Genetic background determines phenotypic strength of METI-II resistance conferred by sdhB S212 mutations in...

Complex II inhibitors (METI-II) are important acaricides for controlling the two-spotted spider mite, Tetranychus urticae, but resistance to this relatively new mode of action is rapidly emerging. Here, we identified a novel sdhB substitution, S212N, in a highly resistant Turkish field strain, while a different substitution at the same position, S212I, has previously been associated with resistance, but without functional validation. We therefore used CRISPR-Cas9 gene editing to introduce S212N and S212I into a susceptible genetic background and revert the resistant-field S212N allele to the susceptible S212 genotype. Functional validation revealed mutation- and compound-specific effects. Introduction of S212I conferred high resistance to all tested METI-II acaricides (resistance ratios >200), whereas S212N caused limited resistance to cyflumetofen and pyflubumide and no detectable resistance to cyenopyrafen. In contrast, reversion of the S212N allele in the resistant field strain significantly increased susceptibility to pyflubumide and cyenopyrafen, demonstrating that the phenotypic effects of sdhB mutations depend strongly on genetic background. However, resistance levels remained elevated after reversion, indicating additional resistance mechanisms. Transcriptomic comparisons between the susceptible GSS strain and the resistant field strain revealed overexpression of detoxification genes previously implicated in METI-II metabolism, suggesting that metabolic detoxification acts synergistically with target-site resistance. Together, our results provide functional validation of two sdhB resistance mutations and show that their phenotypic effects can vary by chemical compound and genetic background. More broadly, our findings demonstrate that fully resolving resistance mechanisms requires not only introducing candidate mutations into susceptible backgrounds but also removing them from resistant populations.