Biological function of RNA interference (RNAi) pathways in the (Hedw.) Bruch & Schimp.

Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau

von Basel Khraiwesh

aus Jinin Camp - Palästina

Freiburg im Breisgau, 2009

Dekan: Prof. Dr. Ad Aertsen Promotionsvorsitzender: Prof. Dr. Eberhard Schäfer Betreuer: Prof. Dr. Ralf Reski, PD Dr. Wolfgang Frank Referent: Prof. Dr. Ralf Reski, PD Dr. Wolfgang Frank Koreferent: Prof. Dr. Wolfgang R. Hess Tag der Verkündigung des Ergebnisses: 24. April 2009

This work has been created in the Department of Plant Institute of II Faculty of Biology Albert-Ludwigs under the guidance of Prof. Dr. Ralf Reski and PD Dr. Wolfgang Frank

To my marvelous mother and dear family To my wife and my lovely boys, For your support, understanding and always being there for me…

Index

Index

List of contents I Publications and manuscripts related to this work II

1 Chapter Ι: Introduction and Overview……………………………….. 1 1.1 Background………………………………………………………………………… 1 1.1.1 RNA Interference: function and technology…………………………………………… 1 1.1.2 Small RNAs and ………………………………………………………… 2 1.1.2.1 (miRNAs)…………………………………………………………………3 1.1.2.2 Trans-acting short interfering RNAs (ta-siRNA)…………………………………… 5 1.1.2.3 Repeat-associated RNAs (ra-siRNA)………………………………………………. 6 1.1.2.4 Natural antisense transcript-derived small interfering RNAs (nat-siRNA)……… 6 1.1.2.5 Piwi-associated RNAs (piRNAs)……………………………………………………. 7 1.1.2.6 Secondary transitive siRNA…………………………………………………………. 7 1.1.3 Dicer proteins……………………………………………………………………………... 9 1.1.4 Physcomitrella patens as a model system…………………………………………… 11 1.2 Results and Discussion………………………………………………………… 14 1.2.1 DICER-LIKE genes in Physcomitrella patens………………………………………...14 1.2.1.1 Generation and molecular analysis of ΔPpDCL1b knockout mutants…………. 16 1.2.1.1.1 Knockout of PpDCL1b causes developmental disorders…………………….. 17 1.2.1.1.2 MiRNA biogenesis is not affected and miRNA-directed cleavage of mRNA- targets is abolished in ΔPpDCL1b mutant lines………………………………..17 1.2.1.1.3 Generation of transitive siRNA in ΔPpDCL1b mutant lines…………………...18 1.2.1.1.4 Analysis of DNA methylation in ΔPpDCL1b mutants and wild type………….19 1.2.1.1.5 Analysis of the ta-siRNA pathway in ΔPpDCL1b mutants…………………….20 1.2.1.1.6 Analysis of ΔPpDCL1b mutants and wild type lines expressing amiR-GNT1…………………………………………………………………………21 1.2.1.1.6.1 Specific methylation of a miRNA1026 target gene in response to the phytohormone abscisic acid (ABA)………………………………………….. 21 1.2.1.1.7 Expression profiling of transcription factor genes in ΔPpDCL1b mutant lines…………………………………………………………………………………22 1.2.2 Highly specific gene silencing by artificial miRNAs in Physcomitrella patens……. 24 1.3 Conclusion………………………………………………………………………… 27 1.4 References………………………………………………………………………… 29

2 Chapter II: Manuscript 1……………………………………………..34 Transcriptional control of gene expression by microRNAs………………35

3 Chapter III: Publication 1…………………………………………..121 Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted ……………………….122

4 Chapter IV: Appendices……………………………………………. 136 4.1 Flow cytometric measurements (FCM)……………………………………...136 4.2 Physcomitrella patens DCL1b (PpDCL1b) mRNA……………………….. 137 4.3 DNA vectors……………………………………………………………………... 140 4.4 Genes downregulated in ΔPpDCL1b mutants…………………………….. 141 4.5 Genes upregulated in ΔPpDCL1b mutants………………………………… 146 4.6 Acknowledgments……………………………………………………………... 152 4.7 Erklärung………………………………………………………………………....153

I Publications

Publications and manuscripts related to this Work:

Manuscript #1

- Khraiwesh, B., M. A. Arif, G. I. Seumel, S. Ossowski, D. Weigel, R. Reski, W. Frank. (2009): Transcriptional control of gene expression by microRNAs. Submitted.

Publication #1

- Khraiwesh, B., S. Ossowski, D. Weigel, R. Reski, W. Frank (2008): Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockouts. Plant Physiology, 148: 684–693.

This work has been presented at the following conferences:

Talks (presented by W. Frank)

− Frank, W., Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R. (2007): Specific epigenetic control of microRNA target genes to compensate for RNAi dysfunctions in a Physcomitrella patens DICER-LIKE mutant. Botanical Congress, September 3-7, 2007, , .

− Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Specific epigenetic control of microRNA target genes to compensate for RNAi dysfunctions in a Physcomitrella patens DICER-LIKE mutant. The Annual International Conference for Moss Experimental Research, August 2-5, 2007, Korea University, Seoul, Korea.

Posters

− Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., Frank, W. (2008): Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockouts. Annual Meeting of the RNA Society, July 28-August 3, 2008, Free University Berlin, Germany.

− Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Knockout of a DICER-LIKE gene causes silencing of microRNA targets in Physcomitrella patens. 5th Colmar Symposium: The New RNA Frontiers, November 8-9, 2007, Colmar, .

− Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Knockout of a DICER-LIKE gene causes silencing of microRNA targets in Physcomitrella patens. The Annual International Conference for Moss Experimental Research, August 2- 5, 2007, Korea University, Seoul, Korea.

II Chapter I Background

1 Chapter Ι: Introduction and Overview 1.1 Background

1.1.1 RNA Interference: function and technology

RNA interference (RNAi) is a mechanism regulating gene transcript levels by either transcriptional gene silencing (TGS) or by posttranscriptional gene silencing (PTGS), which acts in maintenance and the regulation of development (Hannon, 2002; Agrawal et al., 2003). Since the discovery of RNAi in Caenorhabditis elegans (Lee et al., 1993; Fire et al., 1998) extensive studies have been performed focusing on the different aspects of RNAi. In particular, the elucidation of the essential components of RNAi pathways has advanced extensively (Tomari and Zamore, 2005). RNAi has been discovered in a wide range of organisms from plants and fungi to insects and mammals suggesting that it arose early in the of multicellular organisms (Sharp, 2001; Hannon, 2002).

The RNAi pathway is typically initiated by ribonuclease III-like nuclease , called Dicer, that cleave double stranded RNA molecules (dsRNAs; typically >200 nt) into small fragments bearing a 3’ overhang of two nucleotides. One of these two strands is coupled to a second endonuclease called Argonaute (AGO) and then integrated into a large complex (RNA-induced silencing complex, RISC). Subsequently, it has been shown that RISC contains at least one member of the AGO protein family, which is likely to act as an endonuclease and cuts the mRNA. In Drosophila and humans, AGO2 has been identified as being responsible for this cleavage and the catalytic component of the RISC complex. It was proposed that small interfering RNA (siRNA) guide the cleavage of mRNA. SiRNAs are key to the RNAi process and they have complementary nucleotide sequences to the targeted RNA strand. In certain systems, in particular plants, worms and fungi, an RNA dependent RNA polymerase (RdRP) plays an important role in generating siRNA (Cogoni and Macino, 1999). Another outcome are epigenetic changes such as histone modification and DNA methylation (Matzke and Matzke, 2004; Schramke and Allshire, 2004) (Figure1).

In medical research, RNAi is on the way to becoming an important tool to treat HIV, hepatitis C, and cancer (Hannon and Rossi, 2004) and in plants RNAi technology has been used to improve their nutritional value (Tang and Galili, 2004). For science in general it is already a tool of large scale reverse genetic approaches and aids in unravelling gene functions in many species.

1 Chapter I Background

Figure 1: Overview of RNA interference (adapted from Matzke and Matzke, 2004). The Dicer enzymes produce siRNA from dsRNA and mature miRNA from hairpin-like miRNA precursor transcripts. MiRNA or siRNA is bound to an AGO enzyme and an effector complex is formed, either a RISC or RITS (RNA-induced transcriptional silencing) complex. RITS affects the rate of transcription by histone and DNA modifications whereas RISC cleaves mRNA or inhibits its translation.

1.1.2 Small RNAs and gene silencing

Small non-coding RNAs (20-24 nucleotides in size) have been increasingly investigated and they are important regulators of PTGS in eukaryotes (Hamilton and Baulcombe, 1999; Mello and Conte, 2004; Baulcombe, 2005). They were first discovered in the nematode Caenorhabditis elegans (Lee et al., 1993) and are responsible for phenomena described as RNAi, co-suppression, gene silencing or quelling (Napoli et al., 1990; de Carvalho et al., 1992; Romano and Macino, 1992). Shortly after these reports were published, it was shown that PTGS in plants is correlated to small RNAs (Hamilton and Baulcombe, 1999). These small RNAs regulate various biological processes, often by interfering with mRNA translation. Based on their biogenesis and function small RNAs are classified as repeated- associated small interfering RNAs (ra-siRNAs), trans-acting siRNAs (ta-siRNAs), natural- antisense transcript-derived siRNAs (nat-siRNAs) and microRNAs (miRNAs) (Vazquez, 2006) (Table 1).

