Mechanism Of Which IP-L Regulats Its Three Interacting Proteins To Affect TMV Infection In Nicotiana Benthamiana

Tobacco mosaic virus（TMV）is a major virus affecting crops in the world,which can infect all tobacco and various plants including Cucurbits and Crucifers.Once it occurs,it is extremely difficult to control.The purpose of this study was to analyze the effect of IP-L and its interacting proteins NbAS-B,NbCML30 and RIN4 on TMV infection,and to clarify how IP-L,as the junction point between viral CP and host resistance factors,regulates viral infection and induces resistance in the host.First,based on previous studies,we further verified the interaction between IP-L and asparagine synthetase NbAS-B using Co-Immunoprecipitation（Co-IP）and Luciferase Complementation Assay（LCA）.Co-localization assays demonstrated that IP-L and NbAS-B co-localized in the cytoplasm and nucleus.Co-silencing IP-L and NbAS-B accelerated TMV-GFP infection,suggesting that NbAS-B plays a major role in its interaction with IP-L on TMV infection.Further,we found that IP-L can stabilize and promote the expression of NbAS-B,and NbAS-B has the asparagine synthetase activity and can induce the biological synthesis of asparagine（ASN）.ASN treatment could significantly inhibit TMV-GFP infection.Afterwards,q PCR was used to detect the expression of salicylic acid（SA）-,jasmonic acid（JA）-related genes after NbAS-B silencing and external application of ASN,the results showed that silencing NbAS-B significantly inhibited the expression of SA pathway-related genes PR1 and PR2,while external application of ASN significantly enhanced the expression of PR1 and PR2,and external application of Methyl salicylate（Me SA）up-regulated the expression of NbAS-B.However,its antiviral function was lost after exogenous ASN in the SA deletion mutant Nah G,suggesting that NbAS-B-ASN participates in the host resistance response by inducing SA signalling pathway.This section reveals that after the interaction between IP-L and NbAS-B,IP-L stabilizes and promotes the expression of NbAS-B,thereby affecting its asparagine synthetase function,regulating asparagine biosynthesis,and mediating SA signalling pathway to involve in host antiviral defense.Based on previous studies finding that IP-L interacts with NbCML30 in the cytoplasm and nucleus,we first re-verified the interaction by Co-IP,and cauliflower mosaic virus（Ca MV）35S promoter-driven NbCML30 overexpression transgenic plants showed increased resistance to TMV-GFP infection.However,yeast two hybrid（Y2H）showed that NbCML30 did not interact with TMV CP and MP.Therefore,we speculated that IP-L may act as a bridge between TMV CP and NbCML30.Therefore,we first simultaneously silenced IP-L and NbCML30 using tobacco rattle virus（TRV）-mediated co-silencing technology,and found that TMV-GFP infection was attenuated after co-silencing IP-L and NbCML30,indicating that IP-L played a leading role.However,NbCML30 overexpression lost its antiviral function after external treatment of the calcium inhibitor Lanthanum chloride（La Cl₃）,indicating that the disease resistance function of NbCML30 is calcium-dependent.Calcium signals can promote the interaction between IP-L and NbCML30,but IP-L and NbCML30 inhibit each other,and the IP-L can reduce the NbCML30 expression by 26S protease.Futhermore,we detected the expression of oxidative stress pathway-related genes AOX,AAO,GST and PARA after NbCML30 overexpression,and the results showed that NbCML30 overexpression significantly promoted the above genes,and DAB staining showed that NbCML30overexpression significantly enhanced reactive oxygen species（ROS）burst,and significantly increased the activities of antioxidant-related enzymes CAT,POD and SOD,indicating that NbCML30 overexpression significantly enhanced plant oxidative stress pathways.This section revealed a new model for the manipulation of calcium ions by viral proteins:TMV CP,a viral protein without any silencing suppressor function,hijiacks IP-L to inhibit the expression of NbCML30,thereby affecting the calcium-dependent antiviral function of NbCML30,affecting oxidative stress pathway,thus promoting viral infection.RIN4 is a widely studied immune factor.As the only protein identified so far that involved in both PTI and ETI defense,it is recognized and modified by multiple effector proteins,which in turn cause a series of defense responses.Previous studies have shown that IP-L and RIN4 interact in vitro and in vivo.In this section,Co-IP and LCA assays were used to further demonstrate the interaction between IP-L and RIN4,while localization assays showed that RIN4 localized in the cell membrane and co-localizes with IP-L in the cell membrane.Infection with TMV,Tu MV,TRV and PVX activated RIN4 expression to varying degrees,and the expreesion of RIN4 was most significantly increased after TMV infection,but TMV MP and CP did not interact with RIN4.Ca MV35S-driven RIN4-overexpressing transgenic N.benthamiana exhibited promotion of TMV-GFP infection.Co-silencing IP-L and RIN4 showed a stronger function of inhibiting TMV-GFP infection,while co-expression IP-L and RIN4 promoted TMV-GFP infection and supprssed callose deposit and ROS burst,indicating that IP-L and RIN4worked together to affect virus infection.Avr Rpt has no effect on RIN4’s toxic and membrane positioning.We screened the differentially expressed genes of RIN4downstream from the IP-L silenced transcriptome,and verified their expression in IP-L/RIN4 co-silenced plants.The results showed that after co-silencing IP-L and RIN4,the expressions of RPM1,PR1,and RPS2 were significantly increased,while the expression of HSP90 was significantly decreased.RPM1 localized to the cytoplasm and autophagosomes,and TMV infection significantly enhanced its expression,while TRV-mediated silencing of RPM1 promoted TMV-GFP infection,suggesting that RPM1 plays a positive regulatory role in anti-TMV defense.In conclusion,the results in this section demonstrate that RIN4,without interacting with TMV effectors,uses IP-L as a bridge to communicate viral components and immune defense.TMV CP hijacks IP-L to phosphorylate RIN4,thereby inhibiting PTI defense and jointly promoting TMV infection.However,phosphorylated RIN4 is recognized by downstream RPM1 and elicits RPM1-mediated antiviral defense to inhibit further TMV infection.The study revealed that after TMV CP interacts with IP-L,it regulates IP-L and then induces a series of downstream immune defense responses to influence virus infection and activate immune responses.It is vital important to understand the pathogenicity of the virus and resistance of the hosts,as well as the genetics and breeding of antiviral crops to analyze the regulation chain of virus infection based on IP-L.