Study Of Biological Function Of RXLR207 And BPA Family Proteins In The Interaction Between Phytophthora Capsici And Plants

Phytophthora capsici,an oomycete pathogen,causes many devastating diseases on a broad range of plant species,including model plant Arabidopsis thaliana and plenty of vegetable crops such as cucumber,pepper,tomato,etc.P.capsici was first described in 1922 after it was recovered from chilli pepper at the New Mexico Agricultural Experiment Station field plots in 1918.The symptoms caused by P.capsici are complex and vary according to the different environments,hosts and infection sites.In an environment with low humidity,it causes black or rot at the plant root and stem.In an environment with high humidity,infection of P.capsici can be expanded to the whole plant and leads to growth retardation,wilting and finally death.P.capsici is a soil-borne disease,as it appears that P.capsici only survives in soil for extended periods as thick-walled oospores,and thus it is difficult to eliminate P.capsici once epidemic area was formed.The genetic diversity of P.capsici is complex as many physiological races exists caused by rapid mutation in the natural environment through sexual reproduction.Few high-resistance crop lines are available in production,and usage of chemical pesticides are mainly management method to control P.capsici.However,drugresistant P.capsici strains are easy to appear and spread.Because of the above reasons,P.capsici is an important pathogen which is difficult to control for many crops all over the world,and it also caused huge economic losses in Chinese agricultural production.P.capsici is a hemibiotrophic pathogen,which has a biotrophic stage in the early stage of infection and then transit to necrotrophic stage in the late stage of infection.During infection,P.capsici secretes a plenty of effectors to attack the key immune pathways of hosts and promote infection.Among them,RXLR effectors are a kind of effectors that have attracted wide attention and are also the research focus of our laboratory.It is still unknown whether the RXLR effector of P.capsici participates in the interaction between P.capsici and its host and regulates the transition of P.capsici from biotrophic stage to necrotrophic stage.Understanding the mechanism behind the transition of P.capsici from biotrophic stage to necrotrophic stage will provide new clues for the management of P.capsici.Explaining the interaction mechanism between P.capsici and hosts,and digging out resistance-related genes is effective way to facilitate plant protection.However,some resistance-related genes are function redundant and thus hardly to be identified with traditional genetic methods.To solve the problem,we raised a new strategy that using pathogen effectors as “probes” to help to identify resistance-related genes with the preconception that effectors tend to target vital hosts immunity components.To solve the problems mentioned above,in this study,we did four aspects of research work and the major results are:1.RXLR207 mediates the transition from biotrophic stage to necrotrophic stage of P.capsiciP.capsici is a hemibiotrophic pathogen.The mechanism behind the transition of P.capsici from biotrophic stage to necrotrophic stage and whether RXLR effectors involved in this process is still unknown.A part of RXLR effectors could induce cell death in plants,which is potentially regulator of biotroph-to-necrotroph transition of P.capsici.To explore it,we conducted a preliminary screening for cell death-inducing RXLR effectors in N.benthamiana using Agrobacteria infiltration.Here,we found ectopic expression of a P.capsici effector RXLR207 in planta could activate cell death in N.benthamiana yet accelerate cell death in Arabidopsis,leading to reduced pathogen infection.RXLR207 was induced during transition from biotrophic stage to necrotrophic stage of P.capsici.RXLR207 knock-out P.capsici mutant was impaired in the virulence and the ability to induce cell death in host,indicated that RXLR207 involved in transition from biotrophic stage to necrotrophic stage of P.capsici.2.RXLR207 promotes cell death by inducing degradation of BPA family proteins and ACD11To further explore the mechanism how RXLR207 manipulate plant immunity,we identified RXLR207’s target by Yeast-two-hybrid screening.We demonstrated that RXLR207 interacted with BPA family proteins.Further analysis indicated that BPA family proteins were negative regulator of plant immunity.Plants are less susceptible to the oomycete pathogen in the absence of BPAs with enhanced hypersensitive response cell death.bpl3 single mutant displayed enhanced resistance with other BPAs were functional redundant.Furthermore,BPAs directly interact with and stabilize accelerated cell death 11(ACD11),while RXLR207 promotes the degradation of BPAs and ACD11 in a 26 S proteasomedependent way and promote plant cell death.3.BPL3 negatively regulates plant immunity through a natural anti-sense long noncoding RNAOnly bpl3 single mutant displayed enhanced resistance with other BPAs were functional redundant,indicated that BPL3 was a dominant gene.It is still largely unknown how BPL3 regulates plant immunity.We speculated that BPL3 affect RNA processing because it has an RNA binding domain.Strand-specific RNA-seq demonstrated that BPL3 may suppressed FL7 transcript accumulation and elevate levels of a cis-natural antisense lnc RNA of FL7(nalnc FL7).BPL3 directly bound nalnc FL7 but not FL7,stabilized nalnc FL7.nalnc FL7 suppresses FL7 at the transcriptional level and by which BPL3 destabilized FL7.FL7 positively regulated plant immunity to P.capsici while nalnc FL7 was a negative regulator of resistance.4.BPL3 negatively regulates plant immunity through a natural anti-sense long noncoding RNAFL7 belong to FL(FORKED1-like)gene family which act redundantly in the formation of reticulate leaf vein pattern by regulating PIN1 localization.It was unknown whether FL7 involved in plant immunity.In this study,we found that FL7 interacts with HAI1,a type 2C protein phosphatase(PP2C)and suppressing HAI1 phosphatase activity in vitro and in vivo.HAI1 could dephosphorylate MPK3/6 and suppress plant immunity and FL7 enhanced plant immunity through HAI1-dependent pathway.These results indicated that BPL3-nalnc FL7-FL7 is a cascade which regulated plant resistance response to P.capsici through HAI1.Phylogenic analysis indicated that BPL3 and FL7 were conserved in land plant while nalnc FL7 was conserved at least in Brassicaceae.Together,we found RXLR207 was a vital effector involved in the transition from biotrophic stage to necrotrophic stage of P.capsici.RXLR207 interacted with BPA family protein,promoted their degradation and subsequently induced ACD11 degradation,and thus facilitate plant cell death.BPA family proteins belong to a conserved plant RNA-binding protein family,and negatively regulate ROS accumulation and cell death under biotic stresses.Among them,the bpl3 single mutant shows higher resistance to P.capsici.By exploring the function of BPL3,we found BPL3 was a negative regulator of FL7 transcript levels by promoting accumulation of the natural antisense lnc RNA of FL7,nalnc FL7.nalnc FL7 suppressed accumulation of FL7 and BPL3 could directly bound to and stabilized nalnc FL7.We found FL7 positively while nalnc FL7 negatively regulates plant immunity.Furthermore,we observed that FL7 directly bound to and suppressed a PP2 CA protein,HAI1,which normally decreases the phosphorylation levels of MPK3 and MPK6.Collectively,we reported a cascade of BPL3-nalnc FL7-FL7 that synergistically manipulates plant immunity.The cascade is a novel regulator of MPK3/6 activity and the finding will deepen our understanding of plant immunity.