A single plant resistance gene promoter engineered to recognize multiple TAL effectors from disparate pathogens Patrick Ro¨ mer, Sabine Recht, and Thomas Lahaye1 Institute of Biology, Department of Genetics, Martin-Luther-University Halle-Wittenberg, D-06099 Halle (Saale), Germany Edited by Jeffery L. Dangl, University of North Carolina, Chapel Hill, NC, and approved October 2, 2009 (received for review August 6, 2009) Plant pathogenic bacteria of the genus Xanthomonas inject tran- factor UPA20, which induces hypertrophy (i.e., enlargement) of scription-activator like (TAL) effector proteins that manipulate the mesophyll cells, as well as to the promoters of other host genes hosts’ transcriptome to promote disease. However, in some cases that appear to contribute to susceptibility (14). In addition, plants take advantage of this mechanism to trigger defense re- AvrBs3 triggers a programmed cell death response, referred to sponses. For example, transcription of the pepper Bs3 and rice Xa27 as the hypersensitive response (HR), in pepper plants that resistance (R) genes are specifically activated by the respective TAL contain the cognate R gene Bs3 (15, 16). Certain pepper lines effectors AvrBs3 from Xanthomonas campestris pv. vesicatoria have an allele of Bs3 known as Bs3-E, which confers resistance (Xcv), and AvrXa27 from X. oryzae pv. oryzae (Xoo). Recognition of to strains carrying the AvrBs3 derivative AvrBs3⌬rep16 that has AvrBs3 was shown to be mediated by interaction with the corre- a deletion of repeat units 11–14 (15, 17). AvrBs3 and sponding UPT (UPregulated by TAL effectors) box UPTAvrBs3 present AvrBs3⌬rep16 were found to interact specifically with distinct in the promoter R gene Bs3 from the dicot pepper. In contrast, it boxes in the Bs3 and Bs3-E promoters, respectively (16). For was not known how the Xoo TAL effector AvrXa27 transcription- clarity we herein refer to these binding sites collectively as UPT ally activates the matching R gene Xa27 from the monocot rice. (UPregulated by TAL effectors), with a subscript designation for Here we identified a 16-bp UPTAvrXa27 box present in the rice Xa27 the specific TAL effector that targets it. Interaction of AvrBs3 promoter that when transferred into the Bs3 promoter confers or AvrBs3⌬rep16 with their respective UPT sites, UPTAvrBs3, and AvrXa27-dependent inducibility. We demonstrate that polymor- UPTAvrBs3⌬rep16, induces transcription of the Bs3 or Bs3-E coding phisms between the UPTAvrXa27 box of the AvrXa27-inducible Xa27 sequence (cds), leading to HR. Previously we analyzed multiple promoter and the corresponding region of the noninducible xa27 in vitro generated UPTAvrBs3 box mutants and uncovered three promoter account for their distinct inducibility and affinity, with AvrBs3⌬rep16 inducible box derivatives (16). Notably, the rec- respect to AvrXa27. Moreover, we demonstrate that three func- ognition specificity of the mutated boxes always correlated with tionally distinct UPT boxes targeted by separate TAL effectors a loss of the AvrBs3 mediated inducibility and did not result in retain their function and specificity when combined into one UPT boxes with dual recognition specificity. promoter. Given that many economically important xan- Transcription of the rice R gene Xa27 is specifically induced thomonads deliver multiple TAL effectors, the engineering of R by AvrXa27, a TAL effector from X. oryzae pv. oryzae (Xoo) (18). genes capable of recognizing multiple TAL effectors provides a The products of rice Xa27 and pepper Bs3 share no sequence potential approach for engineering broad spectrum and durable homology. However, transcriptional activation of these R genes disease resistance. by their matching TAL effectors suggests that this mechanism of disease resistance is common to both mono- and dicotyledonous AvrBs3 ͉ AvrXa27 ͉ transcription-activator like effector proteins ͉ plant species. Xanthomonas In the present study, we wanted to clarify if activation of the rice Xa27 promoter by the matching Xoo TAL effector AvrXa27 is lant pathogens are a major threat to crop production world- mechanistically similar to activation of the pepper Bs3 promoter by Pwide and durable disease resistance is a major goal in plant the Xcv TAL effector AvrBs3. Furthermore we test if UPT boxes biotechnology (1–3). A key to achieving durable disease resis- matching distinct TAL effectors retain their specificity and func- tance is to elucidate the function of effector proteins that various tionality when combined into one complex promoter. microbial pathogens (bacteria, fungi, oomycetes, and nema- todes) secrete into their hosts (4, 5). Although the primary Results function of microbial effectors is in virulence, some are known The Pepper Bs3 and Rice Xa27 Resistance Genes Use Identical Mech- to trigger plant resistance (R) gene mediated resistance and have anisms for Detection of Their Matching TAL Effectors. Previously we therefore been termed avirulence (Avr) proteins. For example, showed that AvrBs3 binds to the pepper Bs3 promoter, resulting AvrBs3 from the