-ATCGACACACGCTGGAAGTT-3′	187 to 523rd, 337 bp
	raxP2-R	5′-TGCGCGCGTTGTAGAGAT-3′	187 to 523rd, 337 bp
	raxP3-F	5′-GTGGAATCTCTACAACGCGC-3′	501 to 800th, 300 bp
	raxP3-R	5′-GCTTCCAGCGTGTCGGCACTG-3′	501 to 800th, 300 bp
raxC	raxC1-F	5′-GCCACCGACCTGCTGCAGGT-3′	61 to 409th, 349 bp
	raxC1-R	5′-GAAATACGCCGCCGAGGTAC-3′	61 to 409th, 349 bp
	raxC2-F	5′-CCAACAACGATCTGATGACG-3′	368 to 654th, 287 bp
	raxC2-R	5′-CAATGCACGCAAACCAACTA-3′	368 to 654th, 287 bp
	raxC3-F	5′-TACGAGCAGCAGAAGCGCGC-3′	1021 to 1306th, 285 bp
	raxC3-R	5′-CGGTGATGTCGAGCTTGCCG-3′	1021 to 1306th, 285 bp
raxR	raxR1-F	5′-CGGCTGCTGGTCATTGAAGAC-3′	4 to 337th, 334 bp
	raxR1-R	5′-CTCGGGCAGTGCAAAGGG-3′	4 to 337th, 334 bp
	raxR2-F	5′-GCGCCGGCTGCGCGAACAGCAC-3′	195 to 515th, 321 bp
	raxR2-R	5′-GCATCAGCACTTCGAGCAAC-3′	195 to 515th, 321 bp
	raxR3-F	5′-GCCGCAAGTTGCTCGAAGT-3′	488 to 707th, 220 bp
	raxR3-R	5′-CATACAGCTGCGCCGGTAG-3′	488 to 707th, 220 bp
16S rRNA	rRNA-F	5′-TAGCTCAGGTGGTTAGAGCGC-3′
	rRNA-R	5′-CAACGCGAACATACGACTCAA-3′