An ancient cis-element targeted by Ralstonia solanacearum TALE-like effectors facilitates the development of a promoter trap that could confer broad-spectrum wilt resistance.

Ralstonia solanacearum, a species complex of bacterial plant pathogens that causes bacterial wilt, comprises four phylotypes that evolved when a founder population was split during the continental drift ~180 million years ago. Each phylotype contains strains with RipTAL proteins structurally related to transcription activator-like (TAL) effectors from the bacterial pathogen Xanthomonas. RipTALs have evolved in geographically separated phylotypes and therefore differ in sequence and potentially functionality. Earlier work has shown that phylotype I RipTAL Brg11 targets a 17-nucleotide effector binding element (EBE) and transcriptionally activates the downstream arginine decarboxylase (ADC) gene. The predicted DNA binding preferences of Brg11 and RipTALs from other phylotypes are similar, suggesting that most, if not all, RipTALs target the Brg11-EBE motif and activate downstream ADC genes. Here we show that not only phylotype I RipTAL Brg11 but also RipTALs from other phylotypes activate host genes when preceded by the Brg11-EBE motif. Furthermore, we show that Brg11 and RipTALs from other phylotypes induce the same quantitative changes of ADC-dependent plant metabolites, suggesting that most, if not all, RipTALs induce functionally equivalent changes in host cells. Finally, we report transgenic tobacco lines in which the RipTAL-binding motif Brg11-EBE mediates RipTAL-dependent transcription of the executor-type resistance (R) gene Bs4C from pepper, thereby conferring resistance to RipTAL-delivering R. solanacearum strains. Our results suggest that cell death-inducing executor-type R genes, preceded by the RipTAL-binding motif Brg11-EBE, could be used to genetically engineer broad-spectrum bacterial wilt resistance in crop plants without any apparent fitness penalty.

Targeted knockout of a conserved plant mitochondrial gene by genome editing.

Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I. By generating a large number of TALEN-mediated mitochondrial deletion lines, we isolated, in addition to mutants with rearranged genomes, homochondriomic mutants harbouring clean nad9 deletions. Characterization of the knockout plants revealed impaired complex I biogenesis, male sterility and defects in leaf and flower development. We show that these defects can be restored by expressing a functional Nad9 protein from the nuclear genome, thus creating a synthetic cytoplasmic male sterility system. Our data (1) demonstrate the feasibility of using genome editing to study mitochondrial gene functions by reverse genetics, (2) highlight the role of complex I in plant development and (3) provide proof-of-concept for the construction of synthetic cytoplasmic male sterility systems for hybrid breeding by genome editing.

A promoter trap in transgenic citrus mediates recognition of a broad spectrum of Xanthomonas citri pv. citri TALEs, including in planta-evolved derivatives.

Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator-like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc-TALE-trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death-inducing executor gene, avrGf2, was strictly TALE-dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc-TALE-trap mediates resistance to this global panel of Xcc isolates. We also studied in planta-evolved TALEs (eTALEs) with novel DNA-binding domains and found that these eTALEs also activate the Xcc-TALE-trap, suggesting that the Xcc-TALE-trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc-TALE-trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc-TALE-trap offer a promising sustainable approach to control CBC.

The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones.

Inositol phosphates (InsPs) are ubiquitous in all eukaryotes. However, since there are 63 possible different phosphate ester isomers, the analysis of InsPs is challenging. In particular, InsP1, InsP2, and InsP3 already amass 41 different isomers, of which some occur as enantiomers. Profiling of these "lower" inositol phosphates in mammalian tissues requires powerful analytical methods and reference compounds. Here, we report an analysis of InsP2 and InsP3 with capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS). Using this method, the bacterial effector RipBL1 was analyzed and found to degrade InsP6 to Ins(1,2,3)P3, an understudied InsP3 isomer. This new reference molecule then aided us in the assignment of the isomeric identity of an InsP3 while profiling human samples: in urine and kidney stones, we describe for the first time the presence of defined and abundant InsP3 isomers, namely Ins(1,2,3)P3, Ins(1,2,6)P3 and/or Ins(2,3,4)P3.

The eINTACT system dissects bacterial exploitation of plant osmosignalling to enhance virulence.

