The Mechanism Analysis Of Wheat Blue Dwarf Phytoplasma Effector SWP1 Inducing Witches’ Broom Symptom

Wheat blue dwarf(WBD)disease causes dwarfism,witches’ broom,and yellow leaf tips in wheat plants,resulting in severe yield loss in northwestern China.This disease is caused by WBD phytoplasma,which belongs to the class Mollicutes,phylum Tenericutes,16 SrI group.WBD phytoplasma is transmited by Psammotettix striatus to diverse plant species,including members of the Gramineae,Brassicaceae,and Solanaceae families.In previous study,WBD phytoplasma draft genome was obtained.Subsequently,a total of 37 secreted proteins were predicted by bioinformatics analysis.Through Agrobacterium-mediated transient expression screen,a candicate effector-SWP1 was identified for inducing witches’ broom symptoms in N.benthamiana.The witches’ broom symptom,which is characterized as the production of large numbers of axillary shoots,is induced by most phytoplasmas,makes it one of the typical symptom to discover new phytoplasma candidates.This common symptom may play vital roles in the pathogenesis of phytoplasma.The purpose of this study was to investigate the mechanism of SWP1 inducing witches’ broom symptom and to provide a theoretical basis for revealing the pathogenesis of WBD.Firstly,SWP1 was over-expressed in Arabidopsis thaliana to observe whether the witches’ broom symptom could be induced in stable genetic SWP1 transgenic plants.Additionally,using yeast two-hybrid(Y2H)and bimolecular fluorescence complementation assays(BiFC),we demonstrated that SWP1 binding to A.thaliana transcription factor Branched 1(BRC1),a key negative regulator of branching signals in various plant species.Moreover,in planta co-expression expriments showed that SWP1 promotes the degradation of BRC1 via 26 S proteasome system.The main results are described as follows:1.Over-expression of SWP1 induces witches’ broom in Arabidopsis.The secretory function of the predicted signal peptide of SWP1 was identified using a yeast signal sequence trap(YSST)screen,which indicated that SWP1 was a secreted protein.To further determine where SWP1 functions,a NLS of SV40 Large T-antigen and a nuclear export signal(NES)of HIV-Rev were fused to the C-termini of SWP1.As expected,GFP-SWP1-NLS and GFP-SWP1-NES located in nucleus and cytoplasm,respectively.However,witches’ broom-inducing function was abolished when SWP1 was excluded from plant cell nuclei by the NES.Taken together,these results indicate that the nuclear localization is required for the functions of SWP1 in inducing witches’ broom symptom.To further analyze the effect of SWP1 on plant development,transgenic Arabidopsis lines expressing the mature SWP1 protein(without signal peptide)were generated.Four weeks after flowering,homozygous SWP1-transgenic plants exhibited significantly more primary rosette-leaf branches than wild-type plants(Col-0).2.SWP1 targets Arabidopsis shoot branching integrator BRC1 and BRC1 homologues.The interactions between SWP1 and Arabidopsis TCP class II subfamily members were tested by BiFC assay.It was found that SWP1 interacted with one of their members-BRC1,an integrator of branching signals within axillary buds.Furthermore,co-expression of GFP-SWP1 and mCherry-BRC1 showed that SWP1 co-localized with BRC1 in plant cell nucleus.Subsequently,the interaction between SWP1 and BRC1 was further comfirmed by Y2 H assay.Most WBD host plants such as some monocots and Madagascar periwinkle exhibit a typical witches’ broom phenotype,however,a few host plants do not,such as tomato.To test whether this phenomenon was associated with the interaction of SWP1 and BRC1 s,we cloned BRC1 homologous genes from a monocotyledonous model plant Brachypodium distachyon,Madagascar periwinkle,and tomato.Subsequently,the interaction of SWP1 and these BRC1 homologs was examined by Y2 H assays.It was shown that BRC1 s of Brachypodium distachyon and Madagascar periwinkle had strong interaction with SWP1.However,the interaction of BRC1 b from tomato and SWP1 was not observed.These results suggested that the witches’ broom symptom caused by WBD phytoplasma infection probably mediated by the interaction of SWP1 with BRC1 s.3.SWP1 destabilizes BRC1 through the 26 S proteasome system.Shoot architecture of plants expressing SWP1 resembled the phenotype of Arabidopsis brc1 mutant.Thus,we speculated that SWP1 probably induces witches’ broom by destabilizing BRC1.To test this hypothesis,we co-expressed BRC1 with GFP-SWP1 and GFP(a negative control)in N.benthamiana leaves.Western blot analyses revealed that the protein abundance of BRC1 co-expressed with GFP-SWP1 was remarkably lower than that co-expressed with GFP,and the degradation of BRC1 was greatly inhibited by 26 S proteasome inhibitors treatments.These results elucidated that WBD phytoplasma effector SWP1 induced witches’ broom phenotype in Arabidopsis through targeting BRC1 and promoting its degradation.