Mitochondrial heteroplasmy as a transitional state in the evolution of fungicide resistance

Mitochondrial DNA (mtDNA) can exist in homoplasmic or heteroplasmic states, but the dynamics and stability of heteroplasmy remain poorly understood in fungal plant pathogens. Because a key fungicide target (cytochrome b ) is encoded in the mitochondrial genome, understanding these mtDNA dynamics is crucial for predicting resistance development and evolution in agricultural pathosystems. In Zymoseptoria tritici, mutations G143A and Y132C in the quinone outside inhibitor-binding site of the cytochrome b subunit confer a high level of resistance to the fungicides pyraclostrobin and metyltetraprole, respectively. Under fungicide selection, these mutations were observed to coexist only transiently. Using experimental evolution and single-cell analysis, we demonstrate the presence of a temporarily heteroplasmic state in which both G143A and Y132C alleles coexist within individual cells at variable ratios. Allele predominance within the heteroplasmic populations shifted rapidly in response to fungicide pressure, resulting in a mitochondrial mutation switch that ultimately led to either G143A or Y132C homoplasmy. This finding provides evidence that transient heteroplasmy represents a critical intermediate phase during the transition between resistance genotypes in Z. tritici . To our knowledge, this is the first single-cell demonstration of mitochondrial heteroplasmy in a pathogenic fungus, offering new insights into mitochondrial genome dynamics that shape adaptive responses to fungicides. IMPORTANCE Mitochondrial genomes in fungal pathogens are generally considered to be genetically uniform. However, our work shows that mitochondrial heteroplasmy can form a transient and unstable state that enables switching between resistance-associated haplotypes under changing selective pressures. Using single-cell analysis, we demonstrate that individual cells harbor varying proportions of resistance alleles, which can rapidly shift depending on fungicide exposure. These findings provide new insight into how mitochondrial genome dynamics contribute to microbial adaptation and highlight a previously underappreciated mechanism that may influence the emergence and development of antifungal resistance.