Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.

Summary• Pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) depends on a type III secretion system that translocates a cocktail of > 25 type III effector proteins into the plant cell.• In this study, we identified the effector AvrBsT as a suppressor of specific plant defense. AvrBsT belongs to the YopJ ⁄ AvrRxv protein family, members of which are predicted to act as proteases and ⁄ or acetyltransferases.• AvrBsT suppresses the hypersensitive response (HR) that is elicited by the effector protein AvrBs1 from Xcv in resistant pepper plants. HR suppression occurs inside the plant cell and depends on a conserved predicted catalytic residue of AvrBsT. Yeast two-hybrid based analyses identified plant interaction partners of AvrBs1 and AvrBsT, including a putative regulator of sugar metabolism, SNF1-related kinase 1 (SnRK1), as interactor of AvrBsT. Intriguingly, gene silencing experiments revealed that SnRK1 is required for the induction of the AvrBs1-specific HR.• We therefore speculate that SnRK1 is involved in the AvrBsT-mediated suppression of the AvrBs1-specific HR.

Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es) directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance (R) genes or R proteins and induces effector triggered immunity (ETI) that often results in programmed cell death reactions. The identification of R genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86 Solanaceae lines mainly of the genus Nicotiana were screened for phenotypical reactions after Agrobacterium tumefaciens-mediated transient expression of 21 different Xcv effectors to (i) identify new plant lines for T3E characterization, (ii) analyze conservation/evolution of putative R genes and (iii) identify promising plant lines as repertoire for R gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential R genes. In some cases, putative R genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several Nicotiana spp. lines, and Xcv infection assays revealed that XopQ is a host range determinant in many Nicotiana species. Non-host resistance against Xcv and XopQ recognition in N. benthamiana required EDS1, strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing RxopQ as candidate for targeted crop improvement.

Summary 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.

Plants must continually balance the influx of CO 2 for photosynthesis against the loss of water vapor through stomatal pores in their leaves. This balance can be achieved by controlling the aperture of the stomatal pores in response to several environmental stimuli. Elevation in atmospheric [CO 2 ] induces stomatal closure and further impacts leaf temperatures, plant growth and water-use efficiency, and global crop productivity. Here, we review recent advances in understanding CO 2 -perception mechanisms and CO 2 -mediated signal transduction in the regulation of stomatal movements, and we explore how these mechanisms are integrated with other signaling pathways in guard cells.

Summary• The oomycete Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato. The importance of vesicle fusion processes and callose deposition for defense of potato against Phytophthora infestans was analyzed.• Transgenic plants were generated, which express RNA interference constructs targeted against plasma membrane-localized SYNTAXIN-RELATED 1 (StSYR1) and SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FACTOR ADAPTOR PROTEIN 33 (StSNAP33), the potato homologs of Arabidopsis AtSYP121 and AtSNAP33, respectively.• Phenotypically, transgenic plants grew normally, but showed spontaneous necrosis and chlorosis formation at later stages. In response to infection with Phytophthora infestans, increased resistance of StSYR1-RNAi plants, but not StSNAP33-RNAi plants, was observed. This increased resistance correlated with the constitutive accumulation of salicylic acid and PR1 transcripts. Aberrant callose deposition in Phytophthora infestans-infected StSYR1-RNAi plants coincided with decreased papilla formation at penetration sites. Resistance against the necrotrophic fungus Botrytis cinerea was not significantly altered. Infiltration experiments with bacterial solutions of Agrobacterium tumefaciens and Escherichia coli revealed a hypersensitive phenotype of both types of RNAi lines.• The enhanced defense status and the reduced growth of Phytophthora infestans on StSYR1-RNAi plants suggest an involvement of syntaxins in secretory defense responses of potato and, in particular, in the formation of callose-containing papillae.

Signaling networks are at the heart of almost all biological processes. Most of these networks contain a large number of components, and often the connections between these components are either not known or the rate equations that govern the dynamics of soluble signaling components are not quantified. This uncertainty in network topology and parameters can make it challenging to formulate detailed mathematical models. Boolean networks, in which all components are either on or off, have emerged as viable alternatives to detailed mathematical models that contain rate constants and other parameters. Therefore, open source platforms of Boolean models for community use are desirable. Here we present Boolink, a freely available graphical user interface (GUI) that allows users to easily construct and analyze existing Boolean networks. Boolink can be applied to any Boolean network. We demonstrate its application using a previously published network for abscisic acid-driven stomatal closure in Arabidopsis (Arabidopsis thaliana). We also show how Boolink can be used to generate testable predictions by extending the network to include CO2 regulation of stomatal movements. Predictions of the model were experimentally tested and the model was iteratively modified based on experiments showing that abscisic acid effectively closes Arabidopsis stomata at near-zero CO2 concentrations (1.5 ppm CO2). Thus, Boolink enables public generation and use of existing Boolean models, including the prior developed ABA signaling model with added CO2 signaling components.

Summary Low concentrations of CO2 cause stomatal opening, whereas [CO2] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+ and protein phosphorylation in CO2‐induced stomatal closing. Calcium‐dependent protein kinases (CPKs) and calcineurin‐B‐like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+ into specific phosphorylation events. However, Ca2+‐binding proteins that function in the stomatal CO2 response remain unknown. Time‐resolved stomatal conductance measurements using intact plants, and guard cell patch‐clamp experiments were performed. We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO2, light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO2‐induced stomatal closure and, unexpectedly, also in low CO2‐induced stomatal opening. Furthermore, K+‐uptake‐channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light‐mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO2‐regulated stomatal movement kinetics were not clearly affected in plasma membrane‐targeted cbl1/4/5/8/9 quintuple mutant plants. Our findings describe combinatorial cpk mutants that function in CO2 control of stomatal movements and support the results of classical studies showing a role for Ca2+ in this response.