Department of Plant Pathology, University of California, One Shields Avenue, Davis, CA 95616, USA
Correspondence
Bryce Falk
bwfalk{at}ucdavis.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Valencia, Spain.
Present address: Department of Nematology, University of California, Davis, CA 95616, USA.
Present address: University of California Cooperative Extension, 4437 S. Laspina St, Ste B, Tulare, CA 93274, USA.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cucumber mosaic virus (CMV) is one of the most economically important plant viruses and has a very wide host range including plants from approximately 365 genera and at least 85 families (Palukaitis et al., 1992). A number of CMV isolates have been described previously and classified into two subgroups, I and II, according to serological relationships, peptide mapping of the coat protein (CP), nucleic acid hybridization and nucleotide sequence identity (Palukaitis et al., 1992
). More recently, phylogenetic analysis of a number of CMV isolates led to a further subdivision of subgroup I into subgroups IA and IB (Roossinck, 2002
; Roossinck et al., 1999
). CMV is endemic in cucurbit planting areas in California and other parts of the world, and effective genetic resistance is lacking in most cucurbits (Provvidenti, 1993
). Transgenic yellow crookneck squash (Cucurbita pepo cv. Destiny III) plants engineered with the CP genes of CMV, Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV) have been developed and sold commercially in the USA, and have shown good resistance against a number of CMV isolates (Tricoli et al., 1995
). However, whether resistance-breaking CMV isolates/variants already exist in virus populations, and whether or not specific CMV genotypes may be associated with transgenic or conventional host plant resistance, are unknown. In this paper, we used host range analysis, RT-PCR, single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses to estimate the biological and genetic diversity of California CMV isolates collected before and after the development of CMV-resistant transgenic Destiny III plants.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
ELISA.
Indirect ELISA was carried out according to the method of Sasaya & Yamamoto (1995). Antibodies to CMV, WMV, ZYMV and Papaya ringspot virus (PRSV) were from our laboratory collection of polyclonal antisera.
RT-PCR amplification.
Oligonucleotide primers were designed for the CMV CP gene and 3' non-translated region (3'NTR) based on the nucleotide sequences of CMV-Fny RNA 3 (subgroup I, GenBank accession no. D10538) and CMV-Q RNA 3 (subgroup II, GenBank accession no. M21464). These primers are highly conserved within each subgroup and are subgroup-specific. Primers F1 and F3 are complementary to the RNA 3 3'-terminal nucleotides 21972216 and nucleotides 19021922 of CMV-Fny, respectively. Primer F4 corresponds to nucleotides 12451266 of CMV-Fny RNA 3. Primers Q1 and Q3 are complementary to the RNA 3 3'-terminal nucleotides 21742193 and nucleotides 18501872 of CMV-Q, respectively. Primer Q4 corresponds to nucleotides 12351256 of CMV-Q RNA 3.
Total RNAs were extracted from healthy and CMV-infected plants using TRI reagent (Molecular Research Center) according to the manufacturer's instructions. First-strand cDNA was synthesized (primer F1 for subgroup I, primer Q1 for subgroup II) using total RNA as template. cDNA synthesis and PCR amplifications were carried out following the method of Rubio et al. (2001), except that the annealing temperature for both the CP gene (primers F3 and F4, or Q3 and Q4) and the CP+3'NTR (primers F1 and F4) were 45 °C.
SSCP analysis.
SSCP analysis was performed following the method of Rubio et al. (2001) with slight modifications. Five µl of the RT-PCR product was first digested by restriction endonuclease AflIII for the CP gene, or XhoI for the CP+3'NTR, in a 10 µl reaction volume at 37 °C for 60 min in order to obtain smaller size DNA fragments more suitable for SSCP analysis. Two µl of the digested DNA was mixed with 8 µl denaturing solution (95 % formamide, 20 mM EDTA, pH 8, 0·05 % bromophenol blue and 0·05 % xylene/cyanol) and denatured by heating at 95 °C for 10 min. Denatured DNA was subjected to electrophoresis at 4 °C in a non-denaturing 12 % polyacrylamide minigel (for the CP gene, the gel contained 5 % glycerol), using a constant voltage of 200 V for 3 h. The gels were stained with silver nitrate (Rubio et al., 2001
).
cDNA cloning and nucleotide sequence analysis.
