UhAVR1, an HR-Triggering Avirulence Effector of Ustilago hordei, Is Secreted via the ER-Golgi Pathway, Localizes to the Cytosol of Barley Cells during in Planta-Expression, and Contributes to Virulence Early in Infection.

The basidiomycete Ustilago hordei causes covered smut disease of barley and oats. Virulence effectors promoting infection and supporting pathogen lifestyle have been described for this fungus. Genetically, six avirulence genes are known and one codes for UhAVR1, the only proven avirulence effector identified in smuts to date that triggers complete immunity in barley cultivars carrying resistance gene Ruh1. A prerequisite for resistance breeding is understanding the host targets and molecular function of UhAVR1. Analysis of this effector upon natural infection of barley coleoptiles using teliospores showed that UhAVR1 is expressed during the early stages of fungal infection where it leads to HR triggering in resistant cultivars or performs its virulence function in susceptible cultivars. Fungal secretion of UhAVR1 is directed by its signal peptide and occurs via the BrefeldinA-sensitive ER-Golgi pathway in cell culture away from its host. Transient in planta expression of UhAVR1 in barley and a nonhost, Nicotiana benthamiana, supports a cytosolic localization. Delivery of UhAVR1 via foxtail mosaic virus or Pseudomonas species in both barley and N. benthamiana reveals a role in suppressing components common to both plant systems of Effector- and Pattern-Triggered Immunity, including necrosis triggered by Agrobacterium-delivered cell death inducers.

Comparative Analysis Highlights Variable Genome Content of Wheat Rusts and Divergence of the Mating Loci.

Three members of the Puccinia genus, Pucciniatriticina (Pt), Pstriiformis f.sp. tritici (Pst), and Pgraminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi, and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; in comparison, repeats occupy 31.5% for Pst and 36.5% for Pgt We find all three genomes are highly heterozygous, with Pst [5.97 single nucleotide polymorphisms (SNPs)/kb] nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt Of 1358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host-induced gene silencing (HIGS) of the HD and STE3 alleles reduced wheat host infection.

An immunity-triggering effector from the Barley smut fungus Ustilago hordei resides in an Ustilaginaceae-specific cluster bearing signs of transposable element-assisted evolution.

The basidiomycete smut fungus Ustilago hordei was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant avirulence locus UhAvr1 which triggers immunity in barley cultivar Hannchen harboring resistance gene Ruh1, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish UhAvr1 candidates. Systematic deletion and complementation analyses revealed that UhAvr1 is UHOR_10022 which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common U. hordei transposable element (TE), interrupting the promoter of UhAvr1 and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that UhAvr1 is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. UhAvr1 does not contribute significantly to overall virulence. UhAvr1 is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related U. maydis and Sporisorium reilianum. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since U. maydis is not known to trigger immunity in its corn host.

The transition from a phytopathogenic smut ancestor to an anamorphic biocontrol agent deciphered by comparative whole-genome analysis.

Pseudozyma flocculosa is related to the model plant pathogen Ustilago maydis yet is not a phytopathogen but rather a biocontrol agent of powdery mildews; this relationship makes it unique for the study of the evolution of plant pathogenicity factors. The P. flocculosa genome of ~23 Mb includes 6877 predicted protein coding genes. Genome features, including hallmarks of pathogenicity, are very similar in P. flocculosa and U. maydis, Sporisorium reilianum, and Ustilago hordei. Furthermore, P. flocculosa, a strict anamorph, revealed conserved and seemingly intact mating-type and meiosis loci typical of Ustilaginales. By contrast, we observed the loss of a specific subset of candidate secreted effector proteins reported to influence virulence in U. maydis as the singular divergence that could explain its nonpathogenic nature. These results suggest that P. flocculosa could have once been a virulent smut fungus that lost the specific effectors necessary for host compatibility. Interestingly, the biocontrol agent appears to have acquired genes encoding secreted proteins not found in the compared Ustilaginales, including necrosis-inducing-Phytophthora-protein- and Lysin-motif- containing proteins believed to have direct relevance to its lifestyle. The genome sequence should contribute to new insights into the subtle genetic differences that can lead to drastic changes in fungal pathogen lifestyles.

Do TE activity and counteracting genome defenses, RNAi and methylation, shape the sex lives of smut fungi?

The availability of three genomes from smut fungi differing in mating, TE load, and genome defense mechanisms, allowed a comparative analyses and a discussion on evolutionary forces shaping them. A complex balance of selective forces seems at play. A bipolar mating system in Ustilago hordei promotes selfing, advantageous for successful niche occupation but favoring accumulation of repetitive DNA, including TEs. TE activity may have caused genome variations necessary for these obligate parasites under high host selection pressures. Higher TE activity is balanced by genome defenses through recombination, RNAi, methylation and RIP mutagenesis. In tetrapolar U. maydis, lacking silencing and possibly methylation mechanisms, reduced inbreeding potential favors removal of repetitive DNA, presumably by its highly-efficient recombination system.

