Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5538-5542, June 1994 Genetics Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing (gene expresn reglanmodfer locus/Ambidopsis hla) CHRISTOPH DEHIO*t AND JEFF SCHELL*I *Max-Planck-Institut fur Zachtungsforschung, Carl-von-Linn6-Weg 10, 50829 Cologne, Federal Republic of Germany Contributed by JeffSchell, March 4, 1994

ABSTRACT Numerous reports describe phenomena of RoiB phenotype is cell autonomous (13), it was possible to ene ilencisg In plants, yet the underlying genetic and visually monitor rolB gene silencing in somatic tissues. We molecula mechans are poorly understood. We observed describe here a detailed phenotypic, molecular, and genetic that regeneration ofArabidopsis thalana plants tragenlc for characterization of a meiotically reversible transgene-silencing the rMD gene ofAgrobacterinum rhizogenes results In a selection phenomenon acting predominantly at the posttranscriptional for transgene sencing. Transgene silencing could be moni- level. Mutable plantloci are shown to control this phenomenon. tored in this system by reversion ofthe visible RolB phenotype. We report a phenotypc, molecular, and genetic characteriza- MATERIALS AND METHODS tion da melotically reversible transgene sencing phenomenon observed in a roM tranagenic line. In this line, the roW gene is Plant Trnsformation, Mutagenesis, Mutant Decon, and expressed strongly and unformly in seedlings, but in the course Genetic Analysis. Transformation of Arabidopsis thaliana of further development, the roiD gene Is silenced erratically at Col-0 with A. tumefaciens [strain GV3101 (pMP90RK)] car- a frequency that depends on the dosage of roB. The silenced rying plasmid pPCV002-CaMVBT (14) as well as the ethyl state Is mitotically stable, while complete resetting ofroWB gene methanesulfonate (EMS) mutagenesis ofthe generated trans- expression occurs in Seedlings of the following generation. The genic line rolB-2 were performed as described (15). For each s g of roB correlates with a dramatic reduction of of 14 independent M2 families, 5000 seedlings were screened steady-state roWB scripts, while roWB nuclear run-off tran- for a reverted phenotype 8 days after sowing on solidified scripts are only moderately reduced. Therefore, roWB gene Murashige and Skoog medium. The complementation anal- slencing seems to act predomlnantly at the p tional ysis of isolated mutants (Table 1) was done by reciprocal level. The process of roW gene slencing was found to be crosses between each pair of mutants and analysis of the affected by two extragenic modifer loci that influence both the seedling phenotype of the F1 progeny. The frequencies of frequency and the dtming of roWB gene slencing during plant revertant plants (Table 2) were determined by scoring the development. These genetic data demonstrate a direct involve- plants 8 days after sowing for seedlings showing a completely ment of defined plant genes in this form of gene slencing. reverted phenotype as well as 35 days after sowing for plants showing a reverted phenotype of the main shoot. The development oftechniques to introduce foreign DNA into RNA Analysis. The isolation, separation, transfer, and the plant genome has hastened the use of reverse genetics to hybridization of poly(A)+ RNA from plants were described study basic aspects of plant biochemistry, physiology, and before (15). A 2.2-kb EcoRI fragment of plasmid pPCV002- development (for review, see