An Immunity-Triggering Effector from the Barley Smut Fungus <i>Ustilago hordei</i> Resides in an Ustilaginaceae-Specific Cluster Bearing Signs of Transposable Element-Assisted Evolution

<div><p>The basidiomycete smut fungus <i>Ustilago hordei</i> was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant avirulence locus <i>UhAvr1</i> which triggers immunity in barley cultivar Hannchen harboring resistance gene <i>Ruh1</i>, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish <i>UhAvr1</i> candidates. Systematic deletion and complementation analyses revealed that <i>UhAvr1</i> is <i>UHOR_10022</i> which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common <i>U. hordei</i> transposable element (TE), interrupting the promoter of <i>UhAvr1</i> and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that <i>UhAvr1</i> is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. <i>UhAvr1</i> does not contribute significantly to overall virulence. <i>UhAvr1</i> is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related <i>U. maydis</i> and <i>Sporisorium reilianum</i>. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since <i>U. maydis</i> is not known to trigger immunity in its corn host.</p></div