Suayib Üstün

Plastidial Thioredoxin z Interacts with Two Fructokinase-Like Proteins in a Thiol-Dependent Manner: Evidence for an Essential Role in Chloroplast Development in Arabidopsis and Nicotiana benthamiana

This study describes a thioredoxin isoform (TRX z) that is crucial in plant development and functions in regulation of the plastid-encoded RNA polymerase. It shows that at least one TRX z target is regulated by light, indicative of a protein interaction module that might link plastid transcription to light signals. Here, we characterize a plastidial thioredoxin (TRX) isoform from Arabidopsis thaliana that defines a previously unknown branch of plastidial TRXs lying between x- and y-type TRXs and thus was named TRX z. An Arabidopsis knockout mutant of TRX z had a severe albino phenotype and was inhibited in chloroplast development. Quantitative real-time RT-PCR analysis of the mutant suggested that the expressions of genes that depend on a plastid-encoded RNA polymerase (PEP) were specifically decreased. Similar results were obtained upon virus-induced gene silencing (VIGS) of the TRX z ortholog in Nicotiana benthamiana. We found that two fructokinase-like proteins (FLN1 and FLN2), members of the pfkB-carbohydrate kinase family, were potential TRX z target proteins and identified conserved Cys residues mediating the FLN–TRX z interaction. VIGS in N. benthamiana and inducible RNA interference in Arabidopsis of FLNs also led to a repression of PEP-dependent gene transcription. Remarkably, recombinant FLNs displayed no detectable sugar-phosphorylating activity, and amino acid substitutions within the predicted active site imply that the FLNs have acquired a new function, which might be regulatory rather than metabolic. We were able to show that the FLN2 redox state changes in vivo during light/dark transitions and that this change is mediated by TRX z. Taken together, our data strongly suggest an important role for TRX z and both FLNs in the regulation of PEP-dependent transcription in chloroplasts.

The Xanthomonas campestris Type III Effector XopJ Targets the Host Cell Proteasome to Suppress Salicylic-Acid Mediated Plant Defence

The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) requires type III effector proteins (T3Es) for virulence. After translocation into the host cell, T3Es are thought to interact with components of host immunity to suppress defence responses. XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Here we show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity. A C235A mutation within the catalytic triad of XopJ as well as a G2A exchange within the N-terminal myristoylation motif abolishes the ability of XopJ to inhibit the proteasome. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA) and changes in defence- and senescence-associated gene expression. Necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants with reduced expression of NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ dependent phenotypes. Our results suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence response to attenuate onset of necrosis and to alter host transcription. A central role of the proteasome in plant defence is discussed.

HopZ4 from Pseudomonas syringae, a Member of the HopZ Type III Effector Family from the YopJ Superfamily, Inhibits the Proteasome in Plants

The YopJ family of type III effector proteins (T3E) is one of the largest and most widely distributed families of effector proteins, whose members are highly diversified in virulence functions. In the present study, HopZ4, a member of the YopJ family of T3E from the cucumber pathogen Pseudomonas syringae pv. lachrymans is described. HopZ4 shares high sequence similarity with the Xanthomonas T3E XopJ, and a functional analysis suggests a conserved virulence function between these two T3E. As has previously been shown for XopJ, HopZ4 interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity during infection. The inhibitory effect on the proteasome is dependent on localization of HopZ4 to the plasma membrane as well as on an intact catalytic triad of the effector protein. Furthermore, HopZ4 is able to complement loss of XopJ in Xanthomonas spp., as it prevents precocious host cell death during a compatible Xanthomonas-pepper interaction. The data presented here suggest that different bacterial species employ inhibition of the proteasome as a virulence strategy by making use of conserved T3E from the YopJ family of bacterial effector proteins.

The Xanthomonas campestris type III effector XopJ proteolytically degrades proteasome subunit RPT6.

A type III effector from Xanthomonas acts as a protease that interferes with proteasome-mediated turnover of defense signaling components. Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities.

The proteasome acts as a hub for plant immunity and is targeted by Pseudomonas type-III effectors

The proteasome is required for local and systemic immune responses and is targeted by Pseudomonas type III effectors. Recent evidence suggests that the ubiquitin-proteasome system is involved in several aspects of plant immunity and that a range of plant pathogens subvert the ubiquitin-proteasome system to enhance their virulence. Here, we show that proteasome activity is strongly induced during basal defense in Arabidopsis (Arabidopsis thaliana). Mutant lines of the proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae pv tomato DC3000 (Pst) and Pseudomonas syringae pv maculicola ES4326. Both proteasome subunits are required for pathogen-associated molecular pattern-triggered immunity responses. Analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome also plays an important role in defense priming and SAR. In addition, we show that Pst inhibits proteasome activity in a type III secretion-dependent manner. A screen for type III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1, and HopG1 as putative proteasome inhibitors. Biochemical characterization of HopM1 by mass spectrometry indicates that HopM1 interacts with several E3 ubiquitin ligases and proteasome subunits. This supports the hypothesis that HopM1 associates with the proteasome, leading to its inhibition. Thus, the proteasome is an essential component of pathogen-associated molecular pattern-triggered immunity and SAR, which is targeted by multiple bacterial effectors.