2 Chapter I Background

Table 1: Classes of small RNAs identified in eukaryotes (Chapman and Carrington, 2007) Class Description Biogenesis and genomic origin Function miRNA MicroRNA Processing of foldback miRNA gene Posttranscriptional regulation of transcripts by members of the Dicer and transcripts from a wide range of RNaseIII-like families genes Primary Small interfering Processing of dsRNA or foldback RNA by Binding to complementary target siRNA RNA members of the Dicer family RNA; guide for initiation of RdRP dependent secondary siRNA synthesis Secondary Small interfering RdRP activity at silenced loci Posttranscriptional regulation of siRNA RNA (Caenorhabditis elegans) processing of transcripts; formation and RdRP derived long dsRNA or long maintenance of heterochromatin foldback RNA by members of the Dicer family () tasiRNA Trans-acting miRNA-dependent cleavage and RdRP Posttranscriptional regulation of siRNA dependent conversion of TAS gene transcripts transcripts to dsRNA, followed by Dicer processing natsiRNA Natural antisense Dicer processing of dsRNA arising from Posttranscriptional regulation of transcript- sense and antisense transcript pairs genes involved in pathogen derived defense and stress responses in siRNA plants piRNA Piwi-interacting A biogenesis mechanism is emerging Suppression of transposons and RNA which is Argonaute dependent but Dicer- retroelements in the germ lines independent of flies and mammals

1.1.2.1 MicroRNAs (miRNAs)

MiRNAs are ~21nt small RNAs which are encoded by endogenous MIR genes. Their primary transcripts form precursor RNAs exhibiting a partially double-stranded stem-loop structure which are processed by DICER-LIKE proteins to release mature miRNAs (Bartel, 2004). In animals, the primary miRNAs (pri-miRNAs) are cleaved in the nucleus by an enzyme named Drosha to form the pre-miRNAs (Lee et al., 2003). The pre-miRNAs are then transported into the cytoplasm where they are processed into the mature double stranded miRNAs, through cleavage by a second enzyme, Dicer (Bartel, 2004). The first enzyme, Drosha, required for processing of pri-miRNAs in animals, does not exist in plants, so the precursor miRNA is directly cleaved within the nucleus to generate the mature miRNA (Baulcombe, 2004) (Figure 2a). Computational analysis of miRNAs and their potential target mRNAs revealed that many of the miRNA targets belong to the group of transcription factors (Palatnik et al., 2003; Wang et al., 2004). In addition to the control of targets at the post-transcriptional level miRNAs regulate gene expression by epigenetic changes such as DNA and histone methylation (Bao et al., 2004; Lippman and Martienssen, 2004). Overexpression or knockdown of miRNA genes can lead to abnormalities during development (Palatnik et al., 2003; Chen, 2004). For example, plants expressing MIR159, which targets members of MYB transcription factors, exhibit delayed flowering time and male sterility (Achard et al., 2004; Schwab et al., 2005). Plants expressing MIR160, which targets members of the ARF transcription factor family, exhibits agravitropic roots with disorganized root caps and increased lateral rooting (Wang

3 Chapter I Background et al., 2005). Plants expressing MIR166, which targets members of HD-ZIP transcription factors, are arrested in seedling development, and show fasciated apical meristems and femal sterility (Williams et al., 2005).

(g) Piwi-interacting RNA (piRNA)

Figure 2: Small RNA pathways (modified after Vazquez, 2006; Chapman and Carrington, 2007). (a) The miRNA (b) trans-acting siRNA precursors are non-coding RNAs (c) nat-siRNA precursors derive from cis-antisense overlapping coding transcripts. All three precursors are transcribed by RNA polymerase Pol II. (e) Two miRNAs can guide AGO1-mediated cleavage of TAS precursors. The resultant 5’ fragment (TAS1a, 1b, 1c and 2) or 3’ fragment (TAS3) is used by RDR6 and SGS3 as a template for the production of a long dsRNA , which is then cleaved in a phased fashion every 21-nt by DCL4. (f) ta-siRNAs are then 2’-O-methylated at their 3’ ends by HEN1 and guide a slicer-competent AGO protein (AGO7 for TAS3 siRNAs or an unidentified AGO protein for other ta-siRNAs) to their targets for cleavage. (d) A self-amplifying loop believed to depend on RNA Pol IVa is involved in maintaining ra-siRNA-guided methylation of certain DNA repeats. (g) Piwi- interacting RNA (piRNA) biogenesis. Black bars represent genes, with their transcription initiation sites indicated by arrows. Thin black strands represent transcripts of genes encoding small RNAs, and thin blue strands represent target mRNAs. Boxes with broken lines indicate parts of the ta-siRNA and nat-siRNA pathways for which the cellular location is not well established. In small RNA duplexes, the red strands correspond to the guide strand and the black strands correspond to the passenger strand to show that, in the case of siRNAs, the guide strand can originate from either the original sense strand or the newly RDR-synthesized complementary strand.

Target sites in plant miRNAs normally share perfect or nearly perfect complementarity with their target sequence and are often in coding regions (Schwab et al., 2005), whereas in

4 Chapter I Background animals, target sites are often only partially complementary to their miRNAs and are mostly located in the 3'UTR of target genes (Filipowicz, 2005). Currently, hundreds of miRNAs have been identified in plant species and deposited in the miRBase database (http://microrna.sanger.ac.uk/sequences/index.shtml) (Table 2).

Table 2: Plant species and number of miRNAs deposited in miRBase database (Version 12.0, 2008) Species Group Number of miRNAs Arabidopsis thaliana Eudicots 184 Medicago truncatula Eudicots 30 Populus trichocarpa Eudicots 215 Oryza sativa Monocots 243 Zea mays Monocots 96 Physcomitrella patens 220

1.1.2.2 Trans-acting short interfering RNAs (ta-siRNA)

MiRNAs are required for the biogenesis of ta-siRNAs, and both miRNAs and ta-siRNAs regulate mRNA stability and translation (Baulcombe, 2004). Ta-siRNAs arise in plants from specific TAS loci (Figure 2b). TAS transcripts are RNA polymeraseII-dependent and function as highly specialized precursors that feed into an RdRP-dependent siRNA biogenesis pathway. They are targets for cleavage by miRNA-guided mechanisms and yield siRNAs that are in a 21-nt register with the cleavage site (Allen et al., 2005; Rajagopalan et al., 2006; Chapman and Carrington, 2007). Arabidopsis contains different characterized TAS gene families. TAS1a-c and TAS2 ta-siRNA biogenesis is initiated by miR173-guided cleavage on the 5′ side of the ta-siRNA generating region, while TAS3 ta-siRNAs form by miR390-guided cleavage on the 3′ side. MiR390 also interacts in a non cleavage mode with a second site near the 5′ end (Axtell et al., 2006; Montgomery et al., 2008). The resultant 5’ fragment (TAS1a-c and TAS2) or 3’ fragment (TAS3) is used by RDR6 and SGS3 as a template for the production of a long dsRNA, which is then cleaved in a phased fashion every 21-nt by DCL4. This processing involves an interaction between DCL4 with DRB4 for TAS3. Ta-siRNAs are later incorporated into the RISC-like complex and guide cleavage of the complementary mRNAs. However, in Physcomitrella patens, miR173 is absent and therefore miR390 is responsible for the generation of TAS precursors (Axtell et al., 2006). TAS3 ta-siRNAs, but not those from TAS1a-c or TAS2, are dependent on a specialized AGO7 (also called ZIP) (Montgomery et al., 2008). The mechanisms for recognition and routing of transcripts through the ta-siRNA or RDR6/DCL4-dependent pathway are not well understood. Axtell et al. (2006) proposed a

5 Chapter I Background two-hit trigger mechanism, in which transcripts with two or more small RNA target sites are preferentially routed into the RDR6/DCL4 pathway.

1.1.2.3 Repeat-associated RNAs (ra-siRNA)

The mechanism of small interfering RNAs (siRNA) and ra-siRNA production is quite similar. They originate from transgenes, viruses and transposons and may require RdRP for dsRNA formation (Waterhouse et al., 2001; Aravin et al., 2003). Unlike miRNAs, the diced siRNA products derived from the long complementary precursors are not uniform in sequence, but correspond to different regions of their precursor. Because these small RNAs are often single stranded, in fact this means that siRNAs work through the same mechanism, but they are not evolutionary conserved (Bartel and Bartel, 2003; Allen et al., 2004; Jones-Rhoades et al., 2006; Axtell et al., 2007). In plants, the cleavage of siRNA occurs by different DICER-LIKE enzymes than the miRNA processing (Xie et al., 2004). They were first described in plants, where it was shown that the silencing of three transgenes involved a small antisense RNA complementary to each targeted mRNA (Hamilton and Baulcombe, 1999; Hamilton et al., 2002; Bonnet et al., 2006). In plants, siRNA have different functions that can be divided into two broad categories: those that are involved in formation and maintenance of heterochromatin and those that derive from and defend against viruses or sense transgene transcripts (Baulcombe, 2004; Bonnet et al., 2006). Ra-siRNAs (24-nt) are derived from repetitive elements and control the maintenance of DNA and histone modifications (Hamilton et al., 2002; Xie et al., 2004) (Figure 2d). Previous studies on Arabidopsis reported that the transcripts of the two canonical repeats, the retrotransposable element AtSN1 and the 5S ribosomal DNA are converted by RDR2 into long dsRNA as a template for DCL3, which processes 24-nt siRNAs that are O-methylated by HEN1 (Xie et al., 2004; Yang et al., 2006). 24-nt ra-siRNAs guide methylation of AtSN1 and 5S rDNA repeat loci by the action of the AGO4 and they have been implicated in chromatin modifications (Zilberman et al., 2003). The ra-siRNA pathway is a positive feedback loop because methylation of these loci is essential for ra-siRNA accumulation (Xie et al., 2004).

1.1.2.4 Natural antisense transcript-derived small interfering RNAs (nat- siRNA)

Borsani et al. (2005) identified a new class of small RNAs derived from a natural-antisense overlapping transcript pair. The transcripts of P5CDH, a stress-related gene, and SRO5, a gene of unknown function, partially overlap (Figure 2c). In the proposed nat-siRNA pathway, a 760-nt double-stranded region resulting from pairing of cis-antisense transcripts is thought to be processed by DCL2 to generate a unique 24-nt nat-siRNA that guides cleavage of P5CDH transcript through an unidentified AGO protein. In principle, RDR6 and

6 Chapter I Background

SGS3 could synthesize a strand complementary to cleaved fragments of P5CDH mRNA leading to a dsRNA that is processed like TAS duplexes in a phased fashion by DCL1. Each resulting 21-nt nat-siRNA reinforces cleavage of P5CDH mRNAs. NRPD1a could be involved in a reinforcing amplification loop using the dsRNA generated by RDR6 and SGS3 as a template. Accumulation of the 24-nt nat-siRNA is correlated with salt stress-induced transcription of the SRO5 gene and is abolished in dcl2, rdr6, sgs3 and nrpd1a mutants. The finding that 4–20% of the genes in many eukaryotes show cis-antisense overlapping organization raises the possibility that the nat-siRNA could be a major mechanism for gene expression regulation (Borsani et al., 2005).