RT-PCR products of the CP gene were extracted using the QIAquick PCR extraction kit (Qiagen). These were then directly used for sequencing with the respective PCR primers, or were ligated into the pGEM-T vector (Promega) according to the manufacturer's instructions, followed by transformation into Escherichia coli DH5. Positive colonies were screened by the PCR method using the same conditions as the PCR reaction above described. Sequences were determined in both directions by means of an ABI PRISM DNA sequencer 377 (Perkin-Elmer) in the DNA Sequence Facility of UC-Davis. Processing and multiple alignments of the nucleotide sequences (minus primer sequences) were done using the program CLUSTAL W (Thompson et al., 1994
). Alignments were manually adjusted by MacClade 4.0. The nucleotide distance was estimated using the JukesCantor method implemented in DNADIST of the PHYLIP package (Felsenstein, 1989
). The nucleotide sequences of CMV-Fny, -Q and -C were also used for the estimation of nucleotide distance. The CP gene from CMV-C (GenBank accession no. D00462) was that used to engineer CMV resistance into Destiny III (Tricoli et al., 1995
). Phylogenetic relationships were inferred using PAUP* version 4.0b10.0 based on the maximum-parsimony method with a 100 replicate bootstrap search. The first positions of each codon and the transversion substitutions were both weighted as 2. Two other members of the genus Cucumovirus, Peanut stunt virus (PSV) and Tomato aspermy virus (TAV) were defined as outgroups. All branches with <70 % bootstrap value were collapsed.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biological diversity of CMV isolates on transgenic and non-transgenic plants
Although no CMV isolates were recovered from the transgenic, CMV-resistant Destiny III field plants, we still evaluated the biological diversity of several CMV isolates collected from the surrounding non-transgenic plants or plants with transgenic resistance to WMV and ZYMV (Prelude II). Sixty-three CMV group isolates identified above, plus 18 group
CMV isolates collected from various host plants and different geographic areas within California during 19851994 (Table 2
), as well as the standard strains, CMV-Fny (subgroup I), -Q and Ls (subgroup II), were compared by inoculating plants of susceptible (Dixie), conventional-resistant (Freeman) and the transgenic-resistant Destiny III. All CMV isolates were first inoculated onto plants of small sugar pumpkin. However, as most of the field-collected group
isolates were mixed infections with other viruses (e.g. WMV, ZYMV and/or PRSV; Table 1
), they could not be used directly for biological comparisons. Therefore, they were first inoculated to Nicotiana benthamiana plants (a non-host for PRSV) to eliminate PRSV. Our ELISA data showed that PRSV was eliminated, as were most of WMV and/or ZYMV. Only 68 CMV isolates free of the potyviruses were used for further biological analyses (but all 81 isolates were used for initial sequence comparisons, see below). Based on their abilities to infect and cause symptoms on plants of Dixie, Destiny III and Freeman, the 68 California CMV isolates could be classified into five pathotypes (Table 3
).
|
Thirty-three CMV isolates out of 68, including 17 group (collected from Kearney Agricultural Center in 1999) and 16 group
isolates (collected from different California areas during 19851994), infected transgenic Destiny III plants. Symptoms ranged from mild (very similar to the symptoms induced by Ca on Dixie plants; Fig. 1
A), to moderate (stunted plants, more curled leaves with yellow veins and yellow patches around the veins and necrotic lesions on the petioles; Fig. 1B
), to severe (mosaic, leaf curling, crinkling, stunting, more rapid symptom development; Fig. 1C
). Among the Destiny III-infecting isolates, 25 could also infect Freeman plants and were grouped into pathotype III, whereas eight did not infect Freeman plants and were grouped into pathotype IV. Pathotype V contained 17 CMV isolates that infected both Freeman and Dixie plants, but not Destiny III plants. CMV-Fny showed similar pathogenicity to pathotype V, while Ls and Q were similar to isolate Ca, pathotype I. Taken together, these results show that California CMV isolates vary in biological diversity when compared by their ability to infect transgenic- and conventional-resistant plants. Interestingly, some of the Destiny III-infecting group
CMV isolates were collected and stored before transgenic resistance was available, and thus before selection pressure due to transgenic resistance was present. Also of interest is that, even though no CMV was recovered from transgenic CMV-resistant Destiny III or Liberator III field plants, CMV isolates with the ability to infect transgenic-resistant Destiny III plants were recovered from non-transgenic plants in the same overall planting.
