The pathogenicity of many bacteria depends on the injection of effector proteins via type III secretion into eukaryotic cells in order to manipulate cellular processes. TAL (transcription activator-like) effectors from plant pathogenic Xanthomonas are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind to DNA via a central domain of tandem repeats. Here, we show how target DNA specificity of TAL effectors is encoded. Two hypervariable amino acid residues in each repeat recognize one base pair in the target DNA. Recognition sequences of TAL effectors were predicted and experimentally confirmed. The modular protein architecture enabled the construction of artificial effectors with new specificities. Our study describes the functionality of a distinct type of DNA binding domain and allows the design of DNA binding domains for biotechnology.

https://science.sciencemag.org/content/sci/early/2009/10/29/science.1178811.full.pdf

Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors

▪ Abstract In “gene-for-gene” interactions between plants and their pathogens, incompatibility (no disease) requires a dominant or semidominant resistance (R) gene in the plant, and a corresponding avirulence (Avr) gene in the pathogen. Many plant/pathogen interactions are of this type. R genes are presumed to (a) enable plants to detect Avr-gene-specified pathogen molecules, (b) initiate signal transduction to activate defenses, and (c) have the capacity to evolve new R gene specificities rapidly. Isolation of R genes has revealed four main classes of R gene sequences whose products appear to activate a similar range of defense mechanisms. Discovery of the structure of R genes and R gene loci provides insight into R gene function and evolution, and should lead to novel strategies for disease control.

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Plant Disease Resistance Genes

Xanthomonads are bacterial plant pathogens that cause diseases on many plant species, including important crops. Key to pathogenicity of most Xanthomonas pathovars is a Hrp-type III secretion (T3S) system that translocates effector proteins into plant cells. Within the eukaryotic cell, the effectors are thought to perform a variety of tasks to support bacterial virulence, proliferation, and dissemination. We are only beginning to understand the host targets of different effectors. The largest effector family found in Xanthomonas spp. is the AvrBs3/PthA or TAL (transcription activator-like) family. TAL effectors act as transcriptional activators in the plant cell nucleus. Specificity of TAL effectors is determined by a novel modular DNA-binding domain. Here, we describe the discovery of TAL effectors and their structure, activity, and host targets.

Xanthomonas AvrBs3 Family-Type III Effectors: Discovery and Function

Type III secretion systems allow Yersinia spp., Salmonella spp., Shigella spp., Bordetella spp., and Pseudomonas aeruginosa and enteropathogenic Escherichia coli adhering at the surface of a eukaryotic cell to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to destroy or subvert the target cell. These systems consist of a secretion apparatus, made of approximately 25 proteins, and an array of proteins released by this apparatus. Some of these released proteins are "effectors," which are delivered into the cytosol of the target cell, whereas the others are "translocators," which help the effectors to cross the membrane of the eukaryotic cell. Most of the effectors act on the cytoskeleton or on intracellular-signaling cascades. A protein injected by the enteropathogenic E. coli serves as a membrane receptor for the docking of the bacterium itself at the surface of the cell. Type III secretion systems also occur in plant pathogens where they are involved both in causing disease in susceptible hosts and in eliciting the so-called hypersensitive response in resistant or nonhost plants. They consist of 15-20 Hrp proteins building a secretion apparatus and two groups of effectors: harpins and avirulence proteins. Harpins are presumably secreted in the extracellular compartment, whereas avirulence proteins are thought to be targeted into plant cells. Although a coherent picture is clearly emerging, basic questions remain to be answered. In particular, little is known about how the type III apparatus fits together to deliver proteins in animal cells. It is even more mysterious for plant cells where a thick wall has to be crossed. In spite of these haunting questions, type III secretion appears as a fascinating trans-kingdom communication device.

Assembly and function of type iii secretory systems

SUMMARY Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola cause bacterial blight and bacterial leaf streak of rice (Oryza sativa), which constrain production of this staple crop in much of Asia and parts of Africa. Tremendous progress has been made in characterizing the diseases and breeding for resistance. X. oryzae pv. oryzae causes bacterial blight by invading the vascular tissue, while X. oryzae pv. oryzicola causes bacterial leaf streak by colonizing the parenchyma. In rice there are 29 major genes for resistance to bacterial blight, but so far only a few quantitative resistance loci for bacterial leaf streak. Over 30 races of X. oryzae pv. oryzae have been reported. Both pathogens exhibit genetic variation among isolates. Mechanisms of pathogenesis and resistance have begun to be elucidated. Members of the AvrBs3/PthA family of transcription activator-like effectors play a major role in the virulence of X. oryzae pv. oryzae and possibly X. oryzae pv. oryzicola. Cloning of six rice resistance genes for bacterial blight and one from maize effective against bacterial leaf streak has uncovered a diversity of structure and function, some shared by genes involved in defence in animals. This article reviews research that spans a century. It also presents a perspective on challenges for sustainable control, and opportunities that interactions of X. oryzae pathovars with rice present as models for understanding fundamental aspects of bacterial pathogenesis of plants and plant disease resistance, as well as other aspects of plant and microbial biology, with implications also for animal innate immunity.

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Xanthomonas oryzae pathovars: model pathogens of a model crop.

ELUCIDATION of the genetic and molecular basis of plant disease resistance is a major objective in the investigation of plant-micro-bial interactions. Xanthomonas campestris pathovar vesicatoria (Xcv), the causal agent of bacterial spot disease of pepper and tomato, has been developed as a model host–pathogen system to study the genetic interactions that specify the expression of plant disease resistance1–6. Several plant resistance genes (Bsl, Bs2, Bs3) have been genetically characterized from pepper (Capsicum annuum) that determine resistance to particular races of the pathogen carrying specific avirulence genes7–9. For example, pepper plants carrying the resistance locus Bs3 are resistant to Xcv strains expressing the avirulence gene avrBs3. Nucleotide sequence analysis of the avrBs3 gene revealed that the internal portion of the predicted protein product consists of a nearly identical 34 amino acid repeat unit, present in 17.5 copies4. We report here that the repetitive region of the avrBs3 gene determines race-specificity and that deletions of repeat units generate new avirulence specificities and unmask undiscovered resistance genes in pepper and tomato.

Race-specificity of plant resistance to bacterial spot disease determined by repetitive motifs in a bacterial avirulence protein

Xanthomonas campestris pathovar (pv.) vesicatoria (Xcv), a gram negative bacterium, is the causal agent of bacterial spot disease of pepper and tomato. After infection of a plant with Xcv two different types of reactions can be observed. If the plant is susceptible, the infection gives rise to watersoaked lesions (compatible interaction). In a resistant plant a hypersensitive response (incompatible interaction) is induced. The hypersensitive response (HR) is a local defense reaction accompanied by a rapid necrosis of the infected tissue (Klement and Goodman 1967). Such an incompatible interaction requires the presence of a resistance locus in the particular cultivar of pepper or tomato and a corresponding avirulence locus in the particular race of the pathogen (Minsavage et al. 1990).

Karin Herbers

Papers

Race-specificity of plant resistance to bacterial spot disease determined by repetitive motifs in a bacterial avirulence protein

Characterization of Genes from Xanthomas Campestris pv. Vesicatoria That Determine Avirulence and Pathogenicity on Pepper and Tomato