| Sweet potato latent virus-lotus(SPLV-lotus)was first found on lotus in 2017,which belongs to the genus Potyvirus of the family Potyviridae.SPLV-lotus has significant sequence diversity with SPLV-sweet potato isolates(SPLV-sp).In order to establish a serological detection system for SPLV-lotus,SPLV-lotus coat protein(CP)gene was cloned from infected lotus samples in this study,and SPLV-lotus CP recombinant protein was purified by prokaryotic expression system.New Zealand white rabbits were immunized with purified recombinant protein to produce a specific polyclonal antiserum.Based on the high sensitivity of SPLV-lotus CP protein,two rapid,sensitive and high-throughput serological detection methods,Dot blot and ELISA,were established in this study.The results showed that CP antiserum of SPLV-lotus and that of SPLV-sp had weak cross-reactivity and each had high specificity.CP antiserum of SPLV-lotus could not be used in the field test of sweet potato and CP antiserum of SPLV-sp could not be used in the field test of lotus.RNA silencing is a ubiquitous antiviral defense mechanism in plants.In the long course of evolution,the virus has gradually evolved strategies to resist the silencing of host genes.For example,viruses encoded RNA silencing suppressor(RSS)to block or interfere with the ability of the plant to suppress the antiviral silencing by suppressing the immune response of the host in different pathways of plant gene silencing,so as to establish systemic infection in the host.This study investigated whether HC-Pro is a SPLV-lotus RNA silencing suppressor and a pathogenic determinant.First,this study confirmed HC-ProSPLV-lotus is a RNA silencing suppressor of HC-ProSPLV-lotuss by Agrobacterium tumefaciens-mediated transient expression in wild-type Nicotiana benthamiana and illuminated agroinfiltrated leaves under UV light.The experimental results show that HC-ProSPLV-lotus is a gene silencing suppressor encoded by SPLV-lotus,which can effectively suppress the local silence and system silence caused by ssRNAs,but can not effectively suppress the local silence caused by dsRNAs.PVX-HC-ProSPLV-lotus plasmid was constructed by cloning HC-ProSPLV-lotus gene into heterologous expression vector PVX.Recombinant virus inoculation was performed by agroinfiltration.PVX-HC-ProSPLV-lotus could enhance the pathogenicity of PVX.All these results showed that HC-SPLV-lotus protein act as a pathogenic determinant in virus infection.We firstly found that HC-ProSPLV-lotus protein could localized to both cytoplasm and nucleus and two nuclear localization signals of HC-ProSPLV-lotus were predicted by bioinformatics software.When changing HC-ProSPLV-lotus nuclear localization signal,its RNA silencing inhibitory activity was significantly reduced.At the same time changing HC-ProSPLV-lotus nuclear localization signal will also decrease cause its pathogenicity.In order to explore the role of HC-ProSPLV-lotus in RSS,we preliminarily proved HC ProSPLV-lotus cannot form dimer by itself,and may be formed polymer by yeast two-hybrid system,bimolecular fluorescence complementation and glutaraldehyde cross-linking.It is further proved that HC-ProSPLV-lotus cannot interact with AtAGO1.Therefore,further experiments are needed to clarify the inhibition strategy of HC-ProSPLV-Lotus.In conclusion,this study provides a series of high-throughput,sensitive and rapid diagnostic methods based on serology to detect SPLV-lotus.At the same time,the biological function of HC ProSPLV-lotus preliminarily explored provided theoretical basis for understanding the pathogenicity of SPLV-lotus and effective field control of lotus from the molecular level. |
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