Bakkeren Plant Pathology Lab - Ustilago Research

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BACKGROUND

Smut Fungi

Smut fungi are basidiomycete fungi and belong to the order of the Ustilaginales. They are named after the very conspicuous symptoms, black masses of teliospores resembling soot or smut, which they often produce on host plants. Although controlled in our Western societies with fungicides and in some cases resistant crops, smut diseases remain very important plant pathogens world-wide. Indeed, the potential to cause epidemics is lurking even in Western societies, especially with the deregulation of many fungicides.

Over 1,100 smut species have been recognized in over 50 genera infecting more than 4,000 plant species belonging to approximately 75 different plant families. The biggest group infects monocots belonging to the Gramineae which include cereal crops. The smuts encompass among others, the genera Ustilago and Tilletia, the latter causing so-called bunt diseases of various cereal crops.

Phylogeny

We have published a preliminary (Bakkeren et al., 2000) and a more extensive study (Menzies et al., 2003) on phylogenetic relationships between some of the small grain-infecting smut and bunt fungi belonging to the Ustilaginomycetes and pointed to an intriguing correlation with a phylogenetic tree of their respective host plants. In the near future we will see a more extensive study from our collaborators.

Life cycle

Ustilago hordei is representative of a group of fungal pathogens that cause economically important smut diseases world-wide on small grain cereals. These fungi grow as dikaryotic hyphae within developing seedlings, residing mainly in the meristem, without causing dramatic symptoms (I, diagram below). Upon flowering, the fungal cells proliferate, form thick walls and karyogamy takes place, upon which masses of dark, echinulated teliospores are formed that replace the developing seeds (II). Teliospores are survival structures. They can disseminate and survive often under the seed hull of healthy barley seeds. Under the right conditions both seed and teliospores will germinate. Diploid teliospores germinate and undergo meiosis to yield haploid cells which are called basidiospores (III). These meiotic progeny must mate and form a dikaryotic hypha to re-infect the host. The fungus can then enter the emerging seedling by direct penetration (IV; Hu et al, 2002). Infection is a prerequisite for completion of the sexual phase of the life cycle, i.e., the formation of teliospores. Sex and pathogenicity are therefore interconnected in U. hordei and related smut fungi, and the mating-type genes are considered pathogenicity factors.

symptoms | mating interaction

fungal proliferation | infection process

Vital statistics

HIGHER FUNGI
Subdivision: BASIDIOMYCOTINA
Class: HEMIBASIDIOMYCETES (TELIOMYCETIDAE)
Order: Ustilaginales
Genus: Ustilago
Species: hordei

covered smut
Disease	Main hosts	Pathogen
covered smut	barley & oat (Hordeum vulgare & Secale, but also Elymus & Agropyron spp.)	Ustilago hordei (suggested nomenclature: Ustilago segetum f.sp. hordei)

Genome size of U. hordei is approx. 25 Mbp; 250 BAC clones for 1x genome coverage (Bakkeren et al. 2006. Physical mapping of the genome of the fungal pathogen Ustilago hordei and annotation of the 500 kb MAT-1 sequence. Fungal Genet Biol, 43, 655-666)

