Molecular Mechanisms Of BAK1 And BKK1 Depletion-Triggered Autoimmunity In Arabidopsis

During co-evolution of plant pathogens and their hosts,plants utilize a two-tiered immune system consisting of pattern recognition receptor(PRR)-triggered immunity(PTI)and effectortriggered immunity(ETI)to defend themselves against invading microbial pathogens and to maintain plant normal growth and development.Numerous studies have shown that receptor-like protein kinase(RLK)BAK1 acts as a coreceptor,plays a central role in perceiving pathogenassociated molecular pattern(PAMP)and initiating PTI in Arabidopsis via associating with multiple PRRs.Our previous studies showed that,loss-of-function of both BAK1 and its closest paralog BKK1 lead to auto-immune phenotypes including spontaneous cell-death which resemble typical nucleotide-binding leucine-rich repeat(NLR)protein-mediated ETI responses.Meanwhile,mutation of EDS1 or PAD4,two key components in ETI response,can also suppress the autoimmune phenotypes of bak1 bkk1.So,these results suggest BAK1 is not only involved in PTI activation,but also related to ETI when BAK1 is depleted.However,the molecular mechanism of BAK1 regulated plant auto-immune and the biological significance of BAK1 in plant autoimmunity are poorly understood.To solve this puzzle,in this study,compared with Col-0 wild-type plants,we first systematically analyzed the differentially expressed genes in bak1 bkk1 mutant.We found that a large number of genes involved in plant ETI responses are significantly up-regulated in bak1 bkk1 mutant.Since bak1 bkk1 mutant shows an ETI like immune activation,and NLR protein is the key factor mediating the activation of plant ETI response.We analyzed the expression levels of all NLR genes in the bak1 bkk1 mutant and noticed three NLR protein subfamilies in which almost all their encoding genes were highly up-regulated in bak1 bkk1 mutant.One of them is the ADR1 subfamily,the second one was not reported before and we named it a UNR1(Uncharacterized NLR 1)subfamily.The third one is a RPS5 subfamily.By using an RNAi approach,we reduced the expression of three NLR subfamily genes in bak1-3 bkk1-1 respectively.The results showed that the expression of candidate genes was reduced in their RNAi transgenic plants,while the autoimmune phenotypes were only significantly suppressed in ADR1 RNAi in bak1-3 bkk1-1 plants,UNR1 or RPS5 RNAi in bak1-3 bkk1-1 showed no obvious phenotypic alterations from bak1-3-bkk1-1.So,in the subsequent studies we only focused on ADR1 subfamily as candidates for triggering plant immunity in bak1 bkk1.When overexpressing ADR1 s in Arabidopsis wild-type plants,it recapitulated the auto-immune responses observed in bak1 bkk1.More importantly,the auto-immune phenotypes of bak1 bkk1 are dramatically suppressed when ADR1 s are knocked-out,demonstrating the autoimmune activation of bak1 bkk1 is intrinsically the consequence of ETI activation.In order to further explore the biological significance of the auto-immune responses when BAK1 and BKK1 are disrupted,we induced the expression of a Pseudomonas syringae effector protein HopB1 that proteolytically destructs BAK1 and its homologous proteins,to simulate BAK1 and its homologous proteins being attacked and losing their functions during nature pathogen infection.The results showed that induced expression of HopB1 in wild-type plant not only resulted in BAK1 depletion and impaired PTI,but also causes bak1 bkk1 like autoimmunity phenotypes.However,the ADR1 s mutant can significantly suppress the HopB1 mediated plant autoimmunity.Furthermore,introduction of HopB1 via a Pseudomonas fluorescens-delivery system to infect the plant leaves also resulted in an autoimmunity phenotype in an ADR1s-dependent manner.These results indicate BAK1,a common co-receptor mediating multiple PTI signaling pathways,also possesses an important function in a NLR protein dependent immune activation.From an evolutionary point of view,attacking BAK1 and other SERKs by pathogen effectors results in PTI inactivation,but during the evolution of plants and pathogenic microorganisms,BAK1 and other SERKs may also serve as a guardee by NLRs to prevent the invasion of pathogenic microorganisms.Here we propose that the biological significance of BAK1 regulating plant immunity is activation or the depletion of BAK1 all confers plants resistance to microbial pathogens to maintain plant normal growth and development.These studies elucidated a long-lasting question regarding the mechanism of BAK1 in regulating plant auto-immune.This knowledge can also be used to develop strategies for crop improvement with enhanced disease resistance and high yield in the future.