Promoter variants of <i>Xa23</i> alleles affect bacterial blight resistance and evolutionary pattern

<div><p>Bacterial blight, caused by <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> (<i>Xoo</i>), is the most important bacterial disease in rice (<i>Oryza sativa</i> L.). Our previous studies have revealed that the bacterial blight resistance gene <i>Xa23</i> from wild rice <i>O</i>. <i>rufipogon</i> Griff. confers the broadest-spectrum resistance against all the naturally occurring <i>Xoo</i> races. As a novel executor <i>R</i> gene, <i>Xa23</i> is transcriptionally activated by the bacterial avirulence (Avr) protein AvrXa23 via binding to a 28-bp DNA element (<i>EBE</i>_AvrXa23) in the promoter region. So far, the evolutionary mechanism of <i>Xa23</i> remains to be illustrated. Here, a rice germplasm collection of 97 accessions, including 29 rice cultivars (<i>indica</i> and <i>japonica</i>) and 68 wild relatives, was used to analyze the evolution, phylogeographic relationship and association of <i>Xa23</i> alleles with bacterial blight resistance. All the ~ 473 bp DNA fragments consisting of promoter and coding regions of <i>Xa23</i> alleles in the germplasm accessions were PCR-amplified and sequenced, and nine single nucleotide polymorphisms (SNPs) were detected in the promoter regions (~131 bp sequence upstream from the start codon ATG) of <i>Xa23/xa23</i> alleles while only two SNPs were found in the coding regions. The SNPs in the promoter regions formed 5 haplotypes (<i>Pro-A</i>, <i>B</i>, <i>C</i>, <i>D</i>, <i>E</i>) which showed no significant difference in geographic distribution among these 97 rice accessions. However, haplotype association analysis indicated that <i>Pro-A</i> is the most favored haplotype for bacterial blight resistance. Moreover, SNP changes among the 5 haplotypes mostly located in the <i>EBE/ebe</i> regions (<i>EBE</i>_AvrXa23 and corresponding <i>ebes</i> located in promoters of <i>xa23</i> alleles), confirming that the <i>EBE</i> region is the key factor to confer bacterial blight resistance by altering gene expression. Polymorphism analysis and neutral test implied that <i>Xa23</i> had undergone a bottleneck effect, and selection process of <i>Xa23</i> was not detected in cultivated rice. In addition, the <i>Xa23</i> coding region was found highly conserved in the Oryza genus but absent in other plant species by searching the plant database, suggesting that <i>Xa23</i> originated along with the diversification of the Oryza genus from the grass family during evolution. This research offers a potential for flexible use of novel <i>Xa23</i> alleles in rice breeding programs and provide a model for evolution analysis of other executor <i>R</i> genes.</p>