BackgroundRalstonia solanacearum is a soil-borne beta-proteobacterium that causes bacterial wilt disease in many food crops and is a major problem for agriculture in intertropical regions. R. solanacearum is a heterogeneous species, both phenotypically and genetically, and is considered as a species complex. Pathogenicity of R. solanacearum relies on the Type III secretion system that injects Type III effector (T3E) proteins into plant cells. T3E collectively perturb host cell processes and modulate plant immunity to enable bacterial infection.ResultsWe provide the catalogue of T3E in the R. solanacearum species complex, as well as candidates in newly sequenced strains. 94 T3E orthologous groups were defined on phylogenetic bases and ordered using a uniform nomenclature. This curated T3E catalog is available on a public website and a bioinformatic pipeline has been designed to rapidly predict T3E genes in newly sequenced strains. Systematical analyses were performed to detect lateral T3E gene transfer events and identify T3E genes under positive selection. Our analyses also pinpoint the RipF translocon proteins as major discriminating determinants among the phylogenetic lineages.ConclusionsEstablishment of T3E repertoires in strains representatives of the R. solanacearum biodiversity allowed determining a set of 22 T3E present in all the strains but provided no clues on host specificity determinants. The definition of a standardized nomenclature and the optimization of predictive tools will pave the way to understanding how variation of these repertoires is correlated to the diversification of this species complex and how they contribute to the different strain pathotypes.

Calcium-dependent protein kinases (CDPKs) comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule. So far, a biological function in abiotic stress signaling has only been reported for few CDPK isoforms, whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown. Here, we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress. Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation. In transgenic Arabidopsis complementation lines in the cpk21 mutant background, in which either CPK21 wild-type, or a full-length enzyme variant carrying an amino-acid substitution were stably expressed, stress responsitivity was restored by CPK21 but not with the kinase inactive variant. The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain, N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity, suggesting a crucial role for the N-terminal EF-hand pair. Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspensioncultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca 2؉ channels. Inhibition of the oxidative response impaired Cl ؊ efflux, K ؉ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the crosslinking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.

The subversion of plant cellular functions is essential for bacterial pathogens to proliferate in host plants and cause disease. Most bacterial plant pathogens employ a type III secretion system to inject type III effector (T3E) proteins inside plant cells, where they contribute to the pathogen-induced alteration of plant physiology. In this work, we found that the Ralstonia solanacearum T3E RipAY suppresses plant immune responses triggered by bacterial elicitors and by the phytohormone salicylic acid. Further biochemical analysis indicated that RipAY associates in planta with thioredoxins from Nicotiana benthamiana and Arabidopsis. Interestingly, RipAY displays γ-glutamyl cyclotransferase (GGCT) activity to degrade glutathione in plant cells, which is required for the reported suppression of immune responses. Given the importance of thioredoxins and glutathione as major redox regulators in eukaryotic cells, RipAY activity may constitute a novel and powerful virulence strategy employed by R. solanacearum to suppress immune responses and potentially alter general redox signalling in host cells.

In addition to the linear electron flow, a cyclic electron flow (CEF) around photosystem I occurs in chloroplasts. In CEF, electrons flow back from the donor site of photosystem I to the plastoquinone pool via two main routes: one that involves the Proton Gradient Regulation5 (PGR5)/PGRL1 complex (PGR) and one that is dependent of the NADH dehydrogenase-like complex. While the importance of CEF in photosynthesis and photoprotection has been clearly established, little is known about its regulation. We worked on the assumption of a redox regulation and surveyed the putative role of chloroplastic thioredoxins (TRX). Using Arabidopsis (Arabidopsis thaliana) mutants lacking different TRX isoforms, we demonstrated in vivo that TRXm4 specifically plays a role in the down-regulation of the NADH dehydrogenase-like complex-dependent plastoquinone reduction pathway. This result was confirmed in tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. In vitro assays performed with isolated chloroplasts and purified TRXm4 indicated that TRXm4 negatively controls the PGR pathway as well. The physiological significance of this regulation was investigated under steady-state photosynthesis and in the pgr5 mutant background. Lack of TRXm4 reversed the growth phenotype of the pgr5 mutant, but it did not compensate for the impaired photosynthesis and photoinhibition sensitivity. This suggests that the physiological role of TRXm4 occurs in vivo via a mechanism distinct from direct up-regulation of CEF.

The plant pathogen Ralstonia solanacearum possesses two genes encoding a trehalose-6-phosphate synthase (TPS), an enzyme of the trehalose biosynthetic pathway. One of these genes, named ripTPS, was found to encode a protein with an additional N-terminal domain which directs its translocation into host plant cells through the type 3 secretion system. RipTPS is a conserved effector in the R. solanacearum species complex, and homologues were also detected in other bacterial plant pathogens. Functional analysis of RipTPS demonstrated that this type 3 effector synthesizes trehalose-6-phosphate and identified residues essential for this enzymatic activity. Although trehalose-6-phosphate is a key signal molecule in plants that regulates sugar status and carbon assimilation, the disruption of ripTPS did not alter the virulence of R. solanacearum on plants. However, heterologous expression assays showed that this effector specifically elicits a hypersensitive-like response on tobacco that is independent of its enzymatic activity and is triggered by the C-terminal half of the protein. Recognition of this effector by the plant immune system is suggestive of a role during the infectious process.

SummaryHypoosmotic stress activates a phosphorylationdependent oxidative burst. In-gel kinase assays were performed to characterize the protein kinases that could be implicated in osmoregulation and in the activation of the oxidative burst. Hypoosmotic stress activated several kinases among which 50 and 46 kDa proteins displayed mitogen-activated protein kinase (MAP kinase) properties. They phosphorylated myelin basic protein in the absence of calcium, were recognized by antibodies directed against human MAP kinases, and were phosphorylated on tyrosine. Immunoprecipitation with an antibody directed against the tobacco MAP kinase Ntf4 showed that at least one of the activated kinases would be Ntf4-like. Apigenin, a MAP kinase and cyclindependent kinase inhibitor which prevents the hypoosmotically induced oxidative burst (Cazale  et al., 1998; Plant Physiol. 116, 659±669), inhibited these kinases in vitro suggesting that they may play a role in the activation of the oxidative burst. Like the oxidative response, activation of the kinases depended on extracellular calcium in¯ux and protein kinases sensitive to staurosporine and 6-DMAP. However, kinase activation did not depend on ef¯uxes through anion channels or on the oxidative burst. Two-dimensional in-gel kinase assays revealed the presence of three protein kinases with an apparent molecular mass of 50 kDa and one of 46 kDa, all four being activated by hypoosmotic stress. The same kinases were also activated by oligogalacturonides and salicylic acid, underlying the importance of these MAP kinases as common components of different signaling pathways triggered by different extracellular stimuli.

Phytochelatins represent a major detoxifying pathway for heavy metals in plants and many other organisms. The Arabidopsis thaliana CAD1 ( = AtPCS1) gene encodes a phytochelatin synthase and cad1 mutants are phytochelatin deficient and cadmium hypersensitive. The Arabidopsis genome contains a highly homologous gene, AtPCS2, of which expression and function were studied in order to understand the apparent non-redundancy of the two genes. Low constitutive AtPCS2 expression is detected in all plant organs analyzed. The AtPCS2 gene encodes a functional phytochelatin synthase as shown by expression in Saccharomyces cerevisiae and the complementation of a Schizosaccharomyces pombe phytochelatin synthase knockout strain. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.