The Plant Journal (2005) 43, 929–940
doi: 10.1111/j.1365-313X.2005.02492.x
TECHNICAL ADVANCE
Transcriptional gene silencing as a tool for uncovering gene function in maize A. Mark Cigan*, Erica Unger-Wallace† and Kristin Haug-Collet Agronomic Traits Department, Pioneer Hi-Bred International, 7300 NW 62nd Ave., Johnston, IA 50131, USA Received 18 March 2005; revised 6 June 2005; accepted 10 June 2005. * For correspondence (fax 515 248 2608; e-mail
[email protected]). † Present address: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
Summary Transcriptional gene silencing has broad applications for studying gene function in planta. In maize, a large number of genes have been identified as tassel-preferred in their expression pattern, both by traditional genetic methods and by recent high-throughput expression profiling platforms. Approaches using RNA suppression may provide a rapid alternative means to identify genes directly related to pollen development in maize. The male fertility gene Ms45 and several anther-expressed genes of unknown function were used to evaluate the efficacy of generating male-sterile plants by transcriptional gene silencing. A high frequency of male-sterile plants was obtained by constitutively expressing inverted repeats (IR) of the Ms45 promoter. These sterile plants lacked MS45 mRNA due to transcriptional inactivity of the target promoter. Moreover, fertility was restored to these promoter IR-containing plants by expressing the Ms45 coding region using heterologous promoters. Transcriptional silencing of other anther-expressed genes also significantly affected male fertility phenotypes and led to increased methylation of the target promoter DNA sequences. These studies provide evidence of disruption of gene activity in monocots by RNA interference constructs directed against either native or transformed promoter regions. This approach not only enables the correlation of monocot anther-expressed genes with functions that are important for reproduction in maize, but may also provide a tool for studying gene function and identifying regulatory components unique to transcriptional gene control. Keywords: RNAi, transcriptional gene silencing, maize, anther.
Introduction Gene silencing has been described in both plant and animal systems as a means to suppress gene activity at the level of mRNA expression, providing a powerful tool with which to correlate genes with developmental or biochemical functions (Fire et al., 1998; Mello and Conte, 2004; Vaucheret and Fagard, 2001). In post-transcriptional gene silencing (PTGS), double-stranded RNAs having homology to encoded mRNA transcripts are processed to short RNA duplexes of 21–26 nucleotides through the action of an RNase III endonuclease called Dicer, which guides the RNA-induced silencing complex to suppress target mRNA activity (Hamilton and Baulcombe, 1999; Meister and Tuschl, 2004). Both naturally occurring and transgene-mediated PTGS have been reporª 2005 Blackwell Publishing Ltd
ted to either degrade or translationally repress their target transcript (reviewed in Baulcombe, 2004). The significance of naturally occurring double-stranded RNA suppression has been established by its association with key developmental processes in diverse organisms such as Caenorhabditis elegans, Drosophila and Arabidopsis (Bartel and Bartel, 2003; Carrington and Ambros, 2003; Tijsterman et al., 2002), while the incorporation of approaches involving suppression of transgenic RNA interference (RNAi) provides a powerful method for knocking out gene function at the level of expression in a variety of organisms (McGinnis et al., 2005; McManus et al., 2002; Segal et al., 2003; Wesley et al., 2001). More recently, gene suppression has also been 929
930 A. Mark Cigan et al. achieved by expressing double-stranded RNAs derived from promoter, rather than coding, regions of genes in plants and human cells (Kawasaki and Taira, 2004; Matzke et al., 2004; Melquist and Bender, 2003; Mette et al., 1999; Morris et al., 2004; Sijen et al., 2001). Both constitutive and tissue-preferred plant promoters are capable of being transcriptionally repressed by inverted repeats consisting of promoters from either the Agrobacterium nopaline synthase (Nos) or the soybean beta-conglycinin alpha genes in tobacco and Arabidopsis, respectively (Aufsatz et al., 2002a; Kanno et al., 2004; Park et al., 1996). In dicots, the expression of promoter and other repeats has been shown to lead to the loss of mRNA transcription concomitant with increased methylation of homologous regions of genomic DNA and changes in local chromatin structure (Aufsatz et al., 2002a; Mette et al., 1999, 2000; Ye and Signer, 1996). Importantly, these and other studies have led to the identification of the genes involved in chromatin remodeling and have provided insights into the role of heterochromatin-associated factors during development (Aufsatz et al., 2002b; Bartee et al., 2001; Jackson et al., 2002; Kanno et al., 2004; Vongs et al., 1993). Studies of gene silencing in monocot plants are limited (Holzberg et al., 2002; Iyer et al., 2000; Kumpatla et al., 1997; McGinnis et al., 2005; Schweizer et al., 2000; Segal et al., 2003), and to date there have been no reported examples of transgene-directed transcriptional gene silencing in maize. Thus, transforming and evaluating constructs capable of forming double-stranded RNAs composed of promoter sequences would be the first step in determining whether a transgene-mediated transcriptional silencing mechanism can disrupt native gene function in maize. The genes and promoters encoding Ms45, as well as several other antherexpressed RNAs from maize, have been isolated and characterized at the molecular level. Mutations in the Ms45 coding region result in a male-sterile phenotype due to the absence of expression of MS45 protein in the tapetal layer of anthers during the early vacuolate stage of microspore development (Albertsen et al., 1993a; Cigan et al., 2001). The isolated Ms45 gene restored fertility to ms45 maize in transformed plants, while a fragment of the Ms45 promoter ()264 to þ1, relative to the translational start site) was sufficient to express the Ms45 coding region at levels required for complementation (Cigan et al., 2001). By using ms45 complementation as a functional assay, restoration of fertility by constructs that contained the promoters of the 5126, bs7 and sb200 genes fused to the Ms45 genomic coding region suggested that these genes were also expressed in the tapetum at developmental stages similar to MS45 (Cigan et al., 2001). Little is known about the role that these other anther-expressed genes play during reproduction in maize. However, homology to proteins that participate in the biosynthesis of flavonoids and fatty acids, functions proposed to be important for development of the pollen wall (Piffanelli et al., 1998), suggests necessary roles
