Sequence-based comparative secretome analysis reveals conserved core effectors and host lineage-specific divergence between monocot- and dicot-associated powdery mildew lineages

Powdery mildew fungi are obligate biotrophs that parasitize living plant tissues and deploy secreted proteins to support host colonization. We compared predicted secretomes from 26 powdery mildew isolates representing five genera (<i>Blumeria</i>, <i>Erysiphe</i>, <i>Golovinomyces</i>, <i>Parauncinula</i>, and <i>Podosphaera</i>) and encompassing monocot- and dicot-associated lineages. A standardized prediction and filtering workflow identified 7,545 secretome candidates from 219,897 proteins, which were then analyzed by orthogroup clustering, N-terminal motif screening, subcellular localization prediction, functional annotation, and homology searches against reported powdery mildew effectors. OrthoFinder assigned candidates to 1,399 orthogroups, revealing a conserved shared component across genera together with extensive genus- and isolate-specific diversification. Candidates were biased toward short mature proteins and were dominated by low-to-moderate cysteine ratios; higher cysteine content coincided with an increased proportion of proteins predicted to localize to the apoplast. N-terminal Y/F/WxC motifs were frequent in <i>Blumeria</i> secretomes and showed genus-specific positional preferences in mature sequences. At least one database-supported annotation was obtained for 4,148 candidates, with common categories including Egh16-like virulence factors, proteases, glycoside hydrolases, and ribonuclease-related annotations. Homology mapping of 75 known powdery mildew effectors identified conserved, high-abundance orthogroup-linked modules spanning multiple genera and <i>Blumeria</i>-restricted expansion modules. Proteome-wide searches further supported EqCmu and EqPdt as broadly conserved non-canonical (signal peptide-lacking) effectors with strong sequence and structural conservation across powdery mildew isolates.