Mating systems

U. hordei has a bipolar mating system controlled by one mating-type locus (MAT) and two alleles or alternative specificities exist for the locus, MAT-1 and MAT-2. Hybridization experiments with the well-characterized a and b mating-type genes from the related species, Ustilago maydis, revealed that U. hordei possesses similar mating-type functions located at so-called a and b gene complexes within the MAT locus (Bakkeren et al., 1992). In contrast to U. hordei, U. maydis has a tetrapolar mating system due to the fact that the a and b loci are on separate chromosomes and therefore segregate independently during meiosis. In U. maydis, two specificities exist for the a locus: a1 and a2. The a locus encodes cell-type specific pheromones (mfa) as well as the pheromone receptors (pra) that recognize pheromones from compatible mating partners. The b locus of U. maydis is multiallelic and contains two divergently transcribed genes, bE (bEast) and bW (bWest). The two genes have similar organizations in that each encodes a variable amino-terminal region, a conserved carboxy-terminal region and an intervening homeodomain-related motif. The b locus controls pathogenicity and completion of the life cycle through self versus non-self recognition between bE and bW polypeptides to establish a regulatory factor. The homologues of the a and b genes from U. maydis and U. hordei have been demonstrated to be conserved in structure and function (Bakkeren and Kronstad, 1993; Bakkeren and Kronstad, 1996). In both species, only cells of opposite mating-type, that is, having different specificities at both a and b, successfully mate and form colonies with aerial hyphae (Fuz+ reaction). These combinations are infectious when inoculated into host plants. Conversely, haploid strains or incompatible partners of the same mating type form yeast-like colonies and are non-infectious. These yeast-like cells divide by budding and are amenable to many molecular techniques such as transformation and gene replacement.

We have shown that a and b are physically linked on the largest chromosome of U. hordei and together encode key functions within the MAT locus. Preliminary mapping experiments indicated that these gene complexes were >150 kb apart, yet when MAT-1 (a1b1) and MAT-2 (a2b2) strains were crossed, recombinant progeny with genotypes a1b2 and a2b1 were not found. Mating tests between parental strains and their progeny were performed to search for these recombinant progeny (Bakkeren and Kronstad, 1994).

We showed by tagging the a and b gene complexes within both MAT loci with the recognition sequence for the restriction enzyme I-SceI, that the MAT locus extends over a large region and that the size and organization of the locus differs between MAT-1 and MAT-2 strains. Specifically, we found that the distance between the complexes is 500 kb in a MAT-1 strain and 430 kb in a MAT-2 strain. Characterization of the organization of the known genes within the a and b gene complexes provided evidence for non-homology and sequence inversion between MAT-1 and MAT-2. Antibiotic resistance markers were also used to tag the a gene complex in MAT-1 strains (phleomycin) and the b gene complex in MAT-2 strains (hygromycin). Crosses were performed with these strains and progeny resistant to both antibiotics were recovered at a low frequency suggesting that recombination is suppressed within the MAT region. Overall, the chromosome homologues carrying the U. hordei MAT locus share features with primitive sex chromosomes (Lee et al., 1999).

Recently, a BAC fingerprint map was constructed for the U. hordei genome and the complete 527 kb MAT-1 region was sequenced. Comparison with the respective U. maydis chromosomal regions harbouring the a and b mating-type complexes, revealed synteny but also several translocations, indels, inversions and a large number of transposon-like elements and repeats in U. hordei (Bakkeren et al., 2006). Comparison of mating-type regions and loci among basidiomyctes reveales interesting evolutionary trends and points to the generation of larger loci or even sex chromosomes (Bakkeren et al., 2008).

Matings between related smut species which do not occur naturally, can be forced by the introduction of heterologous pheromone and cognate pheromone-receptor genes. This was demonstrated between cells of two natural non-maters, U. hordei and U. maydis (Bakkeren and Kronstad, 1996).

Ustilago maydis

Many aspects of the mating interactions, the molecular components involved and their downstream effectors, targets and genes, as well as many aspects of pathogenicity and interaction with host plants, have been studied in the model smut fungus, Ustilago maydis, a close relative of U. hordei. For example, one of the key features is the morphological switch from growth by budding of the basidiospores to filamentous, pathogenic growth of the dikaryotic cell type produced after mating. This process involves interesting kinase signal transduction pathways. In addition, the complete genome of U. maydis has been sequenced and annotated (see Kamper et al, 2006; accessible through the Broad Institute website or the Munich Information Centre for Protein Sequences). Several Ustilago laboratories around the world are actively pursuing research on this fungus.

Smutted corn cobs or "huitlacoche", are considered a delicacy in many Middle- and South-American countries.

