a b s t r a c tMitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

The phytopathogenic bacterium Ralstonia solanacearum encodes a family of seven type III secretion system (T3SS) effectors that contain both a leucine-rich repeat and an F-box domain. This structure is reminiscent of a class of typical eukaryotic proteins called F-box proteins. The latter, together with Skp1 and Cullin1 subunits, constitute the SCF-type E3 ubiquitin ligase complex and control specific protein ubiquitinylation. In the eukaryotic cell, depending on the nature of the polyubiquitin chain, the ubiquitintagged proteins either see their properties modified or are doomed for degradation by the 26S proteasome. This pathway is essential to many developmental processes in plants, ranging from hormone signaling and flower development to stress responses. Here, we show that these previously undescribed T3SS effectors are putative bacterial F-box proteins capable of interacting with a subset of the 19 different Arabidopsis Skp1-like proteins like bona fide Arabidopsis F-box proteins. A R. solanacearum strain in which all of the seven GALA effector genes have been deleted or mutated was no longer pathogenic on Arabidopsis and less virulent on tomato. Furthermore, we found that GALA7 is a host-specificity factor, required for disease on Medicago truncatula plants. Our results indicate that the GALA T3SS effectors are essential to R. solanacearum to control disease. Because the F-box domain is essential to the virulence function of GALA7, we hypothesize that these effectors act by hijacking their host SCF-type E3 ubiquitin ligases to interfere with their host ubiquitin͞proteasome pathway to promote disease.plant pathogen ͉ SCF complex ͉ type III secretion system ͉ virulence

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solanacearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 x 10(5) in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.

We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed into a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrate the LSPR peak shift due to protein binding to the membrane surface and then characterize the lipid-binding specificity of a pleckstrin-homology domain protein.

The anisotropic organization of plasma membrane constituents is indicative of mechanisms that drive the membrane away from equilibrium. However, defining these mechanisms is challenging due to the short spatiotemporal scales at which diffusion operates. Here, we use high-density single protein tracking combined with photoactivation localization microscopy (sptPALM) to monitor Cdc42 in budding yeast, a system in which Cdc42 exhibits anisotropic organization. Cdc42 exhibited reduced mobility at the cell pole, where it was organized in nanoclusters. The Cdc42 nanoclusters were larger at the cell pole than those observed elsewhere in the cell. These features were exacerbated in cells expressing Cdc42-GTP, and were dependent on the scaffold Bem1, which contributed to the range of mobility and nanocluster size exhibited by Cdc42. The lipid environment, in particular phosphatidylserine levels, also played a role in regulating Cdc42 nanoclustering. These studies reveal how the mobility of a Rho GTPase is controlled to counter the depletive effects of diffusion, thus stabilizing Cdc42 on the plasma membrane and sustaining cell polarity.