|
We next used SSCP analysis of the CP gene RT-PCR product as a first approach to differentiate CMV isolates having different nucleotide sequences. To achieve greater sensitivity and resolution, we used the restriction endonuclease AflIII to cleave the CP DNA product into two smaller DNA fragments of 214 bp and 464 bp. Fourteen distinct SSCP patterns were obtained after analysing the AflIII-digested PCR products of all 81 CMV isolates (Fig. 2). When SSCP patterns for all isolates were examined together, patterns F and I were the most predominant, found for 23 and 40 isolates, respectively. Among 63 group
isolates, 23 showed pattern F and 32 showed pattern I (Fig. 2
). Pattern I also represented the majority of group
isolates (8 of 18 isolates, Fig. 2
). Samples of the same group
isolates, but dried and stored at different times, showed the same SSCP pattern (e.g. see isolates 144I, I90, 116, SJ91, 113 and 160, Table 2
). This suggested that these isolates had not changed significantly during laboratory and greenhouse manipulations, at least in CP sequence.
|
|
Phylogenetic analysis of the CP gene
To analyse and compare these CMV isolates further, phylogenetic analysis of the CP gene was performed for the 14 isolates that represented different SSCP patterns, together with 13 nucleotide sequences from GenBank, as well as two other members of the genus Cucumovirus, PSV and TAV, defined as outgroups. The result confirmed our RT-PCR data showing that these 14 CMV isolates belonged to subgroup I (Fig. 3). In addition, and somewhat surprisingly, we found that these isolates were split into two subgroups. Isolate 113B belonged to subgroup IB and was most closely related to isolates NT9 and Tfn from Taiwan and Italy, respectively. These three isolates formed a single clade with 100 % bootstrap support. Isolate C94T5, which showed the same SSCP pattern as isolate 113B (see Fig. 2
), was then also sequenced and used for phylogenetic analysis. Isolate C94T5 was found to have only one nucleotide change from 113B, and by phylogenetic analysis it also fell into the same clade as isolates 113B, NT9 and Tfn (data not shown). The remaining 13 CMV isolates fell into subgroup IA. Among these, isolates C94M3 and V27 formed a clade having 88 % bootstrap support. All group
isolates (CK26, 27, 31, 33, 41, 51 and 57) and a group
isolate (MD284) were closely related to each other and formed a clade having 98 % bootstrap support. Overall, group
isolates were more diverse than group
isolates (Fig. 3
).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results also showed that 42 CMV isolates were able to overcome the conventional resistance of Freeman plants (Table 3). Among these isolates, 17 did not infect transgenic Destiny III (pathotype V), and among 33 isolates capable of overcoming the transgenic resistance, eight did not infect plants of Freeman (pathotype IV). These results suggest that the mechanisms involved in overcoming Freeman resistance and transgenic Destiny III resistance are different, and that California CMV populations are biologically diverse and contain variants with the ability to overcome both types of resistance.
