Uwe K. Simon

Intragenomic Variation of Fungal Ribosomal Genes Is Higher than Previously Thought

Nuclear ribosomal genes in most eukaryotes are present in multiple copies and often used for taxonomic and phylogenetic analyses. We comprehensively examined intragenomic polymorphism levels of three nuclear ribosomal loci for four important plant pathogenic fungi by polymerase chain reaction amplification and cloning. Here, we show that single nucleotide polymorphisms are present in an unexpectedly high amount. This might have implications for studies of fungal evolution, phylogenetics, and population genetics. Furthermore, our work demonstrates that the majority of all ribosomal sequences obtained from one individual and gene is identical to the majority rule consensus sequence of all detected sequence variants. Due to the large number of polymorphisms found and the fact that the polymorphism level differed markedly even between ribosomal genes of one and the same individual, we assume that nuclear ribosomal genes might not always evolve in a strictly concerted manner.

Cytokinins Mediate Resistance against Pseudomonas syringae in Tobacco through Increased Antimicrobial Phytoalexin Synthesis Independent of Salicylic Acid Signaling

Cytokinins are phytohormones that are involved in various regulatory processes throughout plant development, but they are also produced by pathogens and known to modulate plant immunity. A novel transgenic approach enabling autoregulated cytokinin synthesis in response to pathogen infection showed that cytokinins mediate enhanced resistance against the virulent hemibiotrophic pathogen Pseudomonas syringae pv tabaci. This was confirmed by two additional independent transgenic approaches to increase endogenous cytokinin production and by exogenous supply of adenine- and phenylurea-derived cytokinins. The cytokinin-mediated resistance strongly correlated with an increased level of bactericidal activities and up-regulated synthesis of the two major antimicrobial phytoalexins in tobacco (Nicotiana tabacum), scopoletin and capsidiol. The key role of these phytoalexins in the underlying mechanism was functionally proven by the finding that scopoletin and capsidiol substitute in planta for the cytokinin signal: phytoalexin pretreatment increased resistance against P. syringae. In contrast to a cytokinin defense mechanism in Arabidopsis (Arabidopsis thaliana) based on salicylic acid-dependent transcriptional control, the cytokinin-mediated resistance in tobacco is essentially independent from salicylic acid and differs in pathogen specificity. It is also independent of jasmonate levels, reactive oxygen species, and high sugar resistance. The novel function of cytokinins in the primary defense response of solanaceous plant species is rather mediated through a high phytoalexin-pathogen ratio in the early phase of infection, which efficiently restricts pathogen growth. The implications of this mechanism for the coevolution of host plants and cytokinin-producing pathogens and the practical application in agriculture are discussed.

Post-Translational Derepression of Invertase Activity in Source Leaves via Down-Regulation of Invertase Inhibitor Expression Is Part of the Plant Defense Response

There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Post-translational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports a role for extracellular invertase in plant defense. The acarbose-mediated increase in susceptibility was also detectable in sid2 and cpr6 mutants and resulted in slightly elevated levels of salicylic acid, demonstrating that the effect is independent of the salicylic acid-regulated defense pathway. These findings provide an explanation for high extractable invertase activity found in source leaves that is kept inhibited in situ by post-translational interaction between invertase and the invertase inhibitor proteins. Upon pathogen infection, the invertase activity is released by repression of invertase inhibitor expression, thus linking the local induction of sink strength to the plant defense response.

Cymadothea trifolii, an obligate biotrophic leaf parasite of Trifolium, belongs to Mycosphaerellaceae as shown by nuclear ribosomal DNA analyses.

The ascomycete Cymadothea trifolii, a member of the Dothideomycetes, is unique among obligate biotrophic fungi in its capability to only partially degrade the host cell wall and in forming an astonishingly intricate interaction apparatus (IA) in its own hyphae, while the attacked host plant cell is triggered to produce a membranous bubble opposite the IA. However, no sequence data are currently available for this species. Based on molecular phylogenetic results obtained from complete SSU and partial LSU data, we show that the genus Cymadothea belongs to the Mycosphaerellaceae (Capnodiales, Dothideomycetes). This is the first report of sequences obtained for an obligate biotrophic member of Mycosphaerellaceae.