Some other reviews for further reading:

§ Feldbrugge, M., J. Kamper, G. Steinberg and R. Kahmann (2004) Regulation of mating and pathogenic development in Ustilago maydis. Curr Opin Microbiol, 7: 666-672

• Kamper et al (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature, 444: 97-101
• Special issue on U. maydis, August 2008. Fungal Genetics & Biology, 45, Supplement 1, pp S1-S94
• Brefort, T., G. Doehlemann, et al. (2009). Ustilago maydis as a Pathogen. Ann. Rev. Phytopathol. 47: 423-445

Pathogenicity, virulence & avirulence

Many plant pathogens have been shown genetically to harbour single dominant genes whose presence is recognized by specific host cultivars possessing single, 'matching', dominant resistance genes (‘gene-for-gene interaction'). These pathogen genes have come to be known as avirulence or Avr genes since recognition of their product by a host harbouring the cognate resistance or R-gene (product) triggers a defence reaction (incompatible interaction) which is epistatic over the potential of the pathogenic species to infect the host (compatible interaction).

Much research has focused on the understanding of the molecular workings of Avr genes as well as their isolation because they a) represent single, often dominant genes that are easy to track genetically, b) restrict host range and c) are recognizable pathogenic factors that trigger host defence. Defence mechanisms are often correlated with a conspicuous, necrotic resistance reaction known as the hypersensitive response (HR), which is thought to keep localized infections at bay. Initial responses during resistance but also during compatible interactions, include a variety of molecular events in both host and pathogen which are often the result of induced gene expression (e.g., membrane depolarization, calcium influx, the production of reactive oxygen species (ROS), pathogenesis related (PR-) proteins, etc.

Many bacterial Avr genes have been isolated via classical bacterial genetic techniques such as the transformation of a virulent receptor strain with a genomic library from an avirulent strain. Avr-containing clones are selected as those resulting in a visual hypersensitive response. Larger genomes and inefficient transformation techniques have resulted in fewer such fungal genes being isolated by similar methods. As a consequence, researchers have had to resort to more complex techniques like reverse genetics and marker-assisted genome walking. To date, a significant number of Avr genes have also been isolated from fungal and oomycete species, aided by the analysis of total genome sequences of several model species. Many Avr gene products are small proteins with signal peptides meant for secretion into plant host cells or apoplast where most likely they fulfill a more-or-less essential (virulence or fitness) function. In incompatible interactions they additionally and inadvertently elicit defence responses. Mutation or deletion of the Avr genes yields avr alleles which can escape recognition by the R-gene mediated surveillance system, but might thereby invoke virulence or fitness penalties on its bearer. These Avr genes therefore presumably allow a competitive advantage to the pathogen which seems logical in light of evolutionary pressures. It follows that microbial “Avr” gene products likely have been co-opted by some hosts as triggers for active defence. The molecular basis for this type of resistance might be direct physical interaction of the Avr gene product and the product of its cognate resistance gene (receptor-ligand model), or indirectly with a host component in a complex over which an R-gene product stands guard (guard hypothesis; reviewed in Bakkeren and Gold, 2004, more recent: Jones and Dangl. 2006. The plant immune system. Nature 444, 323-329).

U. hordei avirulence genes

U. hordei displays race-cultivar specialization. Fourteen races have been described which harbour at least 5 single dominant Avr genes. In barley, a visible HR reaction is not observed in an incompatible interaction. Therefore, currently, incompatible interactions are scored after two to three months at heading by the absence of disease symptoms in a significant number of plants compared to a compatible control. A marker-based cloning approach for the isolation of Avr genes was selected because a) although optimized, transformation is still inefficient, b) the genome complexity is estimated at approx. 20 Mbp per haploid genome, making integration of random clones and subsequent pathogenicity tests of at least three independent transformants on at least 50 plants each unfeasible and c) no race-specific elicitor has been found to date making a reverse genetics approach impossible. As a first step a virulent parent (MAT-2) was crossed with an avirulent parent (MAT-1) on the universal susceptible barley cultivar ‘Odessa’, to construct a population in which three unlinked Avr genes were segregating. Each progeny was tested by back crosses on three differentials carrying cognate resistance genes to test for Avr genotype. Marker analysis on genomic DNAs from eight segregants pooled for Avr1 revealed one AFLP and two RAPD markers. No positive clones were obtained from cosmid libraries necessitating the construction of a Bacterial Artificial Chromosome (BAC) library (Linning et al., 2004). Biological activity tests of subclones require their transformation into the virulent strain (MAT 2, v1), subsequent mating of the transformants with a compatible, virulent strain (MAT-1, v1), and inoculation on seedlings of barley cultivar ‘Hannchen’ (Ruh1). Transformants that have received an active Avr1 gene will not produce any sori at heading.