1.1.2.5 Piwi-associated RNAs (piRNAs)

Recent studies have revealed a new class of 24-30-nt RNAs that are generated by a Dicer- independent mechanism and that interact with members of the Piwi subfamily of AGO proteins (Chapman and Carrington, 2007; Klattenhoff and Theurkauf, 2008). The proposed piRNA-biogenesis model involves initial targeting of transcripts from transposons and retroelements by a Piwi-like protein that is programmed with a small RNA (Figure 2g). PiRNAs are important for spermatogenesis in mammals and insects. Plants appear to lack these piRNAs (Faehnle and Joshua-Tor, 2007). In Drosophila, the previously identified ra- siRNAs have been shown to bind the Piwi family members Piwi and Aubergine (Aub) and thus represent a subset of Drosophila piRNAs. Piwi-associated ra-siRNAs (now referred to as piRNAs) are 24−29nt long than AGO-bound siRNAs/miRNAs and are derived predominately from repetitive genomic loci like transposons or satellite repeats (Aravin et al., 2003; Chapman and Carrington, 2007).

1.1.2.6 Secondary transitive siRNA

Previous studies on A. thaliana and C. elegans reported the amplification of silencing related RNA via transitivity, and explain how strong, persistent silencing can be initiated with small amounts of initiator dsRNA (Axtell et al., 2006; Pak and Fire, 2007; Sijen et al., 2007). The emergence of transitivity has an important role for RNAi in controlling gene expression and for understanding the effects of silencing RNAs on cell function and organism development. The initiator of transitivity is a dsRNA which is produced by RdRP activity and then processed by DICER-LIKE family into secondary siRNA or a related type of RNA referred to as miRNA, these 21-25 nucleotide single stranded RNAs are the primary silencing RNAs in the transitive process (Baulcombe, 2007; Moissiard et al., 2007). Upon binding to target transcripts, siRNAs can not only trigger their cleavage and subsequent destruction, but also serve as primers for RdRP. These extend the local RNA double strands and generate templates for production of secondary siRNAs by Dicer action. These secondary siRNAs are

7 Chapter I Background unrelated in sequence to the initial trigger (Moissiard et al., 2007; Mlotshwa et al., 2008). In plants, the transitivity occurs in both directions of the initial dsRNA trigger whereas in animals spreading of the initial signal occurs only upstream of the trigger (Figure 3).

Figure 3: Models for amplification of silencing signals in C. elegans and A. thaliana (adapted from Chapman and Carrington, 2007). (a) Processing of trigger dsRNA in C. elegans by Dicer 1 (DCR-1) releases primary siRNAs.The primary siRNA associates with an AGO protein, such as RDE-1, and the complexes bind to complementary target RNA. The bound complexes might then recruit RdRP, which uses the target transcript as template for synthesis of the secondary siRNA. Abundant secondary siRNAs are formed by independent initiation events (rather than by Dicer-mediated processing), are complementary to the target RNA and accumulate in phased pools. (b) Processing of trigger dsRNA in A. thaliana by one or more Dicer-like enzymes releases primary siRNAs, which associate with an AGO protein, such as AGO1, and guide cleavage of the target RNA. This event is proposed to recruit an RdRP, such as RDR6, which uses the target transcript as template for synthesis of a long dsRNA. This dsRNA precursor is processed by DCL enzymes to release abundant secondary siRNAs.

8 Chapter I Background

1.1.3 Dicer proteins

Dicer and DICER-LIKE (DCL) proteins are RNAaseIII-type enzymes that cleave RNA molecules with dsRNA features into small fragments bearing a 3’ overhang of two nucleotides during PTGS (Elbashir et al., 2001). Dicer is a large multidomain protein conserved in most eukaryotes, consisting of DExD-helicase, helicase-C, Duf283, PAZ (Piwi- Argonaute-Zwille), dual RNAase III , and double stranded RNA-binding (dsRB) domains (Bartel and Bartel, 2003) (Figure 4).

Figure 4: Crystal structure of Dicer (adapted from Macrae et al., 2006). The linear arrangement of domains typically found in DCL or DCR proteins is depicted above the figure. (a) Front and side view ribbon representations of Dicer showing the N-terminal platform domain (blue), the PAZ domain (orange), the connector helix (red), the RNase IIIa domain (yellow), the RNase III b domain (green) and the RNase-bridging domain (gray). Disordered loops are drawn as dotted lines. (b) Close-up view of the Dicer catalytic sites; conserved acidic residues (sticks); erbium metal ions (purple); and erbium anomalous difference electron density map, contoured at 20s (blue wire mesh).

The DExD and helicase-C domains are found towards the N-terminal and C-terminal regions, respectively. There are always two RNase III domains (termed A and B) in a Dicer protein, and the Duf283 is a domain of unknown function but which is strongly conserved among Dicer proteins. The role of the dsRB domain in human Dicer is generally thought to mediate unspecific reactions with dsRNA, with the PAZ, RNase III A and RNase III B domains being crucial for the recognition and spatial cleavage of dsRNAs into siRNA or miRNA (Zhang et al., 2004). In organisms with only one Dicer, this enzyme, with its associated proteins, is presumably the only generator of siRNAs and miRNAs. In organisms with two or more Dicers, there is probably a division of labour. Plants have at least four main types of DCLs (Margis et al., 2006). In A. thaliana, the four different DICER-LIKE proteins (DCL1-DCL4) exhibit predominant functions in particular small RNAs pathways, even

9 Chapter I Background though functional redundancies among these proteins were identified (Henderson et al., 2006). DCL1 produces mature miRNAs which direct cleavage of transcripts containing sequence elements in reverse complementary orientation (Kurihara and Watanabe, 2004). DCL2 mediates the generation of siRNA from RNA of exogenous sources (Xie et al., 2004). DCL3 is required for the formation of heterochromatin-associated endogenous siRNA (Xie et al., 2004) and DCL4 is needed for the formation of ta-siRNA involved in systemic cell-to-cell transmission of silencing signals (Xie et al., 2005). Also all DCLs have been shown to be involved in generation of viral-derived RNAs in coordinated hierarchical actions (Moissiard and Voinnet, 2006). Human, mice and nematodes each contain one Dicer gene, involved in miRNA biogenesis and the generation of siRNAs. Insects and fungi possess two Dicer genes, the two Dicers have related but different roles, one processes miRNAs and the other is necessary for siRNA-mediated RNAi (Margis et al., 2006). Knockout and knockdown experiments indicate that Dicer is essential for vertebrate development (Jaskiewicz and Filipowicz, 2008). Disruption of Dicer in mice arrests embryogenesis (Bernstein et al., 2003). Dicer function was also found to be essential for Zebrafish development and many processes in C. elegans. In Drosophila, Dicer involved in miRNA biogenesis is likewise an essential gene (Jaskiewicz and Filipowicz, 2008). In mammals, Dicer is important for protection against influenza A virus infection (Matskevich and Moelling, 2007). Dicer is also required for the maintenance of epigenetic silencing in human to protect against cancer (Ting et al., 2008). Loss-of-function mutants of DCL1 in Arabidopsis revealed its role in a number of developmental processes including embryogenesis and flower morphogenesis (Golden et al., 2002; Park et al., 2002). The pleiotropic effects observed in these mutants were ascribed to the lack of miRNA which was caused by the loss of miRNA biogenesis. However, other mechanisms controlled by Dicer and related to RNAi, such as DNA methylation, chromatin structure and centromeric silencing, may also contribute to developmental or cellular defects. Although recombinant Dicer is active as a dsRNA endonuclease in vitro, in cells it generally functions in association with other proteins as a component of multiprotein complexes. In animals, Dicer proteins were shown to be associated with other proteins forming complexes which act as RISC or RISC loading complexes (RLC) (Tomari et al., 2004). Thus, miRNA processing and target-RNA cleavage could be coupled. In Drosophila, it was shown that Dcr- 2, which produces siRNA, also acts in the RISC assembly together with its partner R2D2 by loading one of the two siRNA strands into RISC (Tomari et al., 2004). The C. elegans homologue of this protein, RDE-4 was also found to interact with Dicer (Tabara et al., 2002). Similarly, human Dicer may function in loading siRNA into RISC, as siRNA does not cause PTGS in human cells lacking Dicer (Doi et al., 2003). However, this is dependent on

10 Chapter I Background particular cell types as siRNA triggers gene silencing in Dicer knockout embryonic stem cells (Kanellopoulou et al., 2005). Moreover, human Dicer, TRBP and AGO2 were present in a protein complex which is able to perform siRNA or miRNA directed target RNA cleavage (Gregory et al., 2005). In plants, members of the HYL1/DRB family proteins were identified as DCL-interacting dsRBD (dsRNA-binding domain) partners and implicated in small RNA pathways in Arabidopsis (Hiraguri et al., 2005). Another group of well characterized Dicer partners in animals is represented by PPD or AGO proteins. Members of the PPB protein family contain two signature domains: a PAZ domain in the center and a PIWI domain at the carboxyl terminus (Carmell et al., 2002; Tolia and Joshua-Tor, 2007). Several other proteins have been found to interact with Dicer. RNA-helicase-related protein, which is required for RNAi, was found to interact with RDE-4 and Dicer in C. elegans (Tabara et al., 2002). FMRP, an mRNA-binding protein involved in the pathogenesis of fragile X syndrome, has been shown to interact with Dicer and AGO-1 in mammalian cells (Jin et al., 2004). Biochemical analysis of fission yeast Dicer (Dcr1) revealed its physical and functional association with RNA-directed RNA polymerase complex (RDRC) in transcriptional silencing (Shiekhattar, 2007). Identification of so many Dicer-interacting proteins indicates that Dicer participates in many cellular processes (Jaskiewicz and Filipowicz, 2008).

1.1.4 Physcomitrella patens as a model system

The moss Physcomitrella patens is a member of the bryophytes. Physcomitrella patens occupies an important phylogenetic position for the elucidation of the development of higher plants (Figure 5), including other model organisms, such as Arabidopsis, and plants of commercial importance, such as poplar, corn, soybean, sorghum, and rice. In terms of evolutionary distance, Physcomitrella is to the flowering plants what fish is to humans.

Figure 5: Land plant evolution (adapted from Rensing et al., 2008). Bryophytes comprise three separate lineages which, together with the vascular plants (including the flowering plants), make up the embryophytes (land plants). These four lineages, remnants of the initial radiation of land plants in the Silurian, began to diverge from each other about 450 million years ago.

11 Chapter I Background

The gametophytic phase is divided into the protonema and the gametophore stages, which produce the upon particular conditions. Typical for mosses is the heteromorphic- heterophasic alteration of generations, which is responsible for the predominant haploid phase of the (Figure 6), and a diploid phase that produces haploid spores. Physcomitrella patens is a monoecious, self-fertile species, i.e. one plant carries both the male (antheridia) and the female (archegonia) sex organs.