A number of studies have shown that CP-mediated transgenic resistance generally provides more effective protection against challenging viruses with higher amino acid sequence identity as opposed to distantly related viruses or virus strains (Anderson et al., 1989; Nelson et al., 1987
, 1988
; Van Dun & Bol, 1988
; Van Dun et al., 1988
). The resistance of plants of Destiny III is believed to be mediated at the CP protein level, and not by post-transcriptional gene silencing (Quemada & Tricoli, 1995
). Therefore, if this scenario applies to plants of Destiny III expressing the CMV-C CP gene, it would be expected that plants of Destiny III would show better protection against infection by closely related isolates (i.e. subgroup I isolates) than by distantly related isolates (i.e. subgroup II isolates). This trend was observed when Destiny III was challenged by CMV-Fny, which has 98·69 % amino acid identity with CMV-C. However, Destiny III also showed high protection against infection by CMV-Q and CMV-Ls (subgroup II), which only have 80·51 % amino acid sequence identity with CMV-C. When the group
isolates having 100 % amino acid sequence identity with one another, but only 97·37 % amino acid sequence identity with CMV-C, were compared, we found that some group
isolates could infect plants of Destiny III (or Freeman) and some could not. Thus, there was no direct correlation in our studies between the CP amino acid sequence identity of the CMV isolates used here and their ability to infect plants of Destiny III or Freeman. The lack of correlation between the degree of amino acid sequence identity and the level of protection was also observed in the transgenic tobacco plants expressing the CP gene of CMV-C (Quemada et al., 1991
). Thus, other factors (e.g. non CP-viral genomic sequences, CMV satellite RNAs) could contribute to the biological variation.
SSCP proved here to be very sensitive for evaluating the molecular variation of CMV isolates. It was also used to compare the major sequences of the CMV isolates infecting transgenic Destiny III and non-transgenic plants. A rapid change of major sequence within a population of an individual isolate might result from selection of sequence variants with a higher adaptive potential after a host shift, or could be due to the appearance of a more competitive new genotype generated via recombination between the transgene and the infecting virus (see introduction). Our data showed that most CMV isolates analysed did not show significant major sequence changes after infecting transgenic Destiny III plants. For isolate CK41, which did show a different major sequence variant (SSCP pattern) in transgenic and non-transgenic plants, further analyses showed that this difference was due to neither recombination nor selection by transgenic resistance, but most likely reflected a random colonization on new host plants from a mixed population inoculum, because the subdivision of sequences A and B to different host plants was not consistently observed in three repeated inoculations and SSCP analyses (data not shown). More detailed evaluation of the effects of host species and transgenic resistance on the population structure within virus isolates is needed.
Sequence analysis of the CP genes of 27 CMV isolates with distinct and indistinguishable SSCP patterns (Table 2 and Fig. 2
) showed that these CMV isolates all belonged to CMV subgroup I. Based on the sensitivity and accuracy of our SSCP analysis, it is reasonable to conclude that all 81 California CMV isolates analysed here belonged to subgroup I. It is unlikely that subgroup II isolates were also present as: (i) mixed infections with subgroups I and II isolates have not been reported from North America; and (ii) we also analysed the CMV MP gene of these same isolates by RT-PCR using CMV subgroup-universal primers and no subgroup II sequences were found by SSCP and nucleotide sequence analyses (unpublished data). Nucleotide sequence data also showed group
isolates collected from Kearney Agricultural Center in 1999 had very high sequence identity (>98·84 %; Table 4
), and they formed a single clade in the phylogenetic analysis (Fig. 3
), suggesting that the extant CMV population at this location was most likely derived from a common ancestor and only recently spread over the area. In contrast, group
isolates were more diverse,with nucleotide identities as low as 93·24 %. This diversity was further confirmed by phylogenetic analysis (Fig. 3
). It is also interesting to point out that two subgroup IB isolates (113B and C94T5) were found among the California CMV isolates analysed here, and they were most closely related to isolate NT9 from Taiwan and isolate Tfn from Italy. Indeed, these four isolates only had one to three nucleotide differences (Fig. 3
and data not shown). Subgroup IB isolates were believed to be only distributed in Asia (Roossinck et al., 1999
) and Italy (Roossinck, 2002
). Thus, this represents the first report that subgroup IB isolates occur in the Americas. The diversity seen here among group
isolates is not so surprising as they were collected from different plant species, in different areas and in different years. However, the genetic variation of CMV observed here was not correlated with geographic region, collection date or plant species. This is similar to the population structure of CMV described in Spain, where the genetic structure of 17 CMV subpopulations was not correlated with location or year, but showed a metapopulation structure with local extinction and random recolonization from local or distant virus reservoirs (Fraile et al., 1997
; Garcia-Arenal et al., 2001
).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allison, R. F., Schneider, W. L. & Greene, A. E. (1996). Recombination in plants expressing viral transgenes. Semin Virol 7, 417422.
