Project Title: Molecular methods for virus detection in fruit plants
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Pichia pastoris expression system (Invitrogen)Pichia pastoris maintenancePichia pastoris strains and recombinant clones were streaked onto YPD slopes (1% yeast extract, 2% peptone, 2% dextrose + 2% agar), grown at 30C for 2 days and stored at 4C. Construction of expression constructs and transformation of Pichia pastorisP.pastoris cells (strains GS115 and KM71) were made competent using chemical treatment according to the Invitrogen EasyComp transformation kit instructions. Transformation of the chemically competent P.pastoris cells was also carried out according to the Invitrogen EasyComp transformation kit. A no DNA control and the appropriate plasmid were transformed alongside the Pme I linearised Pichia expression vector DNA and plated onto selective media (YPD agar + 100g/ml Zeocin) to allow the growth of Zeocin resistance P.pastoris transformants. Direct PCR screening of the P.pastoris clones was carried out to check for the presence of the viral coat protein according to Linder et al., 1996. All KM71 recombinant colonies have a MutS phenotype but the GS115 transformants were screened for only the Mut+ phenotype. Expression studiesSmall scale expression of recombinant P.pastoris clones was carried out using buffered media (GMGY & BMMH) according to the EasySelect Pichia Expression kit instructions. The culture conditions used were dependent on the P.pastoris Mut phenotype.Extraction of yeast proteinsTwo methods of protein extraction were investigated. However, the protocol recommended by Invitrogen proved more successful, although more complicated than using the Y-PER reagent (Pierce). Polyacrylamide gel electrophoresisAn equal volume of cell lysate was added to 2x SDS PAGE buffer (186mM Tris, 6% SDS, 30% glycerol, 15% mercaptoethanol, 0.003% bromophenol blue), boiled for 5 min before loading onto two 12.5% polyacrylamide gel. One gel was Western blotted and the other Coomassie blue stained to check the protein extraction method. Western AnalysisSemi-dry western blotting (BioRad) was carried out using a single buffer system, 1X Towbin transfer buffer (25mM Tris, 192mM Glycine, 20% methanol). The membrane was probed with commercially bought antisera according to the manufacturers instructions, using NBT/BCIP to detect the presence alkaline phosphatase. E.coli expression system (Qiagen)E.coli maintenanceE.coli host strains, M15 and SG13009, containing the repressor plasmid pREP4 were maintained on LB agar (10g/litre tryptone, 5g/litre yeast extract , 10g/litre NaCl and 15g/litre agar) containing kanamycin. Recombinant clones, E.coli containing both the expression (pQE) and the repressor (pREP4) plasmids, were maintained on LB agar containing 25g/ml Kanamycin and 100g/ml ampicillin. ASPV-expression vector constructs The ASPV coat protein gene was re-amplified from the yeast expression vector using primers (SP70F & R; SP40F & R) designed for direct cloning into the QIAexpress vectors pQE40 and pQE70 (Qiagen). Expression studiesSmall-scale expressions of recombinant clones were carried out following growth on LB medium and its modifications induced with 1mM IPTG over a 4-hour period. No striking differences between the level of expression in different media or E.coli host strain were observedLarge-scale expression studies were carried out using LB medium plus antibiotics with the ASPV-pQE70 expression construct in the E.coli strain SG13009. Cell lysates were prepared from 200ml of culture harvested (4000 xg for 20 min, 4C) after 4 hours induction with 1mM IPTG. Cell pellets were resuspended in 4ml lysis buffer containing 1mg/ml lysozyme and incubated on ice for 30min. Cells were sonicated