Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells.

The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNA(Ile)UAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA.

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.

Functional characterization of CgCTR2, a putative vacuole copper transporter that is involved in germination and pathogenicity in Colletotrichum gloeosporioides.

Copper is a cofactor and transition metal involved in redox reactions that are essential in all eukaryotes. Here, we report that a vacuolar copper transporter that is highly expressed in resting spores is involved in germination and pathogenicity in the plant pathogen Colletotrichum gloeosporioides. A screen of C. gloeosporioides transformants obtained by means of a promoterless green fluorescent protein (GFP) construct led to the identification of transformant N159 in which GFP signal was observed in spores. The transforming vector was inserted 70 bp upstream of a putative gene with homology to the Saccharomyces cerevisiae vacuolar copper transporter gene CTR2. The C. gloeosporioides CTR2 (CgCTR2) gene fully complemented growth defects of yeast ctr2Delta mutants, and a CgCTR2-cyan fluorescent protein (CFP) fusion protein accumulated in vacuole membranes, confirming the function of the protein as a vacuolar copper transporter. Expression analysis indicated that CgCTR2 transcript is abundant in resting conidia and during germination in rich medium and downregulated during "pathogenic" germination and the early stages of plant infection. CgCTR2 overexpression and silencing mutants were generated and characterized. The Cgctr2 mutants had markedly reduced Cu superoxide dismutase (SOD) activity, suggesting that CgCTR2 is important in providing copper to copper-dependent cytosolic activities. The Cgctr2-silenced mutants had increased sensitivity to H2O2 and reduced germination rates. The mutants were also less virulent to plants, but they did not display any defects in appressorium formation and penetration efficiency. An external copper supply compensated for the hypersensitivity to H2O2 but not for the germination and pathogenicity defects of the mutants. Similarly, overexpression of CgCTR2 enhanced resistance to H2O2 but had no effect on germination or pathogenicity. Our results show that copper is necessary for optimal germination and pathogenicity and that CgCTR2 is involved in regulating cellular copper balance during these processes.

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen Colletotrichum gloeosporioides.

BACKGROUND: In order to initiate plant infection, fungal spores must germinate and penetrate into the host plant. Many fungal species differentiate specialized infection structures called appressoria on the host surface, which are essential for successful pathogenic development. In the model plant pathogen Magnaporthe grisea completion of mitosis and autophagy cell death of the spore are necessary for appressoria-mediated plant infection; blocking of mitosis prevents appressoria formation, and prevention of autophagy cell death results in non-functional appressoria. RESULTS: We found that in the closely related plant pathogen Colletotrichum gloeosporioides, blocking of the cell cycle did not prevent spore germination and appressoria formation. The cell cycle always lagged behind the morphogenetic changes that follow spore germination, including germ tube and appressorium formation, differentiation of the penetrating hypha, and in planta formation of primary hyphae. Nuclear division was arrested following appressorium formation and was resumed in mature appressoria after plant penetration. Unlike in M. grisea, blocking of mitosis had only a marginal effect on appressoria formation; development in hydroxyurea-treated spores continued only for a limited number of cell divisions, but normal numbers of fully developed mature appressoria were formed under conditions that support appressoria formation. Similar results were also observed in other Colletotrichum species. Spores, germ tubes, and appressoria retained intact nuclei and remained viable for several days post plant infection. CONCLUSION: We showed that in C. gloeosporioides the differentiation of infection structures including appressoria precedes mitosis and can occur without nuclear division. This phenomenon was also found to be common in other Colletotrichum species. Spore cell death did not occur during plant infection and the fungus primary infection structures remained viable throughout the infection cycle. Our results show that the control of basic cellular processes such as those coupling cell cycle and morphogenesis during fungal infection can be substantially different between fungal species with similar lifestyles and pathogenic strategies.

Bcl-2 proteins link programmed cell death with growth and morphogenetic adaptations in the fungal plant pathogen Colletotrichum gloeosporioides.

Proteins belonging to the Bcl-2 family regulate apoptosis in mammals by controlling mitochondria efflux of cytochrome c and other apoptosis-related proteins. Homologues of human Bcl-2 proteins are found in different metazoan organisms where they play a similar role, while they seem to be absent in plants and fungi. Nonetheless, Bcl-2 protein members can induce or prevent yeast cell death, suggesting that enough functional conservation exists between apoptotic machineries of mammals and fungi. Here we show that induction or prevention of apoptosis by Bcl-2 proteins in the fungal plant pathogen Colletotrichum gloeosporioides is tightly linked with growth and morphogenetic adaptation that occur throughout the entire fungal life cycle. Isolates expressing the pro-apoptotic Bax protein underwent cell death with apoptotic characteristics, and showed alterations in conidial germination that are associated with pathogenic and non-pathogenic life styles. Isolates expressing the anti-apoptotic Bcl-2 protein had prolonged longevity, were protected from Bax-induced cell death, and exhibited enhanced stress resistance. These isolates also had enhanced mycelium and conidia production, and were hyper virulent to host plants. Our findings show that apoptotic-associated machinery regulates morphogenetic switches, which are critical for proper responses and adaptation fungi to different environments.

cAMP regulation of "pathogenic" and "saprophytic" fungal spore germination.

We report on the elucidation of two separate pathways of spore germination in a plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene. Conidia of the fungus can germinate either from one side or from both sides, depending on external conditions. In shake culture that includes an extract made up from fresh peas, the unicellular conidium divides and one of the two cells develops a germ tube. On a solid surface this germ tube differentiates an appressorium. In rich medium without pea extract, germination is highly similar to Aspergillus spore germination: the conidium swells, forms a single germ tube and then divides and forms a second germ tube. Conidia that germinate in a rich medium do not form appressoria even on a solid surface and are non-pathogenic. In rich medium, cAMP stimulates germination in rich liquid cultures and induces appressoria formation on a hard surface. In pea extract cAMP induces swelling and formation of irregular germ tubes and appressoria. Our results suggest that plant surface signals induce pathogenic-specific spore germination in a cAMP-independent manner. cAMP is required for saprophytic germination and for appressorium formation.