- Email: [email protected]
Transmission of Maize streak virus by vascular puncture inoculation with unit-length genomic DNA
Journal of Virological Methods 109 (2003) 95 /98 www.elsevier.com/locate/jviromet
Brief report
Transmission of Maize streak virus by vascular puncture inoculation with unit-length genomic DNA M.G. Redinbaugh * USDA-ARS Corn and Soybean Research and Department of Plant Pathology, Ohio Agriculture Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691, USA Received 19 September 2002; received in revised form 7 January 2003; accepted 8 January 2003
Abstract The infectivity of cloned unit-length genomes of Maize streak virus (MSV) was tested using vascular puncture inoculation (VPI). VPI of kernels with plasmid DNA (pUC19) carrying a tandem repeat of the MSV genome produced 339/8% infection. Similar plasmids carrying the unit-length MSV genome were not infectious. If the MSV genome was released from the plasmid prior to VPI, 169/4% of plants became infected. Ligation of the free linear MSV genome did not increase infectivity. The three infective inocula produced symptoms of similar severity in maize. Bioassay of systemically infected leaves indicated the virus was equally infectious regardless of inoculum. In Southern blots of bioassay plants, no differences in MSV genome restriction endonuclease sites were observed. Thus, inoculation with the free linear or circularized MSV unit-length genome produced infections similar to those with plasmids carrying tandemly repeated genomes. The infectivity of free linear MSV unit-length genomes will facilitate molecular analysis of MSV, because cloning steps are minimized. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Maize streak virus ; Vascular puncture inoculation; Genomic DNA; Geminivirus
Geminiviruses are plant viruses with characteristic circular single-stranded DNA genomes and geminate virions (Bosque-Pe´rez, 2000). The viruses are classified into three genera based on their genomic organization, insect vector and plant host range. Maize streak virus (MSV), the type member of the genus Mastrevirus , is a monopartite geminivirus that causes significant agronomic problems on corn in sub-Saharan Africa. MSV is transmitted by leafhoppers (Circadulina species) to the phloem sieve tubes of host plants, and cannot be transmitted by leaf-rub inoculation. For many years, study of MSV outside of Africa was limited by difficulties with transmitting the virus and maintaining virus cultures. Similarly to other geminiviruses, MSV may be transmitted after inoculation of Agrobacterium tumefaciens carrying binary plasmids harboring a tandem repeat of the MSV genome in a process referred to as agroinoculation (Grimsley et al.,
* Tel.: /1-330-263-3965; fax: /1-330-263-3841. E-mail address: [email protected] (M.G. Redinbaugh).
1987). While there have been several improvements in agroinoculation (Martin and Rybicki, 2000), it requires manipulation of relatively complex binary vectors, and has a high degree of specificity for the Agrobacterium strain and the orientation of the viral genome in the plasmid vector. MSV can also be mechanically transmitted from infected leaf extracts using vascular puncture inoculation (VPI) (Louie, 1995). In VPI, a jeweler’s engraving tool is used to push small pins through a droplet of virus inoculum toward the major vascular bundle in the scutellum of germinating kernels (Louie, 1995). The technique is effective for transmission of all tested maize viruses without using arthropods or other biological vectors (Louie, 1995; Louie et al., 2000). Recently, MSV was transmitted by VPI from Escherichia coli plasmids carrying a tandem repeat of the MSV genome (Redinbaugh et al., 2001). In general, clones carrying tandem repeats of geminivirus genomes are infectious after agroinoculation, but constructs with partially repeated genomes are also infective. For Beet curly top virus (BCTV) and Wheat dwarf virus (WDV), the viral sequences between the two
0166-0934/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 3 ) 0 0 0 4 4 - 2
96
M.G. Redinbaugh / Journal of Virological Methods 109 (2003) 95 /98