for six 10 second bursts with a 10 second cooling period between each burst. The lysate was centrifuged (10,000 xg for 20 min at 4) and the supernatent removed for protein purification.Protein purificationSmall-scale production: Proteins were purified under both denaturing and native conditions using Ni-NTA spin columns (Qiagen). Large scale production: Two methods of protein purification under native conditions were assessed; batch purification under native conditions using Ni-NTA matrix (Qiagen) and HisTrap columns (Pharmacia Biotec). To obtain optimum purity of the ASPV recombinant protein the HisTrap columns were used with a lysis buffer (10mM Imidazole, 20mM phosphate, 0.5M NaCl). The recombinant protein was eluted with 5 ml of elution buffer (300mM Imidazole, 20mM phosphate, 0.5M NaCl). Protein Assay Protein assays were carried out using BSA standards following the Microassay procedure (Bio-Rad). Isolation of RNA for Strawberry crinkle virus work Plant material was collected and frozen in liquid nitrogen. RNA was extracted immediately or leaf material was stored at –80 ˚C. RNA was extracted by one of the methods described below or with the commercial RNeasy Plant Mini Kit (Qiagen Ltd., Crawley, West Sussex). Extreme care was taken not to contaminate the samples with ribonucleases (RNases). Gloves were worn throughout the extraction procedure and changed frequently. Glassware and metal spatulas were baked at 180 ˚C for at least 8 hours. Plastic ware was submerged in a 0.1 % diethyl pyrocarbonate (DEPC) solution and autoclaved at 121˚C for 15 min. To all solutions, except those containing Tris, 0.1% DEPC was added, the solution was shaken vigorously and autoclaved at 121˚C for 15 min. Tris solutions were made up from a uncontaminated pot of Tris crystals with DEPC-treated water. Adjustment of the pH was with the aid of pH measuring strips, pH range 4 to10 (Sigma-Aldrich Ltd., Poole, Dorset). Disposable tips, pipettes and centrifuge tubes were considered to be RNase free. Martin methodThis method for the extraction of RNA from plant material was based on Martin and Northcote (1981). Plant material was ground into a fine powder in liquid nitrogen using mortar and pestle. The powdered leaf was transferred to a 50 ml screw cap tube and taken up in twice its volume RNA extraction buffer (50 mM Tris-HCl, pH 9.0; 150 mM LiCl; 5mM EDTA; 5% lauryl sulphate (SDS)). An equal volume of phenol: chloroform: IAA (25: 24: 1) was added and the sample was mixed by vigorous vortexing for at least 3 min. The phases were separated by centrifugation at 5000 g for 10 min. The upper aqueous layer was transferred to a new 50 ml screw cap tube. Phenol: chloroform extraction was repeated once more, followed by a single extraction with an equal volume of chloroform. The aqueous layer was transferred to a 30 ml Corex tube (Sorvall, Newtown, CT, USA). RNA was precipitated with a third volume of 8 M lithium chloride (LiCl) (Sigma) and incubated at –80 ˚C for at least 1 hour. RNA was spun down by centrifugation at 12,000 g at 4 ˚C for 30 min. The pellet was washed twice in 10 ml of 0.15 M NaCl/ 70 % ethanol. The pellet was recovered by centrifugation at 12,000 g at 4 ˚C for 15 min. The pellet was air-dried and re-suspended in an appropriate amount of nuclease-free water (Sigma). The quantity and purity of the isolated RNA was determined by absorbence at 320 to 240 nm. Covey methodRNA was extracted from P. pubescens according to the method described by Covey and Hull (1981) with the following alterations: RNA and DNA fractions were not separated but total nucleic acid was precipitated with 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of ethanol and incubated at –80 C for 1 hour. Nucleic acid was collected by centrifugation