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Viruses making plants greener
Outlook
LETTERS
Mono-allelic expression
choice at the transcriptional level6,7, not unlike the choice made for the olfactory receptor genes in human cells8,9. The African trypanosome T. brucei has about 20 different expression sites for variant surface glycoproteins and usually only one of these is expressed at a time10,11. It was long thought that these expression sites are independently controlled by stochastic silencing events akin to telomeric silencing in yeast12–14. Recent work has shown, however, that there is some kind of cross-talk between these sites and that a trypanosome cannot stably maintain high-level transcription of two expression sites simultaneously (Ref. 11, and I. Chaves and P. Borst, unpublished). Trypanosomes have obvious advantages for a study of mechanisms of mono-allelic gene expression. Allelic exclusion of expression sites is available. Expression sites References 1 Ohlsson, R. et al. (1998) Monoallelic expression: ‘there can only be one’. Trends Genet. 14, 435–438 2 Borst, P. (1991) Molecular genetics of antigenic variation. Immunol. Today 12, A29–A33 3 Borst, P. et al. (1997) Mechanisms of antigenic variation in African trypanosomes. Behring Inst. Mitt. 99, 1–15 4 Borst, P., Bitter, W., McCulloch, R., Van Leeuwen, F. and Rudenko, G. (1995) Antigenic variation in malaria. Cell 82, 1–4 5 Deitsch, K.W. and Wellems, T.E. (1996) Membrane modifications in erythrocytes parasitized by Plasmodium falciparum. Mol. Biochem. Parasitol. 76, 1–10 6 Chen, Q. et al. (1998) Developmental selection of var gene expression in Plasmodium falciparum. Nature 394, 392–395 7 Scherf, A. et al. (1998) Antigenic variation in malaria: in situ
Outlook
8 9 10
11 12
13
can be made individually recognizable by inserting unique selectable markers. Trypanosomes expressing these markers can be selected in vitro and the markers can be localized in the nucleus15. In this way putative intermediates in switching from one expression site to another one have been isolated (I. Chaves and P. Borst, unpublished). Trypanosomes even have a modified DNA base J, found in inactive, but not active expression sites16,17. Although we do not want to suggest that all mechanisms of mono-allelic expression of genes in mammals have been copied from the wonderfully diverse and versatile world of protozoa, some mechanisms may turn out to be conserved in evolution. It could therefore be useful for mammalian molecular geneticists to keep an eye on the protozoal scene.
switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum. EMBO J. 17, 5418–5426 Efstratiadis, A. (1995) A new whiff of monoallelic expression. Curr. Biol. 5, 21–24 Chess, A. (1998) Expansion of the allelic exclusion principle? Science 279, 2067–2068 Cross, G.A.M. et al. (1998) Regulation of vsg expression site transcription and switching in Trypanosoma brucei. Mol. Biochem. Parasitol. 91, 77–91 Borst, P. et al. (1998) Control of VSG gene expression sites in Trypanosoma brucei. Mol. Biochem. Parasitol. 91, 67–76 Gottschling, D.E. et al. (1990) Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63, 751–762 Rudenko, G. et al. (1995) A ribosomal DNA promoter replacing
14
15
16
17
JOURNAL CLUB
Viruses making plants greener
Tom Brutnell tom.brutnell@ plant-sciences. oxford.ac.uk 96
the promoter of a telomeric variant surface glycoprotein gene expression site can be efficiently switched on and off in Trypanosoma brucei. Cell 83, 547–553 Horn, D. and Cross, G.A.M. (1995) A developmentally regulated position effect at a telomeric locus in Trypanosoma brucei. Cell 83, 555–561 Chaves, I. et al. (1998) Subnuclear localisation of the active variant surface glycoprotein gene expression site in Trypanosoma brucei. Proc. Natl. Acad. Sci. U. S. A. 95, 12328–12333 Van Leeuwen, F. et al. (1997) Localisation of the modified base J in telomeric VSG gene expression sites of Trypanosoma brucei. Genes Dev. 11, 3232–3241 Van Leeuwen, F. et al. (1998) Beta-D-Glucosylhydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres. Proc. Natl. Acad. Sci. U. S. A. 95, 2366–2371
The expression levels of transgenes introduced into plants can vary greatly between lines. In some cases, the levels of transgene transcripts that accumulate are below the limit of detection for northern analysis despite the use of strong constitutive promoters. Furthermore, if the transgene contains sequence homology to an endogenous gene, both transgene and endogenous gene transcripts are sometimes greatly reduced. This reduction in both transgene and endogenous gene transcripts is sometimes due to a post-transcriptional reduction of steady-state transcript levels. Termed post-transcriptional gene silencing (PTGS) this mechanism has recently been proposed as a means of detecting and combating viral infections in plants. Support for this idea has come from the finding that viral proteins can suppress PTGS thereby increasing the likelihood of viral infection. To investigate the role of viral proteins in suppressing PTGS, Brigneti et al.1 utilized transgenic lines of Nicotiana benthamiana that had been transformed with a green
