腐生型共生菌Clitopilus hobsonii促进杨树不定根和侧根发育的生长素通路解析

Clitopilus hobsonii, as a common soil saprophytic fungus, can also found in the ectomycorrhizal root tips of several Quercus species. Interestingly, there is clear evidence that the fungus is capable of colonizing the roots of other trees and forming microsclerotia-like structures extensively distributed in the cortical cell tissues. These findings indicate that a unique symbiotic interaction pattern may take place in C. hobsonii-tree associations, differing greatly from typical mycorrhizal fungi. Surprisingly, it is effective in promoting the development of adventitious and lateral roots. It has been well recognized that the auxin signaling is a core pathway regulating root architecture. Our data provide an ideal model for a better understanding of the saprophytic-symbiotic fungal mediated root architecture modification. In this application, first we aim to examine the dynamics of auxin content in poplar roots during the interaction with C. hobsonii at different stage. Second, we further proceed to investigate the expression pattern of auxin signaling-related genes in the symbiotic system. Third, we identify the key genes involved with auxin pathway and validate their functions by reverse genetics approaches. Briefly, results from this work will shed light on the contributions of the soil symbiotic fungi regulate root system architecture, and reveal co-evolutionary mechanism of fungus-tree symbioses. From the view of application, it will provide a basis for generating robust microbial inoculants which meets the requirement of wood plants rooting in cutting seedlings.

荷伯生氏斜盖伞（Clitopilus hobsonii）是常见的土壤腐生菌，但申请人发现该菌能定殖在多种栎树外生菌根根尖组织。不仅如此，该菌也能侵染其他树木根系形成“微菌核”结构并显著促进不定根和侧根发育。这表明C. hobsonii与树木根系之间可能存在独特的互作模式，区别于典型的菌根真菌。生长素是根系构型调控的核心激素。因此，该菌如何通过生长素通路影响根系构型为破解腐生型共生菌与树木互作机制提供了切入点。重点开展以下内容：解析C. hobsonii与杨树共生过程中生长素含量变化与动态分布，阐明生长素通路基因在C. hobsonii调控杨树根系构型过程中的表达模式，筛选生长素通路关键基因并通过反向遗传学等策略进行功能验证。研究结果将深入认知土壤腐生型共生菌对树木根系构型的调控机制，对揭示真菌与树木共生的进化机制具有重要的科学意义，也为创制新型菌剂解决扦插苗生根困难等技术瓶颈奠定理论基础。

在自然生态系统中，树木根系存在复杂的真菌群落，目前对真菌物种多样性和功能多样性的认知还十分有限。荷伯生氏斜盖伞（Clitopilus hobsonii）是常见的土壤腐生菌，该菌也能定殖在多种栎树外生菌根根尖组织。但是目前对该菌的科学内涵及生态服务功能认识还十分匮乏。通过该项目，对该菌与苗木建立的共生关系中营养调控和互惠共生机制展开了研究。首先，构建了C. hobsonii与苗木的共生体系，发现该菌显著促进了侧根和不定根的发育；此外，在有机氮条件下，C. hobsonii不仅能够促进杨树无性系幼苗根系发育，还能形成外生菌根结构。进一步研究发现，C. hobsonii可以产生生长素并且调控植物生长素信号通路相关基因表达，这可能是C. hobsonii调控植物根系构型的作用机制。在前期研究基础上，发现了C. hobsonii显著促进苗木钾离子的吸收和积累，转录组数据表明三个真菌源的钾离子吸收基因（ChACU、ChSKC和ChHAK）参与了共生体钾离子吸收，并且发现与正常钾（5 mM）相比，低钾（0.05 mM）诱导ChACU和ChHAK基因的高表达，促进苗木钾吸收。通过C. hobsonii与杨树幼苗共培养实验，发现有机氮利于共生关系的维持，促进氮同化，而无机氮打破这种共生关系。解析真菌基因表达谱发现，NH4+诱导大量葡萄糖苷水解酶家族（GH）上调表达，使真菌具有更强的侵染性（致病性）；NO3-激活次级代谢相关基因，可能导致有毒物质的形成；有机氮诱导共生相关基因表达，促进氨基酸水解（矿化），益于植物与真菌建立互惠共生的共生关系。通过基因组组装后发现，C. hobsonii同核体菌株基因组共有13条contigs，基因组组装结果接近染色体水平。本项目将有益于进一步认识腐生型共生菌与植物的共生互作机制，对揭示真菌与植物的共生关系具有重要科学指导意义。