Talks: Signal Transduction in Yeasts, Fungi and Plants

Developmental signal pathways in Dictyostelium

Rick Firtel. University of California, San Diego

The cellular slime mold Dictyostelium discoideum grows as single-celled, mononucleated amoebae. When starved, the amoebae initiate a multicellular developmental program that results in the production of a mature fruiting body comprised of a stalk with a mass of spores on top. Approximately 4 hrs after removal or exhaustion of the food source, cells within the population produce and secrete extracellular cAMP. This interacts with G protein-coupled receptors that leads to chemotactic aggregation of ~10(5) cells to form a multicellular organism, activation of adenylyl cyclase and relay of the cAMP signal, and the activation of gene expression. Aggregation and several aspects of multicellular development have been shown to be controlled through several classes of serpentine receptors coupled to multiple heterotrimeric G proteins. Recent results have suggested that some of the responses that are regulated by cAMP receptors are G protein-independent, suggesting novel mechanisms by which the same ligand can elicit different developmental responses. Genes encoding eight distinct Ga protein subunits have been cloned and disrupted by homologous recombination. The function of some of these and the novel G protein-independent pathways will be discussed.

Signal transduction in Saccharomyces cerevisiae and Ustilago maydis

Flora Banuett and Ira Herskowitz. Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143

Cells respond to external stimuli by transducing these signals from surface to nucleus where gene activation then results in a variety of physiological responses. The MAPKs (mitogen activated kinases) and their upstream regulatory kinases are key players in signal transduction. They constitute a functional module consisting of the MAPK, its activator (MEK), and an activator of MEK (MEKK). S.-cerevisiae has several different MAPKs, each involved in a different pathway. The most intensively studied is the pheromone response pathway. Pheromones produced by haploid a or alpha cells bind to membrane receptors causing dissociation of a trimeric G protein into Galpha and Gbeta gamma. The latter activates the kinase cascade in an unknown fashion, which may involve Ste20 (another kinase), and Ste5 (a scaffold for the MAPK module). Ste20 is proposed to activate Ste11 (MEKK), which in turn activates Ste7 (MEK), which then activates Fus3/Kss1 (MAPK). These MAPKs then activate the transcriptional activator Ste12, which regulates expression of genes for cell fusion, cell cycle arrest, and morphological changes. Recent work indicates that pseudohyphal growth requires several components of this pathway. U. maydis codes for peptide pheromones and receptors that play a role in mating and filamentous growth. A MEK homolog (Fuz7) was identified necessary for the pheromone response and for tumor induction, which may reflect response to a plant signal.

Involvement of leucine-rich-repeat and protein kinases in disease resistance signal transduction in tomato

John Salmeron(1), Caius Rommens(2), David Baulcombe(3) and Brian Staskawicz(2), (1)Ciba Agricultural Biotechnology, Research Triangle Park, NC 27709, (2)Dept. of Plant Pathology, University of California, Berekely, CA 94720, and (3)The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK.

Eleven tomato mutants were isolated with altered response to the bacterial phytopathogen Pseudomonas syringae. Five lines carried mutations at Pto, a member of a clustered gene family predicted to encode serine/threonine protein kinases. Six lines carried mutations a second locus termed Prf and showed loss of sensitivity to the insecticide Fenthion as well as disease susceptibility. In contrast, pto mutants retain Fenthion sensitivity. Tight linkage between the Prf and Pto loci allowed cloning of candidate genomic DNA for the Prf locus. Within a 200-kbp contig spanning the Pto locus, a fragment was identified which detected a 1-kbp deletion in a prf mutant line. Sequencing this DNA revealed a gene predicted to encode a protein with nucleotide triphoshate binding (P-loop) and leucine-rich-repeat motifs, as contained in signalling proteins from a wide range of eukaryotes. To rapidly study members of Pto gene family, an assay for transient expression of these genes was developed based on infection of tomato leaves with recombinant derivatives of the Potato Virus X. Using this assay a Pto family member, designated Fen, was identified that confers Fenthion sensitivity. The predicted protein product of the Fen gene shows 80% amino acid identity to Pto and autophoshorylates in vitro, as does Pto. Therefore, tomato contains distinct kinases (Fen and Pto) specific for transduction of Fenthion and pathogen elicitor signals, and the Prf protein which functions in both signal transduction pathways.

