Olaf Müller

Pathogenicity Determinants in Smut Fungi Revealed by Genome Comparison

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. Biotrophic pathogens, such as the related maize pathogenic fungi Ustilago maydis and Sporisorium reilianum, establish an intimate relationship with their hosts by secreting protein effectors. Because secreted effectors interacting with plant proteins should rapidly evolve, we identified variable genomic regions by sequencing the genome of S. reilianum and comparing it with the U. maydis genome. We detected 43 regions of low sequence conservation in otherwise well-conserved syntenic genomes. These regions primarily encode secreted effectors and include previously identified virulence clusters. By deletion analysis in U. maydis, we demonstrate a role in virulence for four previously unknown diversity regions. This highlights the power of comparative genomics of closely related species for identification of virulence determinants.

A H2O2-producing glyoxal oxidase is required for filamentous growth and pathogenicity in Ustilago maydis

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (<C4) and produces H2O2. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.

Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis

Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens.

A note on closed isometric embeddings

A famous theorem due to Nash ([1]) assures that every Riemannian manifold can be embedded isometrically into some Euclidean space En. An interesting question is whether for a complete manifold M we can find a closed isometric embedding. This note gives the affirmative answer to this question asked to the author by Paolo Piccione. In his famous 1956 article John Nash proved that every Riemannian metric on an ndimensional manifold M can be constructed as a pullback metric for an embedding of M into some Euclidean space. He gave also an estimate of the smallest possible dimension N of the Euclidean space as N = 12 · n · (n + 1) · (3n + 11). Now one can try to find some stronger derivates of this theorem if strengthening the assumptions. In this note, we want to examine the question whether every complete manifold admits a closed isometric embedding. Although folk wisdom apparently has a positive answer to this question already, there does not seem to be any proof in the literature up to now. The question is more difficult than it might seem at first sight as there are plenty of non-closed isometric embeddings of complete manifols, e.g. spirals converging to 0 or to a circle as isometric embeddings of R. For a Lipschitz function f on a metric space M , we denote by L(f) := sup{ |f(p)−f(q) d(p,q) | p, q ∈ M, p 6= q} its Lipschitz number. We will need the following nice theorem from [2] about approximation of Lipschitz functions by smooth functions (even on infinite-dimensional Riemannian manifolds): Theorem 0.1 Let (M, g) be a separable Riemannian manifold, let f : M → R be a Lipschitz function, let ρ : M → (0,∞) be a continuous function, and let r > 0. Then there is a C and Lipschitz function g : M → R with |f(p)− g(p)| ≤ ρ(p) for every p ∈ M , and L(g) ≤ L(f) + r. Now let us state and prove our theorem. The basic idea of the proof is to look at balls of increasing radius and to define an imbedding which lifts the larger and larger balls into an additional direction thereby resolving a possible spiralling. As the distance itself is not differentiable in general, we have to be a little bit more careful and thus we will need the theorem above. Theorem 0.2 If (M, g) is a complete n-dimensional Riemannian manifold, then there is a closed isometric C-embedding of (M, g) into E, where N := 12 · n · (n + 1) · (3n + 11). Instituto de Matemáticas, Universidad Nacional Autónoma de México (UNAM) Campus Morelia, C. P. 58190, Morelia, Michoacán, Mexico. email: olaf@matmor.unam.mx

Improved expression of single-chain antibodies in Ustilago maydis.

To produce the full repertoire of biopharmaceutical proteins, alternative expression platforms are required. Systems that enable secretion of the target protein are favored because this facilitates downstream processing. Ustilago maydis is a promising fungal model organism for future applications in protein expression. Recently, we described the exploitation of a novel unconventional secretion mechanism for the export of heterologous proteins. In this mode of secretion, the endochitinase Cts1 functions as a carrier for export with the main advantage of avoiding potentially harmful N-glycosylation. The major limitation until now was a low yield of secreted full-length protein. For optimization, we identified two bottlenecks: mRNA amount and extracellular proteolytic activity. By generating novel expression vectors harboring a strong constitutive promoter as well as eliminating harmful proteases, yields were increased significantly. A scFv antibody fragment against the cMyc epitope served as proof-of-principle and could be purified in its active, full-length form from the culture supernatant. Thus, we improved the novel expression system in U. maydis such that it can now be investigated with respect to other targets with potential applications for instance in diagnostics and medicine.

Riemannian geometry of the space of volume preserving immersions

Given a compact manifold

LORENTZIAN SPECTRAL GEOMETRY FOR GLOBALLY HYPERBOLIC SURFACES

M

Every conformal class contains a metric of bounded geometry

and a Riemannian manifold

A Note on Invariant Temporal Functions

N

Special Temporal Functions on Globally Hyperbolic Manifolds

of bounded geometry, we consider the manifold