The "Stubbs Collection", began in 1956 by the late Dutch plant pathologist R.W. Stubbs, refers to a unique historic collection of urediniospore samples of Puccinia striiformis that had been stored in liquid nitrogen for decades. Since 2010 the collection has been maintained by the Global Rust Reference Center (GRRC) in Denmark. Part of the collection is now being in a study of past pathogen diversity. A subset of samples collected between 1958 and 1991 from 35 countries was investigated to assess recovery rate, race identity, and previously undetected virulences. A new method for recovery using an airbrush sprayer and NovecTM 7100 fluid as dispersal agent in inoculating host plants was highly successful, resulting in a 96% recovery from 231 isolates. Phenotyping on the World and European differential host sets and additional wheat genotypes revealed 181 apparently uniform isolates, of which race identities were confirmed for 102. Race identities were updated for additional isolates based on improved resolution due to updated and more informative differential lines. Additional virulences corresponding to Yr17, Yr25, and Yr27 were added, as these were not assayed earlier. The past population structure was investigated by genotyping 212 isolates using 19 multilocus microsatellites. Seven distinct populations were detected, including clonal populations and recombinant populations. These results were compared with recent studies and demonstrated an overall consistent population subdivision at the global scale with clear migration events between populations. The outcome of the study facilitates conclusions about long-term temporal dynamics and overall migration patterns within and among world-wide populations of Pst.
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Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. Fungal secreted proteins termed effectors play an important role in modulating the host cellular environment and suppressing the plant defense response to enable fungal growth. They also become targets of plant resistance (R) proteins. We have taken a genomics approach to initially identify candidate effectors. We have built a draft genome for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced North American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly resulted in a 92 Mbp reference genome for Pgt isolate 21-0. This draft genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ~10% of the genes in germinated urediniospores as well as haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 586 were classified as haustorial secreted proteins (HSPs). We are currently exploring effector gene expression during infection of wheat to reduce this candidate list based on a common expression profile identified for Avr genes in the flax rust fungus. Comparison of 21-0 with two presumed clonal field derivatives (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 13 HSP effector candidates. These candidate effectors are being assessed for recognition in wheat accessions with the corresponding R genes using a bacterial type three secretion delivery system based on an engineered Pseudomonas fluorescence strain (Upadhyaya NM et al. Mol Plant Microbe Interact 27:255-264).
An important component of the management of wheat stem rust is an understanding of the population diversity of the pathogen, Puccinia graminis f. sp. tritici (Pgt). The discovery of “Ug99” resulted in renewed efforts on pathogen surveys, sample collections and pathotyping of Pgt, with a primary focus on Africa. In the last few years these efforts have been expanded to include other targeted regions, however a global effort is needed. The aims of the “Global Pgt Initiative” is: to capture and maintain living cultures that collectively reflect the entire global diversity of Pgt in the years 2014 - 2016; pathotype and genotype this collection; develop DNAbased diagnostic tools that will be able to rapidly detect shifts in Pgt populations, and provide an early warning system of the vulnerability of wheat to new virulent strains; and provide a genetic baseline for comparison of Pgt populations over time, both forward and backwards. This initiative will provide the wheat rust community with a geographically distributed, well characterized, living culture collection that represents the global diversity of Pgt; a global open access knowledge bank on Pgt pathotypes and genotypes; and advanced molecular diagnostic tools for rapid detection and tracking of Pgt populations. The Global Pgt Initiative represents the most comprehensive effort to capture and characterize the global diversity of Pgt and provide a unique resource to the global wheat rust community.
Oomycetes and fungi facilitate pathogenesis via secretion of effector proteins that have apoplastic and intracellular localizations. These effector proteins have a diverse array of functions that aid in pathogenesis, including modification of defense responses. In the oomycetes, well characterized effector proteins that can translocate into the host cells share a pair of conserved N-terminal motifs known as RXLR and dEER. The RXLR motif has been shown to mediate translocation of the oomycete avirulence proteins Avr1b and Avr3a into host cells. Detailed mutagenesis of the RXLR motif of Avr1b revealed that the motif is tolerant to several amino acid substitutions while retaining functional translocation activity, resulting in the definition of a broadened RXLR-like motif, [R,K,H] X[L/M/I/F/Y/W]X. This motif has been used to identify functional translocation motifs in several fungal effector proteins, AvrL567, Avr2, and AvrLm6. Effectors with both RXLR and RXLR-like motifs bind phosphatidylinositol- 3-phosphate (PI-3-P) to mediate translocation via lipid raft mediated endocytosis. Mutations in RXLR or RXLRlike motifs result in loss of phospholipid binding and translocation by effectors. Effector entry into plant cells can be blocked by proteins and inositides that disrupt binding to PI-3-P, suggesting effector-blocking technologies that could be used in agriculturally important plant species.
A better understanding of the fundamental principles of host-pathogen interactions should enable us to develop new strategies to control disease and to eliminate or at least manage their causative agents. This is especially true for obligate biotrophic parasites like the rust fungi. One vital aspect in the field of obligate biotrophic host-pathogen interactions is the mobilization, acquisition and metabolism of nutrients by the pathogen. This includes transporters necessary for the uptake of nutrients as well as enzymes necessary for their mobilization and metabolism. In a broader sense effector molecules reprogramming the host or triggering the infected cell into metabolic shifts favorable for the pathogen also play an important role in pathogen alimentation.