Conserved loci of leaf and stem rust fungi of wheat share synteny interrupted by lineage-specific influx of repeat elements.

BACKGROUND: Wheat leaf rust (Puccinia triticina Eriks; Pt) and stem rust fungi (P. graminis f.sp. tritici; Pgt) are significant economic pathogens having similar host ranges and life cycles, but different alternate hosts. The Pt genome, currently estimated at 135 Mb, is significantly larger than Pgt, at 88 Mb, but the reason for the expansion is unknown. Three genomic loci of Pt conserved proteins were characterized to gain insight into gene content, genome complexity and expansion. RESULTS: A bacterial artificial chromosome (BAC) library was made from P. triticina race 1, BBBD and probed with Pt homologs of genes encoding two predicted Pgt secreted effectors and a DNA marker mapping to a region of avirulence. Three BACs, 103 Kb, 112 Kb, and 166 Kb, were sequenced, assembled, and open reading frames were identified. Orthologous genes were identified in Pgt and local conservation of gene order (microsynteny) was observed. Pairwise protein identities ranged from 26 to 99%. One Pt BAC, containing a RAD18 ortholog, shares syntenic regions with two Pgt scaffolds, which could represent both haplotypes of Pgt. Gene sequence is diverged between the species as well as within the two haplotypes. In all three BAC clones, gene order is locally conserved, however, gene shuffling has occurred relative to Pgt. These regions are further diverged by differing insertion loci of LTR-retrotransposon, Gypsy, Copia, Mutator, and Harbinger mobile elements. Uncharacterized Pt open reading frames were also found; these proteins are high in lysine and similar to multiple proteins in Pgt. CONCLUSIONS: The three Pt loci are conserved in gene order, with a range of gene sequence divergence. Conservation of predicted haustoria expressed secreted protein genes between Pt and Pgt is extended to the more distant poplar rust, Melampsora larici-populina. The loci also reveal that genome expansion in Pt is in part due to higher occurrence of repeat-elements in this species.

Genome comparison of barley and maize smut fungi reveals targeted loss of RNA silencing components and species-specific presence of transposable elements.

Ustilago hordei is a biotrophic parasite of barley (Hordeum vulgare). After seedling infection, the fungus persists in the plant until head emergence when fungal spores develop and are released from sori formed at kernel positions. The 26.1-Mb U. hordei genome contains 7113 protein encoding genes with high synteny to the smaller genomes of the related, maize-infecting smut fungi Ustilago maydis and Sporisorium reilianum but has a larger repeat content that affected genome evolution at important loci, including mating-type and effector loci. The U. hordei genome encodes components involved in RNA interference and heterochromatin formation, normally involved in genome defense, that are lacking in the U. maydis genome due to clean excision events. These excision events were possibly a result of former presence of repetitive DNA and of an efficient homologous recombination system in U. maydis. We found evidence of repeat-induced point mutations in the genome of U. hordei, indicating that smut fungi use different strategies to counteract the deleterious effects of repetitive DNA. The complement of U. hordei effector genes is comparable to the other two smuts but reveals differences in family expansion and clustering. The availability of the genome sequence will facilitate the identification of genes responsible for virulence and evolution of smut fungi on their respective hosts.

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi.

BACKGROUND: Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology. RESULTS: To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs. CONCLUSIONS: The current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.

Proteome analysis of wheat leaf rust fungus, Puccinia triticina, infection structures enriched for haustoria.

Puccinia triticina (Pt) is a representative of several cereal-infecting rust fungal pathogens of major economic importance world wide. Upon entry through leaf stomata, these fungi establish intracellular haustoria, crucial feeding structures. We report the first proteome of infection structures from parasitized wheat leaves, enriched for haustoria through filtration and sucrose density centrifugation. 2-D PAGE MS/MS and gel-based LC-MS (GeLC-MS) were used to separate proteins. Generated spectra were compared with a partial proteome predicted from a preliminary Pt genome and generated ESTs, to a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) and to various plant resources. We identified over 260 fungal proteins, 16 of which matched peptides from Pgt. Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six have effector protein signatures, some are related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. Measuring gene expression over several life cycle stages of ten representative candidates using quantitative RT-PCR, all were shown to be strongly upregulated and four expressed solely upon infection.

Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis.