High Resolution Imaging of Temporal and Spatial Changes of Subcellular Ascorbate, Glutathione and H2O2 Distribution during Botrytis cinerea Infection in Arabidopsis

In order to study the mechanisms behind the infection process of the necrotrophic fungus Botrytis cinerea, the subcellular distribution of hydrogen peroxide (H2O2) was monitored over a time frame of 96 h post inoculation (hpi) in Arabidopsis thaliana Col-0 leaves at the inoculation site (IS) and the area around the IS which was defined as area adjacent to the inoculation site (AIS). H2O2 accumulation was correlated with changes in the compartment-specific distribution of ascorbate and glutathione and chloroplast fine structure. This study revealed that the severe breakdown of the antioxidative system, indicated by a drop in ascorbate and glutathione contents at the IS at later stages of infection correlated with an accumulation of H2O2 in chloroplasts, mitochondria, cell walls, nuclei and the cytosol which resulted in the development of chlorosis and cell death, eventually visible as tissue necrosis. A steady increase of glutathione contents in most cell compartments within infected tissues (up to 600% in chloroplasts at 96 hpi) correlated with an accumulation of H2O2 in chloroplasts, mitochondria and cell walls at the AIS indicating that high glutathione levels could not prevent the accumulation of reactive oxygen species (ROS) which resulted in chlorosis. Summing up, this study reveals the intracellular sequence of events during Botrytis cinerea infection and shows that the breakdown of the antioxidative system correlated with the accumulation of H2O2 in the host cells. This resulted in the degeneration of the leaf indicated by severe changes in the number and ultrastructure of chloroplasts (e.g. decrease of chloroplast number, decrease of starch and thylakoid contents, increase of plastoglobuli size), chlorosis and necrosis of the leaves.

Accession-Specific Haplotypes of the Internal Transcribed Spacer Region in Arabidopsis thaliana—A Means for Barcoding Populations

Eukaryote genomes contain multiple copies of nuclear ribosomal DNA (nrDNA) harboring both highly conserved and variable regions. This has made nrDNA the most popular genetic marker for phylogenetic studies and the region of choice for barcoding projects. Furthermore, many scientists believe that all copies of nrDNA within one nucleus are practically identical due to concerted evolution. Here, we investigate the model plant species Arabidopsis thaliana for intragenomic variation of the internal transcribed spacer (ITS) region of nrDNA. Based on a modified deep sequencing approach, we provide a comprehensive list of ITS polymorphisms present in the two most widely used accessions of A. thaliana-Col-0 and Ler. Interestingly, we found that some polymorphisms are shared between these genetically very distinct accessions. On the other hand, the high number of accession-specific polymorphisms shows that each accession can be clearly and easily characterized by its specific ITS polymorphism patterns and haplotypes. Network analysis based on the detected haplotypes demonstrates that the study of ITS polymorphism patterns and haplotypes is an extremely powerful tool for population genetics. Using the methods proposed here, it will now be possible to extend the traditionally species-bound barcoding concept to populations.

A Key Enzyme of the Leloir Pathway Is Involved in Pathogenicity of Leptosphaeria maculans Toward Oilseed Rape

Agrobacterium tumefaciens-mediated random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m186, is analyzed in detail here. Microscopic analyses of infected plant tissues revealed that m186 is specifically blocked at the invasive growth phase after an unaffected initial penetration stage and is unable to switch to the necrotrophic lifestyle. In addition, m186 exhibits an altered cell wall and seems to be affected in its ability to produce cell-wall-degrading enzymes. The T-DNA insertion occurs in the intergenic region between two head-to-tail genes, leading to a constitutive upregulation of their expression. Complementation experiments showed that only one of these two genes, Lmepi, fully accounts for the mutant phenotype. Bioinformatics and expression analyses along with functional studies suggested that the Lmepi gene encodes for the highly conserved UDP-glucose-4-epimerase, a key enzyme of the Leloir pathway involved in galactose metabolism. For the third time, this study highlights the intimate connection between primary metabolism and pathogenicity in L. maculans. This finding, along with similar data obtained from the related species Stagonospora nodorum, indicates the importance of in planta nutrition for the success of infection of plants by fungi belonging to class Dothideomycete.

The unique cellular interaction between the leaf pathogen Cymadothea trifolii and Trifolium repens

Cellular interactions between the ascomycete Cymadothea trifolii and Trifolium repens (white clover) were analyzed using high-pressure freezing and freeze substitution. Cymadothea trifolii, a biotrophic leaf pathogen, forms a unique structure within its own hyphae, presumably for nutrient uptake from its host. This structure, called an interaction apparatus, consists of long, thin, often net-like cisternae surrounded by a membrane continuous with the fungal plasma membrane. The plant plasmalemma opposite the interaction apparatus invaginates to produce a host bubble. The interaction apparatus and host bubble are apoplastic and are linked by a tube with an electron dense sheath that may channel nutrients from the host to the pathogen. Within the tube, the cell walls of host and parasite appear altered. The interaction apparatus and host bubble may be analogous to haustoria in other obligately biotrophic fungi while the electron dense sheath of the tube may be equivalent to the haustorial neckband.

The Lmgpi15 gene, encoding a component of the glycosylphosphatidylinositol anchor biosynthesis pathway, is required for morphogenesis and pathogenicity in Leptosphaeria maculans.

Random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m20, was analysed in detail here. The mutant phenotype was investigated by microscopic analyses of infected plant tissues and in vitro growth assays. Complementation and silencing experiments were used to identify the altered gene. Its function was determined by bioinformatics analyses, cell biology experiments and functional studies. The mutant was blocked at the invasive growth phase after an unaffected initial penetration stage, and displayed a reduced growth rate and an aberrant hyphal morphology in vitro. The T-DNA insertion occurred in the intergenic region between two head-to-tail genes, leading to a complex deregulation of their expression. The unique gene accounting for the mutant phenotype was suggested to be the orthologue of the poorly conserved Saccharomyces cerevisiae gpi15, which encodes for one component of the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway. Consistent with this predicted function, a functional translational fusion with the green fluorescent protein (GFP) was targeted to the endoplasmic reticulum. Moreover, the mutant exhibited an altered cell wall and addition of glucosamine relieved growth defects. It is concluded that the GPI anchor biosynthetic pathway is required for morphogenesis, cell wall integrity and pathogenicity in Leptosphaeria maculans.

A Three-Lesson Teaching Unit Significantly Increases High School Students' Knowledge about Epilepsy and Positively Influences Their Attitude towards This Disease.

Epilepsy is not a regular topic in many countries’ schools. Thus many people harbor misconceptions about people suffering from this disease. It was our aim to a) examine what grade ten students know and believe about epilepsy, and b) to develop and test a teaching unit to improve their knowledge and attitude. The test group comprised eight grade ten classes from six different Austrian high schools (54 girls and 51 boys aged 14–17), the control group (no intervention) five grade ten classes from the same schools (26 girls and 37 boys aged 14–17). The teaching unit consisted of three 45-min lessons using different methods and material. Changes in knowledge about and attitude towards epilepsy as a result of the intervention were psychometrically assessed in a pre-test intervention post-test design (along with a follow-up assessment two months after the intervention) by means of a questionnaire capturing different facets of epilepsy-related knowledge and attitude. Across all knowledge/attitude domains, students of the test group had a significantly improved knowledge about and a more positive attitude towards epilepsy and people suffering from it after the teaching unit. However, starting levels were different between the five knowledge/attitude domains tested. Medical background knowledge was lowest and consequently associated with the highest increase after the intervention. This study shows that epilepsy-related knowledge of many grade ten high school students is fragmentary and that some harbor beliefs and attitudes which require improvement. Our comprehensive but concise teaching unit significantly increased knowledge about epilepsy and positively influenced attitude towards individuals with epilepsy. Thus we recommend implementing this unit into regular school curricula.