Three sensitive and reliable serological assays for detection of potato virus A in potato plants

Vegetative propagation of seed potato often allows passaging of viruses to seed tubers,resulting in significant yield losses and reduction of potato tuber quality.Thus,virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.Among the reported potato-infecting viruses,potato virus A(PVA)is considered as one of the most important viruses in potato-growing regions worldwide.This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies(MAbs)(2D4,8E11,14A6 and 16H10)using purified PVA virions as an immunogen.Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.Using these four MAbs,this study developed antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA),Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327680(w/v,g m L^(-1))by ACP-ELISA or up to1:10240 by Dot-ELISA.The Tissue print-ELISA is the quickest and easiest approach among the three serological assays,and is more suitable for onsite large-scale potato screening programs.Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.Hence,the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys.

Establishment of Multiplex PCR for Three Virus of Potato

[Objective]The aim was to establish the multiplex PCR method for three virus of potato：PVA（potato virus A）,TMV（Tobacco mosaic virus）and PVY（potato virus Y）.[Method]According to the PVA,TMV and PVY sequences available in GenBank,pairs of primer were designed for establishing a multiplex PCR method,and constructing recombinant plasmid of target genes by PCR amplified of three viruses as reference standard simple to be used in sensitivity test;PVX（Potato virus X）,PVM（Potato virus M）,PVS（Potato virus S）,PVV（Potato virus V）and CMV（Cucumber mosaic virus）were used to carry out the specificity test and detection of 11 samples which were suspected of virus infected.[Result]The detection limit for PVA,TMV and PVY was 14,14 and 14 copies/ml,respectively.No cross-reactivity was observed with other viruses.Seven of 11 samples were infected by three viruses.[Conclusion]The multiplex PCR for PVA,TMV,PVY three viruses of potato was established successfully,which had provided basis for the detection technology of potato virus.

Simulation modelling of potato virus Y spread in relation to initial inoculum and vector activity

Potato virus Y(PVY)is a non-persistent virus that is transmitted by many aphid species and causes significant damage to potato production.We constructed a spatially-explicit model simulating PVY spread in a potato field and used it to investigate possible effects of transmission efficiency,initial inoculum levels,vector behavior,vector abundance,and timing of peak vector activity on PVY incidence at the end of a simulated growing season.Lower PVY incidence in planted seed resulted in lower virus infection at the end of the season.However,when populations of efficient PVY vectors were high,significant PVY spread occurred even when initial virus inoculum was low.Non-colonizing aphids were more important for PVY spread compared to colonizing aphids,particularly at high densities.An early-season peak in the numbers of noncolonizing aphids resulted in the highest number of infected plants in the end of the season,while mid-and late-season peaks caused relatively little virus spread.Our results highlight the importance of integrating different techniques to prevent the number of PVY-infected plants from exceeding economically acceptable levels instead of trying to control aphids within potato fields.Such management plans should be implemented very early in a growing season.

Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line

RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.

Cloning and Sequencing of Kappa Light Chain Gene of a Mouse Monoclonal Antibody Directed Against Potato Virus Y

A cDNA library was constructed in λgt11 vectors, complementary to the mRNA isolated from a mouse hybridoma raised against potato virus Y(PVY). Thirty cDNA clones were selected from the cDNA library by in situ immunohybridization with goat anti-mouse kappa-chain-specific antibody conjugated to alkaline phosphatase, from which one clone, k6, having the largest insert was characterized by sequence analysis. The result shows that the immunoglobulin messenger RNA corresponding to k6 is 956 nucleotides in length excluding the poly(A) region, among which 31 bases code for the 5’ non-coding region, 57 for the leader sequence of the protein, 657 for the mature protein and 211 for the 3’ non-coding region. Comparison of deduced amino acid sequences of the protein and other kappa light chains shows that they share a 100% identity in their constant regions(CL) and 93.7% identity in their variable regions(VL). The kappa light chain encoded by k6 is considered to be specific to PVY since only one type of light chain is expressed in the hybridoma.

Screening and Identification of Tobacco Mutants Resistant to Potato Virus Y( PVY)

[ Objective] This study aimed to screen and identify tobacco mutants resistant to potato virus Y （PVY）, thus laying the foundation for obtaining PVY resistance genes. [ Method ] At seedling stage, tobacco mutant materials were inoculated with PVY virus and preliminarily screened by naked-eye observation. Enzyme-linked immunosorbent assay （ELISA）, real-time fluorescence quantitative PCR and test strip assay were performed to further identify the pre-screened PVY- resistant seedlings. [ Result ] In 2011, two highly PVY-resistant tobacco mutants （MZE2-15 and MZE2-16） and three PVY-tolerant mutants （MZE2-70, MZE2- 207 and MZE2-228） were obtained, which were further screened and identified in 2012. According to the results, tobacco mutant materials MZE2-407 and MZE2- 428 were susceptible to PVY; mutant materials MZE2-16 and MZE2-15 were resistant to PVY. [ Conclusion] This study provide theoretical basis for the control of tobacco PVY disease.

Effects of Synergism Between PVY and PVX on Virus Titer and Cell Ultrastructure in Tobacco Plants

The viruses titer and the ultrastructure of infected cells in tobacco host (Nicotiana tabacum cv. Samsun), which doubly infected with potato virus Y necrosis strain (PVYN) and potato virus X (PVX), were studied under greenhouse conditions. The results indicated that PVYN and PVX interacted synergistically. and tobacco plants which doubly infected with PVX and PVYN could greatly increase symptom severity as compared with that induced by the single virus. As determined by triple antibody sandwich enzyme-linked immunosor-bent assay (TAS-ELISA), the titer of PVX in the tobacco leaves infected with both PVYN and PVX was up to 9.10 times higher than the plants infected with PVX only. No significant differences in PVYN titer were detected between singly and doubly infected plants. The enhancement of PVX titer in doubly infected plants was evident in greenhouse and was independent of the virus strains, tested seasons, intervals between PVYN and PVX inoculation. When sections of doubly infected leaves were examined with an electron microscope, it could be frequently found that cells contained pinwheels and large masses of PVX-like particles, pinwheels and laminate inclusions, or all three structures, most of them were swollen, and their chloroplast and other organella were destructed heavily. This suggested that doubly infected cells were involved in the enhancement phenomenon, which seemed to be the result of an increase in the amount of PVX synthesized in them.

Effect of SPVD on Sweet Potato Yield Formation

This study was to investigate the influence of SPVD on the growth devel- opment and yield formation of sweet potato, The virus seeding, landrace, virus-free seedlings of high starch sweet potato XichengO07 were inoculated with SPVD for revealing the interaction mechanism, The results showed that SPVD could result in clustering and dwarfing of sweet potato plant type, smaller leaves and lower effec- tive of leaf area, reduced chlorophyll content, and smaller source, lowered assimila- tive ability and photosynthetic capacity. The flow became smaller, further finally led to the reduced biological yield, and the desorption of SPVD could increase leaf ＂source＂ and the chlorophyll content, improve photosynthetic and flow ability, thus raising the output of production. SPVD could reduce the activities of SOD, POD and CAT in sweet potato plant, increase the content of MDA, decrease antioxidant activity and production, damage the cell membrane. However, virus-free treatment could increase the activities of SOD, POD and CAT in plants, which was helpful to reduce the harm of MDA. After the desorption of SPVD, the production of above- ground was increased by 3.4% and the production of fresh sweet potato was up by 2.9%, while SPVD dissemination could result in the reduction of the aboveground and fresh tubers by 69.9% and 49.1%, respectively.

Activation of MAPK-mediated immunity by phosphatidic acid in response to positive-strand RNA viruses

Increasing evidence suggests that mitogen-activated protein kinase(MAPK)cascades play a crucial role in plant defense against viruses.However,the mechanisms that underlie the activation of MAPK cascades in response to viral infection remain unclear.In this study,we discovered that phosphatidic acid(PA)repre-sents a major class of lipids that respond to Potato virus Y(PVY)at an early stage of infection.We identified NbPLDa1(Nicotiana benthamiana phospholipase Da1)as the key enzyme responsible for increased PA levels during PVY infection and found that it plays an antiviral role.6K2 of PVY interacts with NbPLDa1,lead-ing to elevated PA levels.In addition,NbPLDa1 and PA are recruited by 6K2 to membrane-bound viral repli-cation complexes.On the other hand,6K2 also induces activation of the MAPK pathway,dependent on its interaction with NbPLDa1 and the derived PA.PA binds to WIPK/SIPK/NTF4,prompting their phosphoryla-tion of WRKY8.Notably,spraying with exogenous PA is sufficient to activate the MAPK pathway.Knock-down of the MEK2-WIPK/SIPK-WRKY8 cascade resulted in enhanced accumulation of PVY genomic RNA.6K2 of Turnip mosaic virus and p33 of Tomato bushy stunt virus also interacted with NbPLDa1 and induced the activation of MAPK-mediated immunity.Loss of function of NbPLDa1 inhibited virus-induced activation of MAPK cascades and promoted viral RNA accumulation.Thus,activation of MAPK-mediated immunity by NbPLDa1-derived PA is a common strategy employed by hosts to counteract positive-strand RNA virus infection.