RNA interference (RNAi) acts through transcriptional and post-transcriptional gene silencing of homologous sequences. With the goal of using RNAi as a tool for studying gene function in the related basidiomycete cereal pathogens Ustilago hordei and Ustilago maydis, we developed a general purpose RNAi expression vector. Tandem, inverted fragments of the GUS gene were inserted into this vector flanking an intron and used to transform engineered GUS-expressing haploid cells. Down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei, even though corresponding GUS double-stranded RNA was detected in both species. Similarly, when the endogenous bW mating-type gene was targeted by RNAi, mating was reduced only in U. hordei. Our work demonstrates the feasibility of using RNAi in U. hordei and provides experimental support for the observed lack of RNAi components in the U. maydis genome. We hypothesize that the sharply limited transposon complement in U. maydis is a biological consequence of this absence.

Complementation of Ustilago maydis MAPK mutants by a wheat leaf rust, Puccinia triticina homolog: potential for functional analyses of rust genes.

From a large expressed sequence tag (EST) database representing several developmental stages of Puccinia triticina, we discovered a mitogen-activated protein kinase (MAPK) with homology to kinases with known pathogenic functions in other fungi. This PtMAPK1 is similar to the Ustilago maydis MAPK, Ubc3/Kpp2, but has a longer N-terminal extension of 43 amino acids (aa) with identities to U. maydis Kpp6, a homolog of Ubc3/Kpp2 with a 170-aa N-terminal extension. Ubc3/Kpp2 is involved in mating and subsequent pathogenic development, whereas Kpp6 functions during invasive growth in corn tissue. PtMAPK1, expressed from a Ustilago sp.-specific promoter, was able to complement a ubc3/kpp2 deletion mutant and restore mating. It also substantially increased virulence on corn, measured as tumor formation, of a kpp6 deletion mutant. Moreover, this construct restored to near-full pathogenicity a ubc3/kpp2 kpp6 nonpathogenic double deletion mutant. Complementation of the ubc3/kpp2 mutant with the complete PtMAPK gene and verification of expression by reverse-transcription polymerase chain reaction indicated that the rust promoter is recognized in U. maydis. Phylogenetically, these basidiomycete plant pathogens are related, which was reflected in comparison of P. triticina ESTs to U. maydis gene sequences. The U. maydis heterologous expression system allows functional analysis of rust genes, currently frustrated by the lack of efficient transformation and selection procedures.

Generation of a wheat leaf rust, Puccinia triticina, EST database from stage-specific cDNA libraries.

SUMMARY: Thirteen cDNA libraries constructed from small amounts of leaf rust mRNA using optimized methods served as the source for the generation of 25 558 high-quality DNA sequence reads. Five life-cycle stages were sampled: resting urediniospores, urediniospores germinated over water or plant extract, compatible, interactive stages during appressorium or haustorium formation just before sporulation, and an incompatible interaction. mRNA populations were subjected to treatments such as full-length cDNA production, subtractive and normalizing hybridizations, and size selection methods combined with PCR amplification. Pathogen and host sequences from interactive libraries were differentiated in silico using cereal and fungal sequences, codon usage analyses, and by means of a partial prototype cDNA microarray hybridized with genomic DNAs. This yielded a non-redundant unigene set of 9760 putative fungal sequences consisting of 6616 singlets and 3144 contigs, representing 4.7 Mbp. At an E-value 10(-5), 3670 unigenes (38%) matched sequences in various databases and collections but only 694 unigenes (7%) were similar to genes with known functions. In total, 296 unigenes were identified as most probably wheat and ten as rRNA sequences. Annotation rates were low for germinated urediniospores (4%) and appressoria (2%). Gene sets obtained from the various life-cycle stages appear to be remarkably different, suggesting drastic reprogramming of the transcriptome during these major differentiation processes. Redundancy within contigs yielded information about possible expression levels of certain genes among stages. Many sequences were similar to genes from other rusts such as Uromyces and Melampsora species; some of these genes have been implicated in pathogenicity and virulence.

Mating factor linkage and genome evolution in basidiomycetous pathogens of cereals.

Sex in basidiomycete fungi is controlled by tetrapolar mating systems in which two unlinked gene complexes determine up to thousands of mating specificities, or by bipolar systems in which a single locus (MAT) specifies different sexes. The genus Ustilago contains bipolar (Ustilago hordei) and tetrapolar (Ustilago maydis) species and sexual development is associated with infection of cereal hosts. The U. hordei MAT-1 locus is unusually large (approximately 500 kb) and recombination is suppressed in this region. We mapped the genome of U. hordei and sequenced the MAT-1 region to allow a comparison with mating-type regions in U. maydis. Additionally the rDNA cluster in the U. hordei genome was identified and characterized. At MAT-1, we found 47 genes along with a striking accumulation of retrotransposons and repetitive DNA; the latter features were notably absent from the corresponding U. maydis regions. The tetrapolar mating system may be ancestral and differences in pathogenic life style and potential for inbreeding may have contributed to genome evolution.