for the products of the 5126, bs7 and sb200 genes during microsporogenesis. In this paper, male fertility was used as a phenotypic assay to examine whether both endogenous and transformed copies of the Ms45, 5126, bs7 and sb200 promoters could be transcriptionally suppressed. RNA analysis and in vitro transcription run-on assays were used to examine gene expression from the targeted promoters, while the presence of small RNAs and changes in DNA methylation within target promoters were analyzed to correlate fertility phenotypes with a mechanism consistent with transcriptional gene silencing (TGS). These studies showed that constitutive expression of double-stranded RNAs consisting of promoter sequences resulted in the transcriptional silencing of both endogenous genes and transgenes, providing a new approach for associating regulatory sequences in maize with function. Results Constitutive expression of Ms45 promoter inverted repeats results in male-sterile maize To determine if the expression of inverted repeats (IRs) consisting of promoter sequences would result in suppression of an endogenous monocot gene, different IR containing constructs were introduced into maize (Figure 1). In the first experiments, the endogenous Ms45 promoter was targeted. A segment of the Ms45 promoter shown to be sufficient to restore fertility, including the 5¢ untranslated region (UTR) and 21 nucleotides of the Ms45 coding region, was placed in inverted orientations with a fragment of the Nos gene as a spacer between the inverted repeats. This transcription unit was placed under the control of either the maize anther-specific promoter 5126 (5126MS45pIR) or the constitutive promoter Ubiquitin (MS45pIR), and linked to the herbicide resistance marker 35S:PAT. The constructs were introduced into maize and their fertility phenotypes analyzed (Unger et al., 2001). As shown in Figure 2(a,b), in contrast to either wild-type or transformed plants containing 5126MS45pIR, only constitutive expression of MS45pIR resulted in a male-sterile phenotype. In total, of the 40 independent T0 transformants containing MS45pIR (PHP18770), 34 were male sterile (Table 1). Anthers from sterile plants contained microspores that failed to develop beyond the early vacuolate stage, consistent with the arrest observed in ms45 plants (Albertsen et al., 1993b). With the exception of a male-sterile phenotype, the primary transformants of MS45pIR developed normally. Leaf RNA hybridization analysis of single-copy MS45pIR insertions revealed the presence of a stable MS45pIR transcript in male-sterile plants (Figure 2c). Small RNAs ranging from 22 to 26 nucleotides in length were detected in male-sterile MS45pIR plants, while male-fertile primary T0 plants did not contain these small RNAs (Figure 2d). These ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
Transcriptional gene silencing in maize 931
(a) Promoter
Nos
Pro IR
Pro IR
35S PAT
Plasmid
Name
Promoter
Pro IR
IR end points
IR size
PHP18769
5126MS45pIR
5126
MS45
–369 to +21
390 bp
PHP18770
MS45pIR
Ubiquitin
MS45
–369 to +21
390 bp
PHP20088
MS45p ∆1IR
Ubiquitin
MS45d1
–369 to –80
289 bp
PHP21106
MS45p ∆2IR
Ubiquitin
MS45d2
–264 to –80
184 bp
PHP20089
5126pIR
Ubiquitin
5126
–367 to +3
370 bp
PHP20090
BS7pIR
Ubiquitin
BS7
–300 to +3
303 bp
PHP20091
SB200pIR
Ubiquitin
SB200
–497 to +3
500 bp
(b) Promoter
MS45
35S PAT
Plasmid
Name
Promoter
Gene
Promoter end points
PHP12000
UBIpMS45
Ubiquitin
MS45
–2013 to –1
PHP11982
5126pMS45
5126
MS45
–505 to –1
PHP12025
BS7pMS45
BS7
MS45
–300 to –1
Figure 1. Maize transformation vectors. (a) Schematic diagram of T-DNA vectors for the expression of promoter-inverted repeats (Pro IR) juxtaposed to the herbicide resistance marker 35SPAT. The 5126 or Ubiquitin promoter was used to express the Pro IR-Nos-Pro IR expression cassette. The transcriptional start site is shown with a small arrow above the diagram. The large arrows beneath the diagram depict the orientation of the promoter with the start of the arrow corresponding to the 5¢ end of the native promoter. The end points of the promoter IR are relative to the sequences described in Experimental procedures with þ1 corresponding to the adenine nucleotide of the translational start codon. (b) Schematic diagram of T-DNA vectors for the expression of the Ms45 gene using non-MS45 promoters.
data suggest that the MS45pIR transcripts were processed to small RNA species, indicative of RNA-mediated gene silencing. Male-sterile plants containing single-copy MS45pIR inserts were fertilized and progeny plants from eight independent events (10 plants per event) were screened for herbicide resistance and fertility phenotype. Plants in this generation segregated 1:1 for herbicide resistance and only herbicide-resistant plants were male sterile. Herbicide-sensitive plants were male fertile, suggesting that presence of MS45pIR was required for the male-sterile phenotype in subsequent generations. To determine whether reduced Ms45 gene activity accounted for the male-sterile phenotype, plants containing MS45 constructs under the transcriptional control of the maize Ubiquitin (UBIpMS45) or 5126 (5126pMS45) promoters, were crossed onto male-sterile MS45pIR plants. UBIpMS45 and 5126pMS45 were translational fusions of the Ubiquitin and 5126 promoter regions, respectively, to the Ms45 coding region and lacked the MS45 5¢-UTR sequences (Cigan et al., 2001). All of the progeny from these crosses were selected to contain MS45pIR, while a subset of plants also contained either UBIpMS45 or 5126pMS45. RNA analysis from leaf tissue of progeny plants crossed with ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
UBIpMS45 revealed that in the male-sterile plants only the MS45pIR transcript was detected, while male-fertile plants expressed both MS45pIR and transgene MS45 constitutively (Figure 2e,f). Similarly, male-fertile segregants were also observed when progeny of MS45pIR plants, that also harbored 5126pMS45, were advanced to the next generation (Figure 2g). Analysis of anther RNA revealed that no MS45 transcript was detectable in male-sterile MS45pIR plants (Figure 2h, lanes 1 and 2), whereas MS45 transcript was readily detected in male-fertile MS45pIR plants containing either 5126pMS45 or UBIpMS45 (Figure 2h, lanes 3 and 4). RT-PCR analysis of anther RNA from MS45pIR/5126pMS45 and MS45pIR/UBIpMS45 plants demonstrated that the MS45 transcripts in these fertile plants were derived from the 5126 and Ubiquitin promoter constructs, respectively, and not the native Ms45 locus (data not shown). The ability of UBIpMS45 and 5126pMS45 to reverse the sterility phenotype conferred by MS45pIR, support a hypothesis that male sterility conferred by MS45pIR was not a consequence of MS45 mRNA degradation by the coding sequences within this construct and that specific suppression of transcription from the Ms45 promoter was responsible for the male-sterile phenotype.
