Abstract: Potato virus X (PVX) and Potato virus Y (PVY) are two of the three most prevalent viruses that cause significant yield declines in potato. Twenty-seven PVX and thirty-seven PVY accessions were analyzed for nucleotide sequence variation of the coat protein gene. The average and variance of genetic distance for PVX were estimated at 0.118 and 0.004 and 0.118 and 0.005 for PVY using the neighbour joining method. Results of phylogenetic trees and their certification via stepwise discriminant analysis led us to classify of PVX sequences in four groups and PVY sequences in three groups. One purpose of this project was to determine suitable conserved regions to make of gene silencing constructs. Length of identified conserved regions were enough to silence of the virus coat protein genes on infected plants, many of which were located consequently with short gap spacers. In this term, some of groups were divided into subgroups to obtain conserved regions under minimum length of 25 nt, enough length to design specific diagnostic-primers.
INTRODUCTION
Potato (Solanum tuberosum) originated in the highlands of South America, where it has been consumed for more than 8000 years. Potato is very low in fat and it has more protein than maize. At present, potato is the fourth most important food crop in the world, in both total production and area cultivated. Also, potato is a good bioreactor for recombinant protein production (Artsaenko et al., 1998) and is a one of the selected plants for production of the edible vaccine for oral virus immunization (Richter et al., 2000). An estimated 22% of potato yield is lost per year due to diseases and pests (Ross, 1986). Therefore, the need for genetic improvements of potato was recognized as a primary target for plant genetic engineering. As immediate needs, virus and insect resistance were recognized as important and attainable goals. Development of resistance to the potato viruses were selected as priority goals, because these are the most economically important pest and diseases of potato in the around of world (Kaniewski and Thomas, 2004). PVX and PVY are two of the three the most widespread potato viruses with significant yield loss. PVX and PVY are positive sense ssRNA viruses. They belong to family-genus of Flexiviridae-potexvirus and Flexiviridae-potyvirus, respectively (Mayo and Brunt, 2005).
Gene silencing results in no expression or very low expression of a gene or RNA sequence that was formerly expressed, or likely would be expressed in the absence of the gene-silencing phenomenon (Atkinson et al., 1998). There is great consensus that gene silencing is an adaptive defense mechanism against viruses and transposable elements. Therefore, application of gene silencing is a useful method for production of plant virus resistance cultivars (Waterhouse et al., 2001). The development of genetically engineered resistance depends on exploiting genes from the pathogen, known as Pathogen-Derived Resistance (PDR) (Hamilton, 1980; Sanford and Johnston, 1985). It has been shown that the PVX Coat Protein (CP) is transported through the phloem of potato, unloads into the vascular tissue and is subsequently transported between cells during the course of infection (Cruz et al., 1998). Two distinct mechanisms for viral cell-to-cell movement have been described (Maule, 1991; Carrington et al., 1996). One strategy is typified by TMV. It is independent of the CP (Siegel et al., 1962; Takamatsu et al., 1987) and requires a single Movement Protein (MP) that can traffic between cells (Waigmann et al., 1994). A second well-described movement strategy used by the various viruses, e.g., comoviruses and nepoviruses is CP-dependent and involves the transport of virions through virus-induced tubules that span the cell walls of adjacent