Figure 6: Life cycle of Physcomitrella patens (adapted from http://www.plant- biotech.net). A haploid spore germinates and grows into the filamentous protonema cells. Starting with a three-faced apical cell bud formation is initiated which gives rise to the leafy adult gametophyte. In monoecious moss species both sex organs (antheridia and archegonia) are present on one and the same plant. Fertilization of the egg cell takes place in the presence of water. From the fertilized egg the sporophyte grows out of the archegonia. Within the spore capsule the cells undergo meiosis and new spores are formed.

These features, among others, make Physcomitrella advantagous for scientific use. Because protonema grows quickly and simultaneously, it can be cultivated in a bioreactor as a genetically stable cell suspension. Another advantage is that Physcomitrella can be easily manipulated using molecular biology methods (Reski, 1998a). A unique feature is the high efficiency of , therefore targeted disruption and manipulation of single genes can be performed easily (strepp et al., 1998; Schaefer and Zryd, 1997). The rate of homologous recombination in Physcomitrella is found to be several orders of magnitudes higher than in any other characterized plant species

12 Chapter I Background

(Reski, 1998b). The high rate of homologous recombination together with the predominant haploid phase make Physcomitrella a highly suitable system to initiate forward and reverse approaches, enabling the study of gene functions related to almost all aspects of plant biology. A considerable collection of mutants (Egener et al., 2002) and around 210.000 expressed sequence tag (EST) sequences are available (Rensing et al., 2002). Analyses have shown that around 95% of Physcomitrella’s transcriptome is covered by these data. The moss Physcomitrella patens genome comprises about 511 Mbp which are dispersed on 27 chromosomes. The sequence contains approximately 30,000 protein coding genes. Most predicted genes are supported by multiple types of evidence, and 84% of the predicted proteins appear to be complete. About 20% of the analyzed genes show alternative splicing, a frequency similar to that of A. thaliana and O. sativa (Rensing et al., 2008). Despite its low evolutionary position at the basis of land plants Physcomitrella shares more features with the seed plant A. thaliana, than Arabidopsis as dicotyledonous plant with the monocotyledon O. sativa (Reski, 1998b). Recently, a small RNA database has been established in Physcomitrella (Arazi et al., 2005; Axtell et al., 2006; Axtell et al., 2007; Fattash et al., 2007) and they are highly conserved in plants. Recent reports have shown that the RNAi machinery is present and working correctly in Physcomitrella. However, in contrast to A. thaliana and other plant species the biological function of the RNAi pathways in Physcomitrella were not studied.

The objectives of this study are:

1. To study the biological function of RNAi pathways in the moss Physcomitrella patens, focussing on the function of the key protein of RNAi, Dicer, by the generation of targeted knockout plants and analyzing the pathways of small RNAs and miRNA target genes in Dicer mutants. 2. To study gene silencing using artificial miRNAs (amiRNAs) in the moss Physcomitrella patens as an alternative tool to targeted gene knockouts.

13 Chapter I Results and Discussion

1.2 Results and Discussion

1.2.1 DICER-LIKE genes in Physcomitrella patens

To identify genes encoding DCL proteins BLAST searches of a Physcomitrella EST database (Rensing et al., 2002) were performed using the four Arabidopsis DCL proteins as query. The corresponding cDNA clones of the identified ESTs were sequenced. Analysis of these partial cDNA sequences suggested the existence of four DCL genes in Physcomitrella (Table 3). Table 3: Identification of DICER-LIKE genes in Physcomitrella patens. Closest A. thaliana homologue obtained by reverse BLAST searches using the deduced amino acid sequences of the four P. patens genes in the GenPept/nr database. The PpDCL1b sequence used to generate ΔPpDCL1b mutant lines are underlined.

A.th. homologues (Acc. No.) AtDCL1 AtDCL2 AtDCL3 AtDCL4 P.p. (Q9SP32) (NP_566199) (NP_189978) (P84634) DCL cDNA (Acc. No.) 69% identity PpDCL1a (EF670436) 81% similarity

65% identity PpDCL1b (DQ675601) 78% similarity

32% identity PpDCL3 (EF670437) 48% similarity

35% identity PpDCL4 (EF670438) 53% similarity

These DCL genes have recently been deduced from the Physcomitrella patens genome sequence independently (Axtell et al., 2007). Two of the Physcomitrella patens DCL proteins (PpDCL1a and PpDCL1b) group together with AtDCL1 (Figure 7), the only A. thaliana DCL involved in miRNA processing (Kurihara and Watanabe, 2004). Prediction of protein domains in the Pfam database (Bateman et al., 2004) revealed the existence of all functional domains in the two Physcomitrella patens DCL1 proteins present in the AtDCL1 protein (Figure S2 and S3, Manuscript 1). However, compared to AtDCL1 the PpDCL1b protein lacks approximately 240 amino acids at the N terminus. The other two PpDCL proteins are homologs of AtDCL3 and AtDCL4, whereas an AtDCL2 homolog is lacking.

Using the homologous recombination system, M. Asif Arif generated and analyzed (in his ongoing Ph.D. work) two targeted PpDCL1a knockout mutants (ΔPpDCL1a) (Figure S2, Manuscript 1). The ΔPpDCL1a mutant lines show severe developmental abnormalities.

14 Chapter I Results and Discussion

Most drastically the ΔPpDCL1a mutant lines are not able to develop leafy gametophores and are developmentally arrested at the protonema stage (Figure 1A and 1B, Manuscript 1). The results show that PpDCL1a is the functional equivalent of the A. thaliana DCL1 protein required for the biogenesis of miRNAs and ta-siRNAs. Compared to wild type the expression levels of miRNA and ta-siRNA target genes were upregulated in ΔPpDCL1a mutant lines (Figure 1C-E, Manuscript 1). However, in Physcomitrella patens miRNAs might be processed by additional DCLs as the detection particular miRNAs albeit at significantly reduced expression levels (Figure 1, Manuscript 1). The additional presence of a second AtDCL1 homolog in Physcomitrella patens suggested potential differences in endogenous RNAi pathways in comparison to the seed plant A. thaliana.

Figure 7: Neighbour-joining tree showing the phylogenetic relationships between DICER-LIKE proteins. DICER-LIKE proteins from animals and plants are indicated by vertical lines. The four groups of DICER-LIKE proteins in plants are marked by coloured boxes. Species abbreviations are At (Arabidopsis thaliana), Ce (Caenorhabditis elegans), Cr (Chlamydomonas reinhardtii), Dm (Drosophila melanogaster), Hs (Homo sapiens), Mm (Mus musculus), Mt (Medicago truncatula), Nc (Neurospora crassa), Os (Oryza sativa), Pp (Physcomitrella patens), Pt (Populus trichocarpa), Sp (Schizosaccharomyces pombe). Pp DCL proteins are highlighted in bold. * The sequence of DCL from Chlamydomonas reinhardtii can be retrieved at: http://genome.jgi-psf.org/chlre2. (Figure S1, Manuscript 1).

15 Chapter I Results and Discussion

1.2.1.1 Generation and molecular analysis of ΔPpDCL1b knockout mutants

To generate PpDCL1b knockout lines a PpDCL1b gene disruption construct was prepared by inserting an nptII selection marker cassette into a 560 bp fragment of the PpDCL1b cDNA which encompasses the coding region of the second RNAseIII domain present in the DCL1b protein (Seumel, 2004; Figure S3, Manuscript 1). The resulting construct was used for transfection of Physcomitrella . After selection of regenerating plants they were analyzed by PCR to identify mutant lines which had integrated the disruption construct at the DCL1b genomic locus. Out of a total of 520 analyzed transgenic lines 8 lines (1.54%) unable to produce PpDCLb1 transcripts were identified. Four lines were used for further studies (ΔPpDCL1b 1-4). The full-length cDNA sequence of PpDCL1b was obtained (Figure 8, Appendix 4.2), the cDNA was termed DCL1b encoding a protein of 1695 amino acids. Furthermore, the haploidy of all ΔPpDCL1b mutant lines was verified by flow cytometry to exclude the possible generation of diploid lines by fusion during the transformation process (Appendix 4.1).

Figure 8: Cloning and sequencing of the PpDCL1b cDNA. The PpDCL1b cDNA is indicated by a black line. The colored arrows above depict cDNA fragments obtained by different cloning steps. The numbers in the arrows refer to the corresponding nucleotide positions in the DCL1 cDNA. First, a cDNA clone comprising the 3’ end of PpDCL1b was sequenced. Subsequently, three 5’ RACE-PCRs using the BD Smart RACE cDNA Amplification Kit (Clontech) and one RT-PCR was performed. The primers for the RT-PCR were derived from available Physcomitrella genomic trace files. All PCR and 5’ RACE primers were selected to give rise to overlapping PCR fragments of already known sequence stretches to confirm that the amplicons were derived from the same cDNA. The following primers were used: 5‘RACE-PCR 1: 5’- GAACTCCCAACGATGGTCGAGACGC-3’ 5’RACE-PCR 2: 5’- CCAGCT CATCGTGATCAGTAAAGTCGGG -3’ 5‘RACE-PCR 3: 5’-TCCCAGCGCCCGTGTCTAGAAATGCAAC -3’ RT-PCR: 5’-GAGAGGCGGTCTGTGTCGAGGTCTAG -3’ and 5’-TTGTAGCCACCAGCAACGTCACCCGT -3’

16 Chapter I Results and Discussion

1.2.1.1.1 Knockout of PpDCL1b causes developmental disorders

The ΔPpDCL1b mutant lines showed developmental disorders throughout all stages of protoplast regeneration including abnormalities in cell division, growth polarity, cell size, cell shape and growth of tissues (Figure 2A, Manuscript 1). Moreover, these mutants developed only a small number of gametophores, which in addition were malformed (Figure 9). The observed developmental effects are consistent with previous studies of Dicer mutants in animals and plants. The pleiotropic effects observed in these mutants were ascribed to the lack of miRNA which was caused by the loss of miRNA biogenesis.

Figure 9: Phenotypic analysis of the ΔPpDCL1b mutants. Electron micrographs of gametophores from wild type plants and ΔPpDCL1b mutant 1 (Figure 2B, Manuscript 1).