932 A. Mark Cigan et al.
(a)
(b)
(c)
S
F
S
S S
S
F
NOS
ACTIN
O S
(d)
S
S
F
S
D
20nt
23nt
(e) MS45pIR
U B I p MS 4 5
+ + + + S S F F S S F F
NOS
(f)
(g)
MS45 ACTIN 1 2 3 4 5 6 7 8
(h)
M S 4 5p I R S
S F F F
MS4 5 5126 ACTIN 5126pMS45 UBIpMS45 Wildtype
1 2 3 4 5
Figure 2. Constitutive expression of Ms45 promoter-inverted repeat resulted in male-sterile maize. (a) Male-fertile tassel of a plant containing 5126MS45pIR (PHP18769). (b) Male-sterile tassel of an MS45pIR plant (PHP18770). (c) Hybridization analysis of leaf total RNA from male-sterile (S) and male-fertile (F) MS45pIR plants. (d) Small RNA analysis. Leaf total RNA from five independent fertile and sterile MS45pIR plants separated by gel electrophoresis and hybridized with promoter IR probe as described in Experimental procedures. The 5¢ end-labeled 23-nucleotide oligonucleotide DO4630 (O) and the 20-nucleotide Decade RNA marker (D, Ambion) served as size markers. (e) Hybridization analysis of leaf poly(A)þ-enriched RNA isolated from plants transformed with MS45pIR. Poly(A)þ RNA was sequentially hybridized with DNA probes. RNA samples in lanes 1–4 and 5–8 were individual T2 plants from two different single-copy MS45pIR events. RNA samples in lanes 3, 4, 7 and 8 were derived from plants that also contain a functional copy of the UBIpMS45 vector. (f) Male-fertile tassels of plants transformed with MS45pIR and containing UBIpMS45. (g) Male-fertile tassels of a MS45pIR/5126pMS45 plant. (h) Hybridization analysis of anther poly(A)þ-enriched RNA isolated from plants transformed with MS45pIR. Poly(A)þ RNA was isolated from 100 anthers staged at tetrad release/early vacuolate microspore development. Lanes 1 and 2, T2 plants containing MS45pIR. Lanes 3 and 4, MS45pIR plants also containing 5126pMS45 or UBIpMS45, respectively. Lane 5, wild-type B73.
It has been previously determined that a small region of the Ms45 promoter ()264 to )80) was sufficient to express MS45 to levels necessary for the restoration of fertility in ms45 plants (Cigan et al., 2001). Therefore, to further resolve which sequences within the MS45pIR construct were capable of suppressing Ms45, smaller portions of the Ms45 promoter were used to form the inverted repeats in constructs MS45D1IR and MS45D2IR (Figure 1). Both repeats ended immediately upstream of the native Ms45 TATA box and eliminated the 5¢-UTR and coding sequences present in the original MS45pIR construct.
MS45D1IR contains the same 5¢ end point as MS45pIR, while the inverted repeat in MS45pD2IR was smaller, having a 184 bp Ms45 promoter inverted repeat. MS45D1IR and MS45pD2IR also conferred a high frequency of malesterile maize (Table 1), and this phenotype was stably inherited in subsequent generations. The ability to suppress Ms45 function by expressing inverted repeats consisting solely of the promoter regions is consistent with transcriptional gene silencing observed in other plant species (Aufsatz et al., 2002a; Matzke et al., 2004; Melquist and Bender, 2003; Mette et al., 1999). ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
Transcriptional gene silencing in maize 933 Table 1 Numbers of independent primary transformants examined for each promoter IR construct and distribution of male fertility phenotypes Construct
T0 plants
5126pMS451R MS45pIR MS45pDIR MS45pD2IR 5126pIR BS7pIR SB200pIR
32 40 31 18 32 30 31
Sterile
Breaker
34 (26) 28 (13) 14 (9) 11 (8)
18 (14)
4 (3)
11 (8)
Shedder
Fertile
22 (16) 2 (1)
32 (24) 6 (4) 3 (1) 4 (4) 3 (3) 8 (6) 14 (12)
Number in brackets corresponds to the number of non-identical single-copy events. Sterile: fertility phenotype where no anthers extrude and pollen is absent. Breaker: fertility phenotype where small anthers, approximately onehalf the size of a wild-type anther, extruded from the lassel and contain negligible amounts of pollen. Shedder: fertility phenotype where extruded anthers, only slightly smaller than wild type, contain small amounts of pollen. Fertile: many extruded anthers along tassel contain large amounts of pollen.