cells (Wellink and Van Kammen, 1989; Van Lent et al., 1990; Suzuki et al., 1991; Dolja et al., 1994, 1995; Forster et al., 1992; Chapman et al., 1992; Sit and AbouHaidir, 1993; Cruz et al., 1998). Therefore, CP of viruses have important role at virulence and applicable to produce transgenic PDR cultivars. Virus-resistant transgenics have been developed in many crops by introducing either viral CP or replicase gene encoding sequences. Resistance obtained by using CP is conventionally called Coat Protein Mediated Resistance (CPMR). Coat protein genes have been shown high efficiency in preventing or reducing infection and disease caused by homologous and closely related viruses (Gonsalves and Slightom, 1993). Coat protein-mediated protection has been reported for Tobacco mosaic virus, (TMV) (Nelson et al., 1988), Tomato mosaic virus, (ToMV) (Sanders et al., 1992), Cucumber mosaic virus, (CMV) (Namba et al., 1991; Quemada et al., 1991), Alfalfa mosaic virus (AlMV) (Loesch-Fries et al., 1987; Tumer et al., 1987), PVX, (Hemenway et al., 1988), PVY, (Perlak et al., 1994) and potato leaf roll virus, PLRV, (Kaniewski et al., 1993). Transgenic potato with the coat protein genes of PVX and PVY that expressed both CP genes were resistant to infection by PVX and PVY by aphid transmission and mechanical inoculation (Lawson et al., 1990). Since CP plays a major role in vector transmission, CPMR confers additional advantage of resistance to vector inoculation in a majority of cases. For example, potato, which expresses PVX and PVY CP and tobacco, tomato and cucumber expressing CMV CP were seen to be highly resistant to aphid transmissions (Lawson et al., 1990; Guo et al., 1999). In potato, expression of the antisense RNA prevented virus infection even after grafting with scions from infected plants and therefore this transformant might be regarded as immune to the virus (Paucha et al., 1998). Here we analyzed the PVX and PVY coat protein gene sequences using a phylogenetic approach in order to determine applicable conserved sequences that could be used to design specific diagnostic-primers, in gene-silencing studies to produce virus or co-virus resistance cultivars and correlation between coat protein gene sequences and geological distribution.
MATERIALS AND METHODS
This study was conducted in Agriculture Biotechnology Research Institute for Northwest and West of Tabriz, Iran.
Sequence alignment and phylogenetic tree construction: In this study, 27 CP PVX and 37 PVY gene sequences from GenBank were submitted to multiple sequence alignment performed by ClustalX (ver 1.8) to find relationships among sequences for gap opening, gap extention, presence of divergent sequences and DNA transition weight of 10.00, 0.20, 30 and 0.50, respectively. Phylogenetic tree construction via the Neighbour Joining (NJ) method of Saitou and Nei (1987) was performed between all pairs of sequence from a multiple alignment. Bootstrapping of sequences was performed 1000 iterations and 500 iterations for random number generator seeds. Genetic pair distances and distances from root of trees obtained by PhyloDraw (ver 0.8). Stepwise discriminant analysis, based on distances from root of trees, (Jennrichr and Sampson, 1985; Pimentel, 1979; Jennrich, 1977; Lanchenbruch and Goldstein, 1979) was carried out using SPSS (ver 9.0) to verify the cluster analysis.
Conserved regions: Conserved regions detected by BioEdit (ver 5.0.6) were defined following parameters of maximum average entropy and maximum entropy per position each being were 0.2 with no gaps tolerated and a minimum segment length defined as 25.
RESULTS
Alignment: Multiple alignment of the 27 PVX coat protein gene sequences indicated a gap from position of 85 to 120 for all of sequences exceptions X88781, X88782 and X88785. Total average and variation of genetic distances of pair sequences were 0.118131 and 0.004304, respectively for these sequences. Maximum distance and minimum similarity between sequences were 0.225 with similarity of 74% (between Fujian isolate and N14 strain) and 55% with distance of 0.165 (between HB and DY strains), respectively. Minimum distance was zero with similarity of 100% between NC001455 and M72416 accessions (Table 1).
Multiple alignment of the 37 PVY coat protein gene sequences indicated an N-terminal gap from position 1 to 180, positions of 236 and 274 to 280 for all of sequences exceptions AY459605, AY459607 and AY459609. Total average and variation of genetic distances were 0.118368 and 0.004776, respectively. Maximum distance and minimum similarity between sequences were 0.448 with similarity of 49% (between Fengyang-8-1isolate-China and VTSBTschilombo isolate-South Africa) and 48% with distance of 0.405 (between VTSBTschilombo isolate-South Africa and XCH43 isolate- China). Minimum distance was 0.12 with similarity of 99% between DQ157179 and AY745492 (Table 2).
