Screening And Identification Of The Biological Function Of The Xanthomonas Campestris Pv.campestris 8004 T3S Effectors

When pathogenic microorganisms infect plants,their pathogen-associated molecular patterns(PAMPs)will be specifically recognized by pattern recognition receptors(PRRs),and then induce a series of downstream immune responses against infection in plants.The recognition triggered the first layer innate immunity so called PAMP-triggered immunity(PTI).For successful invasion,pathogens have evolved Type III secretion system(T3SS).The effectors of bacteria can be directly secreted into plant cells by T3 SS and then infect plants such as Xanthomonas campestris pv.campestris(Xcc).Xcc can affect and cause black rot in cruciferous.Xcc8004 is a pathogenic strain of Xcc,which is able to secrete effectors into plant cellular and effect plant growth by T3 SS.To verify the biological function of Xcc8004 effectors,the research use Arabidopsis protoplast transient expression system and then screen effectors that harbor interference defense signaling pathway.However,Xcc8004 can secrete numerous effectors into cell,which can produce functional redundancy.So the character of single effector of Xcc8004 cannot be studied.Therefore,we cloned 24 predicted T3 S effectors in the Xcc8004 genome and studied their biological function,respectively.We used an Arabidopsis thaliana mesophyll protoplast-based assay to identify Xcc8004 T3 S effectors whether effectively interfere with bacterial peptide flg22-induced PTI signaling.Results of this study are as followed:(1)Of the 24 tested effector proteins,11(Xop K、Xop Q、Hrp W、Xop N、Xop AC、Xop D、Xop Z1、Xop AG、Avr Bs2、Xop L、Xop X-1)and 5(Xop K、Xop G、Xop Q、Xop L 、 Xop X-1)inhibited the activation of FRK1 and WRKY33 downstream of PTI,respectively;(2)Xop K-GFP was detected on the plasma membrane,and Xop D-GFP fluorescence was detected on the plasma membrane,cytoplasm and nucleus.The remaining 7 effectors(Avr Bs2-GFP,Xop G-GFP,Xop N-GFP,Xop X-1-GFP,Xop Q-GFP,GFP-Xop AG,GFP-Hrp W)exhibited similar subcellular localization patterns within the cytoplasm;(3)Of the 12 tested effectors,4(Xop X-1,Xop K,Xop Z1,Xop Q)significantly reduced the phosphorylation of MPK3 and MPK6,while none of the remaining 8 affected the activation of either MPK3 or MPK6.These results indicate that multiple T3 S effectors in the Xcc8004 genome interfere with flg22-induced PTI signaling via different molecular mechanisms that acting upstream or downstream of or in parallel to MAP kinase cascades.