Bacteria inject effector proteins into host cells to manipulate cellular processes that promote disease. Since bacteria deliver minuscule amounts of effectors only into targeted host cells, it is technically challenging to capture effector-dependent cellular changes from bulk-infected host tissues. Here, we report a new technique called effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), which facilitates affinity-based purification of nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors. Analysis of purified nuclei reveals that the Xanthomonas effector XopD manipulates the expression of Arabidopsis abscisic acid signalling-related genes and activates OSCA1.1, a gene encoding a calcium-permeable channel required for stomatal closure in response to osmotic stress. The loss of OSCA1.1 causes leaf wilting and reduced bacterial growth in infected leaves, suggesting that OSCA1.1 promotes host susceptibility. eINTACT allows us to uncover that XopD exploits host OSCA1.1/abscisic acid osmosignalling-mediated stomatal closure to create a humid habitat that favours bacterial growth and opens up a new avenue for accurately elucidating functions of effectors from numerous gram-negative plant bacteria in native infection contexts.

The tomato resistance gene Bs4 suppresses leaf watersoaking phenotypes induced by AvrHah1, a transcription activator-like effector from tomato-pathogenic xanthomonads.

The Xanthomonas transcription activator-like effector (TALE) protein AvrBs3 transcriptionally activates the executor-type resistance (R) gene Bs3 from pepper (Capsicum annuum), thereby triggering a hypersensitive cell death reaction (HR). AvrBs3 also triggers an HR in tomato (Solanum lycopersicum) upon recognition by the nucleotide-binding leucine-rich repeat (NLR) R protein Bs4. Whether the executor-type R protein Bs3 and the NLR-type R protein Bs4 use common or distinct signalling components to trigger an HR remains unclear. CRISPR/Cas9-mutagenesis revealed, that the immune signalling node EDS1 is required for Bs4- but not for Bs3-dependent HR, suggesting that NLR- and executor-type R proteins trigger an HR via distinct signalling pathways. CRISPR/Cas9-mutagenesis also revealed that tomato Bs4 suppresses the virulence function of both TALEs, the HR-inducing AvrBs3 protein and of AvrHah1, a TALE that does not trigger an HR in tomato. Analysis of AvrBs3- and AvrHah1-induced host transcripts and disease phenotypes in CRISPR/Cas9-induced bs4 mutant plants indicates that both TALEs target orthologous transcription factor genes to promote disease in tomato and pepper host plants. Our studies display that tomato mutants lacking the TALE-sensing Bs4 protein provide a novel platform to either uncover TALE-induced disease phenotypes or genetically dissect components of executor-triggered HR.

Targeted introduction of heritable point mutations into the plant mitochondrial genome.

The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.

TALE-induced cell death executors: an origin outside immunity?

Phytopathogenic bacteria inject effector proteins into plant host cells to promote disease. Plant resistance (R) genes encoding nucleotide-binding leucine-rich repeat (NLR) proteins mediate the recognition of functionally and structurally diverse microbial effectors, including transcription-activator like effectors (TALEs) from the bacterial genus Xanthomonas. TALEs bind to plant promoters and transcriptionally activate either disease-promoting host susceptibility (S) genes or cell death-inducing executor-type R genes. It is perplexing that plants contain TALE-perceiving executor-type R genes in addition to NLRs that also mediate the recognition of TALE-containing xanthomonads. We present recent findings on the evolvability of TALEs, which suggest that the native function of executors is not in plant immunity, but possibly in the regulation of developmentally controlled programmed cell death (PCD) processes.

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro.

The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding effector protein AvrBs3 from the bacterial pathogen Xanthomonas. Expression of Bs3 in yeast inhibited proliferation, demonstrating that Bs3 function is not restricted to the plant kingdom. The Bs3 sequence shows striking similarity to flavin monooxygenases (FMOs), an FAD- and NADPH-containing enzyme class that is known for the oxygenation of a wide range of substrates and their potential to produce H2O2. Since H2O2 is a hallmark metabolite in plant immunity, we analyzed the role of H2O2 during Bs3 HR. We purified recombinant Bs3 protein from E. coli and confirmed the FMO function of Bs3 with FAD binding and NADPH oxidase activity in vitro. Translational fusion of Bs3 to the redox reporter roGFP2 indicated that the Bs3-dependent HR induces an increase of the intracellular oxidation state in planta. To test if the NADPH oxidation and putative H2O2 production of Bs3 is sufficient to induce HR, we adapted previous studies which have uncovered mutations in the NADPH binding site of FMOs that result in higher NADPH oxidase activity. In vitro studies demonstrated that recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3. Translational fusions to roGFP2 showed that Bs3S211A also increased the intracellular oxidation state in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta, ultimately revealing that H2O2 produced by Bs3 on its own is not sufficient to trigger HR.

Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition.

The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.

Co-incidence of Damage and Microbial Patterns Controls Localized Immune Responses in Roots.

Recognition of microbe-associated molecular patterns (MAMPs) is crucial for the plant's immune response. How this sophisticated perception system can be usefully deployed in roots, continuously exposed to microbes, remains a mystery. By analyzing MAMP receptor expression and response at cellular resolution in Arabidopsis, we observed that differentiated outer cell layers show low expression of pattern-recognition receptors (PRRs) and lack MAMP responsiveness. Yet, these cells can be gated to become responsive by neighbor cell damage. Laser ablation of small cell clusters strongly upregulates PRR expression in their vicinity, and elevated receptor expression is sufficient to induce responsiveness in non-responsive cells. Finally, localized damage also leads to immune responses to otherwise non-immunogenic, beneficial bacteria. Damage-gating is overridden by receptor overexpression, which antagonizes colonization. Our findings that cellular damage can "switch on" local immune responses helps to conceptualize how MAMP perception can be used despite the presence of microbial patterns in the soil.

A Plant Pathogen Type III Effector Protein Subverts Translational Regulation to Boost Host Polyamine Levels.

Pathogenic bacteria inject effector proteins into host cells to manipulate cellular processes and facilitate the infection. Transcription-activator-like effectors (TALEs), an effector class in plant pathogenic bacteria, transcriptionally activate host genes to promote disease. We identify arginine decarboxylase (ADC) genes as the host targets of Brg11, a TALE-like effector from the plant pathogen Ralstonia solanacearum. Brg11 targets a 17-bp sequence that was found to be part of a conserved 50-bp motif, termed the ADC-box, upstream of ADC genes involved in polyamine biosynthesis. The transcribed ADC-box attenuates translation from native ADC mRNAs; however, Brg11 induces truncated ADC mRNAs lacking the ADC-box, thus bypassing this translational control. As a result, Brg11 induces elevated polyamine levels that trigger a defense reaction and likely inhibits bacterial niche competitors but not R. solanacearum. Our findings suggest that Brg11 may give R. solanacearum a competitive advantage and uncover a role for bacterial effectors in regulating ternary microbe-host-microbe interactions.

Cell Death Triggered by the YUCCA-like Bs3 Protein Coincides with Accumulation of Salicylic Acid and Pipecolic Acid But Not of Indole-3-Acetic Acid.

The pepper (Capsicum annuum) resistance gene bacterial spot3 (Bs3) is transcriptionally activated by the matching Xanthomonas euvesicatoria transcription-activator-like effector (TALE) AvrBs3. AvrBs3-induced Bs3 expression triggers a rapid and local cell death reaction, the hypersensitive response (HR). Bs3 is most closely related to plant flavin monooxygenases of the YUCCA (YUC) family, which catalyze the final step in auxin biosynthesis. Targeted mutagenesis of predicted NADPH- and FAD-cofactor sites resulted in Bs3 derivatives that no longer trigger HR, thereby suggesting that the enzymatic activity of Bs3 is crucial to Bs3-triggered HR. Domain swap experiments between pepper Bs3 and Arabidopsis (Arabidopsis thaliana) YUC8 uncovered functionally exchangeable and functionally distinct regions in both proteins, which is in agreement with a model whereby Bs3 evolved from an ancestral YUC gene. Mass spectrometric measurements revealed that expression of YUCs, but not expression of Bs3, coincides with an increase in auxin levels, suggesting that Bs3 and YUCs, despite their sequence similarity, catalyze distinct enzymatic reactions. Finally, we found that expression of Bs3 coincides with increased levels of the salicylic acid and pipecolic acid, two compounds that are involved in systemic acquired resistance.

Microbial effectors and the role of water and sugar in the infection battle ground.

