RUI: Confirmation of the roles of fungal genes in plant stress tolerance

这个项目的重点是三方共生，因为寄主植物对高达125华氏度的土壤温度具有耐受性，这是大多数植物无法生长的温度。植物之所以能在这种温度下存活，是因为它们与一种感染了特定病毒的特定真菌有联系。与寄主植物共生的病毒感染真菌对包括番茄、玉米和水稻在内的多种作物具有耐热性。在田间试验中，与未感染的植株相比，携带真菌-病毒共生体的番茄植株在高温下表现出更好的生长和果实产量。由于环境因素，美国经济每年在作物生产上损失超过150亿美元，因此了解这种病毒-真菌-植物共生诱导耐热性的机制将有潜在的帮助。从这项研究中获得的见解可能有助于开发在高温胁迫下保持作物生产力的方法。该项目将为未被充分代表的少数族裔和西阿拉巴马大学（一所指定的少数族裔服务机构）的第一代大学生提供动手多学科研究培训和教育机会。此外，还将通过“科学星期六”和“科学咖啡店”分别为贫困的K-12学生和成年人提供教育服务。最后，真菌代谢产物在抗逆性中的生理和分子作用将引起科学界的广泛兴趣。在黄石国家公园的地热土壤中，温度可达65°C，在恐怖草（dihanthelium lanuginosum）和真菌内生菌（弯曲菌耐温病毒（CThTV））之间形成了一种三方互惠关系，使这三种生物能够合作在极端温度下生存。代谢分析表明，我们的模型系统番茄（Solanum lycopersicum）感染了C. protuberata后，在应激过程中发生了显著变化。这些分析中收集的数据提供了强有力的证据，表明真菌海藻糖和黑色素对于3-way共生所赋予的耐热性机制至关重要。此外，CThTV通过与真菌翻译控制的肿瘤蛋白（TCTP）和过氧化氢酶/过氧化物酶（KatG）的相互作用来促进耐热性，这两种酶分别控制真菌的细胞周期和细胞氧化还原。本研究的假设是，携带CThTV的C. protuberata通过以下途径赋予植物耐热性：1)产生大量海藻糖，海藻糖被运输到植物组织中，并作为信号或渗透保护分子调节植物在逆境中的生化过程；2)真菌黑色素，可能在逆境中改变真菌细胞壁，控制海藻糖在植物组织中的转运和积累；3) CThTV通过与真菌TCTP和KatG相互作用，调控胁迫下真菌的细胞周期和细胞氧化还原，从而促进真菌的耐热性。特别是，拟议的研究旨在测试真菌海藻糖、黑色素、TCTP和KatG基因在与真菌-病毒共生体相关的耐热番茄中的作用。本研究的目的是：1)敲除携带CThTV的耐热性巨噬菌的海藻糖、黑色素、TCTP和KatG基因，在敲除海藻糖或黑色素合成的过程中，耐热性将被完全消除，并通过敲除TCTP或KatG的表达而降低；2)在无CThTV的非耐热性C. protuberata中过表达相同的基因，在海藻糖和黑色素生物合成基因过表达的情况下，植物有望获得耐热特性，在TCTP和KatG过表达的情况下，植物表现出更好的耐热性。

The 3-way symbiosis at the focus of this project is intriguing by virtue of the host plants gaining tolerance to soil temperatures of up to 125°F, a temperature at which most plants cannot grow. Plants survive this temperature due to their association with a particular fungus infected with a specific virus. Virally infected fungi living symbiotically with a host plant provide heat tolerance to a wide range of crop plants including tomato, corn, and rice. In a field trial, tomato plants harboring a fungal-viral symbiont showed improved growth and fruit production at elevated temperatures compared to non-infected plants. With the US economy losing more than $15 billion annually in crop production due to environmental factors, it will be potentially helpful to understand the mechanisms by which thermotolerance is induced in this virus-fugus-plant symbiosis. Insights gained from the research are likely to help in the development of methods to retain crop productivity in the face of high temperature stress. The project will provide hands-on multidisciplinary research training and educational opportunities for underrepresented minorities and first-generation college students at the University of West Alabama, a designated Minority Serving Institution. In addition, the project will include an educational outreach program for underprivileged K-12 students and adults through the Science Saturdays and Science Coffee Shop outreach programs, respectively. Finally, the physiological and molecular roles of fungal metabolites in stress tolerance will be of broad interest to the scientific community.In the geothermal soils of Yellowstone National Park, where temperatures can reach 65°C, a 3-way mutualistic association has evolved among panic grass (Dichanthelium lanuginosum) and a fungal endophyte (Curvularia protuberata) bearing a mycovirus, Curvularia thermotolerance virus (CThTV), which allows the 3 organisms to cooperatively survive extreme temperatures. Metabolic analyses showed significant changes during stress in our model system tomato (Solanum lycopersicum) infected with C. protuberata. The data collected in those analyses provide strong evidence that fungal trehalose and melanin are essential for the heat-stress tolerance mechanisms conferred by the 3-way symbiosis. In addition, CThTV contributes to thermotolerance via its interaction with a fungal translationally controlled tumor protein (TCTP) and catalase/peroxidase (KatG), which control fungal cell cycle and cellular redox, respectively. The hypotheses of this research are that C. protuberata carrying CThTV confers thermotolerance in plants via: 1) production of large quantities of trehalose, which is transported into plant tissues and functions as a signalling or osmoprotectant molecule to regulate plant biochemical processes during stress; 2) fungal melanin, which likely alters fungal cell walls to control trehalose transport into and accumulation in plant tissues during stress; and 3) CThTV, promotes thermotolerance by interacting with fungal TCTP and KatG to control fungal cell cycle and cellular redox during stress. In particular, the proposed research is designed to test the involvement of fungal trehalose, melanin, TCTP, and KatG genes in thermotolerant tomato associated with fungal-viral symbionts. The aims of this study are to: 1) knock down the trehalose, melanin, TCTP, and KatG genes in thermotolerant C. protuberata-carrying CThTV, thermotolerance will be completely abolished in trehalose or melanin synthesis knock downs, and reduced by knocking down TCTP or KatG expression; and 2) overexpress the same genes in non-thermotolerant C. protuberata without CThTV, plants are expected to gain thermotolerance characteristics in the case of trehalose and melanin biosynthetic gene overexpression, and exhibit improved thermotolerance in the case of TCTP and KatG overexpression.