微丝骨架调控蛋白OsADF5参与水稻对稻瘟菌免疫应答的分子机制

The re-arrangement and dynamics of actin cytoskeleton play vital roles in plant reponse to fungal pathogen infection, mainly involving in the dynamic transport of important immune receptors and resistance proteins as well as establishing pathogen infection barriers. However, our knowledge to the molecular mechanism about the actin cytoskeleton dynamic re-arrangement and physiological function in plant (especially in crops) immunity were known limited. Preliminary results showed that rice actin depolymerization factor family protein, OsADF5 may function closely involving in rice defense against Magnaporthe oryzae (M. oryzae) infection. The rice Osadf5 mutant exhibited a decreased resistance when challenging with M.oryzae, indicating that it may act as one of the key factors in rice defense against M. oryzae. Further more, in vitro experiment showed that OsADF5 could bind the actin filaments and facilitate the formation of the actin cables. Thus, in the proposed study, we will investigate the physiological roles of OsADF5 in regulating the dynamic polymerization of actin filaments during the response of rice to M. oryzae, including analyze the dynamic re-arrangement of the actin filaments at the interaction interface between rice blast fungus, and also study how OsADF5 regulates the assembly and rearrangement of microfilaments during the immune process of rice. The purpose of this study was to reveal the dynamic response of actin cytoskeleton in the interaction between rice and blast fungus on the one hand, and elucidate the physiological function of OsADF5 in this process on the other hand, providing new thought of cell biology for the molecular mechanism of plant immunity.

微丝骨架的重排及动态变化在植物响应病原菌侵染过程中起重要作用，主要参与植物重要免疫受体、抗性蛋白的动态转运及建立病原菌侵染屏障等。然而，其在植物（尤其是作物）免疫过程中的动态变化规律及调控机制尚不清楚。最近，我们发现水稻微丝骨架剪切蛋白家族成员OsADF5参与水稻对稻瘟病抗性，其突变体对稻瘟菌抗性显著降低，暗示其可能是水稻免疫中的一个关键因子。生化实验表明OsADF5蛋白能够结合微丝骨架，并促使微丝成束。本课题将在前期工作基础上，对OsADF5在调控微丝骨架动态组装及参与植物抗性响应中的生理功能进行深入研究。包括分析微丝骨架在稻瘟菌-水稻活体营养互作界面处的结构及动态组装规律；分析OsADF5如何调控水稻免疫过程中微丝骨架的组装和重排。本研究旨在一方面解析微丝骨架在水稻免疫过程中的动态响应规律，另一方面阐明OsADF5在这一过程中所发挥的生理功能，为植物免疫的分子机制提供细胞生物学新思路。

微丝骨架在植物的病原响应及植物免疫信号建立和传递过程中发挥了重要功能。然而，目前我们对微丝骨架如何参与菌抗性还知之甚少。本研究发现OsADF5能够响应稻瘟病的侵染，其突变体的稻瘟病抗性显著下降。说明其是水稻防御稻瘟病侵染的关键因子。生化实验表明OsADF5蛋白能够结合微丝骨架，并促使微丝成束。通过遗传转化，获得稳定表达OsADF5-mCherry植株，发现OsADF5-mCherry信号能够聚集到稻瘟菌的侵染菌丝周边。同时我们将水稻免疫受体OsCERK1-GFP通过杂交导入到OsADF5水稻突变体中，分析关键免疫受体在OsADF5突变体中的转运是否受到影响。这些实验将揭示OsADF5可能通过调控微丝骨架在病原真菌和植物互作界面的组装，进而调控了植物受体的转运从而参与水稻抗病。进而揭示水稻微丝骨架在水稻稻瘟病抗性建立中，如何参与免疫受体的转运的分子机制。. 另外，我们还发现微丝骨架能够介导稻瘟病的致病性。我们发现稻瘟菌通过组织独特的微丝阵列介导稻瘟菌的生长和侵染。我们发现稻瘟菌通过微丝骨架相关蛋白MoFim1和MoTwf1，能结合和促进微丝成束，进而在菌丝的顶端组装一个致密的微丝阵列。这些微丝骨架被证明和菌丝的极性生长，囊泡运输密切相关。当敲除MoFim1和MoTwf1后，稻瘟菌菌丝中的微丝骨架组装异常，并且致病性显著下降。当把靶标MoFim1的小RNA转化到水稻中，发现水稻抵御稻瘟病的能力显著提高。这说明微丝骨架在稻瘟菌的致病过程中发挥了关键作用，对于水稻的抗病遗传育种具有较大的潜力。