A single plant resistance gene promoter engineered to recognize multiple TAL effectors from disparate pathogens

Abstract

Plant pathogenic bacteria of the genus Xanthomonas inject transcription-activator like (TAL) effector proteins that manipulate the hosts' transcriptome to promote disease. However, in some cases plants take advantage of this mechanism to trigger defense responses. For example, transcription of the pepper Bs3 and rice Xa27 resistance (R) genes are specifically activated by the respective TAL effectors AvrBs3 from Xanthomonas campestris pv. vesicatoria (Xcv), and AvrXa27 from X. oryzae pv. oryzae (Xoo). Recognition of AvrBs3 was shown to be mediated by interaction with the corresponding UPT (UPregulated by TAL effectors) box UPT(AvrBs3) present in the promoter R gene Bs3 from the dicot pepper. In contrast, it was not known how the Xoo TAL effector AvrXa27 transcriptionally activates the matching R gene Xa27 from the monocot rice. Here we identified a 16-bp UPT(AvrXa27) box present in the rice Xa27 promoter that when transferred into the Bs3 promoter confers AvrXa27-dependent inducibility. We demonstrate that polymorphisms between the UPT(AvrXa27) box of the AvrXa27-inducible Xa27 promoter and the corresponding region of the noninducible xa27 promoter account for their distinct inducibility and affinity, with respect to AvrXa27. Moreover, we demonstrate that three functionally distinct UPT boxes targeted by separate TAL effectors retain their function and specificity when combined into one promoter. Given that many economically important xanthomonads deliver multiple TAL effectors, the engineering of R genes capable of recognizing multiple TAL effectors provides a potential approach for engineering broad spectrum and durable disease resistance.

Fig. 1.

AvrXa27 activates the rice Xa27 promoter and triggers a Bs3-dependent HR in N. benthamiana. (A) Graphical representation of constructs used for promoter analysis. The Bs3, Xa27, xa27, and the xa27_mut promoters are displayed as yellow, brown, and orange arrows, respectively. A yellow box represents the Bs3 cds. The UPT_AvrBs3, and UPT_AvrXa27 boxes are shown as blue and black boxes, respectively. A black box with a white bar represents the UPT_AvrXa27* box of the xa27 promoter. Black arrows represent transcription start sites (TSS). The scale indicates distances from the ATG start codon. Numbers below the arrows denote the distances between the 3′ end of a UPT box and its TSS. The Bs3 cds is not drawn to scale. (B) The promoter-Bs3 cds and the 35S promoter-driven avr constructs (avrXa27 [Left] or avrBs3 [Right]) were expressed transiently in N. benthamiana leaves via A. tumefaciens. Asterisks (*) mark areas in which the avr construct but no promoter construct was infiltrated. Dashed lines mark the inoculated areas. Four days after infiltration, the leaves were cleared with ethanol to visualize the HR (dark areas).

Fig. 2.

A 16-bp fragment of the rice Xa27 promoter mediates transcriptional activation via AvrXa27. (A) Graphical representation of the analyzed promoter constructs. Yellow arrows and boxes represent the Bs3 promoter and cds, respectively. The UPT_AvrBs3 and UPT_AvrXa27 boxes are shown as blue and black boxes, respectively. A black box with a white bar marks the UPT_AvrXa27* box of the xa27 promoter. Letters on yellow background represent Bs3 promoter sequence. The asterisk (*) marks the position where Xa27/xa27 promoter sequences were inserted. Bold-face black letters mark nucleotide polymorphisms between Xa27 and xa27. Red letters mark the predicted TATA box. The gray background highlights the minimal UPT_AvrXa27 box. “+” and “−” indicates the presence or absence of an HR in N. benthamiana upon codelivery of 35S promoter-driven avrXa27 gene. (B) In planta analysis of UPT_AvrXa27 box deletion constructs. Promoter constructs were delivered together with a 35S promoter-driven avrXa27 gene via A. tumefaciens into N. benthamiana leaves. Asterisks mark areas where only the avr but no R promoter constructs were infiltrated. Dashed lines mark the inoculated areas. Four days after inoculation, the leaves were harvested and cleared with ethanol to visualize the HR (dark areas).

Fig. 3.

AvrXa27 binds specifically to the Xa27 promoter. (A) Probes derived from Xa27, xa27, and Bs3 promoters used in EMSAs. Numbering is relative to the transcription start site (TSS). The polymorphisms between the Xa27 and xa27 promoter are shown by white letters and black background. The UPT_AvrXa27 box of the Xa27 and the UPT_AvrBs3 box of the Bs3 promoter are underlined (B) AvrXa27 binds with high and low affinity to the Xa27 and xa27 promoters, respectively. EMSA with AvrXa27 and Xa27- or xa27-derived probes and competition experiment between AvrXa27 and different amounts (in fmol) of a nonlabeled competitor probe in a 6% nondenaturing polyacrylamide gel. Positions of the bound and free probe are indicated on the left. (C) AvrXa27 and AvrBs3 bind specifically to the Xa27 and Bs3 promoters, respectively. EMSA with AvrBs3 and AvrXa27 and Xa27- and Bs3-derived probes in a 6% nondenaturing polyacrylamide gel. Protein amounts are in fmol. Positions of the bound and free probe are indicated on the left.

Fig. 4.

Tandemly-arranged UPT boxes with distinct TAL effector specificity retain their function when combined into a single promoter. (A) Graphical representation of promoter constructs. Yellow arrows and boxes represent the promoters and cds, respectively of the Bs3 and Bs3-E genes. Bs3 and Bs3-E promoters differ only in their UPT boxes and are therefore displayed in identical color. Green, brown, and orange color represents the promoters (arrows) and cds (boxes) of the tomato Bs4, rice Xa27, and rice xa27 genes, respectively. Pink, black and blue boxes represent the UPT_{AvrBs3Δrep16}, UPT_AvrXa27, and UPT_AvrBs3 boxes, respectively. The black box with the white vertical line represents the nonfunctional UPT_AvrXa27* box of the rice xa27 gene. With exception of the Bs3 cds all elements are drawn to scale. (B) In planta analysis of the promoter constructs. The promoter constructs depicted in (A) were codelivered together with 35S promoter-driven avr genes into N. benthamiana leaves (displayed in boldface letters above each leaf) via A. tumefaciens. Asterisks (*) mark areas in which A. tumefaciens containing only a TAL effector gene were infiltrated. Dashed lines mark the inoculated areas. Four days after inoculation, the leaves were harvested and cleared with ethanol to visualize the HR (dark areas).