| Table 1: | Similarity and genetic distance (10-2) of PVX coat protein genes |
| NC001455(1), M72416(2), E01310(3), M38655(4), X88783(5), U19790(6), X88787(7), Z34261(8), AF528555(9), M95516(10), AF260641(11), AF260640(12), X88788(13), AF272736(14), AB056718(15), AB056719(16), X88784(17), AF485891(18), AY763582(19), Z23256(20), X72214(21), AF172259(22), M63141(23), X88786(24), X88785(25), X88782(26), X88781(27) | |
It is mentionable that similarity could not be alone as a reliable parameter correspondes to the weak correlation between reported genetic distances and similarities. Therefore, conserved regions and genetic distances are reliable than similarity to analyze sequence based functions or Phylogenetic relationship.
Phylogenetic relationships: Cluster analysis based on 27 and 37 sequences of PVX and PVY coat protein gene achieved by the neighbour joining method (Fig. 1). These sequences segregated into four groups. Stepwise discriminant analysis on groups from the cluster analysis using genetic distances of sequences from root of trees verified the groups of PVX sequences but not for PVY sequences groups.
| Table 2: | Similarity and genetic distance (10-2) of PVY coat protein genes |
| AJ439544(1), AJ390307(2), AJ439545(3), DQ157179(4). AY745492(5), DQ157178(6), AY884985(7), DQ008213(8), AJ889868(9), AJ890350(10), AJ890349(11), AY841265(12), AY841266(13), AJ890345(14), AJ390288(15), AJ890347(16), AJ890344(17), AJ890343(18), AJ890342(19), AY884982(20), AJ390285(21), E07484(22), AY884984(23), AJ390286(24), E03317(25), AY884983(26), AJ890346(27), AY841267(28), AY742719(29), S74813(30), AY841257(31), AY841260(32), AY840082(33), AJ890348(34), AY459607(35), AY459605(36), AY459609(37) | |
| Fig. 1: | Phylogenetic trees of PVX and of PVY coat protein genes. Nucleotide sequences were determined with accession number (Vertical lines are as cutting positions) |
For PVX sequences, difference between groups was determined with Wilks lambda of 0.131 (F3,23 = 50.742, p<0.000). Also, Chi-squared test was significant (F3,23 = 4782.816, p<0.000). When we changed the tree of PVY sequences, discriminant analysis on new made groups verified the new groups with significant Wilks lambda of 0.661 (F2,34 = 8.738, p<0.001) and Chi-squared test (F2,34 = 1242.105, p<.000). The new groups are as follow: group 1 included AJ439544, AJ390307, AJ439545, AJ890348, DQ157179, AY745492, DQ157178, AY884985, DQ008213, AJ889868, AJ890350, AJ890349, AY841265, AY841266, AY459607, AY459605, AY459609, AY840082, AY841260, AY841257, AY742719 and AY841267, group 2 included AJ390285, E07484, AJ890346, AJ390286, E03317, AY884983 and AY884984 and group 3 included AJ890343, AJ890342, AJ890345, AJ390288, S74813, AY884982, AJ890347 and AJ890344.
Among the investigated PVX sequences, those isolates from Korea, Japan and Taiwan sequence diversity was related to geographic origins. Korean isolates were classified in group number 4 and Japanese and Taiwanese isolates were classified in group number 3 (Table 3). This relation was achieved only for six, three and two derived isolates from China, South Africa and French, respectively among 37 subjected PVY sequences (Table 4).
Conserved regions: Conserved regions for PVX and PVY sequences groups were determined separately (Table 5 and 6). Group number 2 for PVX and group number 1 for PVY were divided into 2 and 3 subgroups, respectively, to find at least one conserved region under working conditions. There were not any conserved regions under working conditions for all of the sequences when based on virus type. Therefore, to determine conserved regions
| Table 3: | PVX strains-isolates/cluster comparison |
for all of the sequences, minimum segment length parameter was decreased to 10. Under this condition two conserved regions for PVX sequences and one conserved region for PVY sequences were found (Table 5 and 6).
