【作者】 杜鹃；

【导师】 谢从华；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2013， 博士

【摘要】 马铃薯(Solanum tuberosum L.)原产自南美洲安第斯山脉一带,是世界上第四大重要粮食作物。马铃薯在人类消费和工业用途(主要是淀粉产业)都起着重要作用。然而,马铃薯容易受到各种病害感染,每年都会造成巨大经济损失。导致爱尔兰马铃薯大饥荒的元凶Phytophthora infestans所引起的晚疫病是其中最具毁坏性的病害之一。在过去的几十年间,许多抗病(R)基因已经被克隆,其中有些R基因通过有性杂交的方式渗入到一些马铃薯栽培种中。然而人们发现这些渗入R基因的马铃薯栽培种的抗性不能持续很久。有时新品种还没来得及得到商业推广,抗性就已经被快速进化的晚疫病病原菌攻破。所以我们不能只依靠单个的R基因,寻找更有效地提供持久抗性的方法迫在眉睫。尽管许多克隆的R基因已经被晚疫病菌克服,但并不是各地都已发现毒性生理小种。由于R基因的抗性更强,适当利用已知R基因以及快速克隆新的Rpi基因对抗性育种会起到一定帮助作用。由于传统抗性育种周期长效率不高,育成的品种很容易被快速进化的晚疫病菌克服。令人鼓舞的是,效应子组学近来被证明在认识和获得晚疫病抗性方面是一种很成功的策略。在第二章里,我们以"Do’s and Don’ts"两种形式在应用效应子组学方面提出了一些建议。简要来说,我们在“Do’s”里总结了效应子组学的7个优势,而在“Don’ts”里提出了3个在应用效应子组学时应注意的方面。农杆菌注射和PVX病毒侵染是应用效应子组学的两种常规方法。在第三章里,我们阐述了这两种方法在马铃薯和在本氏烟草上应用的操作方法。操作方法本身并不复杂,而对结果的分析需要更多实践经验。我们分享了在长期实践中的一些经验总结,也讨论了这两种方法在应用效应子组学时的优势和劣势。抗性相关基因在下游信号途径上,不直接和病原物互作。因此这类基因引起的抗性对晚疫病菌不会产生太大选择压力,抗性很有可能更为持久一些。为了鉴定马铃薯晚疫病重要的抗性相关基因,我们在本氏烟草和马铃薯上借助病毒介导的基因沉默的方法(virus-induced gene silencing, VIGS)对63个候选基因进行瞬时沉默,紧接着取基因沉默后的叶片离体接种晚疫病病原菌进行抗性鉴定(见第四章)。结果表明其中有两个基因可能在调控晚疫病数量抗性上发挥功能,它们分别编码一种脂氧合酶(EC1.13.11.12)和一种栓化作用相关的阴离子过氧化物酶。植物一般依靠两层免疫反应来抵抗病原菌。除了被病原菌克服的胞内的R基因,在细胞膜上还有另外一层免疫机制。由于细胞膜受体能识别保守的病原分泌物(PAMPs),这类受体产生的抗性被认为会更持久。在马铃薯和晚疫病病原菌互作系统中,这层抗性还没有被研究过,主要因为PAMP诱导免疫反应产生的数量抗性表型妨碍了用图位克隆法去克隆细胞膜受体。而利用效应子组学的方法,我们可以得到elicitin被识别的很清楚的表型。Elicitin是由晚疫病病原菌分泌的一类蛋白家族,被认为是卵菌的相关分子模式。近期一个类受体蛋白ELR被成功地从一个马铃薯野生种Solanum microdontum中克隆得到,我们通过ELR研究了第一层免疫机制。在第5章里,我们对马铃薯栽培种Desiree的ELR转基因植株进行了超过3次的晚疫病抗性鉴定,可靠结果表明超量表达ELR能增强马铃薯对晚疫病的抗性。此外,我们发现ELR的转基因植株能识别许多种疫霉属病原菌分泌的elicitin产生免疫反应。在第6章,我们进一步研究了ELR在不同植物种类里的自然变异情况。在SolRgene网站的地图中,我们发现能识别INF1的马铃薯野生种分布在中美和南美洲一带。我们从这些野生种中成功克隆到7个直系同源基因,测序比对发现它们与ELR的氨基酸序列高度相似。通过对这8个ELR和不同elicitin免疫反应的分析,我们发现它们在茄属和烟草属中的免疫反应很保守。此外,我们也证明了INFl引起的过敏反应在马铃薯野生种Solanum hjertingii349-3能被AVR3aKI所抑制,此前这一抑制反应曾在烟草中被报道过。这些结果都暗示了elicitin的信号途径在茄属和烟草属中比较保守。这个发现会加速我们对马铃薯和其它作物中elicitin信号途径的认识,进而利用这层非原质体抗性。总的来说,我们的研究得到了以下结果：1)对利用效应子组学方面提出一些建议；2)鉴定出若干可能比较重要的抗性相关基因：3)证明了近期克隆的INF1的受体基因ELR能增强马铃薯晚疫病的抗性并能对不同卵菌分泌的elicitin产生免疫反应；4)发现elicitin信号途径在茄属和烟草属中比较保守。我们得到的这些知识对马铃薯抗性育种会很有价值。更多还原

