Rapidly Evolving Genes Are Key Players in Host Specialization and Virulence of the Fungal Wheat Pathogen <i>Zymoseptoria tritici</i> (<i>Mycosphaerella graminicola</i>)

<div><p>The speciation of pathogens can be driven by divergent host specialization. Specialization to a new host is possible via the acquisition of advantageous mutations fixed by positive selection. Comparative genome analyses of closely related species allows for the identification of such key substitutions via inference of genome-wide signatures of positive selection. We previously used a comparative genomics framework to identify genes that have evolved under positive selection during speciation of the prominent wheat pathogen <i>Zymoseptoria tritici</i> (synonym <i>Mycosphaerella graminicola</i>). In this study, we conducted functional analyses of four genes exhibiting strong signatures of positive selection in <i>Z</i>. <i>tritici</i>. We deleted the four genes in <i>Z</i>. <i>tritici</i> and confirm a virulence-related role of three of the four genes <i>ΔZt80707</i>, <i>ΔZt89160</i> and <i>ΔZt103264</i>. The two mutants <i>ΔZt80707</i> and <i>ΔZt103264</i> show a significant reduction in virulence during infection of wheat; the <i>ΔZt89160</i> mutant causes a hypervirulent phenotype in wheat. Mutant phenotypes of <i>ΔZt80707</i>, <i>ΔZt89160</i> and <i>ΔZt103264</i> can be restored by insertion of the wild-type genes. However, the insertion of the <i>Zt80707</i> and <i>Zt89160</i> orthologs from <i>Z</i>. <i>pseudotritici</i> and <i>Z</i>. <i>ardabiliae</i> do not restore wild-type levels of virulence, suggesting that positively selected substitutions in <i>Z</i>. <i>tritici</i> may relate to divergent host specialization. Interestingly, the gene <i>Zt80707</i> encodes also a secretion signal that targets the protein for cell secretion. This secretion signal is however only transcribed in <i>Z</i>. <i>tritici</i>, suggesting that <i>Z</i>. <i>tritici</i>-specific substitutions relate to a new function of the protein in the extracellular space of the wheat-<i>Z</i>. <i>tritici</i> interaction. Together, the results presented here highlight that <i>Zt80707</i>, <i>Zt103264</i> and <i>Zt89160</i> represent key genes involved in virulence and host-specific disease development of <i>Z</i>. <i>tritici</i>. Our findings illustrate that evolutionary predictions provide a powerful tool for the identification of novel traits crucial for host adaptation and pathogen evolution.</p>