Functional Analysis of β-Tubulins in <i>Ustilaginoidea virens</i> and Their Contribution to Carbendazim Resistance

The rice false smut disease, caused by <i>Ustilaginoidea virens</i>, represents a significant fungal threat to rice production. Our investigation aimed to elucidate the functional roles of two β-tubulin paralogs (<i>Uv</i>β<i>1tub</i> and <i>Uv</i>β<i>2tub</i>) in fungal biology and chemical adaptation. Through targeted gene editing, it was demonstrated that both <i>Uv</i>β<i>1tub</i> and <i>Uv</i>β<i>2tub</i> were essential for vegetative growth, cellular morphology maintenance, conidiation, stress adaptation, and virulence in <i>U. virens</i>. The contrasting resistance phenotypes of Δ<i>Uv</i>β<i>1tub</i> (enhanced carbendazim resistance) versus Δ<i>Uv</i>β<i>2tub</i> (hypersensitivity) demonstrate that Uvβ1tub serves as the primary target of carbendazim. The critical determinant of this differential sensitivity was the natural amino acid divergence at position 198, specifically alanine (A198) in Uvβ2tub versus glutamic acid (E198) in Uvβ1tub. Site-directed mutagenesis confirmed this molecular mechanism: the A198E substitution in Uvβ2tub abolished its intrinsic carbendazim insensitivity, while the reciprocal E198A mutation in Uvβ1tub conferred carbendazim resistance. Expression analysis revealed stable transcript levels of both β-tubulin genes during vegetative growth and infection; however, deletion of either gene triggered compensatory upregulation of its paralog. Collectively, our results elucidate the functional significance of β-tubulin isotypes and their evolutionarily conserved structural motifs in <i>U. virens</i>, thereby advancing the mechanistic understanding of β-tubulin-mediated sensitivity to carbendazim in phytopathogenic fungi.