Genetics and resistance/Génétique et résistance Development of EST-derived simple sequence repeat markers for wheat leaf rust fungus, Puccinia triticina Eriks Molecular markers for wheat leaf rust

Abstract: Gene-associated simple sequence repeat (SSR) markers were developed for Puccinia triticina through the data mining of existing EST libraries. Analysis of 7134 expressed sequence tags (ESTs) from cDNA libraries of P. triticina detected 204 EST-SSRs with a minimum of 12 repeating nucleotides. The majority of EST-SSRs contained short di-or tri-nucleotide repeats. These EST-SSRs were evaluated on 35 P. triticina isolates collected in Canada and 21 EST-SSRs were polymorphic and informative in determining intraspecific genetic diversity. A comparison of virulence and EST-SSR genotypes showed a strong correlation between virulence to Lr2a, Lr2c and Lr17a and EST-SSRs genotypes. The differentiation of the P. triticina population based on EST-SSR genotypes was comparable to that obtained with genomic SSRs, despite differences between two types of SSR markers. Eight of the 21 EST-SSRs produced the cross amplification in Puccinia coronata and Puccinia graminis, suggesting that EST-SSRs are more applicable than genomic SSRs for interspecific analysis. In summary, our study suggests that the data mining of EST databases is a feasible way to generate informative molecular markers for genetic studies of P. triticina. Keywords: population genetics, Puccinia triticina, simple sequence repeat, virulence Résumé: Des marqueurs microsatellites (SSR) associés à des gènes ont été conçus pour Puccinia triticina à partir l'exploration de données contenues dans des banques d'étiquettes de séquences exprimées (EST). L'analyse de 7134 EST issues de banques d'ADNc de P. triticina ont permis de détecter 204 EST-SSR avec un minimum de 12 répétitions de nucléotides. La majorité des EST-SSR contenaient de courtes répétitions di-ou tri-nucléotidiques. Ces EST-SSR ont été évaluées sur 35 isolats de P. triticina collectés au Canada. Parmi celles-ci, 21 étaient polymorphiques et ont fourni de l'information servant à établir la diversité génétique intraspécifique. Une comparaison de la virulence et des génotypes EST-SSR a montré une forte corrélation entre la virulence à l'égard de Lr2a, Lr2c et Lr17a ainsi qu'à l'égard des génotypes EST-SSR. La différenciation de la population de P. triticina, basée sur les génotypes EST-SSR, était comparable à celle obtenue avec les SSR génomiques, malgré les différences entre les deux types de marqueurs. Huit des 21 EST-SSR ont engendré la transférabilité chez Puccinia coronata et Puccinia graminis, ce qui suggère que les EST-SSR sont plus adaptables à l'analyse interspécifique que les SSR génomiques. En résumé, notre étude suggère que l'exploration de données des banques d'EST permet de générer des marqueurs moléculaires informatifs pour les études génétiques portant sur P. triticina

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

© 2011 Xu et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.DOI: 10.1186/1471-2164-12-161Background.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

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

Abstract 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 Xanking 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 doublestranded 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

Genomics Research on Non-Model Plant Pathogens: Delivering Novel Insights into Rust Fungus Biology

Fungi of the order Pucciniales cause rust diseases on many plants including important crops and trees widely used in agriculture, forestry and bioenergy programs; these encompass gymnosperms and angiosperms, monocots and dicots, perennial and annual plant species. These fungi are obligate biotrophs and -except for a few cases- cannot be cultivated outside their hosts in a laboratory. For this reason, standard functional and molecular genetic approaches to study these pathogens are very challenging and the means to study their biology, i.e. how they infect, develop and reproduce on plant hosts, are rather limited, even though they rank among the most devastating pathogens. Among fungal plant pathogens, rust fungi display the most complex lifecycles with up to five different spore forms and for many rust fungi, unrelated alternate hosts on which sexual and clonal reproduction are achieved. The genomics revolution and particularly the application of new generation sequencing technologies have greatly changed the way we now address biological studies and has in particular accelerated and made feasible, molecular studies on non-model species, such as rust fungi. The goal of this research topic is to gather articles that present recent advances in the understanding of rust fungi biology, their complex lifecycles and obligate biotrophic interactions with their hosts, through the means of genomics. This includes genome sequencing and/or resequencing of isolates, RNA-Seq or large-scale transcriptome analyses, genome-scale detailed annotation of gene families, and comparative analyses among the various rust fungi and, where feasible, with other obligate biotrophs or fungi displaying distinct trophic modes. This Research Topic provides a great opportunity to provide an up-to-date account of rust fungus biology through the lens of genomics, including state-of-the-art technologies developed to achieve this knowledge

Genomics Research on Non-Model Plant Pathogens: Delivering Novel Insights into Rust Fungus Biology

Fungi of the order Pucciniales cause rust diseases on many plants including important crops and trees widely used in agriculture, forestry and bioenergy programs; these encompass gymnosperms and angiosperms, monocots and dicots, perennial and annual plant species. These fungi are obligate biotrophs and -except for a few cases- cannot be cultivated outside their hosts in a laboratory. For this reason, standard functional and molecular genetic approaches to study these pathogens are very challenging and the means to study their biology, i.e. how they infect, develop and reproduce on plant hosts, are rather limited, even though they rank among the most devastating pathogens. Among fungal plant pathogens, rust fungi display the most complex lifecycles with up to five different spore forms and for many rust fungi, unrelated alternate hosts on which sexual and clonal reproduction are achieved. The genomics revolution and particularly the application of new generation sequencing technologies have greatly changed the way we now address biological studies and has in particular accelerated and made feasible, molecular studies on non-model species, such as rust fungi. The goal of this research topic is to gather articles that present recent advances in the understanding of rust fungi biology, their complex lifecycles and obligate biotrophic interactions with their hosts, through the means of genomics. This includes genome sequencing and/or resequencing of isolates, RNA-Seq or large-scale transcriptome analyses, genome-scale detailed annotation of gene families, and comparative analyses among the various rust fungi and, where feasible, with other obligate biotrophs or fungi displaying distinct trophic modes. This Research Topic provides a great opportunity to provide an up-to-date account of rust fungus biology through the lens of genomics, including state-of-the-art technologies developed to achieve this knowledge

The Ustilago hordei-Barley Interaction is a Versatile System for Characterization of Fungal Effectors

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei-barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley

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.Science, Faculty ofNon UBCBotany, Department ofReviewedFacult