Three members of the Puccinia genus, Puccinia triticina (Pt), P. striiformis f.sp. tritici (Pst), and P. graminis 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.

BackgroundRust 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.ResultsTo 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.ConclusionsThe 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.

Three members of the Puccinia genus, Puccinia triticina (Pt), P. striiformis f.sp. tritici (Pst), and P. graminis 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.

Aecial and telial host range, interfertility, teliospore dimensions, and amount of nuclear DNA were determined for Puccinia recondita collected either worldwide from species of cultivated wheats (Triticum aestivum and Triticum turgidum ssp. durum and rye (Secale cereale), or from wild emmer (Triticum turgidum ssp. dicoccoides) and four species of wild wheat (Aegilops) in Israel. The results indicate that the collections belong in two major groups: Group I (from cultivated wheats and wild emmer), which has Thalictrum speciosissimum (in the Ranunculaceae) as principal aecial host; and Group II (principally from wild wheats or rye), which has several species in the Boraginaceae, such as Anchusa aggregata, Anchusa italica, Echium glomeratum, and Lycopsis arvensis as aecial hosts. In glasshouse experiments, intercrosses could be made readily among collections within Groups I and II but not between the two groups. Group I consisted of all collections from Triticum aestivum, Triticum turgidum ssp. dicoccoides, and most collections from Triticum turgidum ssp. durum. For Group I collections, four species of Aegilops, Hordeum maritimum, S. cereale, as well as Triticum aestivum and Triticum turgidum ssp. durum and ssp. dicoccoides could all serve as telial host in glasshouse experiments. Group II consisted of four types, all clearly different from Group I. Type A was from Triticum turgidum ssp. durum found in fields near Anchusa italica, which was its only aecial host; Triticum aestivum, Triticum turgidum ssp. durum, and Triticum turgidum ssp. dicoccoides could serve as telial hosts. Type B was from Aegilops ovata and had E. glomeratum, Anchusa undulata, and L. arvensis as aecial hosts. Type C was from Aegilops longissima, Aegilops sharonensis, and Aegilops variabilis and had Anchusa aggregata, Anchusa undulata and L. arvensis as aecial hosts. Type D was from S. cereale and had L. arvensis and Anchusa undulata as aecial hosts. In addition to differences in host range, teliospores were wider and bigger in cross sectional area, and nuclear DNA content of pycniospores was 1.3–1.6 times greater in Group II than in Group I. The results suggest that Groups I and II have evolved separately for an extended period and are now morphologically distinct and genetically isolated from each other. Furthermore, differences in both telial and aecial host species, in teliospore dimensions, and in amount of nuclear DNA indicate that subgroups within Group II are beginning to show genetic divergence. Key words: aecial hosts, Aegilops, Anchusa, Echium, Hordeum, leaf rust, Lycopsis, Puccinia recondita, Puccinia triticina, Secale, Thalictrum, Triticum.