Interactions of Xanthomonas type-III effector proteins with the plant ubiquitin and ubiquitin-like pathways.

In eukaryotes, regulated protein turnover is required during many cellular processes, including defense against pathogens. Ubiquitination and degradation of ubiquitinated proteins via the ubiquitin–proteasome system (UPS) is the main pathway for the turnover of intracellular proteins in eukaryotes. The extensive utilization of the UPS in host cells makes it an ideal pivot for the manipulation of cellular processes by pathogens. Like many other Gram-negative bacteria, Xanthomonas species secrete a suite of type-III effector proteins (T3Es) into their host cells to promote virulence. Some of these T3Es exploit the plant UPS to interfere with immunity. This review summarizes T3E examples from the genus Xanthomonas with a proven or suggested interaction with the host UPS or UPS-like systems and also discusses the apparent paradox that arises from the presence of T3Es that inhibit the UPS in general while others rely on its activity for their function.

A Proteomic Approach Suggests Unbalanced Proteasome Functioning Induced by the Growth-Promoting Bacterium Kosakonia radicincitans in Arabidopsis

Endophytic plant growth-promoting bacteria have significant impact on the plant physiology and understanding this interaction at the molecular level is of particular interest to support crop productivity and sustainable production systems. We used a proteomics approach to investigate the molecular mechanisms underlying plant growth promotion in the interaction of Kosakonia radicincitans DSM 16656 with Arabidopsis thaliana. Four weeks after the inoculation, the proteome of roots from inoculated and control plants was compared using two-dimensional gel electrophoresis and differentially abundant protein spots were identified by liquid chromatography tandem mass spectrometry. Twelve protein spots were responsive to the inoculation, with the majority of them being related to cellular stress reactions. The protein expression of 20S proteasome alpha-3 subunit was increased by the presence of K. radicincitans. Determination of proteasome activity and immuno blotting analysis for ubiquitinated proteins revealed that endophytic colonization interferes with ubiquitin-dependent protein degradation. Inoculation of rpn12a, defective in a 26S proteasome regulatory particle, enhanced the growth-promoting effect. This indicates that the plant proteasome, besides being a known target for plant pathogenic bacteria, is involved in the establishment of beneficial interactions of microorganisms with plants.

The Xanthomonas effector XopJ triggers a conditional hypersensitive response upon treatment of N. benthamiana leaves with salicylic acid

XopJ is a Xanthomonas type III effector protein that promotes bacterial virulence on susceptible pepper plants through the inhibition of the host cell proteasome and a resultant suppression of salicylic acid (SA) – dependent defense responses. We show here that Nicotiana benthamiana leaves transiently expressing XopJ display hypersensitive response (HR) –like symptoms when exogenously treated with SA. This apparent avirulence function of XopJ was further dependent on effector myristoylation as well as on an intact catalytic triad, suggesting a requirement of its enzymatic activity for HR-like symptom elicitation. The ability of XopJ to cause a HR-like symptom development upon SA treatment was lost upon silencing of SGT1 and NDR1, respectively, but was independent of EDS1 silencing, suggesting that XopJ is recognized by an R protein of the CC-NBS-LRR class. Furthermore, silencing of NPR1 abolished the elicitation of HR-like symptoms in XopJ expressing leaves after SA application. Measurement of the proteasome activity indicated that proteasome inhibition by XopJ was alleviated in the presence of SA, an effect that was not observed in NPR1 silenced plants. Our results suggest that XopJ – triggered HR-like symptoms are closely related to the virulence function of the effector and that XopJ follows a two-signal model in order to elicit a response in the non-host plant N. benthamiana.

The proteasome acts as a hub for local and systemic plant immunity in Arabidopsis thaliana and constitutes a virulence target of Pseudomonas syringae type-III effector proteins

Recent evidence suggests that the ubiquitin-proteasome system (UPS) is involved in several aspects of plant immunity and a range of plant pathogens subvert the UPS to enhance their virulence. Here, we show that proteasome activity is strongly induced during basal defense in Arabidopsis and mutant lines defective in proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae DC3000 (Pst), strains in local leaves. Both proteasome subunits are required for PTI events such as production of reactive oxygen species and mitogen-activated protein kinases signaling as well as for defense gene expression. Furthermore, analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic-acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome plays an important role in defense priming and SAR. In addition, we show that Pst inhibits proteasome activity in a type-III secretion dependent manner. A systematic screen for type-III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1 and HopG1 as candidates. Identification of proteins interacting with HopM1 by mass-spectrometry indicate that HopM1 resides in a complex together with several E3 ubiquitin ligases and proteasome subunits, supporting the hypothesis that HopM1 associates with the proteasome leading to its inhibition. We conclude that the proteasome is an essential component of the plant immune system and that some pathogens have developed a general strategy to overcome proteasome-mediated defense. One sentence summary The proteasome is required for local and systemic immune responses and is targeted by Pseudomonas type-III effectors