Mechanism Analysis A Novel Protein Elicitor (BAR11) From Saccharothrix Yanglingensis Hhs.015 Induceing Multiple Defense Responses In Plants

We are now advocating green-ecology and safety-ecology,and agricultural production is pioneered to be nuisance-free and pollution-free.Application of beneficial microbes for controlling plant diseases and pests has become the focus of attention.Saccharothrix yanglingensis Hhs.015,a PGPR strain well-studied for biocontrol in apple valsa cancer,was first isolated from cucumber roots and classified in the genus Saccharothrix.S.yanglingensis strain Hhs.015 is able to colonize in apple tissues and trigger resistance to plant disease.Previously,we have sequenced the whole genome of Hhs.015 strain,and we obtained a candidate protein elicitor BAR11,which can induce apple resistance to Valsa mali,by large-scale screening of predicted secreted proteins of Hhs.015.This article aims to explore the molecular mechanism of plant resistance induced by BAR11,using bioinformatics,histocytology,and molecular biology techniques.The results are as follow:1.Sequence analysis following blast of BAR11 against NCBI showed that,the BAR11 is well conserved in various species of actinomycetes.Purified BAR11 protein was obtained following induced-expression in E.coli BL21（DE3）strain,and purification by a His Trap TM HP column.The recombinant protein showed a single band at expected size（27k Da）by SDS-PAGE analysis.The three-dimensional structure of BAR11 was predicted to be simple,consisting of fourα-helix components.2.BAR11 can induce Arabidopsis thaliana to resistance agaisnt Pst DC3000,and it triggers innate immune responses,like H₂O₂accumulation and callose deposition in Arabidopsis leaves.After BAR11 treatment,Arabidopsis enzyme activity of phenylalanine ammonia-lyase（PAL）,catalase（CAT）,peroxidase（POD）and superoxide dismutase（SOD）rapidly induced at 6 h,of which,PAL and CAT increased by 2.0 and1.75-fold,compared to control treatment.POD and SOD were higher than those of 30U and50U,respectively.Real-time quantitative PCR（q RT-PCR）analysis showed that BAR11distinctly induced the expression of PR1,PR2,PR5,PDF1.2,LOX1,LOX2,GST1,and NPR1 in both Arabidopsis and apple.In Arabidopsis,PR1,PR2,and PR5 were up-regulated at 24 hpt,36 hpt,and 48 hpt;PDF1.2 and NPR1 were up-regulated 48 hpt.Particularly,4 d after BAR11 treatment,PDF1.2 was up-regulated by more than 340-fold.In apple,marker genes of SA-mediated signaling pathway（PR1,PR2,PR5）and the JA-mediated signaling pathway（PDF1.2,LOX1,LOX2）were markedly induced,indicating that the elicitor BAR11 participates in both SA and JA signals of plants.3.Using agrobacterium-mediated transient expression,co-immunoprecipitation,and LC-MS/MS analysis,we identified the Nicotiana benthamiana catalase（Nb CAT1）as a candidate target of BAR11.Bimolecular fluorescence complementarity（BIFC）assay suggested that BAR11 interacts with catalase（Nb CAT1,At CAT1,At CAT2,and At CAT3）in both N.benthamiana and Aarabidopsis.,which was further confirmed by Co-IP analysis.4.We have successfully generated BAR11-transgenic Arabidopsis plants.Growth and development monitoring,q RT-PCR analysis defense-related genes,and resistance evalution of BAR11-transgenic Arabidopsis lines showed that,BAR11 can break the balance between plant growth and immunity,resulting in delayed development but,enhanced resistance.In summary,we demonstrated that BAR11 can induce resistance in both apple and A.thaliana,and it can interact with plant catalase,disrupting growth and immune balance and improving the plant disease resistance.