Anderson, E. J., Stark, D. M., Nelson, R. S., Powell, P. A., Tumer, N. E. & Beachy, R. N. (1989). Transgenic plants that express the coat protein genes of tobacco mosaic virus of alfalfa mosaic virus interfere with disease development of some nonrelated viruses. Phytopathology 79, 12841290.
Beachy, R. N. (1997). Mechanisms and applications of pathogen-derived resistance in transgenic plants. Curr Opin Biotechnol 8, 215220.[CrossRef][Medline]
Daniels, J. & Campbell, R. N. (1992). Characterization of cucumber mosaic virus isolates from California. Plant Dis 76, 12451250.
Domingo, E., Holland, J., Biebricher, C. & Eigen, M. (1995). Quasi-species: the concept and the word. In Molecular Basis of Virus Evolution, pp. 181191. Edited by A. J. Gibbs, C. H. K. Calisher & F. García-Arenal. Cambridge: Cambridge University Press.
Eigen, M. (1996). On the nature of virus quasispecies. Trends Microbiol 4, 216218.[CrossRef][Medline]
Enzie, W. D. (1943). A source of muskmelon mosaic resistance found in the oriental pickling melon, Cucumis melo var. Conomon. Proc Am Soc Hortic Sci 43, 195198.
Felsenstein, J. (1989). PHYLIP Phylogenetic Inference Package (Version 3.2). Cladistics 5, 164166.
Ferreira, S. A., Pitz, K. Y., Manshardt, R., Zee, F., Fitch, M. & Gonsalves, D. (2002). Virus coat protein transgenic papaya provides practical control of Papaya ringspot virus in Hawaii. Plant Dis 86, 101105.
Fraile, A., Alonso-Prados, J. L., Aranda, M. A., Bernal, J. J., Malpica, J. M. & Garcia-Arenal, F. (1997). Genetic exchange by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus. J Virol 71, 934940.[Abstract]
Gal, S., Pisan, B., Hohn, T., Grimsley, N. & Hohn, B. (1992). Agroinfection of a transgenic plant leads to viable cauliflower mosaic virus by intermolecular recombination. Virology 187, 525533.[CrossRef][Medline]
Garcia-Arenal, F., Fraile, A. & Malpica, J. M. (2001). Variability and genetic structure of plant virus populations. Annu Rev Phytopathol 39, 157186.[CrossRef][Medline]
Gonsalves, D. (1998). Control of papaya ringspot virus in papaya: a case study. Annu Rev Phytopathol 36, 415437.[CrossRef]
Greene, A. E. & Allison, R. F. (1994). Recombination between viral RNA and transgenic plant transcripts. Science 263, 14231425.[Medline]
Greene, A. E. & Allison, R. F. (1996). Deletions in the 3' untranslated region of cowpea chlorotic mottle virus transgene reduce recovery of recombinant viruses in transgenic plants. Virology 225, 231234.[CrossRef][Medline]
Holland, J. J., De La Torre, J. C. & Steinhauer, D. A. (1992). RNA virus population as quasispecies. Curr Top Microbiol Immunol 176, 120.[Medline]
Nelson, R. S., Powell, P. A. & Beachy, R. N. (1987). Lesions and virus accumulation in inoculated transgenic tobacco plants expressing the coat protein gene of tobacco mosaic virus. Virology 158, 126132.[CrossRef]
Nelson, R. S., McCormick, S. M. & Dellanay X. & 9 other authors (1988). Virus tolerance, plant growth, and field performance of transgenic tomato plants expressing coat protein from tobacco mosaic virus. Bio/Technology 6, 403409.
Palukaitis, P., Roossinck, M. J., Dietzgen, R. G. & Francki, R. I. B. (1992). Cucumber mosaic virus. Adv Virus Res 41, 281348.[Medline]
Provvidenti, R. (1993). Resistance to viral diseases of cucurbits. In Resistance to Viral Diseases of Vegetables: Genetics and Breeding, pp. 843. Edited by M. M. Kyle. Portland, OR: Timber Press.
Quemada, H. D. & Tricoli, D. M. (1995). Petition for determination of regulatory status of squash line CZW-3 containing the coat protein genes from cucumber mosaic virus (CMV), watermelon mosaic virus 2 (WMV 2), and zucchini yellow mosaic virus (ZYMV). USA: USDA petition number 95-352-01p.
Quemada, H. D., Gonsalves, D. & Slightom, J. L. (1991). Expression of coat protein gene from cucumber mosaic virus strain C in tobacco: protection against infection by CMV strains transmitted mechanically or by aphids. Phytopathology 81, 794802.
Robinson, D. J. (1996). Environmental risk assessment of release of transgenic plants containing virus-derived inserts. Transgenic Res 5, 359362.
Rodriguez-Alvarado, G., Kurath, G. & Dodds, J. A. (1995). Heterogeneity in pepper isolates of cucumber mosaic virus. Plant Dis 79, 450455.
Roossinck, M. J. (2002). Evolutionary history of cucumber mosaic virus deduced by phylogenetic analyses. J Virol 76, 33823387.
Roossinck, M. J., Zhang, L. & Hellwald, K.-H. (1999). Rearrangements in the 5' nontranslated region and phylogenetic analyses of cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups. J Virol 73, 67526758.
Rubio, L., Soong, J., Kao, J. & Falk, B. W. (1999). Geographic distribution and molecular variation of isolates of three whitefly-borne closteroviruses of cucurbits: lettuce infectious yellows virus, cucurbit yellow stunting disorder virus, and beet pseudo-yellows virus. Phytopathology 89, 707711.
Rubio, L., Abou-Jawdah, Y., Lin, H. X. & Falk, B. W. (2001). Geographically distinct isolates of the crinivirus cucurbit yellow stunting disorder virus show very low genetic diversity in the coat protein. J Gen Virol 82, 929933.
Sasaya, T. & Yamamoto, T. (1995). Improvements in non-precoated indirect enzyme-linked immunosorbent assay for specific detection of three potyviruses infecting cucurbitaceous plants. Ann Phytopathol Soc Jpn 61, 130133.
Schoelz, J. E. & Wintermantel, W. M. (1993). Expansion of viral host range through complementation and recombination in transgenic plants. Plant Cell 5, 16691679.
Tepfer, M. (1993). Viral genes and transgenic plants. What are the potential environmental risks? Bio/Technology 11, 11251132.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Tricoli, D. M., Carney, K. J., Russell, P. F. & 7 other authors (1995). Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus 2, and zucchini yellow mosaic virus. Bio/Technology 13, 14581465.
Van Dun, C. M. P. & Bol, J. F. (1988). Transgenic tobacco plants accumulating tobacco rattle virus coat protein resist infection with tobacco rattle virus and pea early browning virus. Virology 167, 649652.[CrossRef][Medline]
Van Dun, C. M. P., Overduin, B., Van Vloten-Doting, L. & Bol, J. F. (1988). Transgenic tobacco expressing tobacco streak virus or mutated alfalfa mosaic virus coat protein does not cross-protect against alfalfa mosaic virus infection. Virology 164, 383389.[CrossRef][Medline]
Wintermantel, W. M. & Schoelz, J. E. (1996). Isolation of recombinant virus between cauliflower mosaic virus and a viral gene in transgenic plants under conditions of moderate selection pressure. Virology 223, 156164.[CrossRef][Medline]
Received 19 June 2002;
accepted 11 September 2002.