Transient Expression of BYDV-MP in Nicotiana benthamiana

[Objective]The aim of this study was to identify transient expression of movement protein （MP） gene in Nicotinana benthaminana rapidly and further investigate the function of this exogenous gene. [Method]The movement protein gene of barley yellow dwarf virus （BYDV） was cloned into potato virus X （PVX） viral vector of pGR107,and PVX-recombinant vector was obtained. After electroporation of Agrobacterium tumefaciens,PVX was inoculated into the lower leaves of tobacco by Agrobacterium infiltration assay to observe the infection of virus on tobacco. [Result]After infection for 7 days,upper non-inoculated leaves of tobacco infected by the PVX-recombinant vector showed the virus infection symptoms,while the control group had no viral infection phenomenon. Daily follow-up observations for two groups revealed that tobacco infected by PVX-recombinant vector had severe symptoms of virus infection and curling leaves,or even led to necrosis both in infiltrated and systemic leaves in late period. However,tobacco infected by PVX vector had only slight symptoms of virus infection and could recover from infection. RT-PCR of the infected tobacco indicated that exogenous gene BYDV-MP had a normal transcription and expression in tobacco. [Conclusion]As a determinant factor for viral disease,BYDV-MP promotes the systemic infection rate of PVX and its symptom. In addition,it is feasible to express exogenous MP gene in Nicotiana benthaminan via PVX expression vector.

Monoclonal antibody-based serological assays for detection of Potato virus S in potato plants

Potato virus S （PVS） often causes significant losses in potato production in potato-growing countries. In this study, the ordinary strain of PVS （PVS 0） was purified from PVS-infected potato plants and used as the immunogen to produce hybridomas secreting monoclonal antibodies （MAbs）. Five highly specific and sensitive murine MAbs （1A3, 16C10, 18A9, 20B12, and 22H4） against PVS were prepared using conventional hybridoma technology. Using these MAbs, tissue print-enzyme-linked immunosorbent assay （ELISA）, dot-ELISA, and double-antibody sandwich （DAS）- ELISA were developed for sensitive and specific detection of PVS infection in potato plants. The results of sensitivity assays revealed that PVS could be reliably detected in PVS-infected leaf crude extracts diluted at 1：10240 and 1：163840 （w/v, g/ml）in phosphate buffer saline （PBS） by dot-ELISA and DAS-ELISA, respectively. Twenty-two samples collected from potato fields in Yunnan Province, China were tested for PVS infection using the serological assays we had developed, and 14 of them were found to be positive. This indicates that PVS is now prevalent in potato fields in Yunnan Province.

Evaluation of potential RNA-interference-target genes to control cotton mealybug, Phenacoccus solenopsis （Hemiptera： Pseudococcuidae）

RNA interference （RNAi） of vital insect genes is a potential tool for targeted pest control. However, selection of the right target genes is a challenge because the RNAi efficacy is known to vary among insect species. Cotton mealybug, Phenacoccus solenopsis, is a phloem-feeding economically important crop pest. We evaluated the RNAi of 2 vital genes, Bursicon （PsBur） and V-ATPase （Ps V-ATPase） as potential targets in P. solenopsis for its control. PCR fragments of PsBur and PsV-ATPase were amplified using cDNA synthesized from the total RNA. The PCR amplicons were cloned into Potato virus X （PVX） to develop recombinant PVX for the inoculation ofNicotiana tabacum plants for bioassays with healthy P. solenopsis. Reverse-transcription-polymerase chain reaction （RT- PCR） was used to validate the expression oftransgenes in the recombinant-PVX-inoculated plants （treated）, and suppression of the target genes in the mealybugs exposed to them. The RT-PCR confirmed the expression of transgenes in the treated plants. Mealybug individuals on treated plants either died or showed physical deformities. Further, the population of mealybug was significantly reduced by feeding on N. tabacum expressing RNAi triggers against PsBur and Ps V-A TPase. The results conclude that RNAi is activated in P. solenopsis by feeding on N. tabacum expressing RNAi triggering elements of PsBur and Ps V-ATPase genes through recombinant PVX vector. Further, V-ATPase and Bursicon genes are potential targets for RNAi-mediated control ofP. solenopsis.

A Novel Approach to Functional Analysis of the Ribulose Bisphosphate Carboxylase Small Subunit Gene by Agrobacterium-Mediated Gene Silencing

A novel approach to virus-induced post-transcriptional gene silencing for studying the function of the ribulose bisphosphate carboxylase small subunit （rbcS） gene was established and optimized using potato virus X vector and Nicotiana benthamiana as experimental material. The analysis of silencing phenomena, transcriptional level, protein expression, and pigment measurement showed that the expression of the rbcS endogenous gene was inactivated by the expression of a 500-bp homologous cDNA fragment carried in the virus vector.