bacterial phytopathogen Xanthomonas campes- in transcriptional activation of the Bs3 cds and subsequent tris pv. vesicatoria (Xcv) confers both virulence (6) and aviru- triggering of HR (15, 16). In the current study, we investigated lence (7). AvrBs3-like proteins have been identified in many if the combination of the Xcv AvrBs3 protein and the pepper Bs3 pathovars of Xanthomonas (8) and Ralstonia solanacearum (9). promoter can be functionally substituted by the Xoo AvrXa27 Owing to their homology to eukaryotic transcription factors, protein and the rice Xa27 promoter to facilitate Bs3 gene AvrBs3-like proteins are also termed transcription-activator like (TAL) effectors (10–12). A characteristic central domain of TAL effectors is comprised of a variable number of tandemly- Author contributions: P.R. and T.L. designed research; P.R. and S.R. performed research; and arranged, near-perfect copies of a 34/35-amino acid motif and P.R. and T.L. wrote the paper. determines virulence and avirulence specificity (8). In addition, The authors declare no conflict of interest. TAL effector proteins generally contain C-terminal nuclear This article is a PNAS Direct Submission. localization signals (NLSs) and an acidic transcriptional activa- Freely available online through the PNAS open access option. tion domain (AAD) (8, 11–13). The repeat domain of the TAL 1To whom correspondence should be addressed. E-mail: [email protected]. effector AvrBs3 interacts with a corresponding UPA (UPregu- This article contains supporting information online at www.pnas.org/cgi/content/full/ lated by AvrBs3) box in the promoter of the pepper transcription 0908812106/DCSupplemental. 20526–20531 ͉ PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908812106 Downloaded by guest on September 26, 2021 The promoters of the pepper Bs3 and Bs3-E genes differ by a single 13-bp insertion-deletion (InDel) polymorphism that de- termines the recognitional specificity of the R genes (Fig. S1) (15). In contrast, the 1.5 kb putative promoter regions of the rice Xa27 and the xa27 alleles differ by 16 polymorphisms (Figs. S2 and S3). Because the Bs3/Bs3-E InDel is located in the vicinity of the TATA box (Fig. S1), we hypothesized that the functionally relevant Xa27/xa27 promoter polymorphism might have a similar location. Using site-directed mutagenesis, we replaced the poly- morphic nucleotides adjacent to the TATA box (one 3-bp InDel polymorphism and an adenine to cytosine substitution) (Fig. S2) in the xa27 promoter by the corresponding nucleotides from the Xa27 promoter. The mutated xa27 promoter (xa27mut) was fused to the Bs3 cds and the transgene coexpressed with avrXa27 or avrBs3. The xa27mut promoter construct triggered HR in N. benthamiana when codelivered with avrXa27, but not when codelivered with avrBs3 (Fig. 1B). This indicates that the xa27mut promoter is functionally identical to the Xa27 promoter and that the specificity-determinant in the Xa27/xa27 promoter is located adjacent to the predicted TATA box. Furthermore, these ob- servations suggest that the xa27 promoter region containing the polymorphisms constitutes the UPTAvrXa27 box. The UPTAvrXa27 Box from the Rice Xa27 Promoter Retains Its Function in the Context of the Pepper Bs3 Promoter. Previously we delimited the UPTAvrBs3 box of the Bs3 promoter to an interval of 18 nucleotides and showed that the box retains its function if placed Fig. 1. AvrXa27 activates the rice Xa27 promoter and triggers a Bs3- dependent HR in N. benthamiana.(A) Graphical representation of constructs at different positions within the promoters of the pepper Bs3 or tomato Bs4 genes (16). Here, we wanted to test if the UPT used for promoter analysis. The Bs3, Xa27, xa27, and the xa27mut promoters AvrXa27 are displayed as yellow, brown, and orange arrows, respectively. A yellow box box from the Xa27 promoter retains its function when trans- represents the Bs3 cds. The UPTAvrBs3, and UPTAvrXa27 boxes are shown as blue ferred into another promoter context. To this end, we introduced and black boxes, respectively. A black box with a white bar represents the the regions encompassing the UPTAvrXa27 box of Xa27 or the UPTAvrXa27* box of the xa27 promoter. Black arrows represent transcription UPTAvrXa27* box of xa27 into the Bs3 promoter and inserted these start sites (TSS). The scale indicates distances from the ATG start codon. sequences in front of the Bs3 cds (Fig. 2A). The former transgene Numbers below the arrows denote the distances between the 3Ј end of a UPT (Xa27/Bs3 [UPTAvrXa27 box of Xa27 in Bs3 promoter]) mediated box and its TSS. The Bs3 cds is not drawn to scale. (B) The promoter-Bs3 cds and HR in N. benthamiana when cotransformed with the avrXa27 the 35S promoter-driven avr constructs (avrXa27 [Left]oravrBs3 [Right]) were expressed transiently in N. benthamiana leaves via A. tumefaciens. Asterisks gene (Fig. 2B). In contrast, cotransformation with the other (*) mark areas in which the avr construct but no promoter construct was construct containing the UPTAvrXa27* box of xa27 in the Bs3 infiltrated.