Molecular Mechanism Of Chinese Wheat Mosaic Virus Coat Protein Promoting Viral Infection

Wheat mosaic disease,primarily caused by the Chinese wheat mosaic virus（CWMV）,poses a major threat to safe wheat production in China.In this regard,the cultivation of disease-resistant varieties is the most economical and effective measure for achieving disease prevention and control.However,due to the long-term and large-scale cultivation of a single variety,resistant varieties was easily loss of its resistance ability,which led to a gradual spread of the disease in winter wheat planting areas in China.Therefore,analysis of the pathogenic mechanism of CWMV and the host defense mechanisms is not only beneficial for uncovering new disease resistance pathways,but also significantly important for finding new genetic resources and providing theoretical support for disease-resistant breeding.Although certain progress has been made in establishing the pathogenesis of CWMV,a complete understanding has yet to be gained.In this study,we sought to elucidate the molecular mechanisms underlying the pathogenicity of CWMV by examining the role of the coat protein（CP）of CWMV in the infection process of this virus,and made the following key research findings:1.The wheat protein thiamine thiazole synthase（THI1/2）positively regulates resistance to CWMV in wheat.CWMV CP interacted with TaTHI2,which was confirmed by yeast two-hybrid（Y2H）and luciferase complementation imaging（LCI）.A homology analysis reveal that the amino acid sequences of THI1/2 from many species are highly similar,indicating that THI1/2 are highly conserved in various species.In addition,overexpression of TaTHI2（TaTHI2-OE）inhibited CWMV infection,while TaTHI2 silencing enhanced viral infection in wheat.These data suggest that TaTHI2 play a crucial role in host resistance to CWMV infection.Besides,TaTHI2-OE plants showed increased seed number per ear and1000-kernel weight compared to susceptible wheat cultivar YM158.2.Plants regulate the accumulation of reactive oxygen species（ROS）via a TaTHI2-TaCPK5-TaCAT1 signaling cascade,which in turn activates plant immune responses.Subcellular localization analysis revealed that TaTHI2 is mainly localized in chloroplasts,while TaTHI2 is deposited at plasma membrane under CWMV infection.Liquid chromatography tandem mass spectrometry（LC-MS/MS）,Co-Immunoprecipitation（Co-IP）,LCI and subcellular localization analyses indicated that TaTHI2 interacts with the Ca²⁺-dependent protein kinase TaCPK5 at the plasma membrane.Phosphorylation analysis revealed that TaTHI2 inhibit the autophosphorylation activity of TaCPK5 in vitro.Further studies revealed that TaCPK5 interact with and phosphorylates TaCAT1 in vitro and in vivo.Ser-258 in TaCAT1 is an essential site for phosphorylation by TaCPK5.The results of diaminobenzidine tetrahydrochloride（DAB）staining and nitroblue tetrazolium（NBT）staining indicated that TaCPK5 enhance TaCAT1 catalase activity,leading to increased ROS accumulation.Phosphorylation experiments and parallel reaction monitoring（PRM）analysis indicate that TaTHI2 promotes ROS accumulation by interrupting the TaCPK5-enacted TaCAT1 catalase activity,thereby inhibiting viral infection.3.Transgenic lines expressing CWMV CP（T-L8,T-L6,and T-L1）exhibited more severe disease symptoms compared to YM158 when planted in a field nursery with a history of CWMV infection.Further analysis using q RT-PCR and Western blot showed that the accumulation levels of CWMV RNA and CP protein in T-L8,T-L6,and T-L1 were significantly higher than that in YM158.Moreover,the spike length and the seed number per ear and 1000-kernel weight of these lines were significantly reduced compared to that in YM158.Subcellular localization and cellular component analysis revealed that CP and TaTHI2 are co-localized in the plasma membrane.Pull-down and phosphorylation assays showed that CWMV CP competes with TaCPK5 to bind to TaTHI2 and promotes the autophosphorylation of TaCPK5.In addition,DAB and NBT staining showed a significant reduction in ROS accumulation in T-L8,T-L6,and T-L1 compared to that in YM158 during CWMV infection.Thus,these data suggest that CWMV CP interrupts the host defense response mediated by TaTHI2 to reduce ROS production,facilitating virus infection.4.LC-MS/MS analysis revealed that S45 and S55 phosphorylation sites are the main phosphorylation modification sites of CP.The CWMV mutant encoding a CP no longer phosphorylatable at S45 and S55(CWMV^S45/55A)was inoculated into the leaves of N.benthamiana did not result in clear mosaic symptoms compared with CWMV wild-type（WT）infection.Moreover,q RT-PCR and Western blot showed that the accumulation levels of CWMV m RNA and CP protein in N.benthamiana plants inoculated with CWMV^S45/55Awere significantly reduced compared to those inoculated with CWMV.These results suggest that the phosphorylation of CP at S45 and S55 may play an important role in the infection of CWMV.Taken together,TaTHI2 interacted with TaCPK5,which is localized in the plasma membrane,and promoted ROS production by repressing TaCPK5-mediated activity of the catalase protein TaCAT1.CWMV CP disrupted the interaction between TaTHI2 and TaCAT1,reducing ROS accumulation and facilitating viral infection.In addition,the phosphorylation of CP at S45 and S55 promote CWMV infection in plant.Our findings provide new insight for the mechanism of CWMV infection as well as the evolutionary arms race between virus and plants.