CLONING AND CHARACTERIZATION OF CUCUMBER MOSAIC VIRUS COAT PROTEIN GENE FROM INFECTED BANANA PLANTS IN EGYPT

Cucumber mosaic virus was detected in banana plants showing typical symptoms of CMV infection using DAS-ELISA. Coat protein cDNA was ampli- fied, by RT-PCR using specific primers, and cloned into pGEM®-T Easy Vector, then transformed into E. coli Top 10 cells. The complete sequence of coat protein gene from CMV-Egy strain has been de- termined and compared to other known CMV-CP sequences. The alignment of the amino acid sequences revealed high ho- mology (98-100%) between CMV-Egy and different CMV strains. Phylogenetic analysis also revealed that CMV-Egy is closely related with CMV-Fny isolated from USA and Sny isolated from Israel.

ucumber mosaic virus (CMV) is an important plant virus, affecting hundreds of plant species and causing numerous diseases (Palukaitis et al., 1992).CMV is the type member of the genus Cucumovirus, family Bromoviridae.It is one of the most economically important plant viruses in the world because it causes great losses in vegetables, ornamentals and fruits.The destructive effect is due to its rapid spread by more than 60 aphid species vectors in the field (Edwardson and Christie, 1991).The CMV genome is organized into three single-stranded messenger-sense genomic RNAs (RNAs 1, 2 and 3) and two major subgenomic RNAs (RNAs 4 and 4A, which serve for the expression of the 3′proximal gene of RNAs 3 and 2, respectively).RNAs 1 and 2 codes for components of the replicase complex.RNA 2 codes for the 2b protein, which is expressed from subgenomic RNA 4A and is involved in the suppression of gene silencing.RNA 3 encodes the 3a protein, which is essential for virus movement (Palukaitis and Garcia-Arenal, 2003).The coat protein (CP) is expressed from RNA4 (Habili and Francki, 1974;Palukaitis and Garcia-Arenal, 2003).The CP is required for host range, encapsidation (Suzuki et al., 1991), systemic virus movement (Canto et al., 1997) and aphid transmission (Ng et al., 2000).CMV strains or isolates have been divided into subgroups I and II on the basis of serological data, peptide mapping of the CP, and nucleic acid hybridization (Edwards and Gonsalves, 1983;Owen and Palukaitis, 1988;Wahyuni et al., 1992).The use of test plant species to reliably distinguish CMV strains between subgroups was unsuccessful.Recent phylogenetic analysis of CMV by use of CP ORF and 5 non-translated region (NTR) sequences confirmed the grouping and also led to further subdivision of subgroup I into IA and IB (Roossinck et al., 1999).Also, recombination between subgroups IA and IB was reported (Chen et al., 2007).Banana (Musa spp.) is infected by several viral agents such as Banana bunchy top virus, Banana bract mosaic virus, Banana streak virus.However, cucumber mosaic virus is the most serious viral diseases and has devastating effect on commercial banana plantations (Niblett et al., 1994).In the present study, we report the CP gene sequence-based characterization of CMV infecting banana plants in Egypt and comparing the obtained se-

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quence with other published sequences to get more reliable phylogenetic relationship of the different strains.

Plant Materials
Banana plants (cv.Williams) with typical symptoms (leaf mosaic, yellow stripes in leaves, leaf distortion and stunting of plant) were collected from major banana growing areas of El-Behera governorate, Egypt.

DAS-ELISA
Virus detection in collected tissues was performed directly from infected banana plants without passage in other hosts.The double antibody sandwich-enzyme linked immunosorbant assay (DAS-ELISA), was performed as described by Clark and Adams (1977), using polyclonal antibodies (PAbs) previously raised against CMV particles at the molecular plant pathogenesis laboratory, AGERI, Giza, Egypt.The ELISA values at 405 nm were determined after 20 and 60 minutes post incubation at room temperature.Sample with OD value two folds the negative control is considered as positive (Sutula et al., 1986).

Primer design
The primers for amplification of the coat protein region of CMV were designed using DNA sequences obtained from the National Center for Biotechnology Information (NCBI) database.The se-quences were aligned using MegAlign, an alignment program in DNASTAR Lasergene 7 software (DNASTAR Inc., USA) and the general consensus regions were taken just within the coat protein.
Primers were selected manually and BamH1 site was added at the 5` end of each primer.