hairpin structures was preferentially released after inoculation (Heyraud et al., 1993; Stenger et al., 1991). Similarly, agroinoculation with binary plasmids carrying partially repeated MSV genome constructs produced infection (Boulton et al., 1989). In addition, several bipartite geminiviruses can be transmitted from unitlength genome constructs using either a biolistic procedure or agroinoculation (Bonilla-Ramı´rez et al., 1997; Elmer et al., 1988; Gilbertson et al., 1991; Morris et al., 1988; Sung and Coutts, 1995). However, the infectivity of unit-length mastrevirus genomes such as that of MSV has not been reported. The construction of plasmids carrying tandemly repeated (Fig. 1A) and unit-length MSV genomes (Fig. 1B) was described previously (Redinbaugh et al., 2001). The free linear MSV genome was released by digestion of plasmid carrying the MSV unit-length genome with Bam HI (Fig. 1C). The circularized MSV genome was obtained by treating the digested DNA with T4 DNA ligase (Fig. 1D). Prior to inoculation, DNA was extracted with phenol/chloroform, precipitated with sodium acetate and ethanol, and resuspended in 10 mM Tris, pH 8, 1 mM EDTA. Maize kernels were inoculated with 0.5 mg DNA by VPI as described (Redinbaugh et al., 2001). After 48 h at 30 8C, the
kernels were planted into greenhouse soil and maize seedlings were evaluated for symptom appearance in a growth chamber with a 16 h/27 8C light (200 mmol/m2/s) and an 8 h/18 8C dark period for 3 weeks post VPI. The plasmid carrying the tandemly repeated MSV genome infected 339/8% (mean9/s.e., n /5) of the seedlings after VPI (Table 1), a slightly lower infection rate than was found in the previous study (Redinbaugh et al., 2001). In contrast, VPI with plasmid carrying the MSV unit-length genome produced no infection. However, inoculation of kernels with the free linear MSV genome (i.e. the Bam HI-digested MSV unit-length genome plasmid in Fig. 1C) produced symptoms on 159/2% of the seedlings. The circularized MSV genome (Fig. 1D) also incited symptoms on 169/4% of seedlings. VPI with an extract prepared from symptomatic leaves of plants inoculated with the MSV genome tandem repeat plasmid gave 529/5% infection (mean9/s.e., n / 3), while extracts of plants inoculated with the free linear MSV genome and circularized MSV genome gave 599/ 11 and 649/2% infection, respectively (Table 2). Thus, there were no major differences in infectivity of extracts prepared from plants inoculated with three constructs. Symptom development and severity were similar on all infected plants, regardless of the inoculum used (data
Fig. 1. MSV DNAs used for vascular puncture inoculation of maize kernels. (A) Plasmid (pUC19) carrying a head-to-tail tandem repeat of the MSV genome inserted into the Bam HI cloning site. (B) The single unit-length copy of the MSV genome in the Bam HI site of pUC19. (C) The unit-length MSV genome plasmid digested with Bam HI (free linear MSV genome). (D) The free linear MSV genome treated with T4 DNA ligase (circularized MSV genome).
M.G. Redinbaugh / Journal of Virological Methods 109 (2003) 95 /98
97
Table 1 Infectivity of MSV DNAs by vascular puncture inoculation of maize kernels Symptomatic/germinateda
DNA inoculum
MSV unit-length genome in pUC19 Free linear unit-length MSV genome Circularized unit-length MSV genome MSV genome tandem repeat in pUC19
Exp. 1
´ xp. 2 E
Exp. 3
Exp. 4
Exp. 5
0/21 3/25 3/23 12/21
0/21 3/19 6/12 9/23
0/23 5/23 4/21 4/24
0/23 2/18 1/20 4/23
0/37 7/43 8/45 17/49
a
The number of symptomatic plants/the number of plants germinated out of 25 (Exp. 1 /4) or 50 (Exp. 5) inoculated kernels. Data are presented for five independent experiments.
Table 2 Bioassay of symptomatic plants inoculated with MSV DNA Extract inoculumb
Free linear unit-length MSV genome Circularized unit-length MSV genome MSV genome tandem repeat in pUC19
Symptomatic/germinateda Exp. 1
Exp. 2
Exp. 3
18/44 26/43 21/47
27/47 25/37 21/43
35/44 26/40 29/47
a The number of symptomatic seedlings and the number of seedlings germinated out of 50 inoculated kernels (Pioneer hybrid 3379). The data are for three independent experiments. b The inoculum was made by grinding symptomatic leaves from plants inoculated with MSV plasmid DNA as indicated.