at 12,000 g at 4 ˚C for 30 min. The pellet was re-suspended in 0.5 ml DNase buffer (50 mM Tris-HCl, pH 7.9, 5mM MgCl2) and incubated with 16.0 g of RNase-free DNase (Sigma) for 15 min at 37 C. Enzymes were removed by phenol: chloroform extraction and RNA was precipitated by addition of LiCl to a final concentration of 3M. The reaction was incubated at – 80 ˚C for at least 30 min, then centrifuged at 10,000 g for 20 min. The pellet was washed in 70 % ethanol, air dried and re-suspended in an appropriate volume of nuclease-free water. MacKenzie method RNA was extracted from tissue high in phenolic components and polysaccharides by an adaptation of the method described by MacKenzie et al. (1997). The homogenisation with 20% sarcosyl was omitted. After the RNA was eluted from the columns, it was precipitated with 0.4 volume of 8M LiCl (Sigma). RNA was recovered by centrifugation at 10,000 g for 20 min. The resulting pellet was washed in 70% ethanol and air-dried. The RNA was re-suspended in 25 μl of nuclease-free water (Sigma) and either stored at –80 ºC or used directly in downstream applications. Isolation of mRNA from total RNAPoly-adenylated messenger RNA was isolated from total RNA using Dynabeads Oligo(dT) (Dynal, Oslo, Norway) according to the manufacturer’s instructions. Messenger RNA was either eluted from the beads, for library construction and hybridisation techniques, or beads were re-suspended in 50 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8.0) for RT-PCR and related techniques. Methods for obtaining Strawberry crinkle virus L gene sequence Comparison of rhabdovirus sequences, design of degenerate primers and RT-PCRDegenerate primers were designed to the conserved regions in rhabdovirus large protein gene. Conserved regions were identified through multiple sequence alignment of the amino acid sequences of the large proteins of seven different rhabdoviruses, in four genera, that infect mammals, fish, insects and plants. These were: Rabies virus (RV) (Acc. no. AB009663), Vesicular stomatitis virus (VSV) (Acc. no. J02428), Snakehead rhabdovirus (SHV) (Acc. no. AF147498), Infectious haematopoietic necrosis virus (IHNV) (Acc. no. L40883), Viral haemorrhagic septicaemia virus (VHSV) (Acc. no. Z93414), Sonchus yellow net virus (SYNV) (Acc. no. L32603) and Rice transitory yellows virus (RTYV) (Acc. no. AB011257). Multiple sequence alignments were carried out by the Clustal Method using the MegAlign programme of the DNAStar software (Lasergene). Degenerate primers were designed to positive-sense RNA (mRNA or positive-sense genomic intermediate) and primer sequences are given in Table 4. Table 4. Primers for amplification of Strawberry crinkle virus
RT-PCR with degenerate primers was carried out as follows: 1 μg of total RNA, extracted from healthy or SCV-infected P. pubescens leaves, was mixed with 20 pmoles of degenerate reverse primer, LPROT3 or LPROT5, and 10 nmoles of each dNTP (Promega). The mixture was heated at 65 ºC for 5 min, then chilled on ice. The cDNA synthesis reaction was carried out in a total volume of 20 μl in 1 x M-MLV buffer (Promega) using 200 U of M-MLV reverse transcriptase (Promega) and 40 U of RNase Inhibitor (LifeTechnologies). The reaction was incubated at 37 ºC for 60 min, followed by 15 min at 70 ˚C to inactivate the reverse transcriptase. The amplification reaction was carried out with 5 μl of the cDNA in a total volume of 50 μl. The reaction mixture contained 1 x PCR buffer (Advanced Biotechnology), 75 nmoles of MgCl2, 20 pmoles forward primer LPROT 1 (Table 4), 20 pmoles reverse primer LPROT 3 or LPROT 5, 10 nmoles of each dNTP and 1 U of Red Hot Taq polymerase (Advanced Biotechnology). The PCR was carried out in a Techne Genius thermal cycler and an initial