Cultivation and gene-expression study links low temperature to Armillaria ostoyae fruiting-body development

Low-temperature treatment was required for fruiting-body primordium initiation in the mushroom-forming fungus Armillaria ostoyae under the artificial cultivation conditions tested, according to an experimental study combining orthogonal cultivation trials, RNA sequencing, co-expression analysis, and targeted gene overexpression. The researchers proposed the term “cryometamorphosis” for morphogenetic reprogramming triggered by a critical low-temperature regime, but the results do not identify a single molecular switch or establish a direct regulatory pathway.

The researchers used the diploid strain JG19016, isolated from Heilongjiang Province, China, and its derived haploid isolate JG19016/8. A three-factor, three-level experiment tested temperature regime, daily light duration, and exogenous supplementation. No primordia formed in the groups maintained continuously at 25 °C, whereas all groups cultured at 15 °C produced primordia, with or without prior exposure to 4 °C. Within these conditions, temperature had the largest effect on total fresh weight, with a range-analysis value of 95.17, compared with 30.87 for light duration and 12.77 for supplementation.

Mycelia maintained at 15 °C formed primordia after 25 days. Three days of pretreatment at 4 °C followed by cultivation at 15 °C reduced the time to primordium formation to seven days, an 18-day shortening relative to continuous 15 °C cultivation. A combination of 4 °C pretreatment, 15 °C cultivation, six hours of light per day, and no supplementation produced a 100% primordium-to-fruiting conversion rate, total fresh weight of 46.27 ± 5.33 g, and biological efficiency of 4.74 ± 0.55%. The same treatment had the smallest mean pileus diameter among treatments that formed fruiting bodies, at 3.80 ± 0.20 cm. These observations establish the importance of low temperature under the tested regime, but the experiment does not show that the same combination is optimal in natural settings or across other strains.

RNA sequencing compared vegetative mycelium, primordia, mature fruiting bodies, melanized rhizomorphs, and the monokaryotic mycelium, with three independent biological replicates per state. Genes were considered differentially expressed at a false discovery rate below 0.05 and an absolute log2 fold change of at least two. The transition from vegetative mycelium to primordium produced the largest set of transcriptional changes. Primordium-enriched genes included hydrophobins, several transcription factors, protein kinases, candidate SET-domain histone methyltransferases, lysine methyltransferases, RING-type zinc-finger proteins, and F-box proteins. The findings indicate stage-associated expression, but do not demonstrate that any of these genes directly controls the transition.

The transition from primordia to mature fruiting bodies involved fewer differential-expression changes, and the two transcriptomes clustered closely. A total of 39 genes were significantly upregulated in both reproductive stages relative to vegetative mycelium. Upregulated genes in primordia were enriched for stimulus responses, protein kinase activity, mitotic and meiotic processes, carbohydrate and lipid metabolism, and pectin catabolism. In mature fruiting bodies, enrichment included stimulus responses, protein kinase activity, endoplasmic-reticulum membrane components, meiosis and sporulation regulation, and G-protein-coupled receptor activity. Primordia also had the largest number of upregulated genes encoding predicted transcription factors among the states examined.

Weighted gene co-expression network analysis retained the 5,000 genes with the highest median absolute deviation and identified 10 modules. The MEturquoise module had the strongest positive association with mature fruiting bodies, at r = 0.665 and P = 0.007, and a moderate association with primordia, at r = 0.517 and P = 0.05. Hub and closely associated candidates included Zn(II)2Cys6 and other zinc-finger transcription factors, SET-domain methyltransferases, and F-box proteins. The researchers identified 23 developmentally regulated Zn(II)2Cys6 factors, AoZCy6_1 through AoZCy6_23, and predicted low-temperature-responsive elements in the promoters of 12. These promoter predictions remain to be experimentally tested.

During a seven-day induction at 4 °C, most candidate transcripts showed no significant change during the first 24 hours, while several increased after prolonged exposure. AoZCy6_17 rose 53.9-fold on day 3, with P = 0.0356, and remained 48.7-fold above the day-0 baseline on day 7, with P = 0.0051. A superoxide-dismutase-annotated candidate also increased on days 3 and 7, while two other candidates showed overall increases with greater replicate variation. These temporal profiles support an association between prolonged low temperature and expression of developmental candidates, but they do not demonstrate direct regulation among the genes.

Functional testing involved generating an AoZCy6_17-overexpressing strain through Agrobacterium tumefaciens-mediated transformation. The overexpression strain had no visible rhizomorph network under the examined culture conditions and instead produced denser aerial mycelium and visible surface condensation. At 25 °C, its mean catalase activity was approximately 2,436 U/g fresh weight, compared with approximately 2,001 U/g in the wild type, while malondialdehyde content was approximately 23.1 nmol/g fresh weight versus 9.6 nmol/g. Both differences had P < 0.001. These measured changes associate elevated AoZCy6_17 expression with altered colony architecture and oxidative-status indicators; they do not prove that the factor directly regulates those traits or acts as a master developmental regulator.

Overall, the study supports a working model in which low-temperature exposure is accompanied by stage-specific transcriptional changes involving candidate transcription factors, chromatin-associated regulators, protein-turnover components, hydrophobins, and oxidative-response genes. Its principal limitations are the artificial cultivation conditions, the use of one diploid strain and its haploid derivative, unresolved direct regulatory relationships, and low transcript abundance that prevented robust temporal qPCR estimates for some SET-domain and F-box candidates. The results therefore provide a testable expression framework for cryometamorphosis in A. ostoyae, rather than a complete molecular explanation of how temperature cues initiate fruiting-body development.

Subject of Research: Low-temperature-associated gene expression and fruiting-body development in Armillaria ostoyae

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

Article References: Zhou, J.-J., Zhao, Y.-J., Liu, Q., Liu, J., Li, S.-H., Wang, Z.-Y., Zhu, Z.-X., Xiong, X.-S., & Ji, R.-Q. (2026). Cryometamorphosis expression patterns of fruiting body development in the mushroom-forming fungus Armillaria ostoyae. IMA Fungus, 17, Article e186942. https://doi.org/10.3897/imafungus.17.186942

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.186942

Keywords: Armillaria ostoyae, cryometamorphosis, fruiting-body development, low-temperature induction, RNA sequencing, co-expression analysis, Zn(II)2Cys6 transcription factors, AoZCy6_17, gene overexpression, fungal morphogenesis, expression, patterns