ref. 1). Although the introduced CaMVBT (14) (rolB probe), a 1.3-kb Pvu I/EcoRI fiagment DNA is physically transmitted as a dominant Mendelian trait of plasmid pPCV002 (16) (nptII probe), and a 0.95-kb EcoRI (2), the expression oftransgene-encoded phenotypes was found fragment of plasmid Pc-UBI 4 (17) (ubiquitin probe) were to be unstable in many cases. Several phenomena oftransgene used for hybridizations. Ioation ofNude and Nulear Run-Off n . silencing have been reported (forreviews, see refs. 3-5), which Nucleiwere isolated andhandledat4C. Plantmaterial (5 g) was correlated with transgene copy number (6), state ofmethylation ground to fine powder in liquid nitrogen, suspended in 40 ml of (6, 7), homology of the transgene to endogenous genes (8, 9), buffer A (250 mM sucrose/10 mM NaCl/10 mM Mes, pH 6.6/5 and the homozygous state ofthe transgene (10, 11). However, mM EDTA/0.15 mM spermine hydrochloride/0.5 mM spermi- ourunderstandingofthebasic mechanisms governing transgene dine phosphate/20 mM 2-mercaptoethanol/0.2 mM phenyl- silencing is still very limited. This is partly due to the lack ofan methylsulfonyl fluoride) supplemented with 0.6% Triton X-100 appropriate system allowing a combined genetic and molecular and 2% dextran T40, and briefly homogenized with an Ultra- study of transgene silencing. Indeed, transgene silencing was Tunrax (IKA-Works, Cincinnati). Afterfiltration through50-pin often found accidentally rather than being the primary focus of nylon mesh the filtrate was centrifuged for 5 min at 1500 x g. the experimental design. We introduced a suitable tester gene The pellet was resuspended in 2 ml ofthe same buffer and was into Arabidopsis to study transgene silencing. The advantages loaded on a stepgradient consistingofconsecutive 3.5-mllayers of Arabidopsis as a model system for plant genetics and mo- of 2 M sucrose and 80%6, 60%6, and 40% (vol/vol) Percoll in lecular biology are well documented (for review, see ref. 12). buffer A, followed by a centrifugation for 15 min at 1000 x g. The rolB gene from the Ri-TL DNA of Agrobacterium rhizo- Nuclei were taken from the interface between the sucrose layer genes, when expressed by a constitutive promoter in transgenic and 80% Percoll, diluted 10-fold in buffer B [50 mM Tris HCl, tobacco callus, was reported to inhibit the regeneration of pH 7.8/5 mM MgCl2/20%o (wt/vol) glycerol/10 mM 2-mercap- transgenicplants (13). InArabidopsis, wetookadvantageofthis 5 x property ofthe rolB gene to selectfortransgene silencingduring toethanol], and centriftued for min at 3000 g. The washing the regeneration oftransgenic plants. Furthermore, because the Abbreviations: EMS, ethyl methanesulfonate; CaMV, cauliflower mosaic virus. The publication costs ofthis article were defrayed in part by page charge tPresent address: Institut Pasteur, 28 rue du Dr Roux, 75724 Paris payment. This article must therefore be hereby marked "advertisement" Cedex 15, France. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5538 Downloaded by guest on September 30, 2021 Genetics: Dehio and Scheff Proc. Natl. Acad. Sci. USA 91 (1994) 5539

