Functional Analysis Of NbNdhM Gene In Turnip Mosaic Virus Infection

The viral disease caused by plant viruses,known as “plant cancer”,causing great damage and loss to the quality and yield of crops,is one of the most serious diseases threatening the safety of agricultural production.It is extremely difficult to prevent and control plant virus diseases in agricultural production because of the wide variety of plant viruses,easy dissemination and lack of effective pesticides.Therefore,the in-depth study of the interaction between plant and viruse not only will be useful for understanding the pathogenic mechanism of viruse and the disease resistance mechanism of the host,but also has important practical significance in finding novel strategies for the prevention and control of viral disease and ensuring the safety of grain production in China.Turnip mosaic virus(TuMV),which belongs to the genus potyvirus of potyviradae,can infect a wide range of host species and spreads all over the world.The viral disease caused by TuMV seriously threaten the safe production of vegetable crops.Chloroplast is the production site of a variety of plant immune signal molecules(e.g.,SA and ROS),and plays a key role in plant defence against viral infection.At present,great advances have been made in the molecular mechanism of TuMV infecting plants.However,the interaction between TuMV and chloroplast is still unclear.In this study,we employed the classical TuMV-Nicotiana benthamiana compatible plant-pathogen interaction system as the model.Based on the transcriptome database of TuMV infected N.benthamiana plants previously constructed in our laboratory,we carried out the following research work:1.Bioinformatics analysis of transcriptome data showed that the expression of chloroplast NAD(P)H dehydrogenase-like(NDH)complex M subunit gene(Ndh M)was significantly downregulated after TuMV infection.In order to verify the expression of Ndh M gene in N.benthamiana plants after TuMV infection,the expression pattern of NbNdhM at different time points of TuMV infection was detected by q RT PCR.The result showed that the expression of NbNdhM was first increased and then decreased in TuMV infected-N.benthamiana plants,suggesting that NbNdhM may have a changing dynamics between virus and host.Subsequently,Ndh M gene was systematically silenced by tobacco fragile virus(TRV)-induced gene silencing(VIGS)system in N.benthamiana.Silencing of NbNdhM had no significant effect on the growth and development of plants.When these NbNdhM-silenced N.benthamiana plants were inoculated with TuMV,the TuMV symptoms were more serious and the accumulation of TuMV RNAs and CP were more compared with the control;in contrast,the transgenic plants expressing NbNdhM enhanced the resistance to TuMV.In addition,to further reveal the role of NbNdhM in TuMV infection cycle,a binary plasmid encoding infectious TuMV-GFP was used to transfect the protoplasts of NbNdhM-silenced and-overexpressed plants.q RT-PCR results showed that the accumulation of TuMV RNAs in the protoplasts of NbNdhM-silenced plants were remarkably increased.On the contrary,TuMV RNAs accumulated at a significantly lower level in the protoplasts of NbNdhM-overexpressed plants.These results indicate that NbNdhM can positively regulate the resistance of N.benthamiana to TuMV,and also play an important role in inhibiting viral replication.2.NbNdhM,a nuclear-encoded chloroplast protein,contains a chloroplast transport peptide(c TP)domain at the N-terminal and a Ndh M superfamily domain at the C-terminal which has a nuclear export signal(NES).Subcellular localization results showed that NbNdhM was localized in the nucleus,chloroplasts,cell periphery,and chloroplast stromules.Overexpression of NbNdhM could significantly lead to chloroplast clustering around the nucleus,and the three leucines in the NES of NbNdhM were necessary for chloroplast perinuclear clustering.In previous studies,the chloroplast perinuclear clustering is a defense response against pathogens,and requires ROS as a trigger and actin as a support.Here,we determined the expression levels of pathogenesis-related genes Nb PRs(Nb PR1,Nb PR2,and Nb PR5)in NbNdhM-silenced or-overexpressed plants.The relative expression levels of Nb PRs(Nb PR1,Nb PR2,and Nb PR5)were significantly downregulated in NbNdhM-silenced plants,and markedly upregulated in transgenic plants overexpressing NbNdhM.To clarify the effect of NbNdhM-induced chloroplast perinuclear clustering on TuMV infection,the ROS scavenger dimethylthiourea(DMTU)and the actin inhibitor cytochalasin D(CTD)were used to treated with the transgenic plants.The results showed that the numbers of chloroplasts around nuclei decreased significantly and plants were more susceptible to virus infection.In addition,transient expression of NbNdhM-Δc TP or NbNdhM-ΔNES could no longer inhibit TuMV infection.These results suggest that NbNdhM-induced chloroplast perinuclear clustering plays a role in the resistance to TuMV.3.Corresponding attack and defense strategies have been established between plants and viruses in the process of natural evolution.To further reveal the interaction between NbNdhM and TuMV,Y2 H,Bi FC,LCI,and Co-IP were used to to detect any potential direct interactions between NbNdhM and each of the viral proteins.The results showed that NbNdhM interacted with the VPg protein of TuMV,and the three leucines in in the NES of NbNdhM were the key sites of the interaction.Ndh M of different plant hosts interacted with VPg of different genus Potyvirus viruses,implying that the interaction between Ndh M and VPg may be universal in nature.Moreover,TuMV VPg could not only co-localized with NbNdhM in the nucleus and nucleolus and significantly increase its accumulation in the nucleus,but also could impaire the chloroplast perinuclear clustering-induced by NbNdhM.In addition,p2 protein encoded by rice stripe virus(RSV)also interacted with NbNdhM and suppressed NbNdhM-mediated chloroplast clustering.These results suggest that Ndh M may be a conserved target of a variety of plant viruses in evolution,and virus inhibits the antiviral role of NbNdhM-induced chloroplast perinuclear clustering through encoding corresponding proteins in the manner of protein-protein interaction.Taken together,our study revealed that NbNdhM-mediated chloroplast perinuclear clustering may participate in the process of plant resistance to TuMV infection.As a counteractive strategy against this response,VPg protein encoded by TuMV interacts with NbNdhM in the nucleus and nucleolus and prevents its transport from nucleus to chloroplast,thus inhibiting the disease resistance of NbNdhM-induced chloroplast perinuclear clustering.This also reflects the coevolution characteristic of plant virus and chloroplast in the long-term “arms race”,and provides a new perspective and reference for in-depth understanding the role of chloroplast in plant virus infection.In addition,because of Ndh M overexpression plants protect against virus infection and adversely affect the process of plant growth and development,the study of the function of Ndh M or other subunits of its complex will provide a theoretical basis for disease resistance breeding.