not shown), indicating that the different incula produced a similar infection of maize. In a previous study, native MSV was produced in plants inoculated with plasmid carrying the tandemly repeated MSV genome, as indicated by the production of the MSV virion and capsid protein (Redinbaugh et al., 2001). To test for rearrangements in the MSV genome in planta, DNA was isolated from leaves from plants inoculated with MSV containing extracts. Total DNA was extracted as described (Redinbaugh et al., 2001), digested with Bam HI, Eco RV, Hind III or Sma I, separated on agarose gels, transferred to a nylon membrane and probed with MSV DNA. For all three samples, the observed banding patterns were those expected based on the sequence of the cloned MSVKm plasmids (data not shown). The data indicate that the MSV infections produced by inoculation with plasmid carrying tandemly repeated MSV genomes, the free linear MSV unit-length genome and the circularized MSV unit-length genome were similar. The free linear MSV genome and the circularized MSV genome were infectious when introduced into maize kernels by VPI, but the MSV unit-length genome in pUC19 was not. Bonilla-Ramı´rez et al., (1997) demonstrated that the infectivity of Pepper huasteco virus (PHV) unit-length genomes in plasmids depended on the position of the cloning site in the viral genome. For this begomovirus, cloning of DNA A using a site in the Rep gene or DNA B using a site in the BV1 gene
greatly reduced virus infectivity, whereas intact plasmids that used cloning sites in the coat protein in DNA A or BC1 gene in DNA B were infectious. The Bam HI site of the MSV-Km genome used in this study is immediately after the first ATG of the movement protein (Boulton et al., 1993; Dickinson et al., 1996). Since this protein has analogous function to the BC1 protein and insertion of plasmid DNA into the BC1 gene had no effect on infectivity, the lack of infectivity of this MSV plasmid was somewhat surprising. However, the lack of infectivity may be related to the specific insertion site or to some, as yet undefined, function of the movement protein (Liu et al., 2001). Alternatively, the MSV genome may be inefficiently released from the plasmid in maize, or the release may result in loss of MSV sequence. Similarly to PHV (Bonilla-Ramı´rez et al., 1997), there was no difference in infectivity between linear unit-length MSV genomes and those that were ligated after digestion, nor were there inoculum-dependent differences in symptom severity or development. For PHV, it was proposed that the linear genomic DNA was ligated by plant enzymes after entry into the cell. The rate of transmission by VPI for MSV unit-length genomes (15%) was relatively low compared with 33/ 55% transmission of the tandemly repeated MSV genome in pUC19 (Table 1 and Redinbaugh et al., 2001). Transmission of the virus by VPI from extracts of MSV-infected leaves was more efficient at 58% (Table 2). MSV transmission rates after agroinoculation varied depending on the virus isolate, the Agrobacterium strain and the maize line used (Boulton et al., 1989; Martin and Rybicki, 2000; Schnippenkoetter et al., 2001). However, the highest rates of transmission exceeded 80% suggesting that agroinoculation might be more efficient for transmission of MSV than VPI. Nonetheless, the ability to transmit MSV by VPI using either extracts of infected leaves or DNA from cloned viral genomes is attractive, given the relative simplicity of the technique. In particular, inoculation with free linear unit-length MSV genomes would be an advantage in experiments involving the generation and analysis of multiple molecular variants (e.g. characterization functional domains within viral proteins), because there is no
98
M.G. Redinbaugh / Journal of Virological Methods 109 (2003) 95 /98
need to construct tandem repeat plasmids for each construct and move them to binary Agrobacterium vectors. In summary, similarly to bipartite geminiviruses, free linear and circularized unit-length genomes of MSV are infectious when maize kernels were inoculated by VPI. The infections produced from these inoculations were not distinguished from those produced by inoculation with plasmids carrying tandem repeats of the MSV genome. This finding can save researchers several steps in the development and testing of genetically modified MSV DNAs.
Acknowledgements I thank Kristen Willie and John Abt for skillful technical assistance, Richard Edema and Don Gordon (Ohio State University) for providing the MSV clones, and Daniel Wilkinson (Pioneer HiBred Int., Inc.) for supplying corn seed. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.