Expression of inverted repeats of promoters from other anther-expressed genes abolished gene expression and reduced male fertility The 5126, bs7 and sb200 genes are predicted to encode proteins having similarities to chalcone synthase, dihydroflavonol reductase and cytochrome P450, respectively. Moreover, these single-copy genes are expressed in anthers at developmental stages coincident but not identical to Ms45 (Cigan et al., 2001). Given the similarities of the products of the 5126, bs7 and sb200 genes to protein functions proposed to be important for formation of pollen walls (Piffanelli et al., 1998), coupled with their temporal and spatial expression patterns, promoter IRs were constructed to determine if suppression of these genes would disrupt the pathways required for male fertility in maize. Primary maize T0 transformants containing constitutively expressed 5126pIR, BS7pIR and SB200pIRs developed normally, with the exception that different fertility phenotypes were observed with each introduced IR when compared with MS45pD1IR tassels (Figure 3a–d). As shown in Table 1, the majority of the independent events containing 5126pIR displayed tassels that were classified as either male sterile or as breakers. Breakers describe a male fertility phenotype where small anthers (approximately onehalf the size of wild-type anthers) extruded from the tassel and contain negligible amounts of pollen. Microspores developed through the late uninucleate stage, after which they aborted. The majority of the BS7pIR T0 plants were classified as shedders (Table 1). Different from breakers, plants classified as shedders extruded anthers along the tassel that were only slightly smaller than wild-type and ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
contain small amounts of pollen when compared with wild-type anthers. SB200pIR T0 plants exhibited a wider range of fertility phenotypes (Figure 3d) when compared with the other promoter IR-transformed plants described above, with nearly one-half of the plants classified as either male sterile or breakers (Table 1). The small amount of pollen observed in the 5126pIR plants produced no more than three kernels per ear (five independent singlecopy events out-crossed), while BS7pIR and SB200pIR plants also demonstrated reduced pollen viability as determined by reduced seed set on out-crossed ears (0–60 kernels per ear, four independent single-copy events from each construct out-crossed). A primary promoter IR transcript was detected (Figure 3e) that coincided with presence of small RNAs (data not shown) in 5126pIR, BS7pIR and SB200pIR T0 plants having reduced male fertility phenotypes. Together, these data indicated that reduced pollen production and viability in these plants was correlated with the constitutive expression of their respective promoter IRs and the presence of small RNAs. Sterile, breaker and shedder plants containing single-copy transfer DNA (T-DNA) inserts from six independent 5126pIR, BS7pIR and SB200pIR events were fertilized with wild-type pollen. A comparison of the fertility phenotypes and target gene expression analysis among the T1 progeny from these promoter IR lines and MS45pD1IR T1 (six single-copy events per construct; six herbicide-resistant plants per event) was performed and is summarized in Figure 4. In contrast to tassels from wild-type plants, all of the herbicide-resistant MS45pD1IR plants were completely male sterile, while all plants containing 5126pIR or BS7pIR were either breakers or shedders, respectively, in the T1 generation (Figure 4a). RNA hybridization analysis conducted on developing anthers from single plants from each of the six single-copy independent events revealed that MS45, 5126 and BS7 transcripts were absent from MS45pD1IR, 5126pIR, BS7pIR plants, respectively (Figure 4b). Immunoblot analysis determined that MS45, 5126 and BS7 proteins were also lacking from their corresponding promoter IR anther extracts (data not shown). To determine whether the absence of MS45, 5126 and BS7 transcripts and proteins was due to transcriptional inactivation of the targeted gene, run-on transcription assays were conducted on nuclei collected from anthers at the tetrad release/early uninucleate stage of microspore development. Analysis of nuclear extracts showed that transcripts for MS45, 5126 and BS7 were synthesized in vitro from anther nuclei derived from wild-type plants (Figure 4c), while extracts from MS45pIR, 5126pIR and BS7pIR anthers did not generate transcripts corresponding to MS45, 5126 and BS7, respectively. Together, these data suggested that production of MS45, 5126 and BS7 mRNA was suppressed by their corresponding promoter IRs at the transcriptional level.
934 A. Mark Cigan et al.
(a)
(b)
(c)
(d)
(e)
S F S S S S
B F B B B
F Sh Sh Sh Sh Sh F F S S
B B
NOS ACTIN MS45p∆IR
5126pIR
BS7pIR
SB200pIR
Figure 3. Constitutive expression of promoter IR results in reduced fertility in primary maize transformants. (a) MS45pD1IR T0 tassels. (b) 5126pIR T0 tassels. (c) BS7pIR T0 tassels. (d) SB200pIR T0 tassels. (e) Leaf total RNA analysis. Fertility phenotypes are shown above each lane. S, sterile; F, fertile; B, breaker; Sh, shedder.
In contrast to the altered male fertility phenotypes observed in the T1 generation when the Ms45, 5126 and bs7 promoters were targeted by IRs, all herbicide-resistant SB200pIR T1 plants were male fertile. SB200 mRNA was detected in these male-fertile herbicide-resistant T1 SB200pIR plants (Figure 4, lanes 12–14) while only twofold reduction of SB200 protein was observed (data not shown), despite the observation that these same events were male sterile in the T0 generation. Moreover, hybridization analysis of leaf RNA revealed levels of sb200 promoter repeat transcript similar to the levels found in the primary trans-
formants; however, small RNAs corresponding to this repeat were not detected in these T1 plants. Promoter IRs inactivated randomly integrated copies of target promoters and were required for maintenance of suppression To test if promoter IR constructs were capable of suppressing transformed copies of their cognate target promoter, 5126pIR and BS7pIR plants in an Ms45/ms45 genetic background were fertilized by ms45 plants containing ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
Transcriptional gene silencing in maize 935
(a)
Wild-type
(a)
MS45p∆1IR
ms45, 5126pMS45
5126pIR BS7pIR SB200pIR
(b)
MS45p∆1IR 5126pIR C
BS7pIR C
SB200pIR
ms45, 5126pMS45, 5126pIR
(b) 5126pMS45 5126pIR
++
+ +
+
BS7pMS45 BS7plR
Ms45p
MS45
5126p 1
5126
2
3
4
5
6
7
Figure 5. Constitutively expressed promoter IR suppresses expression of transformed target promoter. (a) Male-fertile tassel from an ms45 plant containing 5126pMS45 T-DNA compared with male-sterile tassels from ms45 plants containing both 5126pMS45 and 5126pIR. (b) Immunoblot analysis. Anther protein extracts from ms45 plants containing either 5126pMS45 (lanes 1–4) or BS7pMS45 (lanes 5–7) were used for immunoblot analysis using MS45 and 5126 polyclonal rabbit antibodies. Anther extracts in lanes 1, 2 and 4 were from plants that also contained 5126pIR T-DNA. Lanes 5 and 7 were from BS7pMS45/BS7pIR plants.
