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Publications (154)
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...
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...
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 d...
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...
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-in...
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‐...
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 com...
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...
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 simi...
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 whol...
The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding transcription activator-like effector AvrBs3 from the bacterial pathogen Xanthomonas . Bs3 is homologous to flavin monooxygenases (FMOs), an enzyme class that has NADPH oxidase activity and can produce H 2 O 2 , a hallmark met...
Forward genetics is a powerful tool to establish phenotype-genotype correlations in virtually all areas of plant biology and has been particularly successful in the model plant Arabidopsis. This approach typically starts with a phenotype in an M2 mutant, followed by identifying a causal DNA change in F2 populations resulting from a cross between th...
The microbial population size, or load, in a host is a fundamental metric of colonization by commensals or infection by pathogens. Sequence-based measurement of DNA amount contributed by host and microbes in a sample provides a simple way of measuring microbial load, and it also has the ability to estimate microbiome diversity. Unfortunately, it is...
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 different...
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 Br...
Plant genome editing is a major advance in the production of novel plant genotypes. There is, however, only a single previous report of applying this technology to hexaploid wheat using TALEN‐mediated gene editing to produce heritable modifications (Wang et al., 2014). Here we describe highly efficient TALEN editing of a uidA transgene and a second...
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 m...
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 pr...
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 me...
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 ide...
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 functio...
Designer transcription activator-like effectors (dTALEs) are programmable transcription factors used to regulate user-defined promoters. The TALE DNA-binding domain is a tandem series of amino acid repeats that each bind one DNA base. Each repeat is 33–35 amino acids long. A residue in the center of each repeat is responsible for defining DNA base...
Significance
AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that enhances water soaking in its known hosts tomato, pepper, and Nicotiana benthamiana . We observe that the water soaking conferred by AvrHah1 is due to the movement of water into the i...
Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that...
A protein-DNA interface can be engineered in ways that an RNA-DNA interface cannot. Programmable TAL effector derived proteins bind DNA with a series of repeats. Each repeat binds a single DNA base. Repeats can be treated as modules, with a pair of residues in the center defining the target base. A set of 31-33 residues flanking the base specifying...
Xanthomonas citri subsp. 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 expression of downstream su...
Background
Although customized endonucleases [transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs)] are known to be effective agents of mutagenesis in various host plants, newly designed endonuclease constructs require some pre-validation with respect to functionality before investing in the creation of stab...
Editorial: genomics and effectomics of the crop killer [i]Xanthomonas[/i]
Xanthomonas axonopodis pv. manihotis (Xam) employs transcription activator like (TAL) effectors to promote bacterial growth and symptom formation during infection of cassava. TAL effectors are secreted via the bacterial type III secretion system into plant cells where they are directed to the nucleus, bind DNA in plant promoters, and activate the e...
Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat
structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like
proteins are known from Ralstonia solanacea...
In Arabidopsis thaliana a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. CYP71A13 is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with it...
The gene-for-gene concept has historically been applied to describe a specific resistance interaction wherein single genes from the host and the pathogen dictate the outcome. These interactions have been observed across the plant kingdom and all known plant microbial pathogens. In recent years, this concept has been extended to susceptibility pheno...
Transcription activator-like effectors (TALEs) from plant pathogenic Xanthomonas spp. and the related RipTALs from Ralstonia solanacearum are DNA-binding proteins with a modular DNA-binding domain. This domain is both predictable and programmable, which simplifies elucidation of TALE function in planta and facilitates generation of DNA-binding modu...
Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types.
While the specificity of zinc-finger nucleases (ZFNs) and RNA-guided endonucleases has been assessed to some extent, little
data are available for transcription activator-like effector-based nucleases (TALENs). Here, we have eng...
The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a gen...
Whether rice, yeast, or fly there is barely a model organism not yet reached by transcription activator like effectors (TALEs) and their derivative fusion proteins. Insights into fundamental biology are now arriving on the back of work in the last years to develop these proteins as tools for molecular biology. This began with the publication of the...
The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as prom...
Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTA...
Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription acti...
The present invention refers to methods for selectively recognizing a base pair in a DNA sequence by a polypeptide, to modified polypeptides which specifically recognize one or more base pairs in a DNA sequence and, to DNA which is modified so that it can be specifically recognized by a polypeptide and to uses of the polypeptide and DNA in specific...
Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11( hrp B ‐re g ulated 11 ), the first identified member of a class of Rips with high sequence similarity to the transcription a...
Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic
assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem
34-amino acid repeats recognize one base pair each and direct...
Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and ri...