incubation at 94 ºC for 2 min was followed by 5 cycles at 94 ºC for 30 sec, 37 ºC for 30 sec, 72 ºC for 2 min, 35 cycles at 94 ºC for 30 sec, 50 ºC for 30 sec, 72 ºC for 2 min and a final incubation at 72 ºC for 10 min. Rapid amplification of cDNA ends to extend the L gene sequence Rapid amplification of cDNA ends (RACE) was used to extend the known sequence of the SCV large protein mRNA towards the 3’ end. For 3’ RACE, first strand cDNA was synthesised from mRNA extracted from SCV-infected P. pubescens leaves using an oligo (dT)-adapter primer, the 3’ RACE synthesis primer (see Table 4). The reaction mixture contained 0.5 μg mRNA, 1 x First Strand Buffer (LifeTechnologies), 200 nmoles of DTT, 20 pmoles of primer, 10 nmoles of each dNTP, 40 U of RNase Inhibitor (Helena Bioscience) and 200 U of SuperScript II (LifeTechnologies). The reaction was incubated at 45 ºC for 50 min, then at 75 ºC for 15 min. For PCR amplification, 5 μl of the first strand cDNA was used in total reaction volume of 50 μl. The reaction mixture contained 10 nmoles of each dATP, dCTP, dGTP and dTTP, 20 pmoles of gene specific primer, LSP1 (Table 4), 20 pmoles of adapter primer (Table 4), 0.4 x buffer A (LifeTechnologies), 0.6 x buffer B (Life Technologies), 1 x eLONGase polymerase mix (LifeTechnologies). The PCR was carried out in a Techne Genius thermal cycler, using an initial incubation at 94 ºC for 1 min, followed by 35 cycles at 94 ºC for 30 sec, 60 ºC for 30 sec, 68 ºC for 4 ½ min and a final incubation at 72 ºC for 10 min. A second, hemi-nested PCR was carried out with 1 μl of 10 x diluted PCR product in the same manner as the first PCR. The primers used for the hemi-nested PCR were the adapter primer and a second gene specific primer, LSP2 (Table 4). RT-PCR for SCV detection in extracts from Physalis and strawberry SCV mRNA and genomic viral RNA were detected by two-step RT-PCR or nested RT-PCR as described below. For the two-step RT-PCR, the cDNA synthesis mixture contained 100 ng RNA, extracted from healthy or infected strawberry or P. pubescens, 1 x M-MLV buffer (Promega), 10 nmoles of each dNTP, 20 pmoles of reverse primer, SCD1RV or SCD5RV (see Table 4), 40 U of RNase Inhibitor (LifeTechnologies) and 200 U of M-MLV-RT (Promega). The first strand cDNA synthesis reaction was incubated at 37 ºC for 60 min. The PCR reaction mixture contained 5 μl of first strand cDNA, 1 x PCR buffer (Sigma), 10 nmoles of each dNTP (Promega), 20 pmoles of each forward (SCD1FW) and reverse primer (SCD1RV or SCD5RV), 2.5 U of Taq polymerase (Sigma) in a total volume of 50 μl. Amplification was carried out in a thermal cycler (Techne Genius) with and initial incubation at 94 ºC for 2 min, followed by 35 cycles at 94 ºC for 30 sec, 54 ºC for 30 sec, 72 ºC for 1 min and terminated with an incubation at 72 ºC for 10 min. For the detection of the viral negative sense genome, the forward primer was used to synthesise first strand cDNA. The conditions for the first strand cDNA synthesis and the PCR amplification were the same as for the amplification from positive-sense virus RNA. Nested RT-PCR was carried out on RNA extracted from infected strawberry plants. First strand cDNA was synthesised as described above, from the SCD5RV primer. The first amplification step was carried out with primers SCD1FW and SCD5RV. The PCR amplification was carried out in a thermal cycler using an initial incubation at 94 ˚C for 2 min, followed by 30 cycles at 94 ˚C for 30 sec, 54 ˚C for 30 sec, 72 ˚C for 1 min and terminated by 72 ˚C for 10 min. Nested PCR was carried out using the primer combinations SCD2FW + SCD1RV. Nested PCR used 1.0 μl of the first amplification reaction in a total volume of 50 μl. The PCR conditions were the same as for the first amplification reaction.
