Dissecting the journey and functions of barley smut fungus Ustilago hordei avirulence effector UhAvr1

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Effector biology, the study of small secreted proteins from microbial pathogens, is a burgeoning research field in both human/animal and plant sciences. Studying effectors has already revealed insight into ways these proteins interact with and manipulate many basic host processes and signaling pathways. This includes development, protein degradation and gene regulation which include defense and resistance responses. Therefore, effectors teach us how pathogens cause disease but can also be used as probes to elucidate basic biological processes. Fungi have large suites of effectors, revealed by genome projects, but their mode of delivery, host cell targets and functions remain largely unknown. Having contributed to the generation and analyses of genome sequences of several cereal-infecting Basidiomycete pathogens (barley smut fungus Ustilago hordei and the related wheat leaf rust fungus Puccinia triticina), we can now focus on functions of the many predicted effectors. We recently identified in U. hordei an effector that triggers defense when infecting a barley cultivar with resistance gene Ruh1. This avirulence effector UhAVR1 is only expressed upon contact with the host, is secreted from the fungus, and its interaction with Ruh1 or its action leads to local host cell death and immunity to the infection. We genetically located Ruh1 on the short arm of barley chromosome 1 to a ~200-kb region. This proposal aims to shed light on 3 basic questions: 1. What are the mechanisms of UhAVR1 secretion and host cell uptake? 2. What is the (virulence) function of UhAVR1 in barley cells, what does it target? 3. How does UhAVR1 carry out its avirulence function / interaction with Ruh1? In 1, we will investigate known genes in the secretory pathways by deleting them in U. hordei and assay by confocal microscopy their effect on delivery of UhAVR1 linked to a fluorescent protein moiety. Using antibodies against UhAVR1, we will also conduct immunolocalisation experimenets by electron microscopy and detect UhAVR1 after cell compartment fractionations. Other genes involved in effector delivery will be revealed by random mutagenesis with tagged inserts of a solopathogenic haploid strain, relying on the strong selection the avirulence gene will provide. That is, interruption of delivery will result in spore production on Ruh1 plants. In 2, changes in gene expression in barley, when challenged with a wildtype infection versus one where UhAvr1 is deleted, is compared. A yeast 2-hybrid screen with UhAVR1 as bait will be used to identify potential barley target proteins. In a biochemical approach, UhAVR1 will be bound to solid supports to isolate proteins or complexes it interacts with, which will be analysed using proteomic techniques. Available fungal and barley genome sequences allow rapid identification of candidate genes and functions will be investigated using an adapted gene silencing system. In 3, barley gene expression is compared as in 2 but now in a resistant host. Ruh1 will be identified by introducing select barley large-insert genomic DNA clones together with UhAvr1 in barley coleoptiles or protoplasts; when Ruh1 is present on a clone, defense triggering can be assayed. Elucidating mechanisms of effector delivery, identification of their host target(s) and defense elicitation in this model system will aid studying difficult, yet economically more important cereal rust pathosystems. Identified resistance genes can be used in breeding programs and host effector target genes can be modified by genome editing to obtain durable disease resistance in crop plants and less reliance on harmful pesticides. Conserved fungal components essential for effector delivery are prime targets for designing specific control measures such as RNA silencing.