Table 1. Crosses and complementation analysis of EMS mutants that cause rolB gene silencing in seedlings Female Male parent parent Mi-i M6-1 M8-1 M14-1 M15-1 Wild type rolB-2 Mi-i 570/575 0/26 0/14 19/19 2/2 0/149 0/137 M6-1 0/38 119/119 29/29 0/24 0/54 0/54 0/44 M8-1 0/26 34/36 98/98 0/12 0/14 0/43 0/45 M14-1 65/66 0/81 0/88 98/98 100/102 0/26 0/54 MiS-i 90/90 0/86 0/40 60/63 92/93 0/44 0/66 Wild type 0/94 ND ND ND ND 498/498 0/201 rolB-2 0/77 ND ND ND ND 0/265 0/755 Ml-i, M6-1, M8-1, M14-1, and M15-1, mutant designations; ND, not determined. The values given are (number of revertant F1 progeny in 8-day-old seedlings)/(total number of F1 progeny). step was repeated, and nuclei were resuspended in buffer B and bacterium-mediated transformation, we confirmed (13) for stored at -80C. Aliquots ofnuclei were counted in a Neubauer Arabidopsis that rolB transgenic calli were impaired in shoot chamber under a fluorescence microscope after staining with differentiation. Nevertheless, rolB transgenic plants could be 4',6-diamidino-2-phenylindole at 1 jg/ml. To label nascent regenerated at a very low frequency. Most established rolB RNA chains, 3 x 106 nuclei were suspended in 300 p1 ofbuffer transgenic lines displayed a variable penetrance and expres- B, and 250 units of RNasin, 15 p1 of 1 M (NH4OSO4, 10 p1 of sion ofthe RolB phenotype among individual progeny plants, 0.1 M MgCl2, 4 p1 of0.1 M MnCl2, 11 p1 of20 mM ATP, 11 p1 suggesting rolB gene silencing. Using adifferent transforma- of20mM CTP, 11 p1 of20mM GTP, and250 uCiof[a-32P]UTP tion protocol (19), similar results were obtained (B. Master- (400 Ci/mmol; 1 Ci = 37 GBq) were added to a final volume of son and J.S., unpublished results). Hence, the rolB gene 400 p1 and incubated for 30 min at 29°C. The reaction was appears to be a useful tool for monitoring transgene silencing stopped by addition of32 p1 oftRNA (5 mg/ml), 40 p1 ofbuffer in Arabidopsis. C (200 mM Hepes, pH 7.6/5 mM MgC12/5 mM CaCl2), and 150 Characterization ofMeioticafly Reversible RolB Sileng In units of RNase-free DNase I followed by an incubation for 30 Arabidopsis. Expression of the rolB gene in Arabidopsis min at 37°C. Subsequently, 50 p1 ofbufferD (100 mM Tris HCl, results in a variety of phenotypic alterations, including early pH 7.5/50 mM EDTA/10% SDS) and 4 p1 ofproteinase K (10 senescence, pronounced growth retardation, inhibition of mg/ml) were added, and incubation proceeded for another 25 hypocotyl and internode elongation, epinastic growth of min at room temperature. After phenol/chloroform extraction, cotyledons and leaves, altered flower morphology, and ste- RNA was separated from free nucleotides on a NICK column rility (Fig. 1 A and B, compare r/o;+/+ and o/o;+/+; Fig. (Pharmacia). Labeled RNA (3 x 106 cpm) was used for hybrid- ization to identical Southern blot filters containing 0.2 pg of 1D). Silencing of rolB can therefore be visually monitored in DNA ofa 0.88-kb Nru I/EcoRI fragment ofplasmid pPCV002- somatic tissues by the failure of displaying this RolB pheno- CaMBT (14) (rolBprobe) and0.2 pg ofDNA ofa0.95-kbEcoRl type (RolB silencing) (Fig. 1E). Among the different rolB fragment of plasmid Pc-UBI 4 (17) (ubiquitin probe). Hybrid- transgenic lines, line rolB-2 displayed the strongest RolB izations were performed in 1 M NaCl, 1% SDS, 10% (wt/vol) phenotype along with an interesting reversible pattern of dextran sulfate, tRNA (100 pg/ml), poly(A) (100 pg/ml), and RolB silencing and was therefore chosen for detailed analy- salmon sperm DNA (100 pg/ml) for 48 h at 65°C. Washing sis. The rolB-2 locus segregates as a single dominant Men- conditions were as described elsewhere (18). delian factor and consists ofthree concatenated copies ofthe transferred T-DNA (arranged in a head-to-head/head-to-tail configuration) at a single genomic integration