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

植物持续暴露于生物以及非生物胁迫因素，例如病原微生物。与微生物的相互作用和微生物的识别导致在接触部位产生信号，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已被广泛研究。尽管根际的微生物数量多得不成比例，但迄今为止，人们对根与病原微生物和互利共生体接触后的信号传导知之甚少。在拟南芥（Arabidopsis thaliana）中进行的遗传筛选导致了CRT 1（Compromised for Recognition of Turnip Crinkle Virus 1）的发现。CRT 1属于GHKL ATP酶的MORC型亚支，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，在五个免疫水平上起作用，包括效应触发免疫（ETI）、模式触发免疫（PTI）、基础抗性、非宿主抗性和系统获得性抗性（SAR）。本申请人与Mercator研究员的先前合作工作已经表明，CRT 1的亚群i）驻留在细胞核中，ii）结合DNA，iii）表现出内切核酸酶活性，和iv）影响对DNA损伤剂的耐受性，认为它在免疫应答的激活期间具有重要的细胞核功能。这表明它们具有进化上保守的功能。申请人已经在尚未完全注释的大麦（Hordeum vulgare）基因组中鉴定了五种CRT 1家族蛋白，命名为HvCRT 1、HvCRH 1、HvCRH 3、HvCRH 6和HvCRH 7。通过RNAi介导的基因沉默或过表达改变这些基因的表达，改变大麦对由禾谷镰刀菌引起的根腐病和由小麦白粉病菌引起的叶白粉病的抗性。鉴于这些结果，我们强烈怀疑CRT 1和/或其同源物参与拟南芥，大麦和其他植物物种的根微生物相互作用。由于大麦中CRT 1家族成员的沉默导致对坏死营养型的更高抗性，（镰刀菌）和活体营养为了阐明CRT 1家族成员的作用模式，并揭示大麦和拟南芥中CRT 1功能之间明显的种特异性差异的潜在机制，重组AtCRT 1和HvCRT 1将使用遗传、分子、生物化学、生物物理和结构方法表征。此外，将产生具有改变的CRT 1家族基因表达的稳定的转基因大麦系，并就其对根引发的病原体感染的响应进行表征。这些研究的长期目标是作物改良。

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.