BS7 SB200 NOS ACTIN 1
2
3
4
5
6
7
8
9 10 11 12 13 14
(c)
MS45 5126 BS7 UBI W
5 B M S7 S 12 ild pI ty 45p 6p R I pe IR R
Figure 4. Constitutively expressed promoter IRs targeting anther-expressed genes reduced male fertility and suppressed mRNA transcription. (a) Male fertility phenotypes of tassels from T1 plants containing different promoter IR constructs as compared with a wild-type B73 tassel. (b) RNA hybridization analysis of anther poly(A)þ RNA staged at tetrad release/early vacuolate microspore development from wild-type plants (C, lanes 7 and 11) and T1 plants containing MS45pDIR (lanes 1–3), 5126pIR (lanes 4–6), BS7pIR (lanes 8–10) and SB200pIR (lanes 12–14). Three of the six non-identical events analyzed are shown in this panel with identical results observed for the three events not shown. (c) Nuclear run-on analysis. In vitro labeled RNA transcripts were synthesized from nuclear extracts listed along the bottom and hybridized to DNA fragments (shown on the left of the figure) immobilized on nylon membranes.
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
5126pMS45 and BS7pMS45, respectively. Homozygous recessive ms45 progeny were selected by PCR to contain the T-DNA insert harboring the non-MS45 promoter transcribing MS45, while a subset was selected that also contained the corresponding promoter IR T-DNA. In the absence of 5126pIR, fertility was restored by the 5126pMS45 in these ms45 plants (Figure 5a), with detectable expression of MS45 protein in early uninucleate anthers (Figure 5b, lane 3). Mutant ms45 plants that contained both 5126pIR and 5126pMS45 were completely male sterile (Figure 5a) as opposed to the breaker phenotype displayed by 5126pIR plants having a functional allele at the Ms45 locus. Moreover, MS45 and 5126 proteins were absent in ms45/5126pIR/5126pMS45 plants (Figure 5b, lanes 1, 2 and 4). Similarly, the BS7pMS45 T-DNA restored fertility to ms45 plants with detectable MS45 protein (Figure 5b, lane 6). However, ms45/BS7pMS45 progeny that also contained a BS7pIR T-DNA were male sterile and lacked MS45 protein in early uninucleate staged anthers (Figure 5b, lanes 5 and 7). Similar results were observed when the MS45pIR T-DNA was combined by sexual crosses with transformed copies of an MS45pMS45 T-DNA in an ms45 background (data not shown), suggesting that gene function associated with the transformed copies of the 5126, bs7 and Ms45 promoters, like their endogenous genes, was suppressed by the corresponding promoter IR. As described above, Ms45, 5126 and bs7 promoter IR plants fertilized with wild-type pollen demonstrated reduced
936 A. Mark Cigan et al. male fertility phenotypes in subsequent generations in the presence of the promoter IR. As a result of this pollination strategy the allele of the endogenous promoter, targeted in the previous generation, segregated in the progeny populations. Promoter IR plants containing Ms45 complementation constructs were therefore used to examine whether suppressed gene activity could be observed in the absence of the inverted repeat, taking advantage of the recessive ms45 allele in these genetic stocks. Two independent male-sterile single-copy MS45pIR events were fertilized by pollen from either UBIpMS45 or 5126pMS45 (ms45/ms45). All herbicidesensitive MS45/ms45 plants advanced (four sensitive plants per cross) from these crosses were male fertile. In addition, to test whether a targeted transgene promoter maintained its suppressed state in the absence of a promoter IR, male-sterile ms45/5126pMS45/5126pIR plants (two independent singlecopy 5126pIR events) were fertilized by MS45/ms45. Eight progeny ms45/5126pMS45 plants were identified and advanced. In this case, all ms45/5126pMS45 progeny examined were also male fertile. Although the number of progeny examined in these two experiments was small, these studies support the observation that MS45 and 5126 promoters required the expression of their corresponding promoter IR for maintenance of the sterility phenotype. Constitutive expression of promoter IRs resulted in cytosine methylation of target promoters Concomitant increases in cytosine methylation within promoters targeted by inverted repeats has been documented in Arabidopsis (Aufsatz et al., 2002a; Melquist and Bender, 2003). Attempts in this study to use bisulfite genomic sequencing of the maize promoters failed due to an inability to generate reproducible conversion of unmethylated cytosines. DNA restriction endonucleases sensitive to cytosine methylation were therefore used to examine the methylation status of the targeted maize promoters in the presence and absence of the IRs. DNA endonuclease restriction sites corresponding to this type of analysis are absent in the Ms45 and 5126 promoters. However, cytosine methylation of the sequence 5¢-ACGCGT-3¢ found within the bs7 promoter inhibits cleavage by MluI. Genomic DNA isolated from four independent shedder BS7pIR events and two independent sterile BS7pMS45/BS7pIR events was subjected to restriction digestion and DNA hybridization analysis and compared with control fertile plants. The endogenous bs7 gene was detected on a SacI DNA fragment containing approximately 3000 bp (Figure 6a, Fragment A), which, in fertile plants, was reduced to 866 bp fragments upon digestion with both MluI and SacI (Figure 6a, Fragment B, lanes 7 and 12). This small MluI–SacI fragment was not detected when genomic DNA was isolated from the shedder BS7pIR transformed plants (Figure 6a, lanes 8–11). In at least one event (Figure 6a, lane 11), a small amount of the 866 bp MluI–SacI