site (data not RESULTS shown). Seedlings invariably express a stable RolB pheno- Unstable Expression of the RolB Phenotpe In Regenerated type (Fig. 1 A and F: r/r;+/+ and r/o;+/+; Table 2), while Transgenic Arabidopuis Plants. By introducing a chimeric during further development the growing shoot may display construct of the rolB gene driven by the cauliflower mosaic RolB silencing. Plants homozygous for the rolB-2 locus virus (CaMV)-35S promoter (14) into Arabidopsis via Agro- express a strong RolB phenotype, ultimately leading to early senescence. Therefore, RolB silencing in homozygotes is Table 2. Influence of the genetic constitution of rolB-2 and accurately marked by the failure to display early senescence egsl-l on the frequency and developmental timing of rolB (Fig. 1C; sections ofthe plants expressing rolE are senescent gene silencing while those displaying RolB silencing remain green). The rolB gene silencing RolB phenotype ofhemizygous plants is weaker, resulting in patches of senescent tissue (Fig. 1D). Nevertheless, RolB 8-day-old seedlings 35-day-old plants* silencing in hemizygotes is reliably marked by the restoration No. rev./ No. rev./ of a normal phenotype and growth rate (Fig. 1E; a lateral Genotype total Freq., % total Freq., % shoot displaying RolB silencing is marked by an arrow). RolB rolB-2 + 0 /466 0 13/345 4 silencing seems to be triggered by an erratic somatic event o T that commits the whole meristem to the silenced state (Fig. rolB-2 + 0 /755 0 276/S10 54 1C), which is then transmitted stably through mitosis. We rolB-2 + have never observed incomplete RolB silencing leading to rolB-2 egsl-l 23/247t 9 93/102 91 chimeric tissues or sectoring along the shoot axis (20), nor o egsl-l have we observed somatic reappearance of the RolB pheno- rolB-2 egsl-l 570 /575 99 467/470 99 type subsequent to RolB silencing. The frequency of RolB rolB-2 egsl-l silencing is dependent on the genetic state ofthe roLB-2 locus. rev., revertants; o, nontransgenic; +, wild-type allele of egsl Hemizygous plants display a relatively stable RolB pheno- locus; Freq., frequency. type: only 4% of plants show RolB silencing in the main *Phenotypic reversion in the main shoot during rosette formation. shoot. In contrast, 54% ofhomozygous plants displayed RolB tAppearance of intermediate phenotypes. silencing in the main shoot (Table 2). Downloaded by guest on September 30, 2021 5540 Genetics: Dehio and ScheU Proc. Nati. Acad. Sci. USA 91 (1994)

A %it x X. X e ox x.X o .o K e i.xe e B x X e A e0. k K e, lo 0 0 V% ( '4. ~ 0' 4. r r ; +/+

D r;o, +1+ E F r r ;+ r/o; +I+

r r: e e r o; e/e

FiG. 1. Phenotypic manifestation of reversible rolB gene silencing. Genotypes depicted refer to the rolB-2 locus [o = wild-type (nontransgenic) constitution; r = rolB-2 (transgenic) allele] and the egs) locus (+ = wild-type allele; e = egsl-1 mutant allele). Representative phenotypes of corresponding genotypes in 8-day-old seedlings (A) and 35-day-old plants (B) are shown. (C) Twenty-one-day-old plants homozygous for the rolB-2 locus showing RoIB silencing at different stages of rosette formation (sections of the plant expressing roWB are senescent while those displaying RolB silencing remain green). Plants hemizygous for the rolB-2 locus showing a stable RolB phenotype at day 35 (D) and late RolB silencing (E; arrow points to the revertant lateral shoot) at day 56 are pictured. (F) RolB silencing in the egsl-) mutant in 8-day-old seedlings.

