An Ammonium Transporter Gene Contributes to the Aggressiveness of the Dutch Elm Disease Pathogen Ophiostoma novo-ulmi

Molecular mechanisms determining pathogenicity of the Dutch elm disease fungus <i>Ophiostoma novo-ulmi</i> are poorly understood. Prior identification of the pathogenicity locus <i>pat1</i> prompted a chromosome walking approach to elucidate gene function in this region. Among 17 identified genes, <i>ONUg0282</i> (<i>amtA</i>) was predicted to encode a high-affinity ammonium transporter. In silico analyses confirmed the presence of four additional <i>amt</i> genes (<i>amtB</i>, <i>amtC</i>, <i>amtD</i>, and <i>amtE</i>) in both <i>O. novo-ulmi</i> and the less aggressive <i>O. ulmi</i> and that <i>amtA</i> and <i>amtB</i> belong to the <i>Saccharomyces cerevisiae mep2</i> clade. The predicted <i>amtA</i> gene product showed features of Mep2-type transceptors, including amino acid residues corresponding to His-168 and His-318 in <i>Escherichia coli</i> AmtB protein, 11 transmembrane helices, and a conserved 22 amino acid motif immediately downstream of the last transmembrane helix. A knockdown <i>amtA</i> mutant with 25% residual expression was significantly less aggressive than wild-type <i>O. novo-ulmi</i> strain H327 when infecting <i>Ulmus americana</i> × <i>U. parvifolia</i> saplings. Predicted AmtA transporters from two CRISPR-Cas9 knockout mutants contained only five intact transmembrane helices. The <i>ΔamtA</i> mutants retained several wild-type phenotypic traits, including yeast-mycelium dimorphism, but were significantly less aggressive than H327 towards <i>U. americana</i> saplings. We concluded that ONUg0282 is an important determinant of aggressiveness in <i>O. novo-ulmi</i>.