Serological Detection of Tomato Pepino mosaic virus in Morocco

Pepino mosaic virus （PepMV）, monopartite RNA virus, 6,500 pb, belonging to Flexiviridae and Potexvirus group, is highly infectious and easily transmissible. Its economic impact is major for the tomato producer＇s countries. Prevention, based on early virus detection is the only effective control measure. Monoclonal antibodies appeared to be very useful tool. The authors used for the production of monoclonal antibodies hybridomas technique, by fusing spleen cells of immunized BALB/c mice to PepMV and SP2/O cancerous cells. The aim of this work is to produce hybridomas producers of Mab that could be used for ELISA in Morocco. At the same time, these efforts will serve to decrease expenses of producers concerning phytosanitory control. We obtained 16 hybridomas lines producers of Mab specific for PepMV. They were tested for efficiencies in ELISA and two lines were retained for production of Mab on large scale （1B 11-G 10 and 5A l-G5）. Isotyping of these two lines showed that they are belonging to IgG 1 class and easily purified by affinity chromatography in agarose column by protein A. The conjugation of these two antibodies to alkaline phosphatase has been verified by DAS-ELISA. These antibodies will enable to diagnose the disease from infected tomato plants, integrating several serological tests to control it and target the actions of struggles.

Pepino mosaic virus antagonizes plant m^(6)A modification by promoting the autophagic degradation of the m^(6)A writer HAKAI

Autophagy plays an active anti-viral role in plants.Increasing evidence suggests that viruses can inhibit or manipulate autophagy,thereby winning the arms race between plants and viruses.Here,we demonstrate that overexpression of an m^(6)A writer from Solanum lycopersicum,SlHAKAI,could negatively regulate pepino mosaic virus(PepMV)infection,inhibit viral RNA and protein accumulations by affecting viral m^(6)A levels in tomato plants and vice versa.The PepMV-encoded RNA-dependent RNA polymerase(RdRP)directly interacts with SlHAKAI and reduces its protein accumulation.The RdRP-mediated decreased protein accumulation of SlHAKAI is sensitive to the autophagy inhibitor 3-methyladenine and is compromised by knocking down a core autophagy gene.Furthermore,PepMV RdRP could interact with an essential autophagy-related protein,SlBeclin1.RdRP,SlHAKAI,and SlBeclin1 interaction complexes form bright granules in the cytoplasm.Silencing of Beclin1 in Nicotiana benthamiana plants abolishes the RdRP-mediated degradation of SlHAKAI,indicating the requirement of Beclin1 in this process.This study uncovers that the PepMV RdRP exploits the autophagy pathway by interacting with SlBeclin1 to promote the autophagic degradation of the SlHAKAI protein,thereby inhibiting the m^(6)A modification-mediated plant defense responses.

Highly sensitive serological approaches for Pepino mosaic virus detection

Pepino mosaic virus(PepMV)causes severe disease in tomato and other Solanaceous crops around globe.To effectively study and manage this viral disease,researchers need new,sensitive,and high-throughput approaches for viral detection.In this study,we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies(mAbs).A panel of highly specific and sensitive murine mAbs(15B2,8H6,23D11,20D9,3A6,and 8E3)could be produced through cell fusion,antibody selection,and cell cloning.Using the mAbs as the detection antibodies,we established double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA),Dot-ELISA,and Tissue print-ELISA for detecting PepMV infection in tomato plants.Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1310720 and 1:20480(weight/volume ratio(w/v),g/mL),respectively.Among the three methods developed,the Tissue print-ELISA was found to be the most practical detection technique.Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction(RT-PCR)and DNA sequencing,dem on strati ng all three serological methods are reliable and effective for monitoring PepMV.An ti-PepMV mAbs and the newly developed DAS-ELISA,Dot-ELISA,and Tissue print-ELISA can benefit PepMV detection and field epidemiological study,and management of this viral disease,which is already widespread in tomato plants in Yunnan Province of China.

m^(6)A modification of plant virus enables host recognition by NMD factors in plants

N^(6)-methyladenosine(m^(6)A)is the most abundant eukaryotic mRNA modification and is involved in various biological processes.Increasing evidence has implicated that m^(6)Amodification is an important anti-viral defense mechanism in mammals and plants,but it is largely unknown how m^(6)Aregulates viral infection in plants.Here we report the dynamic changes and functional anatomy of m^(6)Ain Nicotiana benthamiana and Solanum lycopersicum during Pepino mosaic virus(PepMV)infection.m^(6)Amodification in the PepMV RNA genome is conserved in these two species.Overexpression of the m^(6)Awriters,mRNA adenosine methylase A(MTA),and HAKAI inhibit the PepMV RNA accumulation accompanied by increased viral m^(6)Amodifications,whereas deficiency of these writers decreases the viral RNA m^(6)Alevels but enhances virus infection.Further study reveals that the cytoplasmic YTH-domain family protein NbECT2A/2B/2C as m^(6)Areaders are involved in anti-viral immunity.Protein-protein interactions indicate that NbECT2A/2B/2C interact with nonsense-mediated mRNA decay(NMD)-related proteins,including NbUPF3 and NbSMG7,but not with NbUPF1.m^(6)Amodification-mediated restriction to PepMV infection is dependent on NMD-related factors.These findings provide new insights into the functionality of m^(6)Aanti-viral activity and reveal a distinct immune response that NMD factors recognize the m^(6)Areaders-viral m^(6)ARNA complex for viral RNA degradation to limit virus infection in plants.