TIG March 1999, volume 15, No. 3
fluorescent protein cassette. These GFP expressing plants were then inoculated with the same GFP cassette to induce PTGS. When silencing was complete (no green fluorescence), plants were infected with potato virus Y (PVY). After two weeks leaves showed symptoms of PVY infection. Importantly, the regions of the leaf that mottled and curled coincided with GFP fluorescence and increased GFP transcript levels. Thus, the PVY virus was able to overcome the RNA silencing mechanism as monitored by GFP expression. To further define the components of viralinduced suppression of PTGS, chimeric viruses were constructed using a potato virus X (PVX) vector. Because PVX does not suppress PTGS, the effects of putative PTGS-suppressing proteins of PVY or cucumber mosaic virus (CMV) could be tested directly. When plants were infected with either the HCPro protein of PVY or the 2b protein of CMV in a PVX cassette, plants showed severe infection symptoms and green fluorescence. Furthermore, these effects
were not observed when frame-shift mutations or premature stop codons were introduced into the HCPro or 2b proteins, respectively, indicating the PTGS silencing was not mediated by the transcripts encoding HCPro or 2b protein. As the authors suggest, although both proteins interfered with PTGS, they are unlikely to affect the same components of the PTGS pathway. HCProinfected plants suppressed PTGS in old and young leaves, while 2b-infected plants only showed increased GFP fluorescence in young leaves. Thus, HCPro may affect the maintenance of the PTGS pathway whereas the 2b protein may interfere with the entry of a gene silencing signal into older regions of the plant. These findings strongly suggest that PTGS has evolved as a means to control the infection and spreading of viruses within the plant and opens up new doors to the engineering viral resistance in crop plants.
1 Brigneti, G. et al. (1998) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17, 6739–6746
LETTERS
Mono-allelic expression
choice at the transcriptional level6,7, not unlike the choice made for the olfactory receptor genes in human cells8,9. The African trypanosome T. brucei has about 20 different expression sites for variant surface glycoproteins and usually only one of these is expressed at a time10,11. It was long thought that these expression sites are independently controlled by stochastic silencing events akin to telomeric silencing in yeast12–14. Recent work has shown, however, that there is some kind of cross-talk between these sites and that a trypanosome cannot stably maintain high-level transcription of two expression sites simultaneously (Ref. 11, and I. Chaves and P. Borst, unpublished). Trypanosomes have obvious advantages for a study of mechanisms of mono-allelic gene expression. Allelic exclusion of expression sites is available. Expression sites References 1 Ohlsson, R. et al. (1998) Monoallelic expression: ‘there can only be one’. Trends Genet. 14, 435–438 2 Borst, P. (1991) Molecular genetics of antigenic variation. Immunol. Today 12, A29–A33 3 Borst, P. et al. (1997) Mechanisms of antigenic variation in African trypanosomes. Behring Inst. Mitt. 99, 1–15 4 Borst, P., Bitter, W., McCulloch, R., Van Leeuwen, F. and Rudenko, G. (1995) Antigenic variation in malaria. Cell 82, 1–4 5 Deitsch, K.W. and Wellems, T.E. (1996) Membrane modifications in erythrocytes parasitized by Plasmodium falciparum. Mol. Biochem. Parasitol. 76, 1–10 6 Chen, Q. et al. (1998) Developmental selection of var gene expression in Plasmodium falciparum. Nature 394, 392–395 7 Scherf, A. et al. (1998) Antigenic variation in malaria: in situ
Outlook
8 9 10
11 12
13
can be made individually recognizable by inserting unique selectable markers. Trypanosomes expressing these markers can be selected in vitro and the markers can be localized in the nucleus15. In this way putative intermediates in switching from one expression site to another one have been isolated (I. Chaves and P. Borst, unpublished). Trypanosomes even have a modified DNA base J, found in inactive, but not active expression sites16,17. Although we do not want to suggest that all mechanisms of mono-allelic expression of genes in mammals have been copied from the wonderfully diverse and versatile world of protozoa, some mechanisms may turn out to be conserved in evolution. It could therefore be useful for mammalian molecular geneticists to keep an eye on the protozoal scene.
switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum. EMBO J. 17, 5418–5426 Efstratiadis, A. (1995) A new whiff of monoallelic expression. Curr. Biol. 5, 21–24 Chess, A. (1998) Expansion of the allelic exclusion principle? Science 279, 2067–2068 Cross, G.A.M. et al. (1998) Regulation of vsg expression site transcription and switching in Trypanosoma brucei. Mol. Biochem. Parasitol. 91, 77–91 Borst, P. et al. (1998) Control of VSG gene expression sites in Trypanosoma brucei. Mol. Biochem. Parasitol. 91, 67–76 Gottschling, D.E. et al. (1990) Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63, 751–762 Rudenko, G. et al. (1995) A ribosomal DNA promoter replacing
14
15
16
17
JOURNAL CLUB
Viruses making plants greener
Tom Brutnell tom.brutnell@ plant-sciences. oxford.ac.uk 96
the promoter of a telomeric variant surface glycoprotein gene expression site can be efficiently switched on and off in Trypanosoma brucei. Cell 83, 547–553 Horn, D. and Cross, G.A.M. (1995) A developmentally regulated position effect at a telomeric locus in Trypanosoma brucei. Cell 83, 555–561 Chaves, I. et al. (1998) Subnuclear localisation of the active variant surface glycoprotein gene expression site in Trypanosoma brucei. Proc. Natl. Acad. Sci. U. S. A. 95, 12328–12333 Van Leeuwen, F. et al. (1997) Localisation of the modified base J in telomeric VSG gene expression sites of Trypanosoma brucei. Genes Dev. 11, 3232–3241 Van Leeuwen, F. et al. (1998) Beta-D-Glucosylhydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres. Proc. Natl. Acad. Sci. U. S. A. 95, 2366–2371
The expression levels of transgenes introduced into plants can vary greatly between lines. In some cases, the levels of transgene transcripts that accumulate are below the limit of detection for northern analysis despite the use of strong constitutive promoters. Furthermore, if the transgene contains sequence homology to an endogenous gene, both transgene and endogenous gene transcripts are sometimes greatly reduced. This reduction in both transgene and endogenous gene transcripts is sometimes due to a post-transcriptional reduction of steady-state transcript levels. Termed post-transcriptional gene silencing (PTGS) this mechanism has recently been proposed as a means of detecting and combating viral infections in plants. Support for this idea has come from the finding that viral proteins can suppress PTGS thereby increasing the likelihood of viral infection. To investigate the role of viral proteins in suppressing PTGS, Brigneti et al.1 utilized transgenic lines of Nicotiana benthamiana that had been transformed with a green
TIG March 1999, volume 15, No. 3
fluorescent protein cassette. These GFP expressing plants were then inoculated with the same GFP cassette to induce PTGS. When silencing was complete (no green fluorescence), plants were infected with potato virus Y (PVY). After two weeks leaves showed symptoms of PVY infection. Importantly, the regions of the leaf that mottled and curled coincided with GFP fluorescence and increased GFP transcript levels. Thus, the PVY virus was able to overcome the RNA silencing mechanism as monitored by GFP expression. To further define the components of viralinduced suppression of PTGS, chimeric viruses were constructed using a potato virus X (PVX) vector. Because PVX does not suppress PTGS, the effects of putative PTGS-suppressing proteins of PVY or cucumber mosaic virus (CMV) could be tested directly. When plants were infected with either the HCPro protein of PVY or the 2b protein of CMV in a PVX cassette, plants showed severe infection symptoms and green fluorescence. Furthermore, these effects
were not observed when frame-shift mutations or premature stop codons were introduced into the HCPro or 2b proteins, respectively, indicating the PTGS silencing was not mediated by the transcripts encoding HCPro or 2b protein. As the authors suggest, although both proteins interfered with PTGS, they are unlikely to affect the same components of the PTGS pathway. HCProinfected plants suppressed PTGS in old and young leaves, while 2b-infected plants only showed increased GFP fluorescence in young leaves. Thus, HCPro may affect the maintenance of the PTGS pathway whereas the 2b protein may interfere with the entry of a gene silencing signal into older regions of the plant. These findings strongly suggest that PTGS has evolved as a means to control the infection and spreading of viruses within the plant and opens up new doors to the engineering viral resistance in crop plants.
1 Brigneti, G. et al. (1998) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17, 6739–6746