Screening and Identifying of Interaction Protein AtL5 in Arabidopsis thaliana

Research background: The Arabidopsis-resistance protein L5 (AT1G12290) can trigger cell death in Nicotiana benthamiana, which is a characteristic function of an NBS-LRR (Nucleotide-Binding Sites and Leucine-Rich Repeat) protein activation. Purpose: To explore the function and molecular regulatory network of L5. Method: We employed yeast two-hybrid technology to search for interacting proteins of L5, combined with laser confocal microscopy to observe the subcellular localization of these candidate proteins, and analyzed the impact of these proteins on L5 function using an Agrobacterium mediated transient expression system. Results: Seven candidate interacting proteins were identified from the Arabidopsis cDNA library, including PPA1 (AT1G01050), RIN4 (AT3G25070), LSU1 (AT3G49580), BZIP24 (AT3G51960), BOI (AT4G19700), RING/U (AT4G22250) and PPA3 (AT2G46860). Functional analysis of these candidate interacting proteins showed that they participated in multiple pathways, including biological and abiotic stress, programmed cell death, protein degradation, material metabolism and transcriptional regulation. The results of laser confocal microscopy manifested that RIN4 was only localized on the plasma membrane (PM), and RING/U was mainly associated with the PM. PPA1, PPA3, LSU1, BZIP24, and BOI all emerged nuclear and cytoplasmic localization. The results of the transient assay proclaimed that both BOI and RING/U can inhibit cell death caused by L5. Conclusions: These results indicate that L5 immune receptors may participate in various pathways, and their protein levels and activities are strictly regulated at multiple levels, providing a basis for elucidating the mechanism of L5 immune receptors in Arabidopsis resistance.

Dissecting Multiple Arabidopsis CC-NBS-LRR Proteins Structure and Localization

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.