Recognition of AvrBs3-Like Proteins Is Mediated by Specific Binding to Promoters of Matching Pepper Bs3 Alleles

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Abstract
The pepper (Capsicum annuum) bacterial spot (Bs) resistance gene Bs3 and its allelic variant Bs3-E mediate recognition of the Xanthomonas campestris pv vesicatoria type III effector protein AvrBs3 and its deletion derivative AvrBs3Δrep16. Recognition specificity resides in the Bs3 and Bs3-E promoters and is determined by a defined promoter region, the UPA (for up-regulated by AvrBs3) box. Using site-directed mutagenesis, we defined the exact boundaries of the UPAAvrBs3 box of the Bs3 promoter and the UPAAvrBs3Δrep16 box of the Bs3-E promoter and show that both boxes overlap by at least 11 nucleotides. Despite partial sequence identity, the UPAAvrBs3 box and the UPAAvrBs3Δrep16 box were bound specifically by the corresponding AvrBs3 and AvrBs3Δrep16 proteins, respectively, suggesting that selective promoter binding of AvrBs3-like proteins is the basis for promoter activation specificity. We also demonstrate that the UPAAvrBs3 box retains its functionality at different positions within the pepper Bs3 promoter and confers AvrBs3 inducibility in a novel promoter context. Notably, the transfer of the UPAAvrBs3 box to different promoter locations is always correlated with a new transcriptional start site. The analysis of naturally occurring Bs3 alleles revealed many pepper accessions that encode a nonfunctional Bs3 variant. These accessions showed no apparent abnormalities, supporting the supposition that Bs3 functions only in disease resistance and not in other developmental or physiological processes.