Genome Re-Sequencing and Functional Analysis Reveal an α-1,3-Glucosyltransferase Conferring Metalaxyl Resistance in Phytophthora sojae

Phytophthora and allied oomycete pathogens pose a perennial challenge to global food security through their devastating impact on crop systems. While metalaxyl has demonstrated remarkable efficacy in controlling Phytophthora diseases since its introduction decades ago, the persistent emergence of metalaxyl-resistant strains has severely compromised its field efficacy. Elucidating the genetic determinants underlying resistance mechanisms is critical to developing surveillance strategies and sustainable countermeasures against evolving oomycete resistance. Through experimental evolution, we generated six metalaxyl-resistant Phytophthora sojae mutants exhibiting extreme resistance levels (resistance factor > 2000). Comparative whole-genome re-sequencing of resistant mutants versus the wild-type parental strain identified 64 candidate genes containing conserved nonsynonymous mutations across all resistant lineages. Among these, PsALG8, encoding a putative alpha-1,3-glucosyltransferase, was identified as the primary determinant, carrying a recurrent homozygous missense mutation across all resistant lineages. CRISPR/Cas9-mediated knockout of PsALG8 in both wild-type and resistant backgrounds significantly reduced metalaxyl tolerance (p < 0.01), confirming its functional involvement in resistance modulation. These results suggest that PsALG8 is associated with metalaxyl sensitivity and mycelial growth in P. sojae under laboratory conditions. The conservation of ALG8 homologs suggests that PsALG8 may have a conserved cellular function related to protein glycosylation across eukaryotes. Although this glucosyltransferase is universally conserved among oomycete species, whether its association with metalaxyl sensitivity constitutes a shared resistance adaptation pathway still requires extensive functional validation in diverse Phytophthora pathogens, which may offer insights into future fungicide resistance management strategies in P. sojae.