【Abstract】 Potato(Solanum tuberosum L.), which originates from the Andes in South America, is the fourth most important food crop in the world. Potato plays an important role for both human consumption and industrial purpose mainly for starch production. However, it suffers from various diseases, which cause huge economic losses every year. One of the most devastating diseases is late blight caused by the Irish potato famine agent Phytophthora infestans. During past years, many resistance (R) genes have been cloned and some of them have been introgressed into several potato cultivars by sexual crossing. Yet it was found that the potato with an introgressed single R gene normally could not last for long. Sometimes, the new cultivar was defeated by the fast evolving P. infestans even without going into the commercial pipelines. So we can not only rely on a single R gene and it is urgent to exploit more effective ways to provide durable resistance.Although most cloned R genes have been defeated by P. infestans, the virulent isolates are not everywhere. Proper use of known R genes and rapid cloning of new Rpi-genes could still make contributions to resistance breeding based on their stronger resistance. The traditional resistance breeding is slow and inefficient, which is hard to follow the evolution speed of P. infestans. Encouragingly, effctoromics has recently been proved a successful strategy to understand and achieve resistance to late blight in agriculture. In Chapter2, we provided suggestions in the application of effectoromics, in the form of "Do’s and Don’ts". Briefly, we summarized seven advantages in the Do’s and three practical notes in the Don’ts.Agroinfiltration and PVX agroinfection are two efficient transient expression assays routinely used for effctoromics. In Chapter3, we presented our protocols of the two assays in both potato and Nicotiana benthamiana. The protocols themselves are not complicated, but one may need more experience to analyze the results. We shared our long-term experience for scoring and also discussed about the advantages and disadvantages of both assays in effectoromics application.Defense-responsive genes are involved in the downstream signaling pathway and not directly interacting with pathogens. Thus they do not put too much selection pressure to P. infestans and are very likely to be more durable. To identify the key defense-responsive genes to potato late blight, we performed a functional screening of63selected candidate genes by VIGS (virus-induced gene silencing) on both Nicotiana benthamiana and potato (Chapter4). This treatment was followed by detached leaf assay and assessment of the resistance level. Our results led to the identification of two genes, a lipoxygenase (LOX; EC1.13.11.12) and a suberization-associated anionic peroxidase, which may play a role in quantitative resistance to potato late blight. Plants rely on two layers of immunity to defend against pathogens. Despite the defeated intracellular R genes, the other layer of immunity occurs at the cell surface. Since surface receptors can recognize conserved pathogen associated molecular patterns (PAMPs), they are considered to be more durable. For the potato-P. infestans pathosystem, this layer of immunity has not yet been explored, mainly because the quantitative resistance phenotype conferred by PAMP-triggered immunity is hampering map-based cloning approaches of surface receptors. Taking advantages of effectoromics assays, clear phenotypes could be obtained for recognition of elicitins, a family of proteins of P. infestans that are recognized as oomycete PAMPs. Recently, a receptor-like protein ELR (elicitin response) was successfully cloned from the wild potato species Solanum microdontum. Based on ELR, we studied the first layer of immunity in potato. In Chapter5, we performed more than three repeats of disease tests on ELR transgenic potato cultivar Desiree. Promising results showed that overexpression of ELR could enhance the potato resistance to late blight. Furthermore, we also found that Desiree::ELR could recognize a broad spectrum of Phytophthora elicitins and induce defense responses.In Chapter6, we further analyzed the natural variation of ELR in different plant species. On the map in SolRgene website, we found that INF1-responding wild potato species are distributed in both Central and South America. From those species, we successfully cloned7ELR orthologs, which show high levels of amino acid sequence identity with ELR. By testing these ELR orthologs with various elicitins, we found that patterns of elicitin-triggered defense are conserved in both Solanum and Nicotiana species. Moreover, we proved that INF1-triggered hypersensitive response (HR) can also be suppressed by AVR3aKI in a wild potato species(Solanum hjertingii349-3), which has been shown in N. benthamiana before. All these data indicate that the elicitin signaling pathway is conserved in Solanum and Nicotiana. This finding would accelerate further study of elicitin signaling pathway and application of apoplastic immunity in potato and other crops.In all, our study has led to1) useful suggestions for applying effectoromics,2) identification of several potential useful defense-responsive genes,3) proving that the newly cloned INF1receptor ELR can enhance resistance to P. infestans and induce broad-spectrum defense responses to different oomycete elicitins, and4) discovering that the elicitin signaling pathway is conserved in Nicotiana and Solanum species. The knowledge we obtained may be valuable for potato resistance breeding.更多还原