Shao Lei, Tan Yumei, Song Shiying, Wang Yuchen, Liu Yongxiang, Huang Yonghui, Ren Xiyi, Liu Zuoyi
College of Agriculture, Guizhou University, Guiyang, Guizhou, China.
Guizhou Key Laboratory of Agricultural Biotechnology, Guiyang, Guizhou, China.
J Basic Microbiol. 2022 Dec;62(12):1487-1503. doi: 10.1002/jobm.202200325. Epub 2022 Oct 3.
Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea, hypotonic conditions only induced its sexual development to produce ascospores, while hypertonic conditions only induced its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the roles of Acpbs2, which is homologous to pbs2 from Saccharomyces cerevisiae, in sporulation, stress responses, the color of colonies, and carbon metabolism were explored in A. cristatus. Deletion mutants of Acpbs2 were obtained by homologous recombination. The time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in hypertonic media in ΔAcpbs2 by phenotypic observations, indicating that Acpbs2 plays a positive role in asexual development. Stress sensitivity tests showed that the order of the sensitivity of ΔAcpbs2 to different osmotic regulators was 3 M NaCl > 3 M sucrose > 3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. The growth of the Acpbs2 deletion mutant was inhibited under alkaline-pH stress, indicating that Acpbs2 is involved in high pH stress tolerance. Additionally, compared with the wild type, the colony color of the Acpbs2 deletion mutant became lighter. All the above developmental defects were reversed by the reintroduction of the Acpbs2 gene in ΔAcpbs2. Transcriptome data showed that Acpbs2 regulated the expression of several genes related to conidial development, osmotic stress, oxidative stress, and carbon metabolism. More importantly, the interaction between Acpbs2 and its downstream gene Achog1 was verified by yeast two-hybrid assays. We speculated that this interaction might regulate the osmotic stress response, the oxidative stress response, and asexual sporulation in A. cristatus, which will be one of the focuses of our future research.
冠突曲霉是茯砖茶发酵过程中的优势真菌，低渗条件仅诱导其有性发育产生子囊孢子，而高渗条件仅诱导其无性发育产生分生孢子，这表明渗透胁迫可调节冠突曲霉的孢子产生。然而，其潜在的调控机制尚不清楚。在本研究中，探讨了与酿酒酵母的pbs2同源的Acpbs2在冠突曲霉的孢子形成、应激反应、菌落颜色和碳代谢中的作用。通过同源重组获得了Acpbs2的缺失突变体。通过表型观察发现，在高渗培养基中，ΔAcpbs2产生分生孢子的时间延迟，且产生的分生孢子数量显著减少，这表明Acpbs2在无性发育中起积极作用。应激敏感性测试表明，ΔAcpbs2对不同渗透调节剂的敏感顺序为3M NaCl＞3M蔗糖＞3M山梨醇。此外，缺失突变体对高氧化应激敏感。Acpbs2缺失突变体在碱性pH胁迫下生长受到抑制，这表明Acpbs2参与了对高pH胁迫的耐受性。此外，与野生型相比，Acpbs2缺失突变体的菌落颜色变浅。在ΔAcpbs2中重新引入Acpbs2基因后，上述所有发育缺陷均得到逆转。转录组数据表明，Acpbs2调节了几个与分生孢子发育、渗透胁迫、氧化应激和碳代谢相关的基因的表达。更重要的是，通过酵母双杂交试验验证了Acpbs2与其下游基因Achog1之间的相互作用。我们推测这种相互作用可能调节冠突曲霉的渗透胁迫反应、氧化应激反应和无性孢子形成，这将是我们未来研究的重点之一。
