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Localization and functional study of Ustilago hordei avirulence effector UhAVR1 using barley and Nicotiana benthamiana plant systems
Ana Priscilla Montenegro Alonso: University of British Columbia; Guus Bakkeren: Agric & Agi Food Canada
<div>UhAVR1 is a 190-aa protein of the barley smut fungal pathogen <i>Ustilago hordei</i> (<i>Uh</i>). It has a predicted 19-aa signal peptide (SP) and this candidate effector was identified as an avirulence factor triggering an HR within 48 hrs after infection (hpi) of cv. Hannchen having resistance gene<i> Ruh1</i>. To decipher its virulence function, host target and host response triggered upon its recognition, transcriptomic analyses and localization studies were performed. Barley seedlings from cvs. Odessa (<i>ruh1</i>) and Hannchen (<i>Ruh1</i>), representing compatible and incompatible interactions respectively, were inoculated with teliospores from wild type and an <i>UhAVR1</i> deletion mutant. <i>UhAvr1</i> expression was quantified via ddPCR during infection from 24 hpi till heading showing expression only at the early biotrophic stage and peaking between 72 and 96 hpi. Global transcriptome analysis via RNA-seq at 48 hpi revealed 151 differentially expressed genes (DEGs) in Odessa including kinases, transcription factors, chloroplast genes among others, whereas in Hannchen 173 DEGs were detected including peroxidases, <i>R</i> genes, and chitinases, among others. To identify putative interactors, localization experiments were performed. In heterologous <i>Nicotiana benthamiana </i>(<i>Nb</i>),<i> </i>chimeric UhAVR1-SP, C-terminally tagged with eGFP, was delivered by <i>Agrobacterium tumefaciens</i> into infiltrated leaves. Confocal microscopy revealed localization to cytosolic bodies at 24 and 48 hpi, compared to free eGFP control which was found in the nucleoplasm and cytosol. Protein analysis from leaf samples at the same time points by Western blots using anti-GFP and anti-UhAVR1 antibodies, confirmed the expression of full length chimeric mature UhAVR1 protein of 46 kDa.</div>

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