Nucleotide-binding site leucine-rich repeat receptors (NBS-LRR/NLRs) are crucial intracellular immune proteins in plants. Previous article reported a novel NLR protein SUT1 (SUPPRESSORS OF TOPP4-1, 1), which is involved in autoimmunity initiated by type one protein phosphatase 4 mutation (topp4-1) in Arabidopsis, however, its role in planta is still unclear. This study employed Nicotiana benthamiana, a model platform, to conduct an overall structural and functional analysis of SUT1 protein. The transient expression results revealed that SUT1 is a typical CNL (CC-NBS-LRR) receptor, both fluorescence data and biochemical results showed the protein is mainly anchored on the plasma membrane due to its N-terminal acylation site. Further truncation experiments announced that its CC (coiled-coil) domain possessed cell-death-inducing activity. The outcomes of point mutations analysis revealed that not only the CC domain, but also the full-length SUT1 protein, whose function and subcellular localization are influenced by highly conserved hydrophobic residues. These research outcomes provided favorable clues for elucidating the activation mechanism of SUT1.
NBS-LRR (nucleotide binding sites and leucine rich repeat) protein plays a crucial role as sentries and as defense activators in plants. The structure and function of NBS-LRR proteins are closely related. Previous articles have announced that the activated ZAR1 (HopZ-Activated Resistance 1) forms a pentamer in the plasma membrane, which is a calcium permeable channel that can trigger plant immune signaling and cell death. However, the structure of galore NBS-LRRs in Arabidopsis is not yet clear. The functional sites of distinct NBS-LRR in cells may vary. In addition, identifying pathogens and activating defense regions may occur in different subcellular compartments. Therefore, dissecting the specific structure and positioning of NBS-LRRs is an indispensable step in understanding their functions. In this article, we exploit AlphaFold to predict the structure of some designed NBS-LRRs, and utilize Agroinfiltration transient expression system, combined with biochemical fractionation, to dissect the localization of these NBS-LRR receptors from Arabidopsis. Structural data indicates that the identified NBS-LRRs share analogous conformation. Membrane fractionation assay demonstrates these NBS-LRRs are mainly associated with the membrane. These data show that the Ca2+-permeable channel activity may be evolutionarily conserved in NBS-LRR of Arabidopsis, and this study provides some reference clues for analyzing the structure and localization patterns of other plant immune receptors.
目的为解析党参NBS-LRR(Nucleotide-binding site and leucine-rich repeat)抗病基因家族,探究党参抗根腐病机制,从而解决党参根腐病害难题,促进党参育种及产业发展。方法基于党参响应根腐病病原菌的转录组数据,通过运用生物信息学方法对党参NBS-LRR家族基因进行理化性质、基因结构、系统发育、表达模式及互作网络分析。结果成功鉴定到88个党参NBS-LRR家族基因,包括N、NL、CN、CNL、TN、TNL、PN共7种类型,分别有50、14、1、14、4、3、2个基因。结果表明,党参CNL及TNL类基因结构比较保守;党参CNL亚家族基因在进化过程中发生扩增;党参NBS-LRR家族基因在尖孢镰刀菌(Fusarium oxysporum)侵染条件下存在时间表达模式差异,且侵染前期(6-24 h)高表达的基因DN64786c1g6、DN64786c1g5、DN48234c0g2、DN54844c1g2、DN59747c0g3、DN56071c1g8、DN64591c1g1、DN48464c1g1、DN59886c0g1在调控党参抗病过程中发挥重要作用。其中党参的抗病蛋白DN54844c1g2可能与GLR家族互作,进而通过调节Ca2+内流参与免疫调控;DN64786c1g5可能与CYTC-1和CYTC-2互作,进而通过参与氧化还原反应参与党参响应根腐病过程;DN59747c0g3可能与MPK3互作,进而通过参与MAP信号级联、磷酸化WRKY转录因子以及参与超敏反应(HR),在党参响应根腐病过程中发挥重要作用。结论党参NBSLRR家族基因的鉴定及表达分析对于探究党参抗根腐病机制、发掘基因功能具有重要意义。