1.2.1.1.2 MiRNA biogenesis is not affected and miRNA-directed cleavage of mRNA-targets is abolished in ΔPpDCL1b mutant lines

The isolated PpDCL1b gene from Physcomitrella shows 65% identitiy and 78% similarity to to the DCL1 gene from Arabidopsis (Table 3), encoding the essential enzyme required for the generation of miRNAs from pre-miRNA precursors. If the high sequence conservation causes considerable overlap in function one would expect the absence of miRNAs in the ΔPpDCL1b mutant lines. Interestingly, the accumulation of miRNAs in ΔPpDCL1b mutant lines compared to the wild-type was present in almost equal amounts (Figure 2C, Manuscript 1), indicating that PpDCL1b is not required for processing of miRNA precursors in Physcomitrella. In contrast, miRNA-triggered cleavage of miRNA target genes, encoding different transcription factors, was abolished in the ΔPpDCL1b mutant lines (Figure 3A, Manuscript 1). The abolished miRNA-directed cleavage of target mRNAs in the ΔPpDCL1b mutant lines suggests a direct involvement of PpDCL1b in this step of miRNA action. The requirement of DCL proteins for target cleavage was not shown in plants. It is unlikely that PpDCL1b directly cleaves mRNA targets as this function is commonly associated with AGO proteins present in the RISC (Liu et al., 2003). Studies in animals have shown Dicer in

17 Chapter I Results and Discussion association with protein complexes (Tabara et al., 2002; Liu et al., 2003; Lee et al., 2004; Pham et al., 2004). Some of these complexes, like the Dcr-2/R2D2 heterodimer from Drosophila act in loading siRNA into RISC (Liu et al., 2003). It is possible that Physcomitrella patens DCL1b functions in RISC loading like Dcr-1 and Dcr-2 from Drosophila. Until now, only the Arabidopsis protein HYL1 was shown to interact with Arabidopsis DCL1 in vitro (Hiraguri et al., 2005). However, even though HYL1 shows high similarity to the Drosophila R2D2, a function in RISC loading is unlikely as dsRNA triggered gene silencing is not affected in hyl1 mutants. Analysis of miRNA expression indicated that HYL1 plays a role in miRNA biogenesis as miRNA levels were reduced in the hyl1 mutant (Han et al., 2004).

1.2.1.1.3 Generation of transitive siRNA in ΔPpDCL1b mutant lines

In Physcomitrella wild type, 5’RACE-PCRs performed from the miRNA targets yielded additional fragments besides the expected cleavage products, indicating additional cleavage of the mRNAs at sites other than the miRNA binding site (Figure 3A, Manuscript 1). The mRNA cleavage products may serve as templates for synthesizing cRNA by RdRP (Vaistij et al., 2002) leading to the formation of dsRNA. Subsequently, the dsRNA may be processed into secondary siRNAs resulting in spreading of the initial miRNA signal (Figure 3B, Manuscript 1). In plants, this mechanism, known as transitivity, is initiated by dsRNA triggers (e. g. viruses and transgene transcripts) and transcripts that are targeted by more than one small RNA (Moissiard et al., 2007). In seed plants, the generation of transitive siRNAs from miRNA cleavage products is the exception. To prove the occurrence of transitivity in Physcomitrella, sense and antisense oligonucleotides derived from PpARF and PpC3HDZIP1 mRNA regions upstream and downstream of the miRNA binding sites were used. Sense and antisense siRNAs were only detected in wild type whereas siRNAs derived from miRNA targets were lacking in the ΔPpDCL1b mutants (Figure 10). In Physcomitrella the generation of siRNAs depends on PpDCL1b function and is specific for miRNA-directed cleavage of target RNAs.

Figure 10: Detection of transitive siRNAs derived from miRNA target genes. Detection of sense and antisense siRNAs derived from PpARF and PpC3HDZIP1 with oligonucleotides derived from regions upstream and downstream the miRNA binding sites. Hybridisation with an antisense probe for U6snRNA served as control to indicate equal loading (Figure 3C, Manuscript 1).

18 Chapter I Results and Discussion

1.2.1.1.4 Analysis of DNA methylation in ΔPpDCL1b mutants and wild type

When miRNA targets are not cleaved, the respective mRNAs are likely to accumulate to higher levels in the ΔPpDCL1b mutant lines. Conversely, in ΔPpDCL1b mutants all miRNA targets analyzed had reduced transcript levels when compared to wild type (Figure 11), although these mRNAs were not cleaved in these mutants. It is tempting to speculate that other RNAi components may sense the defective target cleavage as some of them were shown to direct heterochromatin formation and gene silencing. One probable explanation for these unexpected findings is a yet undiscovered epigenetic control of genes encoding miRNA targets in Physcomitrella. Since methylation of cytosine residues is the most prominent mechanism for transcriptional silencing in eukaryotes (Bender, 2004), this possibility was tested by methylation-specific PCR from the miRNA target genes and the control gene (PpGNT1) which is not regulated by a miRNA.

Figure 11: Expression levels of miRNA target genes in ΔPpDCL1b mutants and wild type. RNA blots analysis of miRNA target genes PpARF, PpC3HDZIP1, PpHB10 and PpSBP3 and two control genes, PpGNT1 and PpEF1α. (Figure 4B, Manuscript 1).

In some cases, endogenous siRNAs have an influence on epigenetic control, DNA methylation and chromatin structure at target loci and are associated with RNA-directed DNA methylation (RdDM) and chromatin remodeling (Hamilton et al., 2002; Zilberman et al., 2003; Xie et al., 2004). In plants, dsRNAs which contain sequences that are homologous promoter regions can trigger promoter methylation and transcriptional gene silencing (Melquist and Bender, 2003; Matzke and Birchler, 2005). A function of miRNA 165/166 in directing DNA methylation was shown in the regulation of the homeodomain-leucine zipper (HD-ZIP) transcription factor genes PHABULOSA (PHB) and PHAVOLUTA (PHV) in Arabidopsis (Bao et al., 2004). Promoter regions of the miRNA target genes and the control gene were unmethylated in wild type, whereas in the ΔPpDCL1b mutants the promoters of the genes encoding miRNA targets were methylated (Figure 4D, Manuscript 1). In the latter, methylation occurred specifically at CpG residues (Figure S7, Manuscript 1). In contrast, the promoter of the control gene PpGNT1 remained unaffected in the mutants. Taken together, this reveals a specific epigenetic control of genes encoding miRNA targets upon PpDCL1b dysfunction and subsequent impeded miRNA-directed mRNA cleavage.

19 Chapter I Results and Discussion

In Physcomitrella the pC3HDZIP1 and PpHB10 harbor an intron within their miRNA binding site (Figure S8, Manuscript 1). Therefore, it is unlikely that DNA methlyation is initiated by the formation of an miRNA:DNA hybrid. The miRNA:mRNA duplex may be required to control the DNA methlyation. In Arabidopsis, the composition of a nucleolar complex involved in the siRNA-directed silencing of endogenous repeat regions has been recently identified (Bao et al., 2004). This complex combines several proteins which have been linked to RdDM including RDR2, DCL3 and AGO4. In the yeast Schizosaccharomyces pombe, the RITS complex containing AGO1, a chromodomain protein (Chp1) and other proteins, was shown to bind siRNAs to direct DNA heterochromatin formation (Verdel et al., 2004). In Physcomitrella, the detection of the miRNA:mRNA duplexes in the ΔPpDCL1b mutant lines (Figure 4G, Manuscript 1) suggests that the miRNAs are not incorporated into the RISC but may form a free duplex. Subsequently, this duplex guides the RITS complex to the corresponding genomic region resulting in the initiation of DNA methylation.

1.2.1.1.5 Analysis of the ta-siRNA pathway in ΔPpDCL1b mutants

To challenge the findings obtained from the transitivity and miRNA-dependent DNA methylation the ta-siRNA pathway was analysed. In Physcomitrella, all four ta-siRNA precursors (TAS1-4 RNAs) analyzed to date are cleaved within two distinct miRNA390 binding sites resulting in the production of ta-siRNAs (Axtell et al., 2006; Talmor-Neiman et al., 2006). The mRNA encoding an EREBP/AP2 transcription factor is targeted by one of the ta-siRNAs derived from the TAS4 precursor (Talmor-Neiman et al., 2006). The abolished miRNA390-directed cleavage of TAS4 precursor resulted the lack of ta-siRNAs in ΔPpDCL1b mutant lines (Figure 5A and B, Manuscript 1) revealing that PpDCL1b is required to initiate the ta-siRNA pathway. According to the findings obtained from miRNA target genes, the lack of ta-siRNAs in the ΔPpDCL1b mutants should abolish cleavage of the EREBP/AP2 mRNA and subsequently transitive siRNAs derived from it should be missing. In agreement with findings for miRNA target genes the level of target TAS4-RNA was down-regulated in the ΔPpDCL1b mutants (Figure 12), and the TAS4 genomic locus was methylated in the ΔPpDCL1b mutants but not in wild type (Figure 5D, Manuscript 1). Indeed, the mRNA level of EREBP/AP2 was increased in the ΔPpDCL1b mutants (Figure 12) and the cognate genomic locus was unmethylated in wild type but methylated in the ΔPpDCL1b mutants (Figure 5D, Manuscript 1).

Figure 12: Analysis of expression levels of PpTAS4 and PpEREBP/AP2 in ΔPpDCL1b mutants and wild type. RNA blots analysis of PpTAS4 and PpEREBP/AP2. Ethidium bromide staining shown as loading control below. (Figure 5C, Manuscript 1).

20 Chapter I Results and Discussion

1.2.1.1.6 Analysis of ΔPpDCL1b mutants and wild type lines expressing amiR-GNT1

To check whether the mechanism of epigenetic silencing occurred in Physcomitrella wild type, an amiRNA targeting the control gene PpGNT1 in Physcomitrella wild type as well as in the ΔPpDCL1b mutants was used. PpGNT1-amiRNA was expressed from the Arabidopsis thaliana miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression construct showed precise processing of the PpGNT1- amiRNA (Figure 6B, Manuscript 1). However, normalization of the PpGNT1-amiRNA hybridization signal to the U6snRNA control revealed amiRNA expression levels which differed between the individual lines (Figure 6B, Manuscript 1). In agreement with the results obtained from miRNA target genes, the cleavage product of PpGNT1 in the ΔPpDCL1b mutant background was not detect (Figure 6C, Manuscript 1), and an efficient knock-down of the PpGNT1 gene in the plants expressing the PpGNT1-amiRNA and the transcript level of PpGNT1 even lower in the ΔPpDCL1b mutant background (Figure 6D, Manuscript 1). The PpGNT1 promoter was methylated in the ΔPpDCL1b mutant background (Figure 6E and Figure S9, Manuscript 1). Also DNA methylation at the PpGNT1 promoter in the wild type background which showed a strong expression of the PpGNT1-amiRNA was detected whereas the PpGNT1 promoter was unmethylated in the wild type background expressing the PpGNT1-amiRNA at a low level (Figure 6E and Figure S9, Manuscript 1). This finding suggests that the ratio of the miRNA and its target mRNA is crucial for the induction of DNA methylation at the target locus. At low concentrations the miRNA might be effectively loaded into a cleavage competent RISC to direct target cleavage. If the miRNA concentration reaches a certain threshold the RISC loading capacity for the miRNA might be limited and excessive miRNAs form a duplex with their targets, the excess miRNA might be loaded immediately into an effector complex such as RITS triggering duplex formation that directs DNA methylation (Figure 7, Manuscript 1).

