Thermal Acclimation in Ustilago esculenta: Multivesicular Body Dynamics and a Working Hypothesis of Transcriptional Decoupling Under Temperature Regimes

Temperature limits jiaobai (Zizania latifolia) production by its symbiont, the smut fungus Ustilago esculenta, yet the cellular mechanisms underlying this thermal sensitivity remain poorly understood. In this study, we combined controlled temperature regimes, transmission electron microscopy, and RNA sequencing to dissect the fungal response to heat. A moderate 30/25 °C day/night regime was associated with the highest proportion of cells containing multivesicular bodies (MVBs; 65.0%) and the only detectable autophagosome-like structures (6.7%), whereas regimes incorporating 35 °C drove extensive vacuolar degradation and cytoplasmic rarefaction. Strikingly, the strongest MVB accumulation occurred with the fewest transcriptional changes among 126 endocytic, autophagic, and vacuolar genes, suggesting as a working hypothesis that temperature may primarily shape MVB homeostasis through post-transcriptional, flux-level regulation rather than wholesale transcriptional reprogramming. However, low numbers of MVB-related differentially expressed genes (DEGs) were not unique to the 30/25 °C regime: AT35/25 also had only eight MVB-related DEGs yet showed severe degradation, and T35 had numerous MVBs with only 14 MVB-related DEGs. These observations indicate that DEG counts alone cannot predict MVB abundance. The 30/25 °C regime, which qualitatively resembled the field phenotype, induced only 418 differentially expressed genes, whereas harsher regimes triggered broader transcriptional responses without corresponding increases in MVB abundance. These findings provide a cellular and transcriptional framework for understanding the heat sensitivity of jiaobai production, highlighting the importance of diurnal thermal rhythms and post-transcriptional regulation in fungal thermal acclimation.