SCD1FW SCD1RV 574 SCD1FW SCD5RV 1050 SCD2FW SCD1RV 574 The quality of the RNA extraction from strawberry was tested by RT-PCR using primers designed to the strawberry alcohol dehydrogenase gene. First strand cDNA synthesis was carried out as described above, using primer FvAdRV (Table 4). Amplification by PCR was carried out as described for RT-PCR from total RNA. Single-tube RT-PCR was carried out with total RNA extracted from healthy and infected P. pubescens and strawberry leaves. RNA (100 ng) was combined with 20 pmoles of forward primer, 20 pmoles of reverse primer and 20 nmoles of each dNTP (Promega) and heated at 65 ˚C for 10 min, then chilled on ice. To this mixture 5.0 μl 10 x PCR-buffer (Amersham), 1.5 μl 50 mM MgCl2, 40 U of RNasin RNase inhibitor (LifeTechnologies), 200 U M-MLV reverse transcriptase, 0.5 U of Taq polymerase (Amersham) and water to a total volume of 50 μl were added. Amplification was carried out in a thermal cycler (Techne Genius) using an incubation at 37 ˚C for 60 min (reverse-transcription), 70 ˚C for 5 min, 94˚C for 2 min followed by 30 cycles at 94 ˚C for 30 sec, 54 ˚C for 30 sec, 72 ˚C for 2 min, and a final incubation at 72 ˚C for 10 min. For the detection of SCV from RNA extracted from strawberry plants, a second, nested PCR was carried out in the same manner as described for nested PCR. Analysis of amplified product Aliquots (8 μl) of the RT-PCR or nested RT-PCR products were analysed by electrophoresis on a 1.5% agarose gel in 0.5 x TBE buffer (45 mM Tris-borate, 1 mM EDTA), containing 0.5 g/ml ethidium bromide at 150 V for 45 min. The amplified DNA fragments were visualised using an UV transilluminator and photographed with a GelDoc 1000 imaging system (BioRad). Sizes of the PCR products were determined by comparison with 1Kb DNA molecular weight marker (LifeTechnologies) Cloning and sequencing amplified products PCR products were cloned into the pTOPO vector (Invitrogen) according to the manufacturer’s instructions. Clones were tested for inserts by blue/white colony screening and colony PCR. Plasmid inserts were sequenced by SequiServe (Germany). Sequences were analysed through similarity search by basic local alignment search tool (BLAST) (Altschul et al., 1997) and multiple alignments with other rhabdovirus proteins by the Clustal Method using DNAStar software (Lasergene). RESULTS Objective 1 – Devise PCR-based assay for Apple stem pitting virus (ASPV) Designing and testing primers for a PCR-based assay for ASPV Apple stem pitting virus infection was confirmed by ISEM in bark extracts from 25 apple trees and three pears, affected by ASPV or related diseases, in December 1998. The effectiveness of an IC-RT-PCR test for ASPV from bark samples of these trees (protocol and primers 1 & 3, Schwartz and Jelkmann, 1997) was assessed. ASPV was detected in only 23 of these trees (21 apples, two pears) by PCR using these primers (ASPV1 and 3) and there were questionable product bands in the two replicates of another sample. A new primer (ASPV5) was therefore designed to a well-conserved region upstream of primer 3, using the sequence information of Schwartz and Jelkmann (1997), and IC-RT-PCR was repeated in February 1999. These tests included samples from an extra seven pear trees. ASPV was confirmed by ISEM in these trees, in all the apples and in one of the two pears tested previously. Using primers ASPV1 and ASPV5, clear product bands were visible with the sample that gave a doubtful result with primers ASPV1 and ASPV3 in December. A further six samples that could not be amplified by those primers were successfully amplified by the new primer pair. However, samples from two apples and seven pear trees could not be amplified by either primer pair, including the stony pit-infected pear tree 7/40. Tests carried out in May (using leaf material) showed little difference in the ability of the 2 primer pairs to amplify these ASPV isolates (Table 6) A number of isolates that were not or poorly detected by the primers ASPV1 and ASPV3 have been sequenced. Although there are sequence differences in these ASPV isolates, the region to which primer ASPV3 was designed is no more varied than ASPV1 and does not really explain why the ASPV5 and ASPV1 combination is sometimes more efficient at amplifying ASPV.
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