Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity.

The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into plant cells by a type III secretion system and interfere with cellular processes to the benefit of the pathogen. In this study, we analyzed eight T3Es from Xcv strain 85-10, six of which were newly identified effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress pathogen-associated molecular pattern (PAMP)-triggered plant defense gene expression. In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing defense responses related to both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in the suppression of ETI-related responses, but still interfered with vesicle trafficking and was only slightly affected with regard to the suppression of defense gene induction. This suggests that XopB-mediated suppression of PTI and ETI is dependent on different mechanisms that can be functionally separated.

Breaking the code of DNA binding specificity of TAL-type III effectors.

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.

Detailed analysis of the DNA recognition motifs of the Xanthomonas type III effectors AvrBs3 and AvrBs3Deltarep16.

The Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) employs a type III secretion system to translocate effector proteins into plant cells where they modulate host signaling pathways to the pathogen's benefit. The effector protein AvrBs3 acts as a eukaryotic transcription factor and induces the expression of plant genes termed UPA (up-regulated by AvrBs3). Here, we describe 11 new UPA genes from bell pepper that are induced by AvrBs3 early after infection with Xcv. Sequence comparisons revealed the presence of a conserved AvrBs3-responsive element, the UPA box, in all UPA gene promoters analyzed. Analyses of UPA box mutant derivatives confirmed its importance for gene induction by AvrBs3. We show that DNA binding and gene activation were strictly correlated. DNase I footprint studies demonstrated that the UPA box corresponds to the center of the AvrBs3-protected DNA region. Type III delivery of AvrBs3 and mutant derivatives showed that some UPA genes are induced by the AvrBs3 deletion derivative AvrBs3Deltarep16, which lacks four repeats. We show that AvrBs3Deltarep16 recognizes a mutated UPA box with two nucleotide exchanges in positions that are not essential for binding and activation by AvrBs3.

How Xanthomonas type III effectors manipulate the host plant.

Pathogenicity of Xanthomonas and most other Gram-negative phytopathogenic bacteria depends on a conserved type III secretion (T3S) system which injects more than 25 different effector proteins into the plant cell. Extensive studies in the last years on the molecular mechanisms of type III effector function revealed that effector proteins with enzymatic functions seem to play important roles in the interaction of Xanthomonas with its host plants, for example, the SUMO protease XopD. In addition, xanthomonads express a unique class of type III effectors to pursue another strategy. Effectors of the AvrBs3 family, so far only identified in Xanthomonas spp. and Ralstonia solanacearum, mimic plant transcriptional activators and manipulate the plant transcriptome.

A bacterial effector acts as a plant transcription factor and induces a cell size regulator.

Pathogenicity of many Gram-negative bacteria relies on the injection of effector proteins by type III secretion into eukaryotic cells, where they modulate host signaling pathways to the pathogen's benefit. One such effector protein injected by Xanthomonas into plants is AvrBs3, which localizes to the plant cell nucleus and causes hypertrophy of plant mesophyll cells. We show that AvrBs3 induces the expression of a master regulator of cell size, upa20, which encodes a transcription factor containing a basic helix-loop-helix domain. AvrBs3 binds to a conserved element in the upa20 promoter via its central repeat region and induces gene expression through its activation domain. Thus, AvrBs3 and likely other members of this family provoke developmental reprogramming of host cells by mimicking eukaryotic transcription factors.

Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence.

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.

Characterization of AvrBs3-like effectors from a Brassicaceae pathogen reveals virulence and avirulence activities and a protein with a novel repeat architecture.

Xanthomonas campestris pv. armoraciae strain 5 is a Brassicaceae pathogen that expresses three members of the highly related avrBs3 gene family of type III effectors. Here, we report on the isolation and characterization of these genes, designated hax2, hax3, and hax4 (homolog of avrBs3 in Xanthomonas). All three Hax proteins are translocated from Xanthomonas spp. into the plant cell via the type III secretion system. Hax3 and Hax4 show the typical structure of AvrBs3-like effectors and contain a repetitive region in their central part consisting of 34-amino-acid (aa) repeats. By contrast, the Hax2 repeat region is composed of 35-aa repeats that are characterized by an additional proline residue. Hax2, Hax3, and Hax4 contain 21.5, 11.5, and 14.5 repeats, respectively. Genetic studies revealed an additive effect of hax2, hax3, and hax4 on disease symptom formation of X. campestris pv. armoraciae strain 5 on radish. The contribution of individual genes to the aggressiveness of strain 5 is quantitatively different, with hax2 showing the strongest effect on the development of chlorosis and necrosis. In addition, hax3 and hax4, but not hax2, have a Bs4-dependent avirulence activity in tomato and in transgenic Nicotiana benthamiana expressing the Bs4 resistance gene.