Cryometamorphosis expression patterns of fruiting body development in the mushroom-forming fungus Armillaria ostoyae

Abstract

Abstract Armillaria ostoyae is an economically important forest pathogen whose reproductive development is regulated by environmental cues. Although low temperature is recognized as an important morphogenetic stimulus, the molecular mechanisms underlying the resulting developmental transition—here termed “cryometamorphosis”—remain poorly understood. Using an orthogonal experimental design, we categorized A. ostoyae as a “cold-pressed” species. Induction at 4 °C significantly accelerated primordium formation, reducing the time required for initiation from 25 to 7 days. Transcriptomic profiling across developmental and morphological stages revealed that primordium initiation was associated with the most extensive transcriptional reprograming across the reproductive trajectory. Weighted gene co-expression network analysis identified a core low-temperature-associated module, MEturquoise, and revealed a putative hierarchical regulatory network. Within this network, a group of fungus-specific Zn(II)2Cys6 transcription factors whose promoters contained predicted low-temperature response elements were tightly co-expressed with putative SET-domain histone methyltransferases and F-box proteins. To functionally validate this regulatory model, we established an optimized genetic transformation system for A. ostoyae and overexpressed the leading candidate gene, AoZCy6_17. Overexpression of AoZCy6_17 at a constant temperature of 25 °C was associated with a pronounced morphological transition: no visible rhizomorph formation was observed in the OE strain under the conditions tested, together with increased production of dense aerial mycelia accompanied by abundant surface droplets. Biochemical assays showed that the overexpression strain exhibited enhanced basal antioxidant activity, with significantly higher catalase activity than the wild type (approximately 2,436 versus 2,001 U g-1 fresh weight; P < 0.001). This constitutive physiological reprograming was also associated with increased malondialdehyde accumulation under non-stress conditions (approximately 23.1 versus 9.6 nmol g-1 fresh weight), indicating a potential physiological cost. Notably, activation of AoZCy6_17 alone recapitulated and, for some biochemical traits, exceeded the changes induced by exposure of the wild-type strain to 4 °C. Collectively, our findings provide functional evidence that AoZCy6_17 is a key regulator linking low-temperature signaling to developmental and physiological reprograming in A. ostoyae, thereby establishing a mechanistic framework for investigating fungal cryometamorphosis.