| Wheat blue dwarf disease（WBD）,a major phytoplasma disease threatening wheat production,caused enormous agricultural losses in northwestern China.Previous study showed that WBD phytoplasma encodes many secreted proteins（SWP）.One of them,SWP1 protein,is a key pathogenic effector which can induce witches’broom in Nicotiana benthamiana and Arabidopsis thaliana.To better understand the mechanism of witches’broom symptom induced by SWP1 and provide support for the further study on pathogenesis of WBD phytoplasm,the interaction between SWP1 and plant was investigated in our study.ClassⅡTCPs transcription factor family has important regulatory effects on many physiological processes,such as leaf morphogenesis and branch development.Some members of ClassⅡTCPs were proved to be targets of SWP1-like proteins from Aster Yellows phytoplasma and SAP11-like protein from apple proliferation phytoplasma.Therefore,we examined interactions between SWP1 and ClassⅡTCP members by yeast two-hybrid,and then carried out a study on interaction mechanism between SWP1 and AtTCP12,which negatively regulate branch development.The main results are described as follows:1.The 11 full-length genes encoding ClassⅡTCPs were amplified and inserted into bait vector pGADT7.Recombinant plasmids p GADT7-TCPs were co-transformed into yeast cell with pGBKT7-SWP1 respectively,then the yeast cultures were plated onto selection medium to determined interactions.The results showed that TCP1,TCP2,TCP4,TCP12,TCP18,TCP24 interacted strongly with SWP1,while other members did not.2.The interaction between SWP1 and AtTCP12 was verified further by bimolecular fluorescence complementation（BiFC）.Next,we co-expressed mCherry-AtTCP12 with GFP-SWP1 in N.benthamiana leaves and observed the fluorescence distribution under laser confocal microscopy.We found SWP1 and AtTCP12 co-localize in cell nucleus,and their respective subcellular localization was not affected by co-expression.3.To seek key interaction domains between SWP1 and AtTCP12,two mutants AtTCP12△TCP and AtTCP12△R were constructed and tested interactions with SWP1 by Y2H assays.Similarly,interactions between three mutants SWP1△NLS,SWP1△CC and SWP1△N and AtTCP12 were determined.Results indicated that the TCP domain of AtTCP12 and coiled coil domain of SWP1 were necessary for the interaction.4.Quantitative real-time PCR showed that the mRNA level of AtTCP12 in SWP1-transgenic Arabidopsis was lower than wild-type.Then we constructed plant expression vector containing 3HA-AtTCP12,and co-expressed it with GFP-SWP1 or a negative control GFP in N.benthamiana.The Semiquantitative RT-PCR and immunoblot analyses revealed that mRNA and protein level of AtTCP12 was reduced when co-expressing GFP-SWP1 than with GFP.In conclusion,effector SWP1 interacts with and down-regulates expression of AtTCP12,which is an important cause of witches’broom symptom in Arabidopsis induced by SWP1. |
