Bakkeren Plant Pathology Lab - Rust Research

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BACKGROUND

Rust diseases

Rust fungi are among the most devastating fungal pathogens world-wide. They are basidiomycete fungi, belong to the order Uredinales and encompass approximately 5,000 species which can be ordered into 140-150 different genera. They infect many plant species and families and cause many known diseases such as coffee rust, bean rust and rust of pine trees. Probably the most economically damaging diseases are the rusts on cereal crops. Several rust fungi have a major economic impact on the farming community in Western Canada. In particular stem and leaf rust of wheat and barley, and crown rust on oats can cause up to 20% loss in yield. This translates into hundreds of millions of dollars in annual lost revenue and cost for preventive fungicide treatments. A conservative estimate puts losses in 1999 due to the wheat leaf rust alone at 90M in Western Canada.

Rust fungi have been known since biblical times and have been studied for over a century. However, because they are obligate biotrophs it is very difficult to culture these in the laboratory. Progress on molecular genetic studies has therefore been very slow. Recently, however, "Genomics technologies” have been applied on several of the economically most important rusts, the wheat stem rust fungus, Puccinia graminis f. sp. tritici, and the wheat leaf rust fungus Puccinia triticina (formerly, Puccinia recondita f.sp. tritici). Exemplary research has also been done on the bean rust (Voegele (2006). "Uromyces fabae: development, metabolism, and interactions with its host Vicia faba." FEMS Microbiol Lett 259, 165-73) and flax rust, Melampsora lini (Ellis, Dodds and Lawrence (2007). "The role of secreted proteins in diseases of plants caused by rust, powdery mildew and smut fungi." Current Opinion in Microbiology 10, 326-331). The generation of genomic resources is already accelerating the pace of research in this field.

Life cycle

Rust fungi have without doubt the most complicated life cycles of all fungi. Some of them, the macrocyclic forms such as P. triticina, have five life-cycle stages and require two different, completely unrelated host plants. Diploid teliospores (2n) form on senescing wheat plants in the fall (see stage III in diagram below) and are resilient survival structures which, upon germination and meiosis under the right conditions, give rise to haploid basidiospores (n, stage IV). These very ephemeral basidiospores will enter a sexual cycle which takes place on the alternate host plant. "Fertilization" requires the mixing of pycnidiospores of different mating types (n, stage 0) embedded in the nectar of the pycnidia, often by insects. This produces aecia from which aeciospores, dikaryotic (n + n) dispersal structures (stage I), are released. Aeciospores will start the infection cycle on the primary host which results in the production of masses of dikaryotic (n + n) urediniospores (stage II) in brown-coloured, "rusty" pustules. The urediniospores can re-infect the same or fresh host plants several times during the growing season resulting in exponential increases of inoculum. Wind currents and proper weather conditions can result in heavily infected wheat producing areas, or even epidemics.

P. triticina life cycle

(cartoon from "Introductory Mycology", used
by permission of John Wiley & Sons, Inc.)

Infection process

Vital statistics

HIGHER FUNGI
Subdivision: BASIDIOMYCOTINA
Class: HEMIBASIDIOMYCETES (TELIOMYCETIDAE)
Order: Uredinales
Genus: Puccinia
Species: triticina

Disease	Host	Pathogen	Alternate Host
wheat leaf rust(brown rust)	Triticum aestivum	Puccinia triticina, formerly P. recondita f. sp. tritici	meadow rue (Thalictrum speciosissimum)

Genome size of P. triticina is estimated at approx. 100 – 120 Mbp.
For comparison:
Genome size of P. graminis f. sp. tritici (Pgt) was estimated to be approx. 67 Mbp ( 64% unique, 30% repetitive sequences; 45.3% G + C (Backlund, J. E. and L.J. Szabo. 1993. Current Genetics 24:89-93).

Pgt genome sequencing to approx. 8x coverage has recently been achieved (Jan. 2007; Broad Institute), including 40,000+ ESTs. Based on the genome sequence, the current Pgt genome size is estimated at approx. 80 Mbp.

Current research projects include: 

· Generation of genomic resources for Puccinia triticina and comparative analyses

Having generated large data sets of Expressed Sequence Tags (ESTs) covering all life cycle stages (Hu et al., 2007; Xu et al., 2011), and a Bacterial Artificial Chromosome (BAC) library, we have contributed to sequencing of complete P. triticina genomes (Puccinia Group Sequencing Project, Broad Institute of Harvard and MIT; collaboration with Dr. C. Cuomo, Broad Institute, Cambridge, MA, Dr. L. Szabo, ARS-USDA, CDL-St. Paul MN and Dr. J. Fellers, ARS-USDA, Manhattan KS), funded through the NSF/USDA CSREES Microbial Genome Sequencing Program. BAC end-sequencing was performed by Scientists at the Michael Smith Genome Sciences Centre, Vancouver, BC, Canada.

Large-scale, next-gen genome re-sequencing of many isolates and races, and RNA-seq to generate transcriptome profiles is conducted at the Michael Smith Genome Sciences Centre, Vancouver, BC, Canada to investigate gene and race/isolate diversity as found in agriculture settings and upon environmental selection bottlenecks (e.g., resistance gene introductions), and to study fungal and host responses during various interaction types (Wang et al, 2012). Research is part of two projects: "Genomics Approaches to Mitigate Fungal Threats to Crops" with B. Saville, Trent University, ON, an Ontario Ministry of Research and Innovation-funded project on large-scale transcriptome analyses of cereal rust and other fungi during host infections, and "Poplar and cereal rust comparative genomics: identification of pathogen determinants to prevent and predict epidemics” with R. Hamelin, Dept. of Forest Sciences, UBC & Canadian Forest Service, Laurentian Forestry Centre in Quebec (funded through the Genome British Columbia Strategic Opportunities Fund, round 3). Collaboration with AAFC researchers, B. McCallum and T. Fetch (AAFC Cereal Research Centre in Winnipeg, MB), and Visiting Scientists, Drs. D. Joly and X. Wang.

· Search for virulence and avirulence factors

Having the generated genomic resources and using genetics, molecular techniques, proteomics and bioinformatic mining, we are identifying pathogenicity and virulence genes, including potential effectors and avirulence genes (Ali et al, 2012; Bakkeren et al., 2012). The generated genomic resources will give us a comprehensive framework upon which EST sequences and Illumina-generated transcript reads and other isolated markers can be localized. To this end, collaboration is ongoing with B. McCallum on the construction of a P. triticina genetic map using crosses in which many known avirulence genes segregate, and SSR markers identified through our EST database (Wang et al., 2010a; Wang et al., 2010b) and by genome sequencing of progeny and identification of SNPs. We are also generating protein profiles in haustorial (Song et al., 2011) and other life cycle stage structures (in collaboration with Dr. C. Rampitsch, AAFC Cereal Research Centre in Winnipeg, MB).

· Generating molecular tools

Rusts are difficult to genetically transform; initial attempts have been encouraging for P. triticina (Webb et al., 2006) and optimization is in progress. The use of a heterologous expression system to allow functional analysis of rust genes was explored. The corn smut fungus, Ustilago maydis, was found to allow the expression of a P. triticina MAP kinase gene and to complement a non-pathogenic U. maydis MAPK deletion mutant to restore full pathogenicity (Hu et al., 2007). Several other functional analysis assays are being developed.