viruses making plants greener

LETTERS Mono-allelic expression

96

Outlook LETTERS

Outlook JOURNAL CLUB

TIG March 1999, volume 15, No. 3

References1 Ohlsson, R. et al. (1998) Monoallelic expression: ‘there can

only be one’. Trends Genet. 14, 435–4382 Borst, P. (1991) Molecular genetics of antigenic variation.

Immunol. Today 12, A29–A333 Borst, P. et al. (1997) Mechanisms of antigenic variation in

African trypanosomes. Behring Inst. Mitt. 99, 1–154 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) Membranemodifications in erythrocytes parasitized by Plasmodiumfalciparum. Mol. Biochem. Parasitol. 76, 1–10

6 Chen, Q. et al. (1998) Developmental selection of var geneexpression in Plasmodium falciparum. Nature 394, 392–395

7 Scherf, A. et al. (1998) Antigenic variation in malaria: in situ

switching, relaxed and mutually exclusive transcription of vargenes during intra-erythrocytic development in Plasmodiumfalciparum. EMBO J. 17, 5418–5426

8 Efstratiadis, A. (1995) A new whiff of monoallelic expression.Curr. Biol. 5, 21–24

9 Chess, A. (1998) Expansion of the allelic exclusion principle?Science 279, 2067–2068

10 Cross, G.A.M. et al. (1998) Regulation of vsg expression sitetranscription and switching in Trypanosoma brucei. Mol.Biochem. Parasitol. 91, 77–91

11 Borst, P. et al. (1998) Control of VSG gene expression sites inTrypanosoma brucei. Mol. Biochem. Parasitol. 91, 67–76

12 Gottschling, D.E. et al. (1990) Position effect at S. cerevisiaetelomeres: reversible repression of Pol II transcription. Cell 63,751–762

13 Rudenko, G. et al. (1995) A ribosomal DNA promoter replacing

the promoter of a telomeric variant surface glycoprotein geneexpression site can be efficiently switched on and off inTrypanosoma brucei. Cell 83, 547–553

14 Horn, D. and Cross, G.A.M. (1995) A developmentally regulatedposition effect at a telomeric locus in Trypanosoma brucei.Cell 83, 555–561

15 Chaves, I. et al. (1998) Subnuclear localisation of the activevariant surface glycoprotein gene expression site in Trypanosomabrucei. Proc. Natl. Acad. Sci. U. S. A. 95, 12328–12333

16 Van Leeuwen, F. et al. (1997) Localisation of the modifiedbase J in telomeric VSG gene expression sites of Trypanosomabrucei. Genes Dev. 11, 3232–3241

17 Van Leeuwen, F. et al. (1998) Beta-D-Glucosyl-hydroxymethyluracil is a conserved DNA modification inkinetoplastid protozoans and is abundant in their telomeres.Proc. Natl. Acad. Sci. U. S. A. 95, 2366–2371

choice at the transcriptional level6,7, not unlike the choicemade for the olfactory receptor genes in human cells8,9.

The African trypanosome T. brucei has about 20 differ-ent expression sites for variant surface glycoproteins andusually only one of these is expressed at a time10,11. It waslong thought that these expression sites are independentlycontrolled by stochastic silencing events akin to telomericsilencing in yeast12–14. Recent work has shown, however,that there is some kind of cross-talk between these sitesand that a trypanosome cannot stably maintain high-leveltranscription of two expression sites simultaneously (Ref.11, and I. Chaves and P. Borst, unpublished).

Trypanosomes have obvious advantages for a study ofmechanisms of mono-allelic gene expression. Allelicexclusion of expression sites is available. Expression sites

can be made individually recognizable by insertingunique selectable markers. Trypanosomes expressingthese markers can be selected in vitro and the markerscan be localized in the nucleus15. In this way putativeintermediates in switching from one expression site toanother one have been isolated (I. Chaves and P. Borst,unpublished). Trypanosomes even have a modified DNAbase J, found in inactive, but not active expressionsites16,17. Although we do not want to suggest that allmechanisms of mono-allelic expression of genes in mam-mals have been copied from the wonderfully diverse andversatile world of protozoa, some mechanisms may turnout to be conserved in evolution. It could therefore beuseful for mammalian molecular geneticists to keep aneye on the protozoal scene.

The expression levels of transgenesintroduced into plants can vary greatlybetween lines. In some cases, the levelsof transgene transcripts that accumulateare below the limit of detection fornorthern analysis despite the use ofstrong constitutive promoters. Further-more, if the transgene contains sequencehomology to an endogenous gene, bothtransgene and endogenous gene tran-scripts are sometimes greatly reduced.This reduction in both transgene andendogenous gene transcripts is some-times due to a post-transcriptionalreduction of steady-state transcript lev-els. Termed post-transcriptional genesilencing (PTGS) this mechanism hasrecently been proposed as a means ofdetecting and combating viral infectionsin plants. Support for this idea has comefrom the finding that viral proteins cansuppress PTGS thereby increasing thelikelihood of viral infection. To investigatethe role of viral proteins in suppressingPTGS, Brigneti et al.1 utilized transgeniclines of Nicotiana benthamiana thathad been transformed with a green

fluorescent protein cassette. These GFPexpressing plants were then inoculatedwith the same GFP cassette to inducePTGS. When silencing was complete(no green fluorescence), plants wereinfected with potato virus Y (PVY).After two weeks leaves showed symp-toms of PVY infection. Importantly, theregions of the leaf that mottled andcurled coincided with GFP fluorescenceand increased GFP transcript levels.Thus, the PVY virus was able to over-come the RNA silencing mechanism asmonitored by GFP expression. To fur-ther define the components of viral-induced suppression of PTGS, chimericviruses were constructed using a potatovirus X (PVX) vector. Because PVXdoes not suppress PTGS, the effects ofputative PTGS-suppressing proteins ofPVY or cucumber mosaic virus (CMV)could be tested directly. When plantswere infected with either the HCProprotein of PVY or the 2b protein ofCMV in a PVX cassette, plants showedsevere infection symptoms and green fluorescence. Furthermore, these effects

were not observed when frame-shiftmutations or premature stop codonswere introduced into the HCPro or 2bproteins, respectively, indicating thePTGS silencing was not mediated by thetranscripts encoding HCPro or 2b pro-tein. As the authors suggest, althoughboth proteins interfered with PTGS,they are unlikely to affect the same com-ponents of the PTGS pathway. HCPro-infected plants suppressed PTGS in oldand young leaves, while 2b-infectedplants only showed increased GFPfluorescence in young leaves. Thus,HCPro may affect the maintenance ofthe PTGS pathway whereas the 2b pro-tein may interfere with the entry of agene silencing signal into older regionsof the plant. These findings stronglysuggest that PTGS has evolved as ameans to control the infection andspreading of viruses within the plantand opens up new doors to the engi-neering viral resistance in crop plants.

Viruses making plants greener

1 Brigneti, G. et al. (1998) Viral pathogenicitydeterminants are suppressors of transgenesilencing in Nicotiana benthamiana. EMBO J.17, 6739–6746

Tom Brutnell

tom.brutnell@ plant-sciences.

oxford.ac.uk