1.2.1.1.6.1 Specific methylation of a miRNA1026 target gene in response to the phytohormone abscisic acid (ABA)

The results obtained from the analysis of PpGNT1-amiRNA expressing lines indicated that in Physcomitrella patens miRNAs control the expression of target RNAs at the post- transcriptional and transcriptional level (Figure 7, Manuscript 1). Expression profiling experiments using a Physcomitrella microarray (unpublished data) revealed an ABA- mediated repression of a gene encoding a basic helix-loop-helix transcription factor (PpbHLH) in wild type and was down-regulated in ΔPpDCL1b mutants (Figure 13). This

21 Chapter I Results and Discussion gene has been predicted to be targeted by the Physcomitrella miRNA1026 (Axtell et al., 2007).

Figure 13: Expression profile of PpbHLH. Expression level of PpbHLH down-regulated in response to 10 µM ABA and in ΔPpDCL1b mutants.

RNA gel blots confirmed the down-regulation of PpbHLH in response to ABA and corresponding ABA-induced increase of miRNA1026 expression levels (Figure 6G and H, Manuscript 1). In agreement with hypothesis that miRNAs control the expression of their targets at the post- transcriptional and transcriptional level, the PpbHLH promoter as well as intragenic regions were found to be methylated in the plants treated with ABA (Figure 6J, Manuscript 1). From these results, epigenetic silencing of miRNA target loci contributes to the control of target gene expression in Physcomitrella was concluded. Although this phenomenon in ΔPpDCL1b mutants was initially discovered, subsequent analyses of the miR1026/PpbHLH regulon confirmed that this type of miRNA-dependent control operates also in wild type.

1.2.1.1.7 Expression profiling of transcription factor genes in ΔPpDCL1b mutant lines

Approximately 6% of the protein coding genes are considered to encode transcription factors in Arabidopsis (Riechmann et al., 2000). In addition, more than 50 % of the predicted miRNA target genes belong to the class of transcription factor encoding mRNAs (Rhoades et al., 2002). In Physcomitrella patens, the comparison of the expression pattern of transcription factor encoding genes between wild type and ΔPpDCL1b mutants will identify putative candidate genes, which are regulated by RNAi. It is likely that more genes which are miss-regulated in the ΔPpDCL1b mutants and direct miRNA and ta-siRNA targets were able to be identified. RNA from wild type and two ΔPpDCL1b mutants was hybridized on a custom Combimatrix 12K oligonucleotide microarray representing 1,427 Physcomitrella patens assembled transcript sequences encoding more than 400 Transcription Associated Proteins (TAPs). Corresponding gene models assigned for 1,200 assembled transcripts (Richardt, 2009). In ΔPpDCL1b mutants, all previously analyzed miRNA targets (PpARF, PpC3HDZIP1, PpHB10 and PpSBP3) were downregulated and the ta-siRNA target gene (PpEREBP/AP2) was upregulated when compared to wild type. I hypothesized that the downregulated genes of transcription factors in Physcomitrella ΔPpDCL1b mutants could be putative miRNA target genes and the upregulated ones could be ta-siRNA target genes. Clustering of expression

22 Chapter I Results and Discussion profiles showed different gene expression between ΔPpDCL1b mutant lines and wild-type plants (Figure 14).

Figure 14: Expression profiling of genes in ΔPpDCL1b mutant lines and Wild type. Differential gene expression in ΔPpDCL1b mutant lines and wild-type plants, 213 genes which were downregulated and 273 genes upregulated in ΔPpDCL1b mutant lines (Appendix 4 and 5). MiRNA and ta-siRNA target genes supposed to be downregulated and upregulated in ΔPpDCL1b mutant lines, respectively.

46 miRNA target genes are present on the Combimatrix 12K oligonucleotide microarray. Normalization and statistical analysis identified 20 miRNA target genes differentially expressed within Physcomitrella ΔPpDCL1b mutant lines; the analysis revealed 13 miRNA target genes downregulated (Table 4) and 7 miRNA target genes upregulated in ΔPpDCL1b mutant lines. By analyzing all upregulated genes in ΔPpDCL1b mutants, the ta-siRNA target genes were predicted using the RNA hybrid program (I. Fattash, personal communication). The parameters used in this program are adopted from Schwab et al. (2005). In agreement with findings for ta-siRNA target genes, the analysis revealed 19 ta-siRNA target genes upregulated in ΔPpDCL1b mutant lines (Table 5).

23 Chapter I Results and Discussion

Table 4: MiRNA target genes downregulated in ΔPpDCL1b mutant lines

Target accession Sequence ID Fold MiRNAs Target description (Annotation) (Gene model) (EST) change miR1026ab Phypa1_132150 † PP015054317R 12-oxophytodienoate reductase (OPR1) -1.5 miR1026ab Phypa1_209063 ‡ PP_12500_C1 basic helix-loop-helix (bHLH) family protein -3.2 miR166 Phypa1_116038 ‡ PP015020123R class III HD-Zip protein HB12 -1.5 miR166 Phypa1_182184 ‡ PP020016117R class III HD-Zip protein HB11 -2.0 miR166 Phypa1_184087 ‡ PP_SD_92_C1 class III HD-Zip protein HB10 -2.0 miR166 Phypa1_192868 ‡ BJ580674 class III HD-Zip protein HB14 -2.0 miR414 Phypa1_167487 ‡ PP_9369_C1 Helix-loop-helix DNA-binding -1.6 miR414 Phypa1_145753 ‡ PP_4238_C1 translation initiation factor 3 subunit 3 / eIF-3 -1.6 miR477a Phypa1_130477 ‡ PP_323_C1 Photosystem subunit V, precursor -1.6 miR538abc Phypa1_109598 ‡ PP020062195R MADS-domain protein PPM2 -2.0 miR538abc Phypa1_94754 ‡ PP_SD_0_C1 agamous-like MADS box protein AGL1 -2.0 miR902f Phypa1_199042 † PP030015063R polyubiquitin (UBQ4), identical to GI:17677 -1.5 miR904 Phypa1_141045 ‡ PP015071162R AGO1-1 (Nicotiana benthamiana) -1.4

Table 5: Ta-siRNA target genes upregulated in ΔPpDCL1b mutant lines

Ta- Target accession Sequence ID Fold Target description (Annotation) siRNAs (Gene model) (EST) change

PpTAS2 Phypa1_160018 † PP_10130_C2 Q8H9A2 Dehydratiion responsive element 1.8 binding protein 1 like protein PpTAS1 Phypa1_188484 † PP_10320_C1 Putative nuclear DNA-binding protein G2p 1.8 PpTAS1 Phypa1_170836 † PP_13554_C1 Q9LKG4 Putative DNA binding protein. 1.3 PpTAS2 Phypa1_53217 † PP_12145_C1 Homolog of hypothetical protein sativa 1.8 PpTAS1 Phypa1_184404 † PP_13985_C1 Arabidopsis thaliana genomic DNA, 1.4 PpTAS1 Phypa1_123311 † PP_15546_C1 Q9LW84 Gb|AAF26996.1. 1.7 PpTAS2 Phypa1_61310 † PP_15997_C1 Q9SI75 F23N19.11 Hypothetical protein 3.0 PpTAS1 Phypa1_168363 † PP_17900_C1 Homolog of (AJ131113) VP1/ABI3-like protein 1.4 PpTAS1 Phypa1_175333 † PP_18393_C1 not annotated Physcomitrella patens 2.0 PpTAS3 Phypa1_203982 † PP_10621_C1 Q9FPV8 Putative methionine aminopeptidase 1.3 PpTAS1 Phypa1_13874 † PP_584_C1 scarecrow-like transcription factor 3 (SCL3) 2.1 PpTAS1 Phypa1_216494 † PP_12254_C1 Homolog of lateral suppressor protein 1.7 PpTAS1 Phypa1_165365 † PP_8332_C1 Homolog of AP2 domain, 7.8 PpTAS1 Phypa1_142162 † PP_8343_C1 Putative 2-isopropylmalate synthase 3.0 PpTAS1 Phypa1_15899† PP004007192R Q9FJ91 Dbj|BAA78737.1 AT5g52010 2.0 PpTAS1 Phypa1_138749 † PP004043210R Q9SGT9 T6H22.8.2 protein. 4.1 PpTAS1 Phypa1_167719 † PP004103024R O99018 Chloroplast protease precursor. 1.7 PpTAS3 Phypa1_79139 ‡ PP015028003R Homolog of zinc finger B-box type family 3.6 PpTAS3 Phypa1_161831 † PP020043294R Mitochondrial transcription termination factor 3.3 ‡ Target validated, † Target predicted

1.2.2 Highly specific gene silencing by artificial miRNAs in Physcomitrella patens

Artificial miRNA (amiRNA) are single-stranded 21-nt small RNAs, which have been used to downregulate single or multiple protein coding genes by guiding their cleavage based on sequence complementarity. Their sequences are designed according to known determinants of target selection for natural miRNAs (Schwab et al., 2005; Schwab et al., 2006). Previous reports have shown that DNA sequences encoding Arabidopsis pre-miRNAs can be expressed from the constitutive CaMV35S promoter in transgenic plants to produce mature