Coat protein of plant viruses affect virulence efficiency via different mechanisms (Cruz et al., 1998; Maule, 1991; Carrington et al., 1996; Siegel et al., 1962; Takamatsu et al., 1987; Waigmann et al., 1994; Wellink and Van Kammen, 1989; Van Lent et al., 1990; Suzuki et al., 1991; Dolja et al., 1994) and CP should be a suitable candidate gene to produce PDR cultivars via RNAi (Loesch-Fries et al., 1987; Tumer et al., 1987; Hemenway et al., 1988; Perlak et al., 1994; Kaniewski et al., 1993; Kaniewski et al., 1990; Van dervlugt et al., 1992; Hoekema et al., 1989; Feher et al., 1992; Kollar et al., 1993; Sudarsono Young et al., 1995).
| Table 4: | PVY strains-isolates/cluster comparison |
| Table 5: | Conserved regions of grouped PVX coat protein genes sequences |
| Group 2 was divided to two subgroups (subgroup 1: X88781, X88782 and X88785, subgroup 2: Z23256, AF172259, M63141, X88786 and AF485891) as well as expelling of X72214 to obtain at least one conserved region | |
| Table 6: | Conserved regions of grouped PVY coat protein gene sequences |
| Group 1 was divided to three subgroups as well as elimination of the AJ390285 for obtaining of at least one conserved region. Sub group 1 includes the AY459605 to 607 and sub subgroup 2 includes the DQ157178, DQ157179, AY841492, AY841266, AY841265, AY884985 and AJ890349 and subgroup 3 includes the others | |
Some of conserved regions that should be useful to provide broad resistance for group 1 PVX might be specific to the endemic geographical regions for group 1. Sub group 2 of PVX group 2 have two candidate conserved regions at positions from 388 to 488 and from 604 to 707 to construct the gene silencing cassettes for developing of virus resistance potato varieties. Also, the positions from 544 to 734 for PVX group 3 seem suitable as well. Positions from 379 to 530 and from 595 to 744 of PVX group 4 are applicable for use in silencing constructs. Applicable of conserved regions of PVY to use in gene silencing constructs are as follow: For subgroup 2 of group 1 are from 413 to 604, from 662 to 747 and from 818 to 984, for subgroup 3 of group 1 are from 296 to 815 and from 842 to 980, for group 2 are from 498 to 705 and from 722 to 997 and for group 3 are from 608 to 722, from 791 to 880 and from 911 to 995.
DISCUSSION
We have subjected 27 PVX and 37 PVY coat protein gene sequences in order to variation analysis. According to alignment results, it is apparent that diversity for PVX and for PVY sequences are approximately the same. Also, genetic distances are low for each virus genus among of coat protein sequences. Therefore, these virus isolates have seemingly relatively slow divergent evolution in the coat protein genes, which might be related to a conserved role in the virus life history.
Also, comparison of geological distribution with obtained cluster groups illustrate the possible artificial transportations of viruses isolates via plant materials or existence of the hot spot mutational positions on coat protein gene sequences which different source isolates could be placed into same cluster groups.
It might be possible to produce general PVX-PVY resistance potato by using dual conserved of both PVX and PVY conserved regions. Minimum length of transgene to silence of a gene is approximately 100 nucleotides (Wesley et al., 2001) and therefore, there are numerous candidate sequences that could be efficient to reduce expression of the virus coat protein gene on infected plants. However, conserved sequences of groups or subgroups must be chosen correspond to geological distribution of viruses isolates to develop co-virus resistance potato at particular place.
Pathogen detection is the other application of conserved regions. The detection of viral pathogens is of critical importance in biology, medicine and agriculture. Presently, molecular detection of pathogens specially based on PCR has become an efficient, rapid and simple method in different area as well as plant viruses detection (Hadidi et al., 1995; Haliloglu and Bostan, 2002; Elnifro et al., 2000). Therefore, these detected conserve regions could be capable to use as candidate regions for specific primer designing in order to virus or isolates detection. According to the small length of conserved region based on all of the sequences for each virus, detection of virus isolates must be via 3' specific based primer corresponds to the conserved region. Also, these conserved regions could be subjected for microarray based detection of viral families (Wang et al., 2002), especially for detection of viral isolates or subgroups.