Expression levels of avrBs3-like genes affect recognition specificity in tomato Bs4- but not in pepper Bs3-mediated perception

Abstract

The tomato Bs4 disease resistance gene mediates recognition of avrBs4-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria to give a hypersensitive response (HR). Here, we present the characterization of the Bs4 promoter and its application for low-level expression of bacterial type III effector proteins in planta. Real-time polymerase chain reaction showed that Bs4 is constitutively expressed at low levels and that transcript abundance does not change significantly upon infection with avrBs4-containing xanthomonads. A 302-bp promoter fragment was found to be sufficient to promote Bs4 gene function. Previous studies have shown that high, constitutive in planta expression of avrBs3 (AvrBs3 and AvrBs4 proteins are 96.6% identical) via the Cauliflower mosaic virus 35S (35S) promoter triggers a Bs4-dependent HR whereas X. campestris pv. vesicatoria-mediated delivery of AvrBs3 into the plant cytoplasm does not. Here, we demonstrate that, when expressed under control of the weak Bs4 promoter, avrBs3 does not trigger a Bs4-dependent HR whereas avrBs4 does. In contrast, the pepper Bs3 gene, which mediates recognition of AvrBs3- but not AvrBs4-delivering xanthomonads, retains its recognition specificity even if avrBs4 was expressed in planta from the strong 35S promoter. Importantly, Bs4 promoter-driven expression of hax3, hax4 (two recently isolated avrBs3-like genes), avrBs3, and avrBs4 resulted in identical reactions as observed upon infection with X. campestris pv. vesicatoria strains that express the respective avr gene, suggesting that the protein levels expressed under control of the Bs4 promoter are similar to those that are translocated by the bacterial type III secretion system.