fragment was observed, suggesting that the absence of a major 866 bp hybridizing fragment was not due to the lack of the MluI site within the promoter of the endogenous gene. Moreover, PCR amplification and subsequent digestion of this region with MluI from these BS7pIR events confirmed the presence of this site within the endogenous bs7 promoter (data not shown). To determine if the methylated MluI site within the targeted endogenous bs7 promoter was inherited, pollen from wild-type plants was used to fertilize two independent, semifertile BS7pIR events (Figure 6a; lanes 4 and 10, 5 and 11). Herbicide-sensitive (lacking BS7pIR) and herbicideresistant (containing BS7pIR) progeny from this cross were subjected to restriction enzyme digestion and hybridization analysis and compared with wild-type plants. As shown in Figure 6(a) (lanes 15, 16 and 19, 20), analysis of progeny that contained the BS7pIR revealed that the majority of the hybridization signal corresponded to a 3000-bp DNA fragment, consistent with methylation at the MluI site. In contrast, in segregating progeny from these same two events that did not inherit the inverted repeat (lanes 13, 14 and 17, 18), a nearly equivalent hybridization signal intensity between the 3000 and 866 bp DNA fragments was observed, which suggested methylation of the MluI site from the maternal bs7 allele. Differences in sensitivity of the DNA restriction site were also observed in the transformed copy of the bs7 promoter in male-sterile ms45/BS7pMS45 plants that also contained BS7pIR. Digestion of either BS7pMS45 plasmid or genomic DNA from male-fertile plants containing only this T-DNA revealed an 816 bp MluI fragment that contained a portion of the bs7 promoter (Figure 6b, lanes 3–5 and 8–10, Fragment A). In contrast, MluI digestion and hybridization analysis of genomic DNA from male-sterile ms45/BS7pMS45/BS7pIR plants revealed only the 3000 bp MluI hybridizing band (Figure 6b, lanes 1 and 2, Fragment C). This large fragment was reduced to the predicted 992 bp HindIII–MluI hybridizing fragment if digestion at the internal MluI site within the transformed copy of BS7pMS45 was inhibited. Together, the inability to digest either the endogenous or transformed copy of the bs7 promoter with MluI supported the idea that constitutive expression of the BS7pIR resulted in increased cytosine methylation within the target promoter. In addition, incomplete digestion by MluI within progeny plants that lacked the IR suggested that methylation of the maternal bs7 promoter (targeted in the previous generation) was inherited. Discussion Constitutive expression of promoter IRs in maize resulted in the suppression of endogenous genes as well as integrated transgene cassettes. Using a fertility assay in maize, promoter IR-mediated transcriptional silencing of the male ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
Transcriptional gene silencing in maize 937 Figure 6. Methylation analysis of endogenous and transgene promoters. (a) Identical genomic DNA samples digested with SacI (lanes 1–6) and MluI–SacI (lanes 7–12): HiType II transformation control (lanes 1 and 7), BS7pIR primary maize transformants containing single-copy T-DNA insertions (lanes 2–5 and 8– 11), wild-type B73 (lane 6 and 12). Lanes 13–16 and 17–20 represent segregating progeny from two independent BS7pIR events fertilized with wild-type pollen. Plants lacking (lanes 13, 14 and 17, 18) or containing BS7pIR (lanes 15, 16 and 19, 20) were digested with MluI–SacI and hybridized as described. The schematic representation of the endogenous bs7 gene below the panel shows the bs7 promoter (stippled arrow), the regions of genomic DNA homologous to the BS7 probe (P) as well as the MluI (M) and SacI (S) restriction sites, 5¢-ACGCGT-3¢ and 5¢-GAGCTC-3¢, respectively. (b) Genomic DNA isolated from male-fertile T2 ms45 plants containing BS7pMS45 T-DNA (lanes 1–4, 6–9). Lanes 1, 2, 6 and 7 were from malesterile T2 ms45 BS7pMS45/BS7pIR plants. The endogenous Ms45 gene was present on an MluI– HindIII DNA fragment (more than about 2500 bp; Fragment D). BS7pMS45 plasmid DNA (P; 0.5 ng) was subjected to restriction digestion and used as a size marker.
(a) BS7pIR
+ +
+ + + +
+ + + +
+ +
A
B 1
2
3 4 5 Sac I
7
6
8 9 10 11 12 Mlu I – Sac I
M*
S
13 14 15 16 17 18 19 20 Mlu I – Sac I
S BS7 GENOMIC
BS7
Probe 866 bp >3000 bp
(b)
BS7pIR BS7pMS45
+ + + + + + P
B A
+ + + + ++P
C D
2 3 4 5 Mlu I
1
M
H
A
B
A
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6 7 8 9 10 Mlu I –Hind III M
MS45 GENOMIC
BS7
35S PAT
Probe 816 bp 992 bp >3000 bp
fertility gene Ms45 and the anther-expressed gene 5126 resulted in defects in pollen formation, while loss of bs7 gene function caused reduced male fertility. This reduction of fertility was coincident with the absence of corresponding mRNA transcripts and lack of transcriptional activity. Reduced transcriptional activity was accompanied by increased DNA methylation within the endogenous bs7 gene and an integrated copy of the promoter. Suppression required that the promoter IR be expressed constitutively, as expression of the IR from a tapetum-specific promoter did not perturb gene function as determined by the fertility assay. One explanation for the phenotypic differences observed when either a constitutive or a tissue-specific promoter was used to transcribe the MS45pIR is that the strength or spatial properties of the 5126 promoter are insufficient to fully transcriptionally suppress Ms45. It may also be likely that the Ms45 promoter is not competent to be transcriptionally suppressed at this stage of tapetum development as the 5126 promoter has been successfully used to express MS45 directly or indirectly in complementation ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
A B C
assays (Cigan et al., 2001; Unger et al., 2002). Constitutive promoter IR expression is likely to facilitate modifications in Ms45 chromatin that are required to establish transcriptional silencing prior to or early in tapetal cell formation. In addition, it is possible that molecular components required for transcriptional gene silencing are simply absent in the tapetum. Studies that examine the expression of the maize RNAi effector complex and chromatin-associated modifications within the Ms45 promoter will provide insights into these observed differences. Although RNAi-mediated transcriptional suppression of Ms45 and 5126 resulted in stable male-sterile phenotypes, validating its utility as a gene discovery tool for maize, correlating a gene with its biological process using this approach has limitations. As observed with Bs7pIR plants, despite the absence of BS7 mRNA and protein, a small amount of viable pollen was produced. It cannot be ruled out that very low levels of BS7 expression are sufficient for pollen formation; however, it is possible that genes with similar enzymatic activity were uncovered by this suppression