RNA extraction and RT-PCR
Extraction of total RNAs was performed using TriPure Isolation Reagent (Roche Diagnostics, USA) according to the manufacturer's instructions.Total RNA was extracted from naturally infected and healthy leaf samples of banana.The amplification of full sequence of CP gene was performed using two specific primers; CMVF BamH1 (5`GGGAATTGGATCCATGGACAAAT CTGAATC`3) and CMVR BamH1 (5` GATTGGATCCCGGAATCAGACTGG GAGCA`3).RT-PCR was carried out using a QIAGEN one step RT-PCR kit (QIAGEN, Germany) according to the manufacturer's instructions.RT-PCR reaction was performed in a reaction volume of 50 µl.The reaction contains 2 µg RNA, 10 µl of 5X RT-PCR buffer, 2 µl of dNTPs mix (10 mM), 2 µl RT-PCR enzyme mix, and 0.6 µM of each primer, then the total reaction volume was completed to 50 µl by RNase free dH 2 O.The reaction was subjected to: one cycle at 50C for 30 min, one cycle at 94C for 15 min; 35 cycles, each consists of: 94C for 1 min, 50C for 45 sec, 72C for 45 sec and the final cycle was extended for 10 min at 72C.Amplified products (10 μl) were separated on 2% (w/v) agarose gel in 1X TAE buffer by electrophoresis at 80V for 2 hrs.Gels were stained in ethidium bromide and photographed on a digital gel documentation system (BioRad, USA).

Cloning and sequencing of PCR amplicon
PCR product was cleaned directly with QIAquick PCR Purification Kit (Qiagen Inc., Germany) and cloned into pGEM®-T Easy Vector System I according to Manufacturer's instructions.After ligation, clones were transformed into competent cells of Escherichia coli Top 10 strain.The clones with recombinant plasmid were identified by blue/white colony screening on LB culture plate and restriction digestion.Plasmids from recombinants were isolated by alkaline lysis (Sambrook et al., 1989).Selected clones were screened by restriction digestion using EcoRI, followed by electrophoresis on 1.5% agarose.One of the positive clones was subjected to sequencing, using universal T7 and SP6 primers.DNA sequence was determined by fluorescent dideoxy chain terminator technology, BigDye Terminator Kit using Applied Biosystem 373A Sequencer (Applied Biosystems Crop., USA).

Sequence analysis and phylogenetic study
Putative CP sequence data were analyzed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and compared with existing sequences of CMV strains available in GenBank data-base.ORF were translated into amino acid residues and aligned with different strains using MegAlign and Expasy tools (www.expasy.org/tools/).Phylogenetic analyses were determined by Neighborhood Joining Bootstrap Method in Clustral X (1.81).

DAS-ELISA
Since banana is an important consumable food crop grown commercially for its high nutritional value, identification of the virus infecting banana became essential so that effective diagnosis/control measures may be developed to minimize the losses and spread of the virus.A set of 18 Plant samples showing CMV like symptoms (Fig. 1), in addition to healthy samples were collected and subjected to virus detection by DAS-ELISA using PAbs specific to the viral particles of CMV.The test revealed three positive samples which represent 16.7%.The mean absorbance values at 405 for negative and positive controls were 0.20 and 2.01, respectively, while for positive samples; the values were 0.70, 0.88 and 0.99.

RT-PCR and cloning of CMV-CP gene
RT-PCR was successful in amplifying CMV-CP gene from one of the positive samples.A PCR product of the expected size (~683 bp) was obtained after amplification, while no amplification was obtained from healthy sample (Fig. 2).The amplified product was purified and cloned by direct ligation into pGEM®-T Easy Vector System I, then transformed into E. coli Top 10 cells.Different white colonies were selected and tested by EcoR1 digestion to release the inserts.Figure (3) shows the released cloned fragments with the same expected size of (~683 bp), which confirming the association of CMV in the infected plants.

