In-vitro Analysis of Chenopodium murale extract for resistance against potato virus Y

Potato (Solanum tuberosum) is one of the largely consumed staple food crop of the world, including Pakistan. In Pakistan, potato shares a large percentage in country’s agriculture GDP. In Pakistan majority of good quality virus free seed potato is imported by multinational and local seed companies. This costs a huge burden on country’s economy. Potato Virus Y (PVY) belongs to Potyviridae family, severely damaging leaves of the Solanaceae family including potato. Plant lectins are the potential proteins that specifically bound and cross linked with carbohydrates and target glycans present on the cell surface of viruses through a pathway termed as lectin activation. The present in-vitro study shows the effect of lectin extracted from the seeds of Chenopodium murale against the downregulation of the CP (coat protein) gene of PVY. Stable interaction was found between PVY coat protein and CML insilico. Full length CP gene from PVY virus was amplified and cloned in mammalian expression vector. CML protein was purified to sub fractions through anion exchange chromatography on SP-sepharose gel. The non-cytotoxic effect and safe limit of CML extract was indicated by CC50 value. The hemagglutination activity of CML against the rabbit erythrocytes was revealed to be 35 μg/ml. The real-time PCR revealed the anti-PVY activity of CML extract. CML extract in concentration of 30, 60 and 90 μg/ml was found effective against PVY CP gene in comparison with control.


Introduction
Lectins belong to the class of sugar binding family of non-immune origin.They can recognize and reversibly bind to glycans of glycoconjugate moieties without transforming their covalent structure [1].This binding is highly precise without needing the enzymatic activity.This property of lectins allows them to act as recognition molecules between organisms, cells and even within cells making them a significant tool in biomedical research [2].Naturally, lectins are distributed among huge number of organisms belonging to invertebrates, vertebrates, plants, fungi, sea corals and algae.Lectins are accountable of carrying out numerous biological processes including host-pathogen interactions, cell-cell communication, cell targeting, apoptosis induction, tumor differentiation and growth.In addition to these, they have also been known for their distinctive antiviral, antibacterial and antifungal activities.They can also inhibit virus replication by interacting with glycoprotein envelope of certain viruses [3] Plant lectins are seen to be involved in the identification of pathogenic microorganisms.High affinity is observed in lectins, isolated from soybean, for β-glucan binding which is a powerful PAMP (

In vitro Analysis Chenopodium murale Lectin
Chenopodium murale (L.) seeds were collected from the Botanical garden, University of the Punjab after the confirmation through taxonomic identification by Dr Zahoor Ahmad Sajid (Assistant Prof.).The seeds were grinded into the fine powder in the pestle mortar and dissolved into the 0.1M phosphate buffer (pH 7.8) with respect to 1:5 (w/v).Mixture was stirred at 4˚C for 2 hours and was passed through the cheese cloth to remove the seed debris and then centrifuged at 11000 × g at 4˚C for 30min.Supernatant was collected and subjected to 60% ammonium sulfate precipitation.The pellet was re-dissolved in 20 mM of phosphate buffer (pH7.8) and dialyzed through dialyzing tube (Spectra/Por RC Biotech membrane, 6-8 k MWCO, 32 mm flat width, 100 ft) against 10mM phosphate buffer of ph7.8 for overnight at 4˚C.The dialyzed protein was stored at 4˚C for SDS PAGE analysis and quantitation.Anion exchange chromatography of the dialyzed protein was performed using Q sepharose (GE Healthcare) column.Column was equilibrated with 5 cv of 50 mM phosphate buffer (pH 7.8) and then partially purified protein was loaded onto column.Protein was eluted using the 0.5 M NaCl gradient and different fractions were obtained and analyzed on SDS-PAGE.The concentration of the purified protein was quantified through Bradford method [14] and bovine serum albumin was used as a standard.

Hemagglutinating activity
The hemagglutinating activity of CML (C.murale Lectin) was assessed in U-shaped 96 wells microtiter plate against rabbit erythrocytes as illustrated by [15].For this, two-fold serial dilutions were prepared of CML in PBS (phosphate buffer saline) and an equal volume of 2% erythrocyte suspension was added to it.Control was prepared by adding PBS to erythrocyte suspension.The mixtures were incubated for 1 hour at room temperature and results were noticed.
Positive assay shows a red-carpet layer whereas a red button formation is seen in negative assay.Total RNA isolation from PVY infected potato plants PVY infected potato plants were surveyed in various regions of Punjab for sample collection.From the collected viral infected plant samples, leaves were used for the isolation of total RNA by suing TRIzol reagent method with some minor changes [16].cDNA was synthesized through cDNA synthesis kit (Thermo Fisher, Lithuania) and stored at -20ºC.

Construction of expression plasmid
For the construction of a plasmid, CP gene fragment conserved in the PVY o strain was taken for primer designing using the primer3 software (http://frodo.wi.mit.edu/primer3/).Using the full length sequence of CP gene, (Accession # AB295475.1)primers were designed and at 5' end of primers HindIII and EcoRI sites were incorporated (Table 1) so, as to carry out amplification of the full length CP gene.For sequencing, the obtained PCR product was first cloned into pCR2.1-TOPO(Invitrogen, USA) followed by reamplification.DNA sequencing facility of CAMB was used for sequencing of isolated CP gene and the sequence was submitted to NCBI (Accession # MK130988).After gel purification of the amplified fragment, it was later inserted into the mammalian expression vector pcDNA 3.1 (+) to attain pcDNA-CP clone.