938 A. Mark Cigan et al. approach thereby masking the role that bs7 has in pollen development. Moreover, it has been recently established that mutations in the coding region of sb200 (referred to as Ms26) confer a male-sterile phenotype to maize demonstrating a functional relationship of this gene to pollen formation (Loukides et al., 1995; M. Albertsen and T. Fox, personal communication). This relationship was supported by the presence of male-sterile and breaker plants within the primary SB200pIR transformants in this paper. Although these phenotypes were not observed in SB200pIR Tn plants, immunoblot analysis of the endogenous SB200 gene and SB200pMS45 transgene construct suggested that steadystate levels of SB200 and MS45 proteins were reduced approximately twofold in the SB200pIR Tn plants (M. Cigan, unpublished observation). As the SB200pIR transcript was detected (Figure 3b), differences associated with this target promoter or the IR construct probably contributed to the reduced silencing efficacy in T1 plants. One factor that might contribute to suppression of a target by an IR is the number and/or location of potential methylation sites close to or within important promoter regulatory elements. Examination of the target promoter regions for nucleotide content did not reveal a significant difference in cytosine percentage in the SB200 promoter relative to the other promoters used in this study (see Table S1). Moreover, mutational analysis of the MS45D1 promoter converting all CG and CnG to TG and TnG, respectively, resulted in a functional promoter capable of complementing MS45 yet still suppressed by MS45pIR (M. Cigan, unpublished observation). However, the inability to detect small RNAs corresponding to the SB200pIR in the Tn generation suggested that the absence of SB200p small RNAs was probably an underlying factor. Given this latter observation, it may be possible that the sequence content of SB200pIR makes this transcript a poor substrate for processing by Dicer and results in nonproductive suppression of the endogenous or transformed copy of the SB200 promoter. Together, though, these observations suggest that properties of target genes and promoter IRs differ and are important considerations in rational design and interpretation of experimental results obtained from this reverse genetics approach. Given these considerations, the promoter IR approach has recently been used to target the maize Pg47 gene (Allen and Lonsdale, 1993) leading to the suggestion that polygalacturonase function is required during pollen development (M. Cigan, unpublished observation). Pg47 is a member of a multigene family with nearly identical nucleotide sequences in their coding and 5¢ flanking regions (Barakate et al., 1993). Thus, like PTGS, TGS is a valuable tool for suppressing the function of a family of genes that maintain extensive sequence conservation. Transcriptional gene silencing could also provide a novel means of uncovering the differential function of highly similar protein family members by targeting their divergent promoters for suppression. The relatively
small promoters of the anther-expressed genes used in this study are in contrast to the often large and highly complex regulatory regions associated with some maize genes (Sidorenko et al., 2000; Stam et al., 2002). Interestingly, though, it may be possible to alter the pattern of expression of a gene by expressing IRs capable of targeting previously identified regions within promoters responsible for conferring differential tissue specificity. Similarly, TGS may be used to identify and map the transcriptional regulatory regions of a gene in planta. Experimental procedures T-DNA constructs and plant transformation To construct Ms45 promoter inverted repeats (IRs), nucleotides 1023–1412 (accession no. AF360356) containing 369 nucleotides of promoter and non-coding DNA and 21 nucleotides of coding region of the Ms45 gene were cloned in inverted orientations and separated by a portion of the Nos gene (nucleotides 259–568; accession no. V00087) into Bluescript SK vector. This MS45p-Nos-MS45p cassette, contained on a 1142 bp NotI–HindIII fragment, was used to replace the Ms45 gene in plasmid PHP12000 (Cigan et al., 2001). The resulting vector, PHP18770, placed the MS45pIR under the transcriptional control of the maize Ubiquitin promoter (ubiquitin-1, including the first intron )899 to þ1092; (Christensen et al., 1992) and upstream of the 35S:PAT selectable marker (Unger et al., 2001). The maize anther-specific 5126 promoter (nucleotides 985–1490; accession no. I75204) was used to replace the maize Ubiquitin promoter in PHP18770 to generate PHP18769. PHP20088 and PHP21106 were similar to PHP18770 except that the Ms45 promoter IRs were 289 bp (nucleotides 1023–1311) and 184 bp (nucleotides 1128–1311), respectively, and truncated just upstream of the Ms45 TATA box. The promoter and leader region, including the translational start site of the 5126 (370 bp, nucleotides 1121–1490; accession no. I75204), bs7 (303 bp, nucleotides 20–322; accession no. AF366294) and sb200 (500 bp, nucleotides 592–1091; accession no. AF366296) genes, were used to replace the Ms45 promoter IR in PHP20088 to generate PHP20089, PHP20090 and PHP20091, respectively. Plasmids PHP12000 (UBIpMS45-35S:PAT), PHP11982 (5126pMS45) and PHP12025 (BS7pMS45-35S:PAT) have been previously described (Cigan et al., 2001). Plasmids were introduced into Agrobacterium strain LBA4404 and used to transform Hi-Type II maize embryos as previously described (Unger et al., 2001). Typically 20 independent events were generated for each construct. Regenerated plants were screened by DNA hybridization analysis and only plants containing single-copy T-DNA inserts were used for further characterization and crosses. The copy number of the T-DNA was determined by digesting T0 and Tn genomic DNA separately with StuI and SacI restriction enzymes that cut uniquely at the 3¢ end of the constructs described in this paper. The digested DNA was sequentially hybridized with 32P-labeled Nos and PAT DNA fragments to identify single-copy events that contained a unique hybridization signal that co-migrated with both probes.