Current research projects:

Ø Cloning and characterization of Ustilago hordei avirulence genes, mode of action

· Using AFLP, SSR and RAPD marker technology on bulked progeny, Avr1, one of five known avirulence genes of the barley smut fungus, Ustilago hordei, was located on BAC clones and delineated on a 40-kb region (Linning et al., 2004). Confirmation of the isolation of Avr1 by functional analysis and characterization of the gene is in progress. Using the published U. maydis and U. hordei genome sequences, we are selecting other markers linked to UhAvr6 which should also allow the isolation of this avirulence gene. Future work will include the functional analysis of these avirulence genes, their potential interaction with host factors and occurrence of their alleles in different smut races, species and populations.

· In collaboration with Drs. G. Scoles, B. Rossnagel and T. Grewal of the Dept. of Plant Sciences, University of Saskatchewan, barley resistance gene, Ruh1, recognizing U. hordei Avr1, was identified and was mapped to the short arm of chromosome 1 (7H) between markers iPgd1A and BCD129 on the ‘Harrington’/TR306 map (Grewal et al., 2008). In addition, we might have revealed novel U. hordei Avr genes and cognate resistance genes by using a large collection of defined new barley cultivars and germplasm.

Ø U. hordei genome sequencing and comparative analyses among smuts

· The generation of a U. hordei BAC library, fingerprint map (in collaboration with Dr. J. W. Kronstad, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada), and the generation of BAC-end sequences (Bakkeren et al., 2008), has laid the basis for the generation of the complete U. hordei genome sequence (Laurie et al., 2012), a collaboration with J. Schirawski (Rheinisch-Westfälische Technische Hochschule Aachen University, Germany), R. Kahmann (Max Planck Institute for Terrestrial Microbiology, Marburg, Germany) and Drs. G. Mannhaupt, M. Muensterkoetter, U. Gueldener and P. Wong at the Münich Information center for Protein Sequences (MIPS, Helmholtz Zentrum, München, Germany). Comparative analyses among the three, curently generated smut genomes (U. maydis, U. hordei and Sporisorium reilianum), with emphasis on the diversity of effector genes, is in progress.

Ø Barley host responses during compatible and incompatible interactions

· Microscopy and gene expression patterns, isolation of involved genes. The isolation of an avirulence gene has allowed us to construct isogenic fungal strains differing only in the absence or presence of individual avirulence genes. Such strains permit the systematic study of the role(s) these avirulence genes play in compatible versus incompatible interactions. In incompatible interactions, no (visible) hypersensitive response (HR) is generated but resistance is nevertheless achieved through arrest of fungal hyphal growth. Preliminary data suggest that different Avr / R-gene combinations "act" at different stages during pathogen invasion. The Avr1-Ruh1 interaction was studied in detail on the microscopic level; a localized, microscopic HR takes place immediately upon penetration of the first cell layer of the barley seedling, including collapse of few cells surrounding the penetration site (Hu et al., 2003). This data has aided in selecting the timing and tissues for RNA isolation for transcript profiling of the barley host response using microarrays. Parallel work on the defence and resistance pathways in wheat and barley is being carried out. Similarly, nonhost interactions have been studied by applying U. hordei to wheat & Triticale (Gaudet et al., 2010; collaboration with Drs. D. Gaudet and A. Laroche, Lethbridge Research Centre, Lethbridge, AB).