Advances in sequencing technologies as well as development of algorithms and workflows have made it possible to generate fully phased genome references for organisms with nonhaploid genomes such as dikaryotic rust fungi. To enable discovery of pathogen effectors and further our understanding of virulence evolution, we generated a chromosome-scale assembly for each of the 2 nuclear genomes of the oat crown rust pathogen, Puccinia coronata f. sp. avenae (Pca). This resource complements 2 previously released partially phased genome references of Pca, which display virulence traits absent in the isolate of historic race 203 (isolate Pca203) which was selected for this genome project. A fully phased, chromosome-level reference for Pca203 was generated using PacBio reads and Hi-C data and a recently developed pipeline named NuclearPhaser for phase assignment of contigs and phase switch correction. With 18 chromosomes in each haplotype and a total size of 208.10 Mbp, Pca203 has the same number of chromosomes as other cereal rust fungi such as Puccinia graminis f. sp. tritici and Puccinia triticina, the causal agents of wheat stem rust and wheat leaf rust, respectively. The Pca203 reference marks the third fully phased chromosome-level assembly of a cereal rust to date. Here, we demonstrate that the chromosomes of these 3 Puccinia species are syntenous and that chromosomal size variations are primarily due to differences in repeat element content.

Oat crown rust caused by Puccinia coronata f. sp. avenae is the most destructive foliar disease of cultivated oat. Characterization of genetic factors controlling resistance responses to Puccinia coronata f. sp. avenae in nonhost species could provide new resources for developing disease protection strategies in oat. We examined symptom development and fungal colonization levels of a collection of Brachypodium distachyon and B. hybridum accessions infected with three North American P. coronata f. sp. avenae isolates. Our results demonstrated that colonization phenotypes are dependent on both host and pathogen genotypes, indicating a role for race-specific responses in these interactions. These responses were independent of the accumulation of reactive oxygen species. Expression analysis of several defense-related genes suggested that salicylic acid and ethylene-mediated signaling but not jasmonic acid are components of resistance reaction to P. coronata f. sp. avenae. Our findings provide the basis to conduct a genetic inheritance study to examine whether effector-triggered immunity contributes to nonhost resistance to P. coronata f. sp. avenae in Brachypodium spp.

Advances in sequencing technologies as well as development of algorithms and workflows have made it possible to generate fully phased genome references for organisms with non-haploid genomes such as dikaryotic rust fungi. To enable discovery of pathogen effectors and further our understanding of virulence evolution, we generated a chromosome-scale assembly for each of the two nuclear genomes of the oat crown rust pathogen, Puccinia coronata f. sp. avenae (Pca). This resource complements two previous released partially phased genome references of Pca, which display virulence traits absent in the isolate of historic race 203 (isolate Pca203) that was selected for this genome project. A fully phased, chromosome-level reference for Pca203 was generated using PacBio reads and Hi-C data and a recently developed pipeline named NuclearPhaser for phase assignment of contigs and phase switch correction. With 18 chromosomes in each haplotype and a total size of 208.10 Mbp, Pca203 has the same number of chromosomes as other cereal rust fungi such as Puccinia graminis f. sp. tritici and Puccinia triticina, the causal agents of wheat stem rust and wheat leaf rust, respectively. The Pca203 reference marks the third fully-phased chromosome-level assembly of a cereal rust to date. Here, we demonstrate that the chromosomes of these three Puccinia species are syntenous and that chromosomal size variations are primarily due to differences in repeat element content.

BackgroundHistological examination using fluorochromes is one of the standard methods for observation of microorganisms in tissues and other compartments. In the study of fungi, especially those that cannot be cultured in axenic media such as biotrophic fungi, histological examination of processes associated with the fungal growth, differentiation, infection and other cellular functions can lead to the better understanding of host-parasite interactions. Fluorescence microscopy coupled with Fluorochrome Uvitex 2B have been extensively utilized to study rust fungi structures and host-pathogen interactions. In this study, we report development of a rapid staining protocol of the rust fungus Puccinia triticina using fluorochrome Uvitex 2B. The newly developed rapid procedure was compared with a standard staining technique to observe in planta fungal infection structures development during the wheat—Puccinia triticina interaction.ResultsWhile significantly reducing the time for staining, the rapid protocol described here was equally efficient or better compared to standard procedure in detecting fungal infection structures using Uvitex 2B. In the rapid staining procedure, pre-heating of the stain increased efficiency to detect all the infection structures including haustoria with highly reduced background noise from plant tissue.ConclusionThis staining process described here is simple and quick. It can be completed in 4 h, which is of 6 times faster than the standard Uvitex 2B staining procedure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0096-0) contains supplementary material, which is available to authorized users.

Oat crown rust caused by Puccinia coronata f. sp. avenae is the most destructive foliar disease of cultivated oat. Characterization of genetic factors controlling resistance responses to Puccinia coronata f. sp. avenae in non-host species could provide new resources for developing disease protection strategies in oat. We examined symptom development and fungal colonization levels of a collection of Brachypodium distachyon and B. hybridum accessions infected with three North American P. coronata f. sp. avenae isolates. Our results demonstrated that colonization phenotypes are dependent on both host and pathogen genotypes, indicating a role for race-specific responses in these interactions. These responses were independent of the accumulation of reactive oxygen species. Expression analysis of several defense-related genes suggested that salicylic acid and ethylene-mediated signaling, but not jasmonic acid are components of resistance reaction to P. coronata f. sp. avenae. Our findings provide the basis to conduct a genetic inheritance study to examine if effector-triggered immunity contributes to non-host resistance to P. coronata f. sp. avenae in Brachypodium species.

The infection process of wheat stem rust (Puccinia graminis f. sp. tritici) on barley (Hordeum vulgare) is often observed as a mesothetic infection type at the seedling stages, and cultivars containing the same major resistance genes often show variation in the level of resistance provided against the same pathogen race or isolate. Thus, robust phenotyping data based on quantification of fungal DNA can improve the ability to elucidate host-pathogen interaction, especially at early time points of infection when disease symptoms are not yet evident. Quantitative real-time polymerase chain reaction (qPCR) was used to determine the amount of fungal DNA relative to host DNA in infected tissue, providing new insights about fungal development and host resistance during the infection process in this pathosystem. The stem rust susceptible 'Steptoe', resistant cultivars containing only Rpg1 ('Beacon', 'Morex', and 'Chevron'), and the resistant line Q21861 containing Rpg1 and the rpg4/Rpg5 complex were evaluated using the traditional 0-to-4 rating scale, fluorescence microscopy, and qPCR. Statistical differences (P<0.05) were observed in fungal development as early as 24 h postinoculation using the qPCR assay. Fungal development observed using fluorescence microscopy displayed the same hierarchal ordering observed using the qPCR assay. The fungal development occurring at 24 and 48 h postinoculation was vastly different than what was expected using the traditional disease phenotyping methodology; with Steptoe appearing more resistant than the barley lines harboring the known Rpg1 and rpg4/Rpg5 resistance complex. These data indicate potential early prehaustorial resistance contributions in a cultivar considered susceptible based on infection type. Moreover, the temporal differences in resistance suggest pre- and post-haustorial resistance mechanisms in the barley-wheat stem rust infection process, indicating potential host genotype contributions related to basal defense during the wheat stem rust infection process.