Viable seeds are formed exclusively after RolB silencing. process of reversible rolB gene silencing, we have screened The resulting seedlings invariably again express the RolB mutagenized populations for mutants displaying RolB silenc- phenotype. This complete resetting of the RolB phenotype ing already at the seedling stage. Screening of 70,000 EMS- seems to occur during, or immediately after, meiosis, since mutagenized M2 seedlings (obtained from 11,000 Mi plants) developing seeds were found to express rolB only in the homozygous for the rolB-2 locus resulted in the isolation of embryo and not in the seed coat as analyzed by in situ five mutants of independent origin, each carrying a single hybridization experiments (H. Meijer and C.D., unpublished nuclear recessive mutation that is not located within the results). rolB-2 locus (see Table 1 for the FI-progeny analysis of Molecular Analysis ofMeiotically Reversible RolB Silencing. crosses; F2-segregation data are not shown). Complementa- RolB silencing was found to be correlated with an =100-fold tion analysis of these mutants (Table 1) allowed definition of reduction of steady-state rolB transcripts, whereas steady- two genetic loci: egs) for mutants Mi-i (allele egsl-)), M14-1 state transcript levels ofthe physically linked nptII gene and (allele egs)-2), and M15-1 (allele egsl-3); and egs2 for mu- the endogenous ubiquitin genes were apparently unaffected tants M6i (allele egs2-1) and M8-1 (allele egs2-2). The (Fig. 2, compare r/r;+/+ in A and B). In contrast, RolB genetic interactions of locus egsl (allele egs)-1) and the silencing resulted in only an =5-fold drop of rolB nuclear rolB-2 locus were studied in detail. Plants heterozygous for run-off transcripts (Fig. 3, compare r/r;+/+ in A and B). egs)-l were not affected in RolB silencing (Figs. 1 and 2), Therefore, rolB gene silencing seems to result from a pre- indicating that the egsl-) allele is recessive. In contrast, the dominantly posttranscriptional mechanism. egs)-) mutant was found to trigger RolB silencing in 99% Arabidopsis Mutants That Modify the Frequency and the (compared to 0% for the corresponding wild-type allele) of Timing of Meloticaly Reversible RolB Silencing During Plant seedlings homozygous for the rolB-2 locus (Table 2; Figs. 1A, Development. Interestingly, no RolB silencing was ever ob- 2A, and 3A, compare r/r;e/e and r/r;+/+) and 9%o (com- served during the seedling stage (Table 2). To dissect the pared to 0%6' for its wild-type allele) of seedlings hemizygous Downloaded by guest on September 30, 2021 Genetics: Dehio and Scheff Proc. Nati. Acad. Sci. USA 91 (1994) 5541 gene silencing observed in the main shoot of unmutagenized A x B X x x x x x plants or in seedlings ofthe egs)-I mutant (both homozygous

0 0 0 X 0, 0 X X X for the roLB-2 locus) displays similar molecular characteris- tics: a dramatic decrease in steady-state rolB transcripts in 0 contrast to only a moderate decrease in rolB nuclear run-off 3| | < rolB transcripts (Figs. 2 and 3, compare r/r;e/e in A with r/r;+/+ in B). Thus, egs) seems to be a modifier locus of reversible rolBgene silencing. The consequences ofthe egs)-I mutation on this process appear to be twofold: rolB gene silencing is triggered at an earlier developmental stage, in the seedling, and the frequency of rolB gene silencing is generally in- creased. Because of this enhancement effect, this modifier locus was named enhancer ofgene silencing (egs; see above). Genetic segregation analysis, Northern blot analysis and nuclear run-off transcription experiments (data not shown) support the notion that the egs2 locus acts on reversible rolB -jubiquitin gene silencing in a similar fashion as the egs) locus. DISCUSSION Fio. 2. Effect of reversible rolE gene silencing on the steady- Transgene silencing in plants has been observed in many state transcript level ofthe rolB-2 locus. Genotypes depicted refer to the lack ofan appropriate model the rolB-2 locus (o = wild-type (nontransgenic) constitution; r = different systems. However, rolB-2 (transgenic) allele] and the egs) locus (+ = wild-type allele; system has hampered progress toward an understanding of e = egs)-) mutant allele). Eight-day-old seedlings (A) and main the molecular and genetic processes involved. By introducing shoots of 35-day-old plants (B) of corresponding genotypes (for the rolB gene into Arabidopsis, we have established a useful representative phenotypes see Fig. 1 A and B) were subjected to model offering a battery oftools to study transgene silencing: Northern blot analysis of poly(A)+ RNA by serial hybridizations of (i) a selection for transgene silencing because stable rolB a single filter with different probes as indicated. expression interferes with plant regeneration; (ii) a visual assay for transgene silencing based on reversion of the RolB for the rolB-2 locus (Table 2). The appearance ofintermediate phenotype in transgenic regenerants; and (iii) Arabidopsis phenotypes in seedlings of the egsl-l mutant hemizygous for may allow a genetic identification of plant functions involved the rolB-2 locus (Fig. iF; r/o;e/e) may reflect the erratic in transgene silencing. The versatility of this model was used occurrence ofrolB gene silencing during embryo and seedling for a detailed analysis of a reversible transgene silencing development. In contrast, the much higher frequency of rolB phenomenon in a particular rolB transgenic line. In this line, gene silencing in seedlings of the egs)-I mutant homozygous rolB gene expression leads to a pleiotropic phenotype includ- for the rolB-2 locus (Table 2, compare r/o;e/e and r/r;e/e) ing pronounced growth retardation and early senescence (see show that at a higher rolB gene dosage this process is favored Fig. 1D). Silencing as seen by the formation ofphenotypically to occur at an early developmental stage, thus leading to normal shoots occurs erratically and is stable through mitosis phenotypically homogenous seedling populations (Fig. 1F, (see Fig. 1E), whereas rolB expression is fully restored during r/r;e/e). During further development (rosette formation), the or immediately after meiosis. To our knowledge, only two egs)-I mutant displayed RolB silencing in 99%6 (compared to transgene silencing phenomena described in the literature are 54% for the corresponding wild-type allele) ofplants homozy- reminiscent ofthis pattern ofmeiotically reversible rolB gene gous for the rolB-2 locus (Table 2; Fig. 1B, 2B, and 3B, silencing: the heterologous expression of a Nicotiana plum- compare r/r;e/e and r/r;+/+) and in 91% (compared to 4% baginifolia 3-1,3-glucanase gene in Nicotiana tabaccum (10) for the corresponding wild-type allele) of plants hemizygous and of a N. tabaccum chitinase gene in Nicotiana sylvestris for the rolE-2 locus (Table 2). (11). Overall similarities between these different systems that egs) is involved in the suggest a similar mechanism, which was analyzed in more Two observations suggest visual assay process of reversible rolE gene silencing as described above, detail in this study thanks to the advantages ofa gene silencing for gene silencing. Whereas in the previous studies gene rather than in a different and independent state of the rolE-2 locus: (i) the egs)-) mutant displays silencing was correlated with the homozygous mechanism at the by Northern blot or Western blot RolB silencing more frequently in plants homozygous for the tranisgene (as monitored than in hemizygous plants (Table 2), and (ii) rolb analysis), we could demonstrate that gene silencing also rolB-2 locus occurs in the hemizygote, albeit with an --1fold lower frequency. Reversible gene silencing, therefore, seems to be A x x x x e dependent on a threshold level of transgene expression, x. x x which is more often reached in plants homozygous for the 0 k( ^0 q transgene than in hemizygotes. The molecular characteristics of reversible gene silencing 4M < ubiqUitn studied in these different systems (refs. 10 and 11 and this a 40 sa roII3 study) are consistent with respect to the dramatic decrease of 3- steady-state transgene mRNAs. However, nuclear run-off transcription ofthe transgene was apparently not affected by FIG. 3. Effect