黑粉菌-茭白植株互作及其对产品器官形成的调控机制

The swollen culm of Zizania latifolia Turcz., used as an aquatic vegetable, is induced by the infection of a basidiomycete smut fungus Ustilago esculenta, which is commonly cultivated in southern China. However, in practical cultivation, some plants often escape the fungus infection, which has been a bottleneck on the cultivation of high yield and quality and breeding program of this crop. Up to date, it is unknown that what is involved in the interplay between the fungus and the host, and how the swollen culm is triggered by the fungus. Previous studies have identified several mechanisms for host cell manipulation by microbial infection and genes involved in tumor formation in maize seedlings by U. maydis infection. In natural conditions, plants have developed multifaceted defence systems, these responses include induction of pathogenesis related genes, production of secondary metabolites as well as the reinforcement of cell walls. Induction of the basal plant defence machinery occurs upon the recognition of conserved molecules which are commonly found in a variety of microbial species, but which are absent in the host. However the direct defense mechanism employed by plant against fungal infection has not been characterized in Z. latifolia-U. esculenta system. In this study, we will reveal a complex interplay of U. esculenta with its host plant,identify genes functionally involved in swollen culm formation after comparing expression in swollen culm and noninfected culm tissue by using the transcriptomic analysis. Moreover, we will visualize expressional changes of distinct biological pathways and their relation to plant defence resonses.We will also reveal the role of callose in plant defence to the fungus by characterizing callose deposits in plant cell wall using histochemical staining, and assaying the expression of callose synthase genes. The level of benzoxazinoid hydroxamic acids such as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), 6-methoxy-2-benzoxazolinone (MBOA) and benzoxazolinone (BOA), the concentration of salicylic acid (SA), jasmonic acid (JA), ethylene and abscisic acid (ABA）will be determined to undestand whether they play a role in the mechanism of plant defence to the fungus. Based on the data obtained, the results will provide molecular evidence that U. esculenta has the capacity to extend the undifferentiated state of infected tissue and yields insight into putative defense responses as well as distinct regulatory programs triggered during U. esculenta infection.This study may unravel the complex interactions of Z. latifolia- U. esculenta, and this may be helpful in decreasing the occurrence of no swelling plant of Z. latifolia crop, which is due to the plant defence to the fungus infection, this will lay a foundation for the rational design of crop cultivation strategies for high yield and quality of this vegetable,and is also of significance in studying the plant-microbe interatctions.

茭白（Zizania latifolia）是我国南方普遍栽培的水生蔬菜，其膨大茎是由黑粉菌（Ustilago esculenta）诱导形成的，有研究认为茎膨大与植物激素有关，但对其机制迄今仍不清楚，限制了茭白高产栽培和育种技术的发展。本项目拟利用转录组测序、细胞学和HPLC分析等技术，重点研究黑粉菌-茭白互作过程中与激素代谢相关基因的表达及其与调控茎膨大的关系；测定黑粉菌侵染后茭白植株不同生长发育时期、不同组织器官中植物激素含量, 揭示茭白茎膨大的生理基础；研究细胞胼胝质累积特点及其合成酶基因表达水平，结合测定异羟肟酸类物质含量和防卫激素（水杨酸、乙烯、茉莉酸等）的水平，明确物理屏障和多信号防卫途径在"雄茭"形成中的作用。本研究结果基本揭示茭白茎膨大和"雄茭"产生的机制，对生产上提高茭白产量、防止"雄茭"产生提供理论基础和指导，同时也对研究植物-微生物互作具有理论意义。

本项目通过对黑粉菌的侵染规律、对植株茎膨大的影响等方面的研究，试图明确黑粉菌-茭白植株的互作及其与茎膨大的关系的分子及生理机制。研究结果表明，冬孢子萌发的最适温度为27℃-30℃，最适pH值为3-4。1.0%的蔗糖溶液对萌发有显著促进作用。异羟肟酸对黑粉菌生长有抑制作用。通过光学和电子显微镜观察发现菌丝在茭白植株主要分布在膨大茎、根节及芽中。菌丝在细胞间或破裂的细胞壁间形成一个大空腔。成熟茭白膨大茎中的冬孢子和“灰茭”中的冬孢子基本相同。菌丝通过薄壁组织和维管束从母体节中进入到新生芽中和根节上，在越冬前后，茎和根节上的几乎所有新生芽都被侵染。.利用HiSeq2000对植株茎进行转录组测序，并与茭白基因组进行比对。用DEGseq软件对差异基因进行分析得到茭白与"雄茭"的差异基因19033个，“灰茭”与雄茭的差异基因17669个。进一步对"灰茭"和茭白在膨大-7天、1天及10天进行表达谱分析发现，茭白膨大茎与"雄茭"差异基因三个时期分别为4873、6996和6063个，而“灰茭”差异基因数分别为4083、6928和4981个。对膨大1天和膨大10天差异基因进行分析，鉴定茭白中可能与调控膨大相关的上调基因有473及782个，而“灰茭”中有1094及983个，同时，茭白中可能与膨大相关的下调基因有742及908个，而“灰茭”中有1130及1111个；茭白黑粉菌的487和569个上调基因，612和512个下调基因；以及“灰茭”黑粉菌的409和375个上调基因，300和409个下调基因可能参与茎膨大过程。最后，鉴定了376个植物基因和187 个黑粉菌基因可能是茎膨大的关键基因。.与雄茭相比，茎膨大中期和后期Cals和BG基因表达显著上升。胼胝质的积累可能是黑粉菌侵染的结果，是植物对病原菌的识别及防御反应的表现。激素合成酶IPT基因和YUCCA基因在膨大茎中表达量均显著高于雄茭，表明二者对茎膨大起着调节作用。.总之，通过本项目研究，初步明确了黑粉菌-植物互作的分子基础和参与调节茎膨大的有关基因，不仅对进一步研究植物-微生物互作提供了理论依据，而且对通过分子操作技术提高茭白的产量和品质有潜在应用价值。