Mechanism and function of the oxidative burst in plant defense

Chris J. Lamb, Plant Biology Laboratory, Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037

Treatment of bean or soybean cells with fungal elicitor causes a rapid insolubulization of pre-existing (hydroxy) proline-rich structural proteins in the plant cell wall. This insolubilization, which involves H2O2 - mediated oxidative cross-linking, is initiated within 2-5 min, and is complete within 10-20 min, and hence precedes the expression of transcription-dependent defenses. Oxidative cross-linking makes the cell wall refractory to digestion by microbial protoplasting enzymes and is strictly dependent on gene-for-gene mediated incompatibility in an isogenic setting. Thus stimulus-dependent oxidative cross-linking of wall proteins likely has an important function in the initial stages of plant defense. Cross-linking is substrate controlled by the rapid generation of H2O2 at the cell surface. We show that oxidative burst H2O2 also acts as a signal to trigger other key aspects of the hypersensitive responses deployed in the early stages of an incompatible interaction. Emerging evidence indicates that elicitor or pathogen stimulation of H2O2 production in plant cells bears a striking mechanistic and functional resemblance to the cytochrome b- mediated plasma membrane oxidase involved in the oxidative burst during macrophage activation.

G protein-linked signal transduction and fungal virulance: virus-mediated or transgenic suppression of a G protein a subunit and attenuation of fungal virulence

Gil H. Choi, Baoshan Chell, Shaojian Gao and Donald L. Nuss, Roche Institute of Molecular Biology, Roche Research Center, Nutley NJ 07110

Strains of the chestnut blight fungus Cryphonectria parasitica harboring RNA viruses of the genus Hypovirus exhibit significantly reduced levels of virulence (hypovirulence). The accumulation of a heterotrimeric GTP-binding protein a subunit of the Gi class, designated CPC-1, was found to be reduced in hypovirus-containing C. parasitica strains. Transgenic co-suppression, a phenomenon frequently observed in transgenic plants, reduced CPC-l accumulation in virus-free fungal strains. Significantly, the resulting transgenic fungal strains were also hypovirulent. These results indicate a crucial role for G protein-linked signal transduction in fungal pathogenesis and suggest a molecular basis for virus-mediated hypovirulence. Subsequent differential mRNA display analysis revealed a battery of potential reporter genes that are identically regulated in hypovirus infected and in the transgenic CPC-1 suppressed strains. Representative cDNA clones obtained from that analysis are currently being employed to dissect this pathogenesis-related, G protein-linked signaling pathway.

cAMP dependent protein kinase catalytic subunit (cpkA) gene is required for appressorium formation in Magnaporthe grisea

T.K. Mitchell and R.A. Dean. Department of Plant Pathology and Physiology, Clemson University, Clemson, SC 29634 USA.

Successful infection of rice by the fungal pathogen Magnaporthe grisea depends upon the formation of a dome shaped, highly melanized infection structure, an appressorium. The differentiation of this unique, specialized cell from the tip of an emerging germ tube is a response to environmental stimuli. The exogenous and endogenous signalling mechanisms involved in surface recognition and the transfer of this information into the cell leading to infection related morphogenic events remain to be elucidated. We have shown that cyclic AMP, a second messenger involved in signal transduction systems, regulates appressorium formation. In other systems, the primary target for cAMP is cAMP dependent protein kinase. Activation of this kinase directly or indirectly results in specific developmental changes. To elucidate the mechanism of cAMP action in M. grisea, we have isolated, sequenced and disrupted the single gene encoding the catalytic subunit of cAMP dependent kinase (cpkA). Strains lacking the cpkA gene appear unaffected in their ability to grow and reproduce sexually and asexually. However, they are unable to form appressoria on rice or in the presence of cAMP and are non pathogenic. This is the first direct evidence that fungal pathogenesis is mediated via cAMP dependent protein kinase.

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