24 Chapter I Results and Discussion miRNAs. Moreover, alterations of several nucleotides within a miRNAs 21-nt sequence do not affect its biogenesis and maturation (Vaucheret et al., 2004). These findings raise the possibility of modifying miRNA sequences according to the determinant miRNA target selection, such that the 21-nt specifically silence their intended target gene(s). In humans miR30 precursor has been modified to generate an amiRNA to downregulate gene expression by translation inhibition (Boden et al., 2004; Dickins et al., 2005). Arabidopsis miRNA precursors have been modified to silence endogenous and exogenous target genes in the dicotyledonous plants Arabidopsis, tomato and tobacco (Parizotto et al., 2004; Alvarez et al., 2006; Niu et al., 2006; Schwab et al., 2006; Qu et al., 2007). Gene silencing in monocotyledon species by amiRNAs has been reported (Warthmann et al., 2008). Previous results have shown that artificial ta-siRNAs (ata-siRNAs) confer consistent and effective gene silencing in Arabidopsis by engineering the TAS1c (ta-siRNAs1c) locus to silence the FAD2 gene (de la Luz Gutierrez-Nava et al., 2008). So amiRNAs and ata-siRNAs make an effective tool for specific gene silencing in plants. In the moss Physcomitrella patens analysis of gene function can be carried out by the generation of targeted gene knockout lines. However, the development of an amiRNA expression system will be a valuable alternative to speed up such analyses. As a proof of concept two amiRNAs, targeting the gene PpFtsZ2-1, which is indispensable for chloroplast division (Strepp et al., 1998), and the gene PpGNT1 encoding an N- acetylglucosaminyltransferase (Koprivova et al., 2003) were designed. Both amiRNAs were expressed from the Arabidopsis thaliana miR319a precursor fused to a constitutive promoter (Figure 1A, Publication 1). Based on the conservation of the miR319 family among land plants and similar secondary structures of miR319 precursor transcripts from Arabidopsis and Physcomitrella (Figure 1B, Publication 1), the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA were correctly processed from the Arabidopsis miR319a precursor. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of the amiRNAs and an efficient knockdown of the cognate target mRNAs (Figure 1D and 2A, Publication 1). Furthermore, chloroplast division was impeded in PpFtsZ2-1- amiRNA lines which phenocopied PpFtsZ2-1 knockout mutants (Figure 15). The formation of macrochloroplasts in the PpFtsZ2-1-amiRNA lines was observed in all tissues and cells analyzed indicating an efficient production of mature amiRNAs from constitutively expressed precursor transcripts. To investigate the possibility of transitivity, sense and antisense oligonucleotides derived from a PpFtsZ2-1 and PpGNT1 mRNA regions downstream the amiRNA recognition site were used for RNA gel blot analysis. Sense and antisense siRNAs were only detected in PpFtsZ2-1-amiRNA and PpGNT1- amiRNA lines, but not in wild type (Figure 2B and C, Publication 1). Additionally, these

25 Chapter I Results and Discussion siRNAs do not seem to have a major effect on sequence-related mRNAs, confirming specificity of the amiRNA approach.

Figure 15: Impeded plastid divison and formation of macrochloroplasts in PpFtsZ2-1-amiRNA overexpressors. A, Light microscopy from protonema and leaves of wild type (WT) and one PpFtsZ2-1-amiRNA overexpression line (Size bars: 100 µm). B, Confocal laser scanning microscopy from protonema and leaves of wild type (WT) and one PpFtsZ2-1-amiRNA overexpression line (Size bars: 50 µm). Red: chlorophyll autofluorescence in plastids. (Figure 3, Publication 1).

26 Chapter I Conclusion

1.3 Conclusion

These findings reveal the existence of novel RNAi pathways in Physcomitrella patens. As opposed to Arabidopsis thaliana, the miRNA-directed posttranscriptional control of target mRNAs in Physcomitrella patens is amplified by transitive siRNAs. Furthermore, we identified a pathway that depends on miRNA:targetRNA duplexes and triggers the silencing of genes encoding miRNA targets. Consequently, ΔPpDCL1b mutants deficient in miRNA target cleavage are not viable in some plants and animals. In contrast, Physcomitrella ΔPpDCL1b mutant lines are viable, although severely affected in several cellular features and in development. From that a model for gene-specific sensing of the levels of specific miRNAs and their target-RNAs (by miRNA:mRNA or miRNA:TAS-RNA duplex formation) was proposed, effective (or ineffective) target cleavage, and subsequent epigenetic control of target-RNA accumulation (Figure 16). In summary, the conclusions are: 1- PpDCL1a is the functional equivalent of Arabidopsis AtDCL1 (miRNA biogenesis). 2- PpDCL1b is essential for miRNA target cleavage (including the ta-siRNA pathway). 3- In Physcomitrella, amplification of miRNA and ta-siRNA signals by transitive siRNAs is a common mechanism. 4- The accumulation of miRNAs and their cognate RNA targets in the ΔPpDCL1b mutants causes a specific hypermethylation of the corresponding genomic loci. 5- MiRNAs induce a specific epigenetic silenicng of miRNA target genes which depends on the miRNA:target ratio and is mediated by the formation of stable miRNA:RNA duplexes. 6- The expression of amiRNAs in Physcomitrella leads to an efficient silencing of their target mRNAs comparable to the effects of targeted gene knockouts. 7- The amplification of the initial amiRNA signal by secondary transitive siRNAs, these siRNAs do not have a major effect on highly conserved gene families, confirming specificity of the amiRNA approach in Physcomitrella.

27 Chapter I Conclusion

Figure 16: Model for the post-transcriptional and epigenetic control of miRNA target genes in Physcomitrella patens. (A) Pathways leading to miRNA target cleavage. The maturation of miRNAs from stem-loop precursors is catalyzed by PpDCL1a. PpDCL1b is required for loading miRNAs into cleavage competent RISC. After loading of miRNAs into RISC (consisting of PpDCL1b, AGO and unknown proteins) transient miRNA:target-RNA duplexes form based on sequence complementarity. Subsequently, target-RNAs are cleaved. From the cleavage products dsRNA is produced by the action of RdRP. Subsequently, the dsRNA is processed to generate transitive siRNAs (from mRNA cleavage products) or ta-siRNAs (from TAS-RNAs).Transitive siRNAs lead to an amplification of the miRNA trigger; ta-siRNAs are directed to their mRNA targets guiding their cleavage. (B) Epigenetic control of miRNA target genes. In the ΔPpDCL1b mutant lines miRNAs are not loaded into cleavage competent RISC and target cleavage is abolished. Also in Physcomitrella patens wild type miRNAs can accumulate which cannot be loaded efficiently into RISC. In both cases miRNAs may be loaded into alternative complexes such as the RITS complex and targeted to cognate target-RNAs. These miRNA:RNA duplexes bound by RITS enter the nucleus and initiate DNA methylation at complementary genomic loci. The RITS complex expands into adjacent regions (e. g. promoters) and completes CpG methylation of the entire genomic locus. In consequence, genomic loci are silenced and accumulation of mRNAs is inhibite

28 Chapter I References

1.4 References

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33 Chapter II Transcriptional control of gene expression by microRNAs

2 Chapter II: Manuscript 1 Transcriptional control of gene expression by microRNAs

Own contribution:

Carried out all molecular and phenotypic analyes of the ΔPpDCL1b mutants, generated and analyzed the amiR-GNT1 overexpression lines, and performed the complete analysis of the miR1026 and its PpbHLH target. Writing parts of the manuscript and preparing the figures. The work was supervised by W. Frank

34 Transcriptional control of gene expression by microRNAs

Basel Khraiwesh1, M. Asif Arif1, Gotelinde I. Seumel1, Stephan Ossowski2, Detlef

Weigel2, Ralf Reski1,3,4, Wolfgang Frank1,3*

1Plant Biotechnology, Faculty of Biology, University of Freiburg, Schänzlestraße 1, D-

79104 Freiburg, Germany

2Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-

72076 Tübingen, Germany

3Freiburg Initiative for Systems Biology (FRISYS), Faculty of Biology, University of

Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany

4Centre for Biological Signalling Studies (bioss), University of Freiburg, Schänzlestr. 1,

D-79104 Freiburg, Germany

* Corresponding author

Phone: +49 (0)761-203-2820

Fax: +49 (0)761-203-6945

Email: [email protected]

1 Summary

MicroRNAs (miRNAs) control gene expression in animals and plants. They share with another class of small RNAs, siRNAs, the ability to post-transcriptionally affect target mRNAs. In contrast to siRNAs, however, the role of miRNAs in transcriptional regulation has been less clear. Here we reveal dual transcriptional and post- transcriptional activities of miRNAs in Physcomitrella patens. In plants lacking activity of one DICER-LIKE gene (PpDCL1b), miRNA target genes are silenced. The specific function of PpDCL1b in miRNA-mediated target cleavage suggests that changes in the ratio of the miRNA and its targets cause miRNA:target-RNA duplex formation, which in turn triggers DNA methylation. We propose that miRNA-mediated transcriptional silencing, which also occurs in wild type plants, provides a mechanism critical for homeostasis of miRNA-dependent gene expression.

Introduction

Small RNAs (sRNAs) are important regulators of post-transcriptional and transcriptional gene expression (Meister and Tuschl, 2004). In plants, microRNAs (miRNAs), which are produced from hairpin-like precursor transcripts, are also required for the biogenesis of trans-acting small interfering RNAs (ta-siRNAs). Both miRNAs and ta- siRNAs regulate mRNA stability and translation, siRNAs, which originate from perfectly double-stranded RNA (dsRNA) precursors post-transcriptionally silence transposons, viruses and transgenes and are important for the establishment and maintenance of cytosine DNA methylation (Baulcombe, 2004). Even though the role of plant siRNAs in the methylation of cognate genomic loci is well understood (Matzke et al., 2007), evidence for a similar function of miRNAs in directing DNA methylation is limited. The biogenesis of sRNAs from dsRNA is catalyzed by Dicer proteins and the size of the

Dicer gene family varies between organisms, reflecting different degrees of specialization of Dicer proteins. For example, in D. melanogaster Dcr1 produces

2 miRNAs from hairpin precursors, whereas Dcr2 generates siRNAs from dsRNA molecules (Tomari and Zamore, 2005). By contrast, in C. elegans the single Dicer protein DCR-1 is directed by accessory proteins such as PIR-1, ER-1 and RRF-3 to produce sRNAs from different dsRNA triggers (Duchaine et al., 2006). Besides their function in dicing dsRNA, animal Dicers are associated with accessory proteins in complexes which act as RISC (RNA-induced silencing complex) or RISC loading complexes (Doi et al., 2003; Pham et al., 2004). Thus, Dicer proteins are also essential components in the executive phase of RNAi, indicating that miRNA/siRNA processing and target RNA cleavage are coupled. Dcr-2 from Drosophila, which produces siRNA, acts together with its partner R2D2 to load one of the two siRNA strands into RISC(Liu et al., 2003; Tomari et al., 2004). Similarly, human Dicer associated with Ago2, TRBP and RHA acts in RISC assembly (MacRae et al., 2008) which is further supported by the observation that siRNAs cannot cause post-transcriptional gene silencing in human cells lacking Dicer (Doi et al., 2003).