References Bonilla-Ramı´rez, G.M., Guevara-Gonza´lez, R.G., Garzon-Tizna´do, J.A., Ascencio-Iban˜ez, J.T., Torres-Pacheco, I., Rivera-Bustamante, R.F., 1997. Analysis of the infectivity of monomeric clones of Pepper huasteco virus . J. Gen. Virol. 78, 947 /951. Bosque-Pe´rez, N.A., 2000. Eight decades of Maize streak virus research. Virus Res. 71, 107 /121. Boulton, M.I., Buchholz, W.G., Marks, M.S., Markham, P.G., Davies, J.W., 1989. Specificity of Agrobacterium -mediated delivery of maize streak virus-DNA to members of the Gramineae. Plant Mol. Biol. 12, 31 /40. Boulton, M.I., Pallaghy, C.K., Chatani, M., Macfarlane, S., Davies, J.W., 1993. Replication of Maize streak virus mutants in maize protoplasts-evidence for a movement protein. Virology 192, 85 /93. Dickinson, V.J., Halder, J., Woolston, C.J., 1996. The product of Maize streak virus ORF V1 is associated with secondary plasmo-
desmata and is first detected with the onset of viral lesions. Virology 220, 51 /59. Elmer, J.S., Sunter, G., Gardiner, W.E., Brand, L., Browning, C.K., Bisaro, D.M., Rogers, S.G., 1988. Agrobacterium -mediated inoculation of plants with Tomato golden mosaic virus DNAs. Plant Mol. Biol. 10, 225 /234. Gilbertson, R.L., Faria, J.C., Hanson, S.F., Morales, F.J., Ahlquist, P., Maxwell, D.P., Russell, D.R., 1991. Cloning of the complete DNA genomes of 4 bean-infecting geminiviruses and determining their infectivity by electric-discharge particle acceleration. Phytopathology 81, 980 /985. Grimsley, N., Hohn, T., Davies, J.W., Hohn, B., 1987. Agrobacterium mediated delivery of infectious Maize streak virus into maize plants. Nature 325, 177 /179. Heyraud, F., Matzeit, V., Kammann, M., Schaefer, S., Schell, J., Gronenborn, B., 1993. Identification of the initiation sequence for viral-strand DNA-synthesis of Wheat dwarf virus . EMBO J. 12, 4445 /4452. Liu, H.T., Boulton, M.I., Oparka, K.J., Davies, J.W., 2001. Interaction of the movement and coat proteins of Maize streak virus: implications for the transport of viral DNA. J. Gen. Virol. 82, 35 / 44. Louie, R., 1995. Vascular puncture of maize kernels for the mechanical transmission of Maize white line mosaic virus and other viruses of maize. Phytopathology 85, 139 /143. Louie, R., Redinbaugh, M.G., Gordon, D.T., Abt, J.J., Anderson, R.J., 2000. Maize necrotic streak virus, a new maize virus with similarity to species of the family Tombusviridae. Plant Dis. 84, 1133 /1139. Martin, D.P., Rybicki, E.P., 2000. Improved efficiency of Zea mays agroinoculation with Maize streak virus . Plant Dis. 84, 1096 /1098. Morris, B.A.M., Richardson, K.A., Andersen, M.T., Gardner, R.C., 1988. Cassava latent virus infections mediated by the Ti-plasmid of Agrobacterium tumefaciens containing either monomeric or chimeric viral DNA. Plant Mol. Biol. 11, 795 /803. Redinbaugh, M.G., Louie, R., Ngwira, P., Edema, R., Gordon, D.T., Bisaro, D.M., 2001. Transmission of viral RNA and DNA to maize kernels by vascular puncture inoculation. J. Virol. Methods 98, 135 /143. Schnippenkoetter, W.H., Martin, D.P., Hughes, F.L., Fyvie, M., Willment, J.A., James, D., Von Wechmar, M.B., Rybicki, E.P., 2001. The relative infectivities and genomic characterization of three distinct mastreviruses from South Africa. Arch. Virol. 146, 1075 /1088. Stenger, D.C., Revington, G.N., Stevenson, M.C., Bisaro, D.M., 1991. Replicational release of geminivirus genomes from tandemly repeated copies-evidence for rolling-circle replication of a plant viral-DNA. Proc. Natl. Acad. Sci. USA 88, 8029 /8033. Sung, Y.K., Coutts, R.H.A., 1995. Mutational analysis of Potato yellow mosaic geminivirus . J. Gen. Virol. 76, 1773 /1780.
Brief report
Transmission of Maize streak virus by vascular puncture inoculation with unit-length genomic DNA M.G. Redinbaugh * USDA-ARS Corn and Soybean Research and Department of Plant Pathology, Ohio Agriculture Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691, USA Received 19 September 2002; received in revised form 7 January 2003; accepted 8 January 2003
Abstract The infectivity of cloned unit-length genomes of Maize streak virus (MSV) was tested using vascular puncture inoculation (VPI). VPI of kernels with plasmid DNA (pUC19) carrying a tandem repeat of the MSV genome produced 339/8% infection. Similar plasmids carrying the unit-length MSV genome were not infectious. If the MSV genome was released from the plasmid prior to VPI, 169/4% of plants became infected. Ligation of the free linear MSV genome did not increase infectivity. The three infective inocula produced symptoms of similar severity in maize. Bioassay of systemically infected leaves indicated the virus was equally infectious regardless of inoculum. In Southern blots of bioassay plants, no differences in MSV genome restriction endonuclease sites were observed. Thus, inoculation with the free linear or circularized MSV unit-length genome produced infections similar to those with plasmids carrying tandemly repeated genomes. The infectivity of free linear MSV unit-length genomes will facilitate molecular analysis of MSV, because cloning steps are minimized. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Maize streak virus ; Vascular puncture inoculation; Genomic DNA; Geminivirus
Geminiviruses are plant viruses with characteristic circular single-stranded DNA genomes and geminate virions (Bosque-Pe´rez, 2000). The viruses are classified into three genera based on their genomic organization, insect vector and plant host range. Maize streak virus (MSV), the type member of the genus Mastrevirus , is a monopartite geminivirus that causes significant agronomic problems on corn in sub-Saharan Africa. MSV is transmitted by leafhoppers (Circadulina species) to the phloem sieve tubes of host plants, and cannot be transmitted by leaf-rub inoculation. For many years, study of MSV outside of Africa was limited by difficulties with transmitting the virus and maintaining virus cultures. Similarly to other geminiviruses, MSV may be transmitted after inoculation of Agrobacterium tumefaciens carrying binary plasmids harboring a tandem repeat of the MSV genome in a process referred to as agroinoculation (Grimsley et al.,
* Tel.: /1-330-263-3965; fax: /1-330-263-3841. E-mail address: [email protected] (M.G. Redinbaugh).
1987). While there have been several improvements in agroinoculation (Martin and Rybicki, 2000), it requires manipulation of relatively complex binary vectors, and has a high degree of specificity for the Agrobacterium strain and the orientation of the viral genome in the plasmid vector. MSV can also be mechanically transmitted from infected leaf extracts using vascular puncture inoculation (VPI) (Louie, 1995). In VPI, a jeweler’s engraving tool is used to push small pins through a droplet of virus inoculum toward the major vascular bundle in the scutellum of germinating kernels (Louie, 1995). The technique is effective for transmission of all tested maize viruses without using arthropods or other biological vectors (Louie, 1995; Louie et al., 2000). Recently, MSV was transmitted by VPI from Escherichia coli plasmids carrying a tandem repeat of the MSV genome (Redinbaugh et al., 2001). In general, clones carrying tandem repeats of geminivirus genomes are infectious after agroinoculation, but constructs with partially repeated genomes are also infective. For Beet curly top virus (BCTV) and Wheat dwarf virus (WDV), the viral sequences between the two
0166-0934/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 3 ) 0 0 0 4 4 - 2
96
M.G. Redinbaugh / Journal of Virological Methods 109 (2003) 95 /98
hairpin structures was preferentially released after inoculation (Heyraud et al., 1993; Stenger et al., 1991). Similarly, agroinoculation with binary plasmids carrying partially repeated MSV genome constructs produced infection (Boulton et al., 1989). In addition, several bipartite geminiviruses can be transmitted from unitlength genome constructs using either a biolistic procedure or agroinoculation (Bonilla-Ramı´rez et al., 1997; Elmer et al., 1988; Gilbertson et al., 1991; Morris et al., 1988; Sung and Coutts, 1995). However, the infectivity of unit-length mastrevirus genomes such as that of MSV has not been reported. The construction of plasmids carrying tandemly repeated (Fig. 1A) and unit-length MSV genomes (Fig. 1B) was described previously (Redinbaugh et al., 2001). The free linear MSV genome was released by digestion of plasmid carrying the MSV unit-length genome with Bam HI (Fig. 1C). The circularized MSV genome was obtained by treating the digested DNA with T4 DNA ligase (Fig. 1D). Prior to inoculation, DNA was extracted with phenol/chloroform, precipitated with sodium acetate and ethanol, and resuspended in 10 mM Tris, pH 8, 1 mM EDTA. Maize kernels were inoculated with 0.5 mg DNA by VPI as described (Redinbaugh et al., 2001). After 48 h at 30 8C, the
kernels were planted into greenhouse soil and maize seedlings were evaluated for symptom appearance in a growth chamber with a 16 h/27 8C light (200 mmol/m2/s) and an 8 h/18 8C dark period for 3 weeks post VPI. The plasmid carrying the tandemly repeated MSV genome infected 339/8% (mean9/s.e., n /5) of the seedlings after VPI (Table 1), a slightly lower infection rate than was found in the previous study (Redinbaugh et al., 2001). In contrast, VPI with plasmid carrying the MSV unit-length genome produced no infection. However, inoculation of kernels with the free linear MSV genome (i.e. the Bam HI-digested MSV unit-length genome plasmid in Fig. 1C) produced symptoms on 159/2% of the seedlings. The circularized MSV genome (Fig. 1D) also incited symptoms on 169/4% of seedlings. VPI with an extract prepared from symptomatic leaves of plants inoculated with the MSV genome tandem repeat plasmid gave 529/5% infection (mean9/s.e., n / 3), while extracts of plants inoculated with the free linear MSV genome and circularized MSV genome gave 599/ 11 and 649/2% infection, respectively (Table 2). Thus, there were no major differences in infectivity of extracts prepared from plants inoculated with three constructs. Symptom development and severity were similar on all infected plants, regardless of the inoculum used (data
Fig. 1. MSV DNAs used for vascular puncture inoculation of maize kernels. (A) Plasmid (pUC19) carrying a head-to-tail tandem repeat of the MSV genome inserted into the Bam HI cloning site. (B) The single unit-length copy of the MSV genome in the Bam HI site of pUC19. (C) The unit-length MSV genome plasmid digested with Bam HI (free linear MSV genome). (D) The free linear MSV genome treated with T4 DNA ligase (circularized MSV genome).
M.G. Redinbaugh / Journal of Virological Methods 109 (2003) 95 /98
97
Table 1 Infectivity of MSV DNAs by vascular puncture inoculation of maize kernels Symptomatic/germinateda
DNA inoculum
MSV unit-length genome in pUC19 Free linear unit-length MSV genome Circularized unit-length MSV genome MSV genome tandem repeat in pUC19
Exp. 1
´ xp. 2 E
Exp. 3
Exp. 4
Exp. 5
0/21 3/25 3/23 12/21
0/21 3/19 6/12 9/23
0/23 5/23 4/21 4/24
0/23 2/18 1/20 4/23
0/37 7/43 8/45 17/49
a
The number of symptomatic plants/the number of plants germinated out of 25 (Exp. 1 /4) or 50 (Exp. 5) inoculated kernels. Data are presented for five independent experiments.
Table 2 Bioassay of symptomatic plants inoculated with MSV DNA Extract inoculumb
Free linear unit-length MSV genome Circularized unit-length MSV genome MSV genome tandem repeat in pUC19
Symptomatic/germinateda Exp. 1
Exp. 2
Exp. 3
18/44 26/43 21/47
27/47 25/37 21/43
35/44 26/40 29/47
a The number of symptomatic seedlings and the number of seedlings germinated out of 50 inoculated kernels (Pioneer hybrid 3379). The data are for three independent experiments. b The inoculum was made by grinding symptomatic leaves from plants inoculated with MSV plasmid DNA as indicated.
not shown), indicating that the different incula produced a similar infection of maize. In a previous study, native MSV was produced in plants inoculated with plasmid carrying the tandemly repeated MSV genome, as indicated by the production of the MSV virion and capsid protein (Redinbaugh et al., 2001). To test for rearrangements in the MSV genome in planta, DNA was isolated from leaves from plants inoculated with MSV containing extracts. Total DNA was extracted as described (Redinbaugh et al., 2001), digested with Bam HI, Eco RV, Hind III or Sma I, separated on agarose gels, transferred to a nylon membrane and probed with MSV DNA. For all three samples, the observed banding patterns were those expected based on the sequence of the cloned MSVKm plasmids (data not shown). The data indicate that the MSV infections produced by inoculation with plasmid carrying tandemly repeated MSV genomes, the free linear MSV unit-length genome and the circularized MSV unit-length genome were similar. The free linear MSV genome and the circularized MSV genome were infectious when introduced into maize kernels by VPI, but the MSV unit-length genome in pUC19 was not. Bonilla-Ramı´rez et al., (1997) demonstrated that the infectivity of Pepper huasteco virus (PHV) unit-length genomes in plasmids depended on the position of the cloning site in the viral genome. For this begomovirus, cloning of DNA A using a site in the Rep gene or DNA B using a site in the BV1 gene
greatly reduced virus infectivity, whereas intact plasmids that used cloning sites in the coat protein in DNA A or BC1 gene in DNA B were infectious. The Bam HI site of the MSV-Km genome used in this study is immediately after the first ATG of the movement protein (Boulton et al., 1993; Dickinson et al., 1996). Since this protein has analogous function to the BC1 protein and insertion of plasmid DNA into the BC1 gene had no effect on infectivity, the lack of infectivity of this MSV plasmid was somewhat surprising. However, the lack of infectivity may be related to the specific insertion site or to some, as yet undefined, function of the movement protein (Liu et al., 2001). Alternatively, the MSV genome may be inefficiently released from the plasmid in maize, or the release may result in loss of MSV sequence. Similarly to PHV (Bonilla-Ramı´rez et al., 1997), there was no difference in infectivity between linear unit-length MSV genomes and those that were ligated after digestion, nor were there inoculum-dependent differences in symptom severity or development. For PHV, it was proposed that the linear genomic DNA was ligated by plant enzymes after entry into the cell. The rate of transmission by VPI for MSV unit-length genomes (15%) was relatively low compared with 33/ 55% transmission of the tandemly repeated MSV genome in pUC19 (Table 1 and Redinbaugh et al., 2001). Transmission of the virus by VPI from extracts of MSV-infected leaves was more efficient at 58% (Table 2). MSV transmission rates after agroinoculation varied depending on the virus isolate, the Agrobacterium strain and the maize line used (Boulton et al., 1989; Martin and Rybicki, 2000; Schnippenkoetter et al., 2001). However, the highest rates of transmission exceeded 80% suggesting that agroinoculation might be more efficient for transmission of MSV than VPI. Nonetheless, the ability to transmit MSV by VPI using either extracts of infected leaves or DNA from cloned viral genomes is attractive, given the relative simplicity of the technique. In particular, inoculation with free linear unit-length MSV genomes would be an advantage in experiments involving the generation and analysis of multiple molecular variants (e.g. characterization functional domains within viral proteins), because there is no
98
M.G. Redinbaugh / Journal of Virological Methods 109 (2003) 95 /98
need to construct tandem repeat plasmids for each construct and move them to binary Agrobacterium vectors. In summary, similarly to bipartite geminiviruses, free linear and circularized unit-length genomes of MSV are infectious when maize kernels were inoculated by VPI. The infections produced from these inoculations were not distinguished from those produced by inoculation with plasmids carrying tandem repeats of the MSV genome. This finding can save researchers several steps in the development and testing of genetically modified MSV DNAs.
Acknowledgements I thank Kristen Willie and John Abt for skillful technical assistance, Richard Edema and Don Gordon (Ohio State University) for providing the MSV clones, and Daniel Wilkinson (Pioneer HiBred Int., Inc.) for supplying corn seed. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.
References Bonilla-Ramı´rez, G.M., Guevara-Gonza´lez, R.G., Garzon-Tizna´do, J.A., Ascencio-Iban˜ez, J.T., Torres-Pacheco, I., Rivera-Bustamante, R.F., 1997. Analysis of the infectivity of monomeric clones of Pepper huasteco virus . J. Gen. Virol. 78, 947 /951. Bosque-Pe´rez, N.A., 2000. Eight decades of Maize streak virus research. Virus Res. 71, 107 /121. Boulton, M.I., Buchholz, W.G., Marks, M.S., Markham, P.G., Davies, J.W., 1989. Specificity of Agrobacterium -mediated delivery of maize streak virus-DNA to members of the Gramineae. Plant Mol. Biol. 12, 31 /40. Boulton, M.I., Pallaghy, C.K., Chatani, M., Macfarlane, S., Davies, J.W., 1993. Replication of Maize streak virus mutants in maize protoplasts-evidence for a movement protein. Virology 192, 85 /93. Dickinson, V.J., Halder, J., Woolston, C.J., 1996. The product of Maize streak virus ORF V1 is associated with secondary plasmo-
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