Phytopathogenic microbes multiply in the apoplast-a plant's intercellular spaces-of infected plants, and hence their success relies on the conditions in this habitat. Despite being extracellular parasites, most microbes translocate effectors into host cells that promote disease by acting inside cells. Initial studies suggested that effectors act predominantly as suppressors of plant immunity. These pioneering studies were trend-setting, causing a strong bias in the functional investigation of effectors. Yet, recent studies on bacterial model pathogens have identified effectors that promote disease by causing either increased sugar or water levels in the apoplast. These studies are likely to initiate a new era of effector research that will clarify the disease-promoting rather than defense-suppressing function of effectors, a molecular rather than genetic distinction.

Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effectors among X. citri strains.

BACKGROUND: Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains. RESULTS: We utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains. CONCLUSIONS: We demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods.

A modular toolbox for Golden-Gate-based plasmid assembly streamlines the generation of Ralstonia solanacearum species complex knockout strains and multi-cassette complementation constructs.

Members of the Ralstonia solanacearum species complex (Rssc) cause bacterial wilt, a devastating plant disease that affects numerous economically important crops. Like other bacterial pests, Rssc injects a cocktail of effector proteins via the bacterial type III secretion system into host cells that collectively promote disease. Given their functional relevance in disease, the identification of Rssc effectors and the investigation of their in planta function are likely to provide clues on how to generate pest-resistant crop plants. Accordingly, molecular analysis of effector function is a focus of Rssc research. The elucidation of effector function requires corresponding gene knockout strains or strains that express the desired effector variants. The cloning of DNA constructs that facilitate the generation of such strains has hindered the investigation of Rssc effectors. To overcome these limitations, we have designed, generated and functionally validated a toolkit consisting of DNA modules that can be assembled via Golden-Gate (GG) cloning into either desired gene knockout constructs or multi-cassette expression constructs. The Ralstonia-GG-kit is compatible with a previously established toolkit that facilitates the generation of DNA constructs for in planta expression. Accordingly, cloned modules, encoding effectors of interest, can be transferred to vectors for expression in Rssc strains and plant cells. As many effector genes have been cloned in the past as GATEWAY entry vectors, we have also established a conversion vector that allows the implementation of GATEWAY entry vectors into the Ralstonia-GG-kit. In summary, the Ralstonia-GG-kit provides a valuable tool for the genetic investigation of genes encoding effectors and other Rssc genes.

Metabolomics of tomato xylem sap during bacterial wilt reveals Ralstonia solanacearum produces abundant putrescine, a metabolite that accelerates wilt disease.

Ralstonia solanacearum thrives in plant xylem vessels and causes bacterial wilt disease despite the low nutrient content of xylem sap. We found that R. solanacearum manipulates its host to increase nutrients in tomato xylem sap, enabling it to grow better in sap from infected plants than in sap from healthy plants. Untargeted GC/MS metabolomics identified 22 metabolites enriched in R. solanacearum-infected sap. Eight of these could serve as sole carbon or nitrogen sources for R. solanacearum. Putrescine, a polyamine that is not a sole carbon or nitrogen source for R. solanacearum, was enriched 76-fold to 37 µM in R. solanacearum-infected sap. R. solanacearum synthesized putrescine via a SpeC ornithine decarboxylase. A ΔspeC mutant required ≥ 15 µM exogenous putrescine to grow and could not grow alone in xylem even when plants were treated with putrescine. However, co-inoculation with wildtype rescued ΔspeC growth, indicating R. solanacearum produced and exported putrescine to xylem sap. Intriguingly, treating plants with putrescine before inoculation accelerated wilt symptom development and R. solanacearum growth and systemic spread. Xylem putrescine concentration was unchanged in putrescine-treated plants, so the exogenous putrescine likely accelerated disease indirectly by affecting host physiology. These results indicate that putrescine is a pathogen-produced virulence metabolite.

TALE-induced bHLH transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato.

AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix-loop-helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1 By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions.

An engineered promoter driving expression of a microbial avirulence gene confers recognition of TAL effectors and reduces growth of diverse Xanthomonas strains in citrus.

Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs31EBE ), and fused this engineered promoter with multiple EBEs (ProBs314EBE ) to either the ß-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs31EBE and ProBs314EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs314EBE by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs314EBE . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants.