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

<p>Abstract</p> <p>Background</p> <p>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 <it>Magnaporthe grisea </it>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.</p> <p>Results</p> <p>We found that in the closely related plant pathogen <it>Colletotrichum gloeosporioides</it>, 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 <it>in planta </it>formation of primary hyphae. Nuclear division was arrested following appressorium formation and was resumed in mature appressoria after plant penetration. Unlike in <it>M. grisea</it>, 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 <it>Colletotrichum </it>species. Spores, germ tubes, and appressoria retained intact nuclei and remained viable for several days post plant infection.</p> <p>Conclusion</p> <p>We showed that in <it>C. gloeosporioides </it>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 <it>Colletotrichum </it>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.</p

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

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen -2

On). Note that the nucleus is still in the hypha out of the incipient appressorium. (b-d) Time lapse of nuclear division within the appressorium: (b) the nucleus migrates from the hypha to the appressorium neck; (c) the nucleus divides within the appressorium neck; (d) one nucleus remains inside the appressorium and the other moves back into the hypha. (B) Effect of inhibitors on appressorium formation: (a) no treatment; (b) HU (fully developed appressorium develops without nuclear division); (c) benomyl. (C) Spores of strain H1-13 were inoculated onto onion epidermis. Pictures were taken after 24 h. (a) A hypha and an appressorium develop on the surface; a primary hypha is formed inside the plant under the appressorium. Note that the primary hypha is formed before nuclear division (arrow). (b, c) Nuclear division occurs in the appressorium after primary hypha formation. A single nucleus remains inside the appressorium: (b) projections of optical sections; (c) side view of the same sample. (d) A picture showing the spore from which infection originated, a hypha that developed on the leaf, an appressorium, and the underlying developing primary hyphae with several nuclei (arrow). Note that the spore and appressorium, which are on top of the onion epidermis, contain intact nuclei. Picture is a projection of optical sections. The scale bar is 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen "</p><p>http://www.biomedcentral.com/1741-7007/6/9</p><p>BMC Biology 2008;6():9-9.</p><p>Published online 14 Feb 2008</p><p>PMCID:PMC2276476.</p><p></p

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen -1

Nomyl; (g, h) LatA. The scale bar is 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen "</p><p>http://www.biomedcentral.com/1741-7007/6/9</p><p>BMC Biology 2008;6():9-9.</p><p>Published online 14 Feb 2008</p><p>PMCID:PMC2276476.</p><p></p

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen -0

Min); (c) germ tube formation (90–120 min); (d,e) germ tube elongation and second nuclear division (150–180 min); (f) appressorium formation and third nuclear division (4–6 h, arrow points to the septum between the germ tube and appressorium); (g, h) formation of the second germ tube and appressorium (7–9 h). Pictures represent sequence events and are projections of optical sections. The scale bar is 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen "</p><p>http://www.biomedcentral.com/1741-7007/6/9</p><p>BMC Biology 2008;6():9-9.</p><p>Published online 14 Feb 2008</p><p>PMCID:PMC2276476.</p><p></p

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen -5

Nomyl; (g, h) LatA. The scale bar is 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen "</p><p>http://www.biomedcentral.com/1741-7007/6/9</p><p>BMC Biology 2008;6():9-9.</p><p>Published online 14 Feb 2008</p><p>PMCID:PMC2276476.</p><p></p

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen -3

Developed on the surface of the onion epidermis contain intact nuclei. (b) After 48 h post inoculation the mycelium and appressoria on the leaf surface still retain intact nuclei. Pictures represent the scan of the surface without optical sections. (c, d) Projection of optical sections showing the hyphae on and inside the leaf 72 h post inoculation: (c) top view; (d) side view of the same image. Upper nuclei line is on the leaf surface. (B) Spores were germinated on a slide with PE. (a) Untreated hyphae of strain H1-13 showing intact nuclei. (b) Spores and hyphae of wild-type strain stained with FDA (positive staining indicates viable cells). (c) Spores of strain H1-13 were germinated and then treated with lovastatin, which induces apoptosis. Picture was taken 24 h after treatment. Note the abnormal development of the hyphae and smearing of the GFP signal, which indicates nuclei disintegration. (d) Spores of the wild-type strain were germinated and then treated with lovastatin. The sample was stained with FDA 24 h after lovastatin application. (C) TUNEL assay of mycelium on the onion epidermis. Spores of the wild-type strain were inoculated onto the onion epidermis. TUNEL staining was performed 48 h post infection. (a) DNAse-treated sample (positive control). (b) Control of a sample that was incubated only with labeling solution without the terminal transferase (negative control). (c) Picture showing a spore (black arrow), a mature appressorium and underlying primary hyphae (white arrow) stained with TUNEL. Lack of staining indicates lack of PCD (viable cells). The scale bar for (b-d) in (A) is 20 μm; for all others it is 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen "</p><p>http://www.biomedcentral.com/1741-7007/6/9</p><p>BMC Biology 2008;6():9-9.</p><p>Published online 14 Feb 2008</p><p>PMCID:PMC2276476.</p><p></p

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 ctr2Δ 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