Identification And Studies On NP-interacting Plant Host Factors Of Rice Stripe Tenuivirus

Rice stripe tenuivirus(RSV)is one of the most important rice viruses in East Asia.NP/Pc3 is the main structural protein of RSV,which accumulates in host plant cells in large quantities.However,there are few reports on which plant host factors NP interacts with.In this paper,we screened,validated and studied the function of plant host factors interacting with RSV NP.The main results are as follows:(1)Screening and validation of plant host factors interacting with NP:The fusion protein of RSV NP and Biotin Ligase Air ID was expressed in Nicotiana benthamiana leaf,and 16 candidate proteins interacting with NP were screened using proximity labeling technology.The 16 candidate protein genes and RSV NP were respectively constructed into p GADT7/p GBKT7,YN/YC and p Early Gate-101/102 vectors.Subsequently,yeast two hybrid(Y2H),bimolecular fluorescence complementary(Bi FC),and subcellular co-localization experiments were conducted.The results show that the interaction of Nb Ra RH1 b,Nb Ra RB1 c and Nb ARP1 with NP can be verified both in Y2 H and Bi FC experiments.The interaction of Nbs RGNc,Nb GR-RBP and Nb DBR6 with NP can be verified in Bi FC experiment,but both fusion expression and self activation of the reporter gene were present simultaneously.Six proteins are individually co-localized with NP.(2)Pull down verification of the interaction and identification of key interaction areas between NP and Nb Ra RH1b/Nb Ra RB1c: Nb Ra RH1 b and Nb Ra RB1 c are homologous small GTP-binding proteins.In order to verify their interaction with RSV NP in vitro,the fusion proteins of Nb Ra RH1b/Nb Ra RB1 c with HIS and NP with GST were severally expressed in Escherichia coli,after which the expression products were used for GST pull-down experiments.The results showed that GST antibody can simultaneously pull down NP-GST and Nb Ra RH1bHIS/Nb Ra RB1c-HIS,which means Nb Ra RH1b/Nb Ra RB1 c and NP also interact in vitro.For the purpose of studying the key region of NP and Nb Ra RH1b/Nb Ra RB1 c interaction,five NP mutants with C-terminal amino acid deletion were constructed,and then the interaction between the mutants and Nb Ra RH1b/Nb Ra RB1 c was studied by Y2 H and Bi FC.The results showed that the N-terminal amino acid position 56 to 163 is necessary for NP to interact with Nb Ra RH1 b /Nb Ra RB1 c.(3)The role of Nb Ra RH1b/Nb Ra RB1 c in RSV infection: Nb Ra RB1 c or Nb Ra RH1 b silenced Nicotiana benthamiana was obtained using the gene silencing technique induced by tobacco rattle virus(TRV)in the cause of investigating the significance of the interaction between RSV NP and Nb Ra RH1b/Nb Ra RB1 c.Symptoms were observed after inoculation with RSV,and q RT-PCR was used to detect the accumulation of viral RNA on days 14,21,and 28 after inoculation.The results showed that RSV caused significantly weaker symptoms on Nb Ra RB1 c and Nb Ra RH1 b silenced Nicotiana benthamiana than the control,and the accumulation of viral RNA was also significantly lower on the 28 th day of inoculation.This suggests that Nb Ra RH1 b and Nb Ra RB1 c may positively regulate RSV infection,and RSV may activate or recruit certain functions of Nb Ra RH1 b and Nb Ra RB1 c through NP.In conclusion,six plant host factors interacting with RSV NP were identified through screening and verification.Further research was conducted on Nb Ra RH1 b and Nb Ra RB1 c among them.The results showed that NP interacts with Nb Ra RH1b/Nb Ra RB1 c at its amino acids position 56-163,and this interaction may be conducive to RSV infection.These studies could lay an important foundation for further analysis of NP’s involvement in RSV and plant host interaction,and offer certain reference significance for the design of RSV control strategy.