In the plant A. thaliana, the four DCL proteins (AtDCL1-4) act in specific sRNA pathways, with some functional redundancies of the four isoforms (Gasciolli et al.,

2005; Henderson et al., 2006). The maturation of miRNAs from imperfect RNA foldbacks relies on AtDCL1 activity. In consequence, A. thaliana dcl1 mutants have significantly reduced miRNA levels and a corresponding increase in target mRNA levels, which causes a multitude of developmental defects (Golden et al., 2002; Park et al., 2002). AtDCL2 mediates the generation of siRNAs from exogenous RNA sources

(Xie et al., 2004), AtDCL3 is required for the formation of heterochromatin-associated endogenous siRNAs (Herr et al., 2005; Xie et al., 2004) and AtDCL4 is needed for the formation of ta-siRNAs involved in systemic cell-to-cell transmission of silencing signals

(Dunoyer et al., 2005; Xie et al., 2005).

The genome of the moss Physcomitrella patens encodes four DCL proteins

(Axtell et al., 2007). PpDCL1a and PpDCL1b are very similar to AtDCL1 (Figure S1).

3 PpDCL3 and PpDCL4 proteins are orthologs of AtDCL3 and AtDCL4, whereas an

AtDCL2 ortholog is lacking. The primary PpDCL1a transcript harbors a miRNA precursor within one intron, which is reminiscent of AtDCL1, and suggests a conserved autoregulatory control of mRNA maturation (Axtell et al., 2007). Together with the slightly greater sequence similarity, this led us to hypothesize that PpDCL1a as the functional equivalent of AtDCL1 is required for miRNA biogenesis, while the additional presence of PpDCL1b suggested also potential differences in sRNA pathways between

P. patens and A. thaliana. Here, we present an analysis of P. patens ΔPpDCL1a and

ΔPpDCL1b knockout mutants, which supports differences in sRNA pathways such as the formation of transitive siRNAs. Moreover, we propose a mechanism for the miRNA- mediated transcriptional silencing of miRNA target genes that relies on miRNA abundance, formation of miRNA:target-RNA duplexes and DNA methylation.

Results

Requirement of PpDCL1a for miRNA biogenesis

Taking advantage of the efficient homologous recombination system in P. patens

(Strepp et al., 1998) we generated two PpDCL1a knockout mutants (ΔPpDCL1a)

(Figure S2). Complete loss of PpDCL1a function resulted in retarded growth and developmental disorders including abnormalities in cell size, and cell shape. The mutants were arrested at the filamentous protonema stage and did not form gametophores (Figure 1A and 1B).

To test whether miRNA biogenesis is affected in the ΔPpDCL1a mutants, we analyzed the accumulation of miR156, 160, 166, and 390 (Arazi et al., 2005; Fattash et al., 2007). Due to the limited amount of plant material of the ΔPpDCL1a mutant lines, miRNA expression was investigated by RT-PCR (Varkonyi-Gasic et al., 2007). PCR products were sequenced to rule out unspecific amplification. Compared to wild type,

4 miR156, 160 and 166 had drastically reduced levels, while miR390 was undetectable in

ΔPpDCL1a mutants (Figure 1C). In P. patens, all known trans-acting siRNA (ta-siRNA) precursors (PpTAS1-4 RNAs) are initially cleaved at two distinct miR390 target sites.

Subsequently, dsRNAs are generated from the cleavage products and processed in a phased manner to generate ta-siRNAs (Axtell et al., 2006; Talmor-Neiman et al., 2006).

Two ta-siRNAs (pptA079444 processed from PpTAS1, and pptA013298 processed from PpTAS3) (Axtell et al., 2006) were detected in wild type, but were absent in the

ΔPpDCL1a mutants, indicating that the lack of miR390 abolishes ta-siRNA production

(Figure 1D). To test whether reduced levels of miRNAs result in elevated transcript levels of miRNA targets, as observed in A. thaliana dcl1 mutants, we analyzed the expression of the miRNA targets PpSPB3 for miR156 (Arazi et al., 2005),

PpC3HDZIP1 and PpHB10 for miR166 (Axtell et al., 2007; Floyd and Bowman, 2004),

PpARF for miR160 (Fattash et al., 2007), and PpTAS1 for miR390(Axtell et al., 2006).

RT-PCR analysis revealed increased transcript levels of all analyzed miRNA targets in the ΔPpDCL1a mutants (Figure 1E). From these results we conclude that PpDCL1a is the major P. patens DCL protein required for the processing of miRNAs and thus the functional equivalent of A. thaliana DCL1.

Requirement of PpDCL1b for miRNA guided target cleavage

The presence of PpDCL1b as a second P. patens AtDCL1 homolog raised the question whether PpDCL1b acts redundantly with PpDCL1a, given that some miRNAs were not completely abolished in ΔPpDCL1a mutants. We generated four targeted PpDCL1b knockout mutants (ΔPpDCL1b) (Figure S3). The PpDCL1b mutants were strongly affected in cell division, growth polarity, cell size, cell shape and tissue differentiation

(Figure 2A and Figure S4A). The developmentally arrested mutants produced only a small number of malformed gametophores (Figure 2B and Figure S4B). Thus, while

5 both ΔPpDCL1a and ΔPpDCL1b mutants suffer from severe developmental defects, the exact mutant phenotypes differed.

Analysis of six different miRNAs, miR156, 160, 166, 390, 535, and 538(Arazi et al., 2005; Fattash et al., 2007), revealed that their levels were unchanged in ΔPpDCL1b mutants (Figure 2C-2E). Thus, PpDCL1b is not essential for miRNA maturation from precursor RNAs. The severe developmental defects of the mutants prompted us nevertheless to examine miRNA targets PpSPB3, PpC3HDZIP1, PpHB10, and PpARF.

We verified cleavage of these miRNA targets in P. patens wild type based on 5’ RACE, cDNA cloning and sequencing (Figure 3A). An unrelated mRNA (PpGNT1) (Koprivova et al., 2003), which is not miRNA regulated, was examined as control (Figure 3A).

Although the ΔPpDCL1b mutants produced apparently normal levels of miRNAs, the miRNA target transcripts were not cleaved (Figure 3A), indicating a surprising requirement of PpDCL1b for miRNA-guided mRNA cleavage. We propose that

PpDCL1b may act in loading miRNAs into an RNA-cleavage competent RISC, in analogy to what has been reported for animal Dicer proteins (Doi et al., 2003; Liu et al.,

2003; MacRae et al., 2008; Pham et al., 2004; Tomari et al., 2004).

Generation of transitive siRNA triggered by miRNA-guided transcript cleavage

In P. patens wild type we had not only detected 5’ RACE products resulting from miRNA-guided cleavage of the target mRNA, but also a variety of shorter and longer products (Figure 3A). In analogy with other plant systems, where targeting of a transcript with dsRNA-derived siRNAs or multiple miRNAs (Axtell et al., 2006; Howell et al., 2007; Vaistij et al., 2002) causes the production of secondary siRNAs, the miRNA cleavage products may serve as templates for synthesizing cRNA by RNA-dependent

RNA polymerase (RdRP). Subsequently, the resulting dsRNA may be processed into secondary siRNAs resulting in spreading of the initial trigger signal (Figure 3B). In

6 flowering plants, this phenomenon, known as transitivity, is, however, rarely observed after targeting of an mRNA with a single miRNA (Axtell et al., 2006; Howell et al.,

2007).

To investigate the possibility of transitivity in P. patens, we asked whether one could synthesize cDNA from both the sense and antisense strand of miRNA target mRNAs (PpARF and PpC3HDZIP1). Indeed, this was the case in wild type, indicating the presence of dsRNA. Such dsRNA molecules were lacking in ΔPpDCL1b mutants

(Figure 3C). To determine whether transitive siRNAs were generated from these dsRNAs, we performed small RNA blots using probes for both upstream and downstream sequences relative to the miRNA targeting site. Such small RNAs corresponding to sense and antisense strands of PpARF and PpC3HDZIP1 mRNAs were detected in wild type, but not in ΔPpDCL1b mutants (Figure 3D). Thus, in P. patens transitive siRNAs arise from regions upstream as well as downstream of the miRNA targeting motif after miRNA-directed cleavage of mRNAs. These siRNAs most likely cause cleavage of the cognate mRNAs at additional sites, which explains why we observed additional mRNA fragments in the 5’ RACE analyses. We did not detect such siRNAs in the ΔPpDCL1b mutants, nor for the control mRNA (PpGNT1) in wild type

(Figure 3C and 3D), indicating that the generation of transitive siRNAs depends on

PpDCL1b and is specific for mRNAs subject to miRNA-directed cleavage.

DNA methylation of miRNA target loci in ΔPpDCL1b mutants

A. thaliana ago1 and dcl1 mutants are defective in miRNA-directed target cleavage or miRNA biogenesis, respectively. Consequently, transcript levels of miRNA targets are elevated in both mutants (Ronemus et al., 2006). Conversely, all miRNA targets analyzed had reduced transcript levels in ΔPpDCL1b mutants (Figure 4A and 4B), even though they were not cleaved. As explanation for these unexpected findings we

7 considered the possibility that miRNA target loci are under epigenetic control in P. patens. Since methylation of cytosine residues is the most prominent mechanism for transcriptional silencing in plants and other eukaryotes (Bender, 2004), we evaluated this scenario by methylation-specific PCR of four miRNA target loci, along with an unrelated locus, PpGNT1 (Figure 4C-4F and Figure S5 and Figure S6). Promoters of all five genes were unmethylated in wild type, but in ΔPpDCL1b mutants the four miRNA target promoters were methylated (Figure 4D). These findings were confirmed by sequencing the PCR products of the PpARF promoter from wild type and

ΔPpDCL1b mutants. In the latter, methylation occurred specifically at CpG residues

(Figure S7). Taken together, we conclude that disruption of PpDCL1b causes specific epigenetic changes in genes encoding miRNA targets, and that this is accompanied by a loss of miRNA-directed mRNA cleavage.

It is well-known that siRNA pathways govern DNA methylation in A. thaliana, e.g. at repeat-associated loci (Herr et al., 2005). However, only one study has suggested a function of miRNAs in the silencing of cognate target genes, at the PHB and PHV loci, which are targeted by miR165/166. Normally methylated DNA sequences downstream of the miRNA complementary motif became hypomethylated in plants with dