Functional Studies Of Selective Autophagic Receptor NbNBR1 In Geminivirus Infection

Begomovirus is the largest genus in the family of Geminivirdae,causing serious damage in agricultural production.Tomato yellow leaf curl China virus(TYLCCNV)and its associated tomato yellow leaf curl China betasatellite(TYLCCNB)is a typical begomovirus,and infects many crops in the field,leading to obvious symptoms such as yellowing,leaf curling and stem distorting.The TYLCCNB?C1 protein has been identified as a multifunctional protein that inhibits plant defense responses through multiple pathways and it can also be targeted by the immune response of plants as well.However,the virus is still able to successfully infect plant hosts and induce severe symptoms,indicating that there exist some anti-defense strategies that have not been found.Based on this,the following studies are carried out in this study:The selective autophagy receptor Nb NBR1 was found to interact with?C1 by yeast two-hybrid(Y2H),bimolecular fluorescence complementation assay(Bi FC)and co-immunoprecipitation(Co-IP)methods,and the interaction complex forming granular-like structures was localized in the cytoplasm.Further study found that the infection of TYLCCNV/TYLCCNB could induce the expression level of Nb NBR1.More experiments were performed to investigate the biological significance of the interaction between Nb NBR1 and?C1 and found that when knock down or knock out Nb NBR1,plants showed more resistant to TYLCCNV/TYLCCNB infection,whereas when transient overexpression or transgenic overexpression of Nb NBR1,TYLCCNV/TYLCCNB-infected plants showed more severe symptoms and increased levels of viral DNA-Accumulation.In order to clarify the molecular mechanism how Nb NBR1 promotes virus infection,PVX-?C1 was inoculated on tobacco rattle virus(TRV)based Nb NBR1-silenced plants or Nb NBR1-konck-out lines.The results showed that the accumulation level of?C1 protein was significantly reduced,and the typical symptoms associated with?C1 were also weakened than that in control plants,vice versa.These results showed that Nb NBR1 may affect the infection of TYLCCNV/TYLCCNB by affect the accumulation level of?C1.Thus,both NBNBR1 and?C1 were co-expressed together,and the presence of NBNBR1 did indeed enhance the accumulation of?C1 protein.The mutation of the fourth lysine on?C1,?C1^K4A showed no interaction with NBNBR1,and NBNBR1 could not increase the accumulation level of?C1^K4A.Meanwhile,a mutant infectious clone TYLCCNV/TYLCCNB^K4Awas constructed,and the TYLCCNV/TYLCCNB^K4Amutant virus caused milder disease symptoms and accumulated much less viral genomic DNAs in the infected plants.Previous studies have reported that?C1 is degraded by Nt RFP1-mediated 26S ubiquitin proteasome system,and Nb NBR1 itself is a selective autophagy receptor that binds ubiquitinated proteins.Further experiments illustrated that Nb NBR1 showed no direct interaction with Nb RFP1,overexpression of Nb NBR1 had no effect on the protein and m RNA levels of Nb RFP1.Yeast-three hybrid experiment showed that Nb NBR1 inhibited the interaction between Nb RFP1 and?C1 and change their interaction site.This competitive binding of Nb NBR1 and?C1 can protect?C1 away from degradation,increase the accumulation level of?C1 protein and promote TYLCCNV/TYLCCNB infection.Furthermore,whether Nb NBR1 shows a similar effect on the infection of other geminiviruses was also examined.Transgenic overexpression of Nb NBR1 can promote the infection of tomato yellow leaf curl virus Beijing isolate,TYLCV-BJ,while knockout of Nb NBR1 inhibited the infection of the virus.Meanwhile,the V2 protein encoded by TYLCV-BJ interacted with NBNBR1 and also showed granular-like structures in the cytoplasm.Here,a novel anti-defense strategy of geminivirus was found.?C1 can escape from Nb RFP1-mediated degradation by interacting with a selective autophagy receptor Nb NBR1 to form a granule-like structure,thus promoting the successful infection of geminivirus,This study expands the interaction network between plants and geminiviruses.