Analysis of sequence data
One of the confirmed clones was purified and sequenced using thermal cycle sequencing with dye terminators and ABI automatic sequencer.DNA sequence analysis revealed a fragment contains an uninterrupted open reading frame (ORF) of 657 nucleotides encoding a polypeptide of 219 amino acid residues (Fig. 4).DNA sequence was compared to those in public databases (Altschul et al., 1990;BLAST comparison with GenBank).The BLAST search revealed a high degree of similarity with other published CMV coat protein sequences.Deduced amino acid sequence was aligned with sixteen CMV coat protein sequences using Clustal W method of the MegAlign procedure supplement within the DNASTAR package.The result of alignment is summarized in Table (1) and Fig. ( 5).The alignment revealed that homology for the deduced amino acid sequence ranged between 98.2-100%.The analysis revealed a very high amino acid homology (100%) with the CMV-Fny strain isolated from Cucurbita pepo in USA and 99.5% with both CMV-Sny isolate from Cucurbita pepo in Israel and J isolate from Cucumis sativus L. in Poland.As shown in Fig. ( 6), a dendrogram was constructed using the Neighbour-Joining method.The phylogram illustrates the phylogenetic relationships between our isolate (CMV-Egy) and other published sequences based on the amino acids sequence.Visual comparison of the phylogenic CP tree showed that the CP of the Egyptian strain was closely grouped with CMV-Fny (USA) and CMV-Sny (Israeli) strains.However it showed divergence with other strains.Several reports indicated that CMV-Fny strain is related to subgroup IA (Canto et al., 1997;Verma et al., 2006;Balaji et al., 2008;Hellwald et al., 2001).These results have clearly established the genetic relatedness of CMV-Egy isolate with the members of IA subgroup.The strains of CMV have been divided into two subgroups (I and II).Further splitting of subgroup I into IA and IB has been proposed on the basis of sequence data, analysis of 5'-non-translated region of RNA3 of several strains and phylogenetic analysis of CP (Roossinck, 2002).An interesting finding that despite the different geographical origin of the selected strains, there is less variation in the CP gene of these strains.It has been reported that the CP interacts predominantly with itself or with the viral RNA and has little interaction with the host.CP interactions with the aphid vector are also probably minimal and mostly nonspecific, since more than 85 species of aphids can transmit CMV (Edwardson and Christie, 1991).Under natural conditions, CMV generally is transmitted by aphids, but the virus also has been transmitted by seed in some plant species (Neergard, 1977;Ali and Kobayashi, 2010).These findings indicate that CMV can be transmitted from country to another through seeds.Therefore, this can explain the high similarity among different CMV isolates.High similarities and conserved regions were observed among the CMV subgroup members at CP level; therefore, this homology may be utilized for specific detection of different strains and for developing pathogen-derive resistance (PDR) in susceptible plants.It is clear that antibodiesbased diagnosis will be efficient tools due to the reactivity of recognizing sharing epitopes which are present in both I and II subgroups which are highly conserved among these CMV strains.Therefore, using antisera against CP for the detection of CMV enabled reliable detection of all CMV isolates.Finally as noted above, Data presented here clearly indicate that CP region is sufficient to provide a simple and reliable method for detection and strain identification of banana CMV.

SUMMARY
Cucumber mosaic virus was detected in banana plants showing typical symptoms of CMV infection using DAS-ELISA.Coat protein cDNA was amplified, by RT-PCR using specific primers, and cloned into pGEM®-T Easy Vector, then transformed into E. coli Top 10 cells.The complete sequence of coat protein gene from CMV-Egy strain has been determined and compared to other known CMV-CP sequences.The alignment of the amino acid sequences revealed high homology (98-100%) between CMV-Egy and different CMV strains.Phylogenetic analysis also revealed that CMV-Egy is closely related with CMV-Fny isolated from USA and Sny isolated from Israel.

Fig
Fig. (1): Naturally infected Banana plants showing typical symptoms of CMV.A: healthy plant.B: infected plant with CMV.

Fig
Fig. (6): Phylogenetic tree constructed from the alignment of amino acid sequences of coat protein gene of 17 CMV strains including Egy-CMV using Neighborhood Joining Bootstrap Method.

Table ( 1
): Coat protein gene sequences of various Cucumber mosaic virus strains used for comparison.