Study On The Interaction Mechanism Between Geminivirus C2 And Host Factor ATG7

Geminiviruses are a kind of single-stranded circular DNA plant viruses which cause tremendous damage to food crops and cash crops worldwide.The AC2/C2 protein encoded by the geminivirus is a multifunctional protein,which is able to regulate the expression of viral late genes,and interacts with several host proteins to help the virus suppress or evade the host immune response and create an environment beneficial for virus replication.Autophagy is an evolutionarily conserved intracellular process that transports cytoplasmic contents to lysosomes or vacuoles for degradation.Increasing studies have revealed the antiviral effect of autophagy in plants.However,to successfully establish the systemic infection,the virus has evolved diverse strategies to counter the autophagy pathway.In this study,tomato leaf curl virus Yunnan(TLCYn V)was used as a model to study the interaction between autophagy and geminivirus.First,q RT-PCR analysis showed that TLCYn V infection induced the autophagy pathway in Nicotiana benthamiana plants.Transient overexpression of autophagy-related genes ATG5,ATG7,or ATG8 and transgenic overexpression of Nb ATG7 exhibited reduced virus symptoms,which were accompanied by decreased virus accumulation compared to controls.Combined with our previous finding that TLCYn V-induced symptoms were aggravated when ATG5,ATG7,or ATG8 was knocked down in Solanum lycopersicum and N.benthamiana plants.These results indicated that the activated autophagy plays an antiviral role in TLCYn V infection.However,the detection of the number of autophagy bodies and ATG8 protein levels in TLCYn V infected plants showed that TLCYn V infection could also inhibit autophagy,implying a perplexing role of autophagy in geminivirus infection.Furthermore,the interactions between TLCYn V C2 and Nb ATG7,and TLCYn V C2 and Sl ATG7 were found by yeast two-hybrid,pull-down,bimolecular fluorescence complementation(Bi FC),and co-immunoprecipitation.The co-localization analysis revealed that C2 was exported from the nucleus and co-localized with Nb ATG7 or Sl ATG7 forming granules in the cytoplasm in the context of viral infection.Through mapping the ATG7 domains interacting with C2,the C-terminal domain of ATG7 was confirmed to play a key role in the interaction between C2 and ATG7.Subsequently,similar experiments demonstrated that ATG7 interacted with ATG8 in S.lycopersicum and N.benthamiana plants,and the C-terminal domain of ATG7 was responsible for their interactions.There was no interaction between C2 and ATG8,and C2 had no impact on the m RNA level of ATG7,and both C2 and ATG8 interacted with ATG7 through the same ATG7 domain,we speculated that C2 might affect the interaction between ATG7 and ATG8.Through Bi FC,pull-down and co-immunoprecipitation experiments,it was proved that the expression of C2 interfered with the interaction between ATG7 and ATG8,and this competitive inhibition exhibited a dose effect.Considering that the interaction between ATG7 and ATG8 participates in the formation of autophagosomes,the number of autophagy bodies and the level of ATG8 protein were further detected in C2 over-expressing plants,and results indicated that C2 could inhibit the autophagy activity of the host.The interactions between ATG7 and the C2 proteins from other geminiviurses(Tomato yellow leaf curl China virus,TYLCCNV;Tomato yellow leaf curl virus,TYLCV;Mungbean yellow leaf curl virus,MYMIV)were also found.Furthermore,we also found that the C2-mediated inhibition mechanism of autophagy in plants was conserved in these geminiviruses.In conclusion,when geminivirus invades the host plant,it will activate the autophagy pathway in plants.The ATG7 protein can bind the viral protein C2 and mediate the nuclear export of C2 to inhibit its function as a viral transcription activator,playing an antiviral role.However,the geminivirus adopts a passive anti-defense strategy: the viral protein C2 interferes with the interaction between ATG7 and ATG8 in plants through competitive binding to ATG8,thereby inhibiting the autophagy activity and successfully infecting the host.