DNA and RNA analysis DNA and poly(A)þ-enriched RNA was isolated, separated by gel electrophoresis and hybridized with 32P-labeled probes as described previously (Unger et al., 2001). DNA probes for hybridization ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
Transcriptional gene silencing in maize 939 analyses were as described (Cigan et al., 2001; Unger et al., 2001). For methylation studies, MS45 and BS7 hybridization probes were generated by PCR using the following oligonucleotide pairs, DO59107 (5¢-GGGGCGGCCGCGGCGTGATGGCATCGTGCAGTA-3¢) and DO59111 (5¢-GGGATCCGTCGTTGAGTTCACTCCATTGGCACAGACT-3¢, and DO59094 (5¢-GGGAAGCTTTGGTGACCTCAAGCAAGGGCAAGG-3¢) and DO59097 (5¢-GGGAATTCCTGATCCTCACCGCAGACGACGA-3¢), respectively, and maize B73 genomic DNA as the template for the reactions. Total RNA was isolated from maize leaf as described (Chomczynski and Sacchi, 1987).
Small RNA analysis For the detection of small RNAs containing homology to the introduced promoter-inverted repeats, 12.5 lg of total RNA was resuspended in running dye [80% formamide, 10 mM EDTA (pH 8.0), 1 mg ml)1 xylene cyanol, 1 mg ml)1 bromophenol blue], heated for 3 min at 90C and separated by gel electrophoresis on a 12% polyacrylamide, 7 M urea gel (Sequagel; National Diagnostics, Atlanta, GA, USA). The RNA was visualized by staining with ethidium bromide (2 lg ml)1 in 0.5· Tris-Boric acid-EDTA) prior to electrotransfer (0.5· TBE, 200 mA, overnight) to Hybond Nþ membrane (Amersham Bioscience, Piscataway, NJ, USA). Ambion Decade markers end-labeled with [c-32P]ATP and a 23 nucleotide oligo DO4630 (Cigan et al., 2001) end-labeled with [c-32P]ATP served as size markers for the small RNA gel electrophoresis. T7 RNA polymerase (Strip-EZ RNA T7; Ambion, Austin, TX, USA) and uridine 5¢-[a-32P]triphosphate (specific activity 3000 Ci mmol)1; Amersham) was used to label in vitro synthesized antisense transcripts of promoter-inverted repeats. The reaction was treated with RNase-free DNaseI (Roche Diagnostics Corporation, Indianapolis, IN, USA) for 15 min at 37C and applied to a G-50 Sephadex column (Roche). Sheared labeled transcripts were generated following the procedure outlined by Mette et al. (2000), neutralized and immediately added to filters pre-hybridized in ExpressHyb (Clontech, Palo Alto, CA, USA) at 65C. Filters were hybridized overnight, washed with 0.1% SDS, 0.5· SSC at 65C and exposed to film with intensifying screens at )70C.
Isolation of nuclei and run-on transcription assays To isolate nuclei for run-on transcription assays, 300 maize anthers from wild-type or promoter-inverted repeat-expressing plants were harvested at the tetrad release/early uninucleate stage of microspore development. Anthers were ground in 2 ml of homogenization buffer (Ye and Signer, 1996) without spermidine. Extract was applied to a Biospin column (Bio-Rad, Hercules, CA, USA; cat. no. 732-6003). Prior to addition of the extract, the tip of the column was removed and two layers of cheesecloth were placed into the bottom of the column. The modified column containing the extract was centrifuged at 1000 g for 2 min to remove cell debris into a 2-ml microcentrifuge tube as previously described (Busk and Pages, 1997). The flow-through was filtered through another Biospin column with its end removed and fitted with a layer of Miracloth (Calbiochem, La Jolla, CA, USA), and centrifuged at 4000 g for 5 min. The pelleted nuclei were washed in 1 ml of nuclei storage buffer (Ye and Signer, 1996) and centrifuged (4000 g for 5 min). The nuclei were resuspended in 100 ll storage buffer and placed at )70C. In vitro synthesis of labeled RNA using isolated anther nuclei was performed essentially as described in Ye and Signer (1996) with the exception that the reaction volumes were reduced to accommodate nuclei in 100 ll storage buffer and the primary reaction allowed to proceed for 60 min at 28C. The labeled RNA was ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 929–940
hybridized to nylon membranes immobilized with denatured DNA fragments (60 pmol) containing gene-specific-sequences in ExpressHyb (Clontech) at 65C overnight. Filters were washed with 0.1% SDS, 0.5· SSC at 65C and exposed to film with intensifying screens at )70C for 4 days.
Immunoblot analysis Whole-cell protein extracts were made from anthers staged at tetrad release/early vacuolate microspore development, immobilized on nitrocellulose filters and incubated with primary and secondary antibodies as previously described (Cigan et al., 2001). Immunoreactive proteins were visualized by chemiluminescence following the instructions supplied by the manufacturer (Amersham). Novel materials described in this publication are available for noncommercial research purposes upon acceptance and signing of a material transfer agreement. In some cases such materials may contain or be derived from materials obtained from a third party. In such cases, distribution of material will be subject to the requisite permission from any third-party owners, licensors or controllers of all or parts of the material. Obtaining any permissions will be the sole responsibility of the requestor. Transgenic materials are available only in accordance with all applicable governmental regulations.
Acknowledgements The authors would like to thank Rui-Ji Xu, Yinghong Li and Joshua Clapp for their excellent technical contributions, Pat Bedinger and Tim Fox for the 5126 antibody, and Gary Huffman, Wes Bruce, Scott Tingey, Vicki Chandler and two anonymous reviewers for providing their comments on this manuscript. We also thank Howard Hershey and Marc Albertsen for their discussions and support of these experiments.
Supplementary Material The following supplementary material is available for this article online: Table S1 Cytosine and guanine content within targeted promoters
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