Characterization of the BNT1 immune receptor in Arabidopsis targeted to plastids

Abstract

Plants depend on a non-adaptive immune system triggered by receptors after the detection of pathogens. Among them, nucleotide-binding leucine-rich repeat receptors (NLRs) family are key immune sensors that recognize specific effectors used by pathogens to promote their virulence. NLR proteins have been found in various subcellular compartments and they can be relocated after activation, indicating that trafficking is involved in their function. However, NLRs acting from endosymbiont organelles such as plastids or mitochondria have not been described. NLRs consist of a N-terminal region that is similar to the "Toll/Interleukin-1 receptor" (TIR) or forms a "coiled coil" (CC), a nucleotide binding domain (NB) and a leucine rich repeat (LRR) (TIR/CC-NB-LRR). Recently, we have found a novel N-terminal localization signal for plastid or mitochondria targeting. Notably, among the Arabidopsis predicted proteins that possess this signature one is the NLR receptor BNT1. In this work, we have characterized the changes in transcripts and localization of BNT1. We carried out an in silico analysis of the transcriptional variations of BNT1 isoforms generated by alternative splicing (AS). By RT-PCR we validated and examined the BNT1 isoforms level in basal and biotic/abiotic stress conditions. We also generated BNT1-GFP fusions for transient expression in Nicotiana benthamiana to determine its localization using confocal microscopy and subcellular fractionations. Several BNT1 N-terminal mutated versions were also analyzed. Together, our results suggest that BNT1 isoforms generated by AS specifically participate in different stress responses. Additionally, the differential localization of BNT1 mutated versions suggest a key role of its N-terminal region during defenses against pathogens. The elucidation of BNT1 biological role could represent a paradigm to understand the function of NLR immune receptors localized in plastids and different subcellular spaces.