##Assembly-Data-START## Assembly Method :: Sequencher v. 4.0 Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
##Assembly-Data-START## Assembly Method :: Sequencher v. 4.0 Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
Specific control of gene activity is a valuable tool to study and engineer cellular functions. Recent studies uncovered the
potential of transcription activator-like effector (TALE) proteins that can be tailored to activate user-defined target genes.
It remains however unclear whether and how epigenetic modifications interfere with TALE-mediated tr...
Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases
(ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription
activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to d...
Transcription activator-like effector (TALE) DNA binding proteins show tremendous potential as molecular tools for targeted
binding to any desired DNA sequence. Their DNA binding domain consists of tandem arranged repeats, and due to this repetitive
structure it is challenging to generate designer TALEs (dTALEs) with user-defined specificity. We pr...
In this study, we present a method for transient expression of the type III effector AvrGf1 from Xanthomonas citri subsp. citri strain A(w) in grapefruit leaves (Citrus paradisi) via Agrobacterium tumefaciens. The coding sequence of avrGf1 was placed under the control of the constitutive CaMV 35S promoter in the binary vectors pGWB2 and pGWB5. Infi...
Proteins that can be tailored to bind desired DNA sequences are key tools for molecular biology. Previous studies suggested that DNA-binding specificity of transcription activator-like effectors (TALEs) from the bacterial genus Xanthomonas is defined by repeat-variable diresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units. We have stud...
Transcription activator like effectors (TALEs) are injected via the type III secretion pathway of many plant pathogenic Xanthomonas spp. into plant cells where they contribute to disease or trigger resistance by binding to DNA and turning on TALE-specific host genes. Advances in our understanding of TALEs and their targets have yielded new models f...
With 1 figure and 1 table
The pepper (Capsicum annuum) disease resistance gene Bs3 mediates recognition of avrBs3-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria. We established the co-dominant DNA marker PR-Bs3 that detects a functional nucleotide polymorphism in the Bs3 promoter. Analysis of 20 F2 segregan...
*Plant pathogenic bacteria of the genus Xanthomonas inject transcription activator-like effector (TALe) proteins that bind to and activate host promoters, thereby promoting disease or inducing plant defense. TALes bind to corresponding UPT (up-regulated by TALe) promoter boxes via tandemly arranged 34/35-amino acid repeats. Recent studies uncovered...
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 s...
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, wher...
The pepper (Capsicum annuum) bacterial spot (Bs) resistance gene Bs3 and its allelic variant Bs3-E mediate recognition of the Xanthomonas campestris pv vesicatoria type III effector protein AvrBs3 and its deletion derivative AvrBs3Deltarep16. Recognition specificity resides in the Bs3 and Bs3-E promoters and is determined by a defined promoter regi...
Many phytopathogenic bacteria inject virulence effector proteins into plant cells. To identify novel virulence effectors of the bacterial plant pathogen Xanthomonas, a worldwide collection of pepper (Capsicum annuum) pathogenic Xanthomonas strains was studied. Xanthomonas gardneri strains produced in pepper enhanced watersoaking, a phenotype that i...
Plant disease resistance (R) proteins recognize matching pathogen avirulence proteins. Alleles of the pepper R gene Bs3 mediate recognition of the Xanthomonas campestris pv. vesicatoria (Xcv) type III effector protein AvrBs3 and its deletion derivative AvrBs3Δrep16. Pepper Bs3 and its allelic variant Bs3-E encode flavin monooxygenases with a previo...
Agrobacterium tumefaciens-mediated genetic transformation is an efficient tool for genetic engineering of plants. VirE2 is a single-stranded DNA binding Agrobacterium protein that is transported into the plant cell and presumably protects the T-DNA from degradation. Using a yeast two-hybrid system, we identified Arabidopsis thaliana VIRE2-INTERACTI...
Several specific problems are encountered when nonradioactive detection methods are used in conjunction with plant nucleic acids. In this chapter, we describe protocols for the isolation of DNA and RNA from plant leaves and the preparation of probe molecules by either PCR or in vitro transcription with different haptens. Furthermore, standard condi...
The pepper (Capsicum annuum) Bs3 gene confers resistance to avrBs3-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria. To physically delimit Bs3, a pepper YAC library was screened with two flanking DNA markers that are separated from Bs3 by 1.0 and 1.2 cM, respectively resulting in the identification of three Y...
Plant disease resistance (R) genes mediate specific recognition of pathogens via perception of cognate avirulence (avr) gene products. The numerous highly similar AvrBs3-like proteins from the bacterial genus Xanthomonas provide together with their corresponding R proteins a unique biological resource to dissect the molecular basis of recognition s...
Non-invasive fluorophore-based protein interaction assays like fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC, also referred to as "split YFP") have been proven invaluable tools to study protein-protein interactions in living cells. Both methods are now frequently used in the plant sciences and are...
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 plant...
