Crosstalk between Ca2+-signaling and the light-sensing pathway during stress responses and hyphal polar growth in Aspergillus nidulans and A. fumigatus

烟曲霉是世界范围内最重要的气传子囊菌病原体和过敏原之一。数百万人患有侵袭性或慢性感染，每年导致60万人死亡。因此，更好地了解这种真菌的生物学特性可能有助于开发新的或改进现有的抗真菌药物。真菌病原体必须适应体内的各种障碍才能生存。这些障碍包括高温、pH变化、氧化应激或质膜应激对唑类药物治疗的影响。与其他丝状真菌一样，真菌病原体已经进化出许多信号转导系统来感知和响应环境压力，以便在恶劣的环境条件下生存和繁殖。HOG信号通路和钙信号通路都参与了应激适应。最近，人们发现光敏色素通过HOG途径感知光信号，在植物和细菌中有证据表明光敏色素起到了分子温度计的作用。在这一建议中，我们假设光敏色素依赖的光敏感可能通过HOG途径与钙信号相互关联。我们的目标是在两个相关的子囊菌中研究这两个信号通路，它们是尼杜拉曲霉和机会致病的烟曲霉菌。我们的初步数据还表明，光敏色素和钙信号通路都参与了根结线虫的温度感应。我们计划研究光敏色素、HOG和钙信号通路在温度和氮唑抗真菌感应中的作用，以及在烟曲霉中的作用。这两条信号通路之间的相互作用将在转录和转录后水平上进行研究。也有很好的证据表明，钙不仅在压力适应中发挥作用，而且在极化生长中也发挥作用。结果表明，在极生菌丝伸展过程中，Ca~(2+)浓度发生振荡，这种振荡可能与泡囊分泌的振荡和菌丝的振荡生长有关。由于应激反应引起细胞内钙离子浓度的剧烈变化，应激对极细胞伸展的影响是可能的。为了研究这种关系，我们将分析胁迫反应途径如何影响这两种真菌的菌丝极地生长，以及它们如何影响烟曲霉的毒力。为此，我们将结合荧光蛋白GECO技术，监测表达钙报告蛋白aequorin的真菌细胞内钙离子浓度的动态变化，并研究其对囊泡运输的影响。我们将应用超分辨率显微镜来显示菌丝中不同的囊泡群体，并记录它们的旅行路线，直到与顶膜融合。

Aspergillus fumigatus is one of the most important airborne ascomycete pathogens and allergen worldwide. Several million people have invasive or chronic infections that lead to >600,000 deaths every year. Hence, a better understanding of the biology of this fungus may help to develop new or improve existing antifungal drugs in the future. Fungal pathogens must adapt to a variety of in vivo obstacles to survive. These obstacles include high temperature, pH variation, oxidative stress or plasma membrane's stress upon azole treatment. As other filamentous fungi, fungal pathogens have evolved numerous signal-transduction systems to sense and respond to environmental stresses to survive and proliferate in harsh environmental conditions. The HOG pathway and the Ca2+ signaling pathway both are involved in stress adaptations. Recently, it was discovered that phytochrome senses the light signal through the HOG pathway, and there is evidence in planta and in bacteria that phytochrome plays a role as molecular thermometer. In this proposal we hypothesize that phytochrome-dependent light sensing might be interlinked with Ca2+ signaling via the HOG pathway. We aim at characterizing the two signaling pathways in two related ascomycetes, Aspergillus nidulans and the opportunistic pathogenic A. fumigatus. Our preliminary data also indicate both phytochrome- and the Ca2+-signaling pathway are involved in thermo-sensing in A. nidulans. We plan to study the role of phytochrome, the HOG and the Ca2+ signaling pathway in temperature and azole antifungal sensing in A. nidulans and in A. fumigatus. The interaction between the two signaling pathways will be studied at the transcriptional and the posttranscriptional level. There is also good evidence that Ca2+ not only plays a role in stress adaptation, but also in polarized growth. It was shown that the concentration of Ca2+ oscillates during polar hyphal extension and that this oscillation may be responsible for oscillations of vesicle secretion and thus oscillatory growth of hyphae. Because stress responses cause dramatic changes of the intracellular Ca2+ concentration, effects of stress on polar cell extension are likely. To study this relationship, we are going to analyze how stress-response pathways affect hyphal polar growth in both fungi and how they affect virulence in A. fumigatus. For this aim, we will monitor the dynamics of the Ca2+ concentration in fungal cells expressing the calcium reporter aequorin combined with fluorescent protein GECO technologies and study the effect on vesicle traffic. We are going to apply super-resolution microscopy to visualize different vesicle populations in the hyphae and document their travel routes until fusion with the apical membrane.