Table 1. Sequences of primers designed against coat protein gene of PVY used in the study
Cytotoxicity assay MTT (3-(4,5-dimethylthiazol-2-yl) -2,5diphenyl tetrazolium bromide) assay kit (Millipore, USA) was used for assessing proliferation and cell viability.A 100ul (1×10 5 ) of Huh-7 cells were cultured in 96 wells microtiter plate using DMEM with added penicillin (100 IU/ml), streptomycin (100ug/ml) and 10% FBS and incubated at 37˚C in CO2 incubator for 24 hrs.Different dilutions were prepared for CML protein, added to microtiter plate and incubated for 24 hrs. in CO2 incubator at 37˚C.Each dilution was then analyzed in three replications.Media was decanted after 24 hours and freshly prepared media (100ul) was added, supplemented with 10ul of MTT solution (5mg/ml in PBS), as per manufacturer's guidelines.The plate was then incubated again at 37˚C for 4 hours in CO2 incubator.Formazan crystals were seen in the plate wells after 24 hours which were dissolved by addition of 0.1ml DMSO.Optical density of formazan product was assessed through ELISA (Enzyme Linked Immunosorbent Assay) plate reader at 570nm as test wavelength and 620nm as reference wavelength.Following formula was used to calculate cell viability [1]: x 100 Transfection HepG2 cells were cultured at 1×10 6 cells per well in 6 wells plate.Cells were grown overnight before transfection.Transfection of the cells was carried out at 50%-70% confluence holding medium of 0.5 ml per well.Cells were co-transfected with the isolated CVL, together with pcDNA-CP construct.Transfection was carried out using a transfection reagent, lipofectamine 2000 (Invitrogen, CA).For the downregulation of mRNA of pcDNA-CP, 50ng-1µg of pcDNA-CP was used whereas three different protein concentrations of CML were tested 25, 50 and 75µg/ml.Experiments were carried out in triplicate.

Real-time PCR analysis
To study real-time PCR analysis of pcDNA-CP, mRNA expression was measured using RT-PCR.TRIzol reagent (Invitrogen) was used to isolate total RNA form HepG2 cells and cDNA synthesis was performed from 1µg isolated RNA using cDNA synthesis kit (Thermo Fisher, Lithuania).Primers were chosen which could amplify a fragment size of 172 bp from full length 810 bp gene segment of pcDNA-CP at optimized temperature.Agarose gel (2%) was performed to check the absence of nonspecific bands through Tm optimization.PikoReal TM Real time PCR system was used to conduct RT-PCR utilizing SYBR Green Mix (Thermo Fisher, Lithuania).cDNA was used to study the mRNA expression of CML treated pcDNA-CP.Using β-actin as control 30 cycles of RT-PCR were run.Cq values of different samples and calculated standard deviation was used to perform relative gene expression analysis.Triplicates were performed for each real-time PCR assay and PikoReal software was used to interpret the results.

Hemagglutination activity
After incubation of one hour, hemagglutination activity of CML was noticed against rabbit erythrocytes with minimum concentration of approximately 35ug/ml.Cytotoxicity assay MTT reagent gave purple formazan crystals with living cells due to mitochondrial succinic dehydrogenase.Cell viability can be assessed through absorbance of formazan crystals in visible region [17].Cytotoxicity of CML against HepG2 cells has been depicted in figure 2 and its IC 50 was observed at 360µg/ml.CP gene amplification and pcDNA-CP construct formation CP gene was ligated in pcDNA 3.1(+) vector between HindIII and EcoRI restriction sites as shown in (Figure 3).Isolation of total RNA was done followed by cDNA synthesis and primers of CP gene were designed as table 1 shows.PCR was performed to amplify CP gene from PVY infected potato leaves (Figure 4) and was inserted in pcDNA 3.1 (+) using the HindIII and EcoRI restriction sites (Figure 5).In the present study, a set of primers were used to isolate and amplify CP gene form PVY infected potato plants.A HepG2 cell line was utilized as it gives the best expression of targeted gene in almost 24 hours, additionally; full length CP gene was being downregulated by the CML as the dose dependent manner which clearly showed the CML can be future target for making the PVY resistant potato crop through transformation.

Conclusion
Conclusively, in this study we have reported the activity of lectin derived from C.murale against coat protein of potato virus Y. Invitro the purified fractions of CML exhibited strong antiviral effect against PVY.In Pakistan, this is the first report of use of CML extract to reduce antiviral activity of PVY.In future experiments the transgenic plants having lectin in potato genome could provide complete protection not only from PVY but also to other viruses.

Figure 1 .
Figure 1.Shows the results of SDS PAGE.PageRuler™ Prestained Protein Ladder (Catalog number: 26616) having bands of 180, 130, 100, 70(red), 55, 40, 35, 25, 15 and 10 kDa from top to bottom was used as a standard in well no. 1 while, well no.2 contains 60% ammonium sulfate precipitated crude protein after dialysis, well no.3, 4 and 5 contain the fractions obtained in the anion exchange chromatography.SDS-PAGE shows two subunits of approximately 37 and 18 kDa of the partial purified protein Another study showed transfection of 480bp of CP from PVY into CHO cells and siRNA was used to conduct its mRNA knockdown assay [8].Moreover, capsids of plant virus have already been used as vectors for gene delivery and expression analysis in mammals [24].Recent studies show production of a photochemically active product when HEK293T got a successful expression of Arabidopsis cryptochrome [25].CP gene is most significant in PVY studies as it causes several events including uncoating of PVY and viral RNA translation, targeting of replication site and potyvirus transmission by aphids/vectors [26].Various studies provided strong evidence of involvement of CP transgene in inducing resistance in transgenic potato crops [9, 27].