Investigation into the secretion and localization of Ustilago hordei avirulence effector UhAVR1
Ana Priscilla Montenegro Alonso: University of British Columbia
<div>We previously identified avirulence effector UhAVR1 in <em>Ustilago hordei</em>, a basidiomycete fungus causing covered smut disease on barley. UhAVR1 is a 171 aminoacids protein with an N-terminal signal peptide (SP). Presence of resistance gene <em>Ruh1</em> in barley cv. Hannchen leads to a strong hypersensitive response upon infection, arresting pathogen growth, whereas cv. Odessa (<em>ruh1</em>) supports disease. To determine the virulence function of UhAVR1, localization and secretion studies were carried out. In <em>Nicotiana benthamiana</em>, using agroinfiltration, transiently expressed UhAVR1:eGFP mature peptide (-SP) was shown to localize to the nucleoplasm and cytosol where it was concentrated in small cytosolic bodies. When testing UhAVR1+SP:eGFP, no apoplastic localization was seen upon plasmolysis. Protein blots confirmed the production of full length fusion protein. Agroinfiltration in barley confirmed a cytosolic localization. In a novel secretion system, full length UhAVR1:mCherry was detected by protein blots when expressed from a constitutive strong Hsp70 promoter in haploid <em>U. hordei</em> cells, as well as in spent medium, indicating secretion. Infiltration in barley leaves of this <em>Ustilago</em> strain showed UhAVR1:mCherry fluorescence in cytosolic bodies in mesophyll cells. With Brefeldin A, secretion of UhAVR1:mCherry seemed unaffected, suggesting secretion through a non-canonical pathway. This would be the first report of non-conventional secretion of a basidiomycete pathogen effector.</div>
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