Suppression Of Plant Immunity By Phytophthora Avr3a Effectors

Oomycete is a kind of unique filamentous eukaryotic microorganisms being evolutionarily distant from fungi,including many notorious plant pathogens,such as Phytophthora spp.,Peronospora spp.,and Pythium spp.P.infestans,P.sojae and P.capsici all cause many destructive crop diseases and threaten agricultural production.A number of effectors,notably the RXLR and CRN effectors that carry N-terminal conserved motifs required for translocation into host plant cells and variable C-terminal sequences have been identified in the sequenced Phytophthora genomes.The P.capsici genome alone encode 357RXLR effectors.Effectors play key roles in infection.However,the underlying mechanisms of the most Phytophthora effectors are not clear.Identification of effector targets and the underlying mechanisms are crucial to the understanding of Phytophthora infection and plant immunity,which facilitate development of novel methods to control crop diseases.PiAvr3a is a typical P.infestans RXLR effector that suppresses PAMP INF1-triggered cell death to faciliate plant infection,and be recognized by potato resistance protein R3a resulting with hypersensitive resistance.The geneome sequences of P.sojae and P.capsici encode many PiAvr3a-like secreted proteins,including P.sojae avirulence protein PsAvr1b.Other studies have shown that multiple PiAvr3a-like proteins neither suppress INF1-triggered cell death nor be recognized by disease resistance protein R3a.In this study,we use these PiAvr3a-like effectors to identify their plant targets and to investigate the underlying mechanisms.The follows were achieved:1.Multiple PcAvr3a genes were cloned from P.capsici,including PcAvr3a1,PcAvr3a3,PcAvr3a5,PcAvr3a11,PcAvr3a12,PcAvr3a14 and PcAvr3a15.These PcAvr3a effectors have similar sequences but different functions from PiAvr3a as indicated by sequence analysis and transcient expression assays in Nicotiana benthamiana.The transcient expression assays also showed that these Pc Avr3a effectors could not disturb the recognition between R3a and PiAvr3a and nor trigger cell death on multiple Nicotiana tabacum lines.2.Among them,PcAvr3a12 is an effector gene up-regulated in the early stage of Arabidopsis thaliana infestation by Phytophthora capsici.By yeast-two-hybrid,co-immunoprecipitation（Co-IP）and bimolecular fluorescence complementation（BiFC）,we found that the endoplasmic reticulum-localized protein FKBP15-2 was targeted by PcAvr3a12.Genetic analysis showed that the FKBP15-2 positive regulated plant resistance to Phytophthora.PPIase activity assay in vitro and PPIase activity attenuated mutant of FKBP15-2 showed that FKBP15-2 has PPIase activity and its PPIase activity is required for its immune function.In vitro experiments further showed that PcAvr3a12 could directly inhibit the activity of PPIase.Real-time fluorescence quantitative PCR experiments after TM treatment and Phytophthora capsici infection,FKBP15-2 was involved in endoplasmic reticulum stress and partially influenced endoplasmic reticulum stress-mediated plant immunity.3.Further,yeast-two-hybrid assay showed that PcAvr3a1 also interacted with FKBP15-2.However,PcAvr3a1 neither attenuate plant immunity when was expressed in plant nor significantly inhibit the PPIase activity of FKBP15-2 in vitro.4.FKBP20,a cytoplasm and cell nucleus protein,could interact with PcAvr3a1,PcAvr3a3,PcAvr3a12,PcAvr3a14,PcAvr3a15 and PiAvr3a^EM.Real time RT-PCR showed that FKBP20 was weakly up-regulated during P.capsici early infection stages.In conclusion,by employing the model Phytophthora-Arabidopsis pathosystem and a range of approaches including biochemistry,molecular biology,cell biology and genetic analysis,we showed that P.capsici RXLR effector PcAvr3a12 could target ER-stress related protein FKBP15-2 and inhibit its PPIase activity to suppress plant immunity.This research also suggested that the ER-stress plays important roles in plant immunity.In addition,we also found that FKBP20 is a potential plant target of multiple Avr3a family proteins through analyzing the interaction relationship between FKBP family and Avr3a family.