ofreversible rolB gene silencing on transcriptional gene silencing in ref. 10, whereas we noted a moderate but initiation at the roLB-2 locus. Genotypes depicted refer to the rolEB-2 significant decrease in independent experiments. Although locus [o = wild-type (nontransgenic) constitution; r = rolB-2 (trans- both results argue for a mechanism affecting mRNA stability, genic) allele] and the egs) locus (+ = wild-type allele; e = egsl-1 the former suggests a purely posttranscriptional mode of mutant allele). Eight-day-old seedlings (A) and main shoots of action, whereas ours may indicate a mechanism acting at the 35-day-old plants (B) of corresponding genotypes (for representative an observable effect on phenotypes see Fig. 1 A and B) were used for nuclear run-off posttranscriptional level but with rolB transcription experiments (for experimental details see Materials nuclear run-off transcripts. Alternatively, the moderate de- and Methods). crease of rolB nuclear run-off transcription is not a direct Downloaded by guest on September 30, 2021 5542 Genetics: Dehio and ScheU Proc. Nati. Acad. Sci. USA 91 (1994) consequence of the gene silencing process but rather reflects To test our hypothetical model, we analyzed the process of the coincident loss of the rolB gene product. It was recently reversible gene silencing by genetic dissection. The absence demonstrated that transcription of the CaMV-35S promoter of rolB gene silencing in seedlings allowed us to perform a is stimulated in rolE transgenics (21). Since in our study the mutagenic screen for early rolB gene silencing events. Two rolE gene was driven by this promoter, a positive feedback recessive extragenic loci, egs) and egs2, were found that loop might be eliminated by rolE gene silencing. Clarification promote rolh gene silencing already at the seedling stage and of this aspect awaits further experimental evidence. that cause, in addition, a general increase in the frequency of In any event, the (predominantly) posttranscriptional na- rolB gene silencing. With respect to the suggested model, ture of reversible gene silencing appears to rule out methyl- wild-type alleles of these loci may be needed to maintain the ation as a mechanism, since methylation is thought to cause functional state of the mRNA-stabilizing mechanism in seed- transgene silencing by transcriptional inactivation (22). Con- lings and to a lesser extent also in later developmental stages. sistent with this conclusion, reversible gene silencing was not Molecular identification of the gene products of these loci in found to be correlated with the methylation state of several concert with analysis ofpossible further genetic loci involved restriction sites analyzed in the transgene locus of ref. 11 or in the control of reversible rolB gene silencing (as defined by with methylation of a particular Msp I/Hpa II restriction site different genetic screens) will result in a more complete in the CaMV-35S promoter (which was shown to be diag- molecular description of reversible gene silencing in nostic fortransgene silencing-e.g., in ref. 23) ofthe chimeric plants. rolB gene in this study (data not shown). We thank Heiner Meyer z.A. for gardening work and Maret Kalda The molecular mechanism causing meiotically reversible for photography. We thank Jeff Dangl and Peter Meyer for helpful gene silencing is not known. However, the biochemical discussions. We are grateful to Jeff Dangl and Peter Morris for switch hypothesis originally formulated by Meins (24) and critically reading the manuscript. This work was supported by the modified by Jorgensen (3) may serve as a working model for Fritz-Thyssen-Stiftung and the Max-Planck-Gesellschaft. C.D. this process since it is in good agreement with the charac- gratefully acknowledges the Fritz-Thyssen-Stiftung for a stipend. teristics ofreversible gene silencing reported thus far (refs. 10 and 11 and this study). This hypothesis postulates two 1. Kahl, G. & Weising, K. (1993) Biotechnology, eds. Rehm, H.-J. alternative stable states. A threshold concentration of some & Reed, G. (VCH, Weinheim, Germany), pp. 547-625. product ofgene expression (e.g., RNA) would be responsible 2. Otten, L., De Greve, H., Hernalsteens, J. P., Van Montagu, for the switch. When the threshold is reached, rapid RNA M., Schieder, O., Straub, J. & Schell, J. (1981) Mol. Gen. would cease. Genet. 183, 209-313. turnover would ensue and gene expression 3. Jorgensen, R. (1992) AgBiotech News Inf. 4, 265-273. Here we expand this hypothesis by suggesting a simple 4. Matzke, M. & Matzke, A. J. M. (1993) Annu. Rev. Plant molecular mechanism for reversible gene silencing. Our Physiol. Plant Mol. Biol. 44, 53-76. model assumes that the stability ofthe transgene mRNA (and 5. Kooter, J. M. & Mol, J. N. M. (1993) Curr. Opin. Biotech. 4, potentially other mRNA species) is dependent on the specific 166-171. binding (e.g., via recognition of defined sequences) of a 6. Linn, F., Heidmann, I., Saedler, H. & Meyer, P. (1990) Mol. diffusible mRNA-stabilizing gene product and, furthermore, Gen. Genet. 222, 329-336. that this mRNA-stabilizing gene product is necessary for its 7. Matzke, M. A., Primig, M., Trnovsky, J. & Matzke, A. J. M. own expression by binding to its own mRNA. If the concen- (1989) EMBO J. 8, 643-649. tration ofthe transgene mRNA would reach a certain thresh- 8. Smith, C. J. S., Watson, C. F., Bird, C. R., Ray, J., Schuch, old, it titrate out the W. & Grierson, D. (1990) Mol. Gen. Genet. 224, 477-481. might eventually mRNA-stabilizing gene 9. Napoli, C., Lemieux, C. & Jorgensen, R. (1990) Plant Cell 2, product, resulting in a cessation of gene expression of the 279-289. locus encoding the mRNA-stabilizing gene product and con- 10. de Carvalho, F., Gheysen, G., Kushnir, S., Van Montagu, M., sequently also of the transgene itself and of other genes, the Inze, D. & Castresana, C. (1992) EMBO J. 11, 2595-2602. expression of which is dependent on this mRNA-stabilizing 11. Hart, C. M., Fischer, B., Neuhaus, J.-M. & Meins, F., Jr. mechanism. Resetting of the mRNA-stabilizing mechanism (1992) Mol. Gen. Genet. 235, 179-188. could then simply result from a transient stabilization of 12. Meyerowitz, E. M. (1989) Cell 56, 263-269. nascent transcripts. 13. Schmilling, T., Schell, J. & Spena, A. (1988) EMBO J. 7, Such a titration model would explain the high basal level of 2621-2629. in all cases of reversible 14. Spena, A., Schmilling, T., Koncz, C. & Schell, J. (1987) EMBO transgene expression found gene J. 6, 3891-3899. silencing reported thus far (refs. 10 and 11 and this study). 15. Dehio, C. & Schell, J. (1993) Mol. Gen. Genet. 241, 359-366. Reversible gene silencing inArabidopsis plants transgenicfor 16. Koncz, C. & Schell, J. (1986) Mol. Gen. Genet. 204, 383-396. the rolA gene was indeed only observed in a transgenic line 17. Trezzini, G. F., Horrichs, A. & Somssich, I. E. (1993) Plant with a very marked RolA phenotype (ref. 15; unpublished Mol. Biol. 21, 385-389. results). Our model further predicts that silencing of an 18. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular ectopically expressed gene should result in cosilencing ofthe Cloning: A Laboratory Manual (Cold Spring Harbor Lab. endogenous copy of this gene (because a diffusible mRNA- Press, Plainview, NY), 2nd Ed. stabilizing gene product is not expected to discriminate 19. Kemper, E., Grevelding, C., Schell, J. & Masterson, R. (1992) between identical mRNAs). Such an instance was indeed Plant Cell Rep. 11, 118-121. 20. Li, S. L. & Redei, G. P. (1969) Radiat. Bot. 9, 125-131. described in ref. 11. Our model also implies that the stability 21. Walden, R., Czaja, I., Schmfilling, T. & Schell, J. (1993) Plant of the rolB transcript depends on hitchhiking a mRNA- Cell Rep. 12, 551-554. stabilizing gene product that may control the expression of an 22. Meyer, P., Heidmann, I. & Niedenhof, I. (1993) Plant J. 4, as yet unidentified class of genes. rolB gene silencing would 89-100. consequently be expected to result in cosilencing of these 23. Prdls, F. & Meyer, P. (1992) Plant J. 2, 465-475. genes. 24. Meins, F., Jr. (1989) Annu. Rev. Genet. 23, 395-408. Downloaded by guest on September 30, 2021