The Molecular Mechanism By Which BOI Negatively Regulates The Activation Of A CC-NBS-LRR Protein In Arabidopsis

In plant immunity,nucleotide-binding site(NBS)and leucine-rich repeat(LRR)receptors,also known as NLRs proteins,play an important role.According to the difference of its N-terminal,NLRs can be simply divided into RPW8-NBS-LRR(RNL),CC-NBS-LRR(CNL)and TIR-NBS-LRR(TNL)proteins.There are about 150 genes encoding NBS-LRR protein in Arabidopsis thaliana genome.To date,only a few NBS-LRRs have been fully studied.Therefore,we first cloned twenty unpublished NBS-LRR genes,and then transiently expressed in the Nicotiana benthamiana leaves.We found that transient expression of AT1G12290(named L5 protein)in N.benthamiana could mediate cell death,which is one of the typical characteristics of NLR protein activation.Subsequently,through fluorescence localization observation and biochemical experimental data,we proved that L5-YFP-HA protein is located on the plasma membrane,and the predicted myristoylation site Gly2 is very important for the plasma membrane localization and cell death activity of L5 protein.Through further structural analysis,we found that the CC domain of L5 protein was sufficient to mediate obvious cell death response in N.benthamiana,and the first 100 amino acids at the N-terminal of L5 protein were the smallest region mediating cell death and self-interaction.These findings provide some important clues for further study of the activation mechanism of L5 protein.Transiently expressed the CC-NBS-LRR protein L5 can trigger cell death in N.benthamiana,indicating that the activation of L5 can induce HR.However,L5 is a protein in Arabidopsis,and it does not induce HR in the normally grown wild-type plant(Columbia ecotype).We reasoned that either L5 is not expressed or does not accumulate enough protein level in normally grown Arabidopsis plant,or there are proteins that negatively suppress the activation of L5 in Arabidopsis.We constructed and obtained the stable 35S::L5-YFP-HA transgenic plants to exclude the possibility that L5 does not express in plant.The transgenic lines with detected L5 protein still showed the wild-type phenotype,suggesting that there are proteins that either negatively regulate the protein level of L5 or suppress the activation of L5 in Arabidopsis.In order to find these possible negative regulatory factors,we used L5 CC domain to screen Arabidopsis c DNA library and obtained several candidate genes.E3 ubiquitin ligase BOI is one of them,which has been found to interact with and ubiquitinate BOS1,but there are no reports about the ubiquitinate degradation of NLRs by BOI.Then we analyzed the relationship between L5 and BOI in detail.Through yeast two-hybrid and pull-down experiments,we verified the interaction between BOI and L5 CC domain.Ubiquitin experiments in vitro show that BOI is indeed an E3 ubiquitin ligase and can directly ubiquitinate L5.The subsequent transient expression experiment in N.benthamiana revealed that L5 was regulated by 26 S proteasome,BOI mediated the ubiquitination degradation of L5,and the stability of BOI itself was also regulated by proteasome.We also investigated the specific subcellular localization of L5 degraded by BOI and the potential receptor ubiquitin sites.Our results show that BOI does not degrade L5 in the nucleus,but outside the nucleus and it is ubiquitinated by the Lys425 of L5.Interestingly,we found that BOI could also mediate the ubiquitination degradation of L5 in transgenic Arabidopsis,but the L5 protein level was not significantly increased in the BOI-deficient(boi-1)plants.This result suggested that E3 ubiquitin ligase which is functionally redundant with BOI exists in Arabidopsis.Subsequent studies found that other members of the BOI family,including BRG1 and BRG3 proteins,but not BRG2,could also reduce the accumulation of L5 proteins in N.benthamiana,indicating suggested that the BOI family has partial specificity and functional redundancy in the negative regulation protein level of L5.These findings add to our understanding of the scope of action of the BOI protein family.