Characterization of CRT1 family members in root-induced basal resistance, SAR, and ISR of Arabidopsis and barley

植物不断地暴露在生物和非生物的胁迫因素中，例如病原微生物。与微生物的相互作用和识别导致接触部位(S)产生信号，这些信号被传递到植物的周围和远端组织。这种非自我识别后的信号转导网络在植物的地上部分得到了广泛的研究。尽管根际微生物数量多得不成比例，但到目前为止，人们对根中与微生物病原体和互生体接触时的信号转导知之甚少。拟南芥的遗传筛选导致了CRT1(识别芜菁皱纹病毒1的折衷)的发现。CRT1属于GHKL ATPase的MORC型亚类，在拟南芥对病毒、细菌和卵菌病原体的抗性中发挥重要作用，作用于5种免疫水平，包括效应触发免疫(ETI)、模式触发免疫(PTI)、基础抗性、非寄主抗性和系统获得性抗性(SAR)。申请人之前与墨卡托研究员的合作表明，CRT1家族的一个亚群i)驻留在核中，ii)结合dna，iii)展示核酸内切酶活性，iv)影响对dna损伤剂的耐受性，认为它在免疫反应的激活过程中具有重要的核功能(S)。迄今为止，CRt1家族成员已在包括单子叶植物在内的所有植物物种中被发现，这表明它们具有进化上的保守功能。5个CRT1家族蛋白，命名为HvCRT1、HvCRH1、HvCRH3、HvCRH6和HvCRH7，已被申请人在尚未完全注释的大麦基因组中鉴定。通过RNAi介导的基因沉默或过表达来改变这些基因的表达，都会改变大麦对禾谷镰刀菌根腐病和白粉病的抗性。鉴于这些结果，我们强烈怀疑CRT1和/或其同系物参与了拟南芥、大麦和其他植物的根-微生物相互作用。由于大麦中CRT1家族成员的沉默导致对坏死性(Fusarium)和生物营养性(Blumeria)病原菌更高的抗性，预计这些基因可能具有很高的农学应用潜力。为了阐明CRT1家族成员的作用方式，并揭示大麦和拟南芥中CRT1功能存在物种特异性差异的潜在机制，我们将利用遗传、分子、生化、生物物理和结构方法对重组AtCRT1和HvCRT1进行鉴定。此外，还将产生稳定的表达CRT1家族基因的转基因大麦品系，并研究它们对根引发的病原菌侵染的反应。这些研究的长期目标是改良农作物。

Plants are continuously exposed to biotic, as well as abiotic, stress factors, such as pathogenic microbes. Interaction with and recognition of microbes leads to production of signals at the site(s) of contact that are transmitted to the surrounding and distal tissues of the plant. This signal transduction network following non-self-recognition has been extensively studied in the aerial parts of the plant. Despite the fact that the rhizosphere is populated by a disproportionately higher number of microbes, to date relatively little is known about signaling in the roots upon contact with microbial pathogens and mutualists.A genetic screen in Arabidopsis thaliana led to the discovery of CRT1 (Compromised for Recognition of Turnip Crinkle Virus 1). CRT1 belongs to the MORC-type subclade of GHKL ATPases and was shown to play an important role in Arabidopsis resistance to viral, bacterial, and oomycete pathogens, acting at five levels of immunity including effector-triggered immunity (ETI), pattern-triggered immunity (PTI), basal resistance, non-host resistance, and systemic acquired resistance (SAR). Previous collaborative work of the applicant together with the Mercator fellow has shown that a subpopulation of CRT1 i) resides in the nucleus, ii) binds DNA, iii) exhibits endonuclease activity, and iv) affects tolerance to a DNA-damaging agent, arguing that it has (an) important nuclear function(s) during the activation of immune responses.CRT1 family members have been identified in all plant species, including monocots, sequenced to date, suggesting they have an evolutionary conserved function. Five CRT1 family proteins, named HvCRT1, HvCRH1, HvCRH3, HvCRH6, and HvCRH7, have been identified by the applicants within the not yet fully annotated barley (Hordeum vulgare) genome. Altering expression of these genes either by RNAi-mediated gene silencing or overexpression changes the resistance of barley to root rot caused by Fusarium graminearum and leaf powdery mildew caused by Blumeria graminis. Given these results, we strongly suspect that CRT1 and/or its homologs participate in root-microbe interactions in Arabidopsis, barley, and other plant species. Since silencing of CRT1 family members in barley leads to higher resistance against necrotrophic (Fusarium) and biotrophic (Blumeria) pathogens, it is anticipated that these genes may have high potential for agronomical applications.To elucidate the mode of action for CRT1 family members and to reveal the underlying mechanisms for the apparent species-specific differences between CRT1 function in barley and Arabidopsis, recombinant AtCRT1 and HvCRT1 will be characterized using genetic, molecular, biochemical, biophysical, and structural methods. Furthermore, stable transgenic barley lines with altered expression of CRT1 family genes will be generated and characterized with respect to their response to root-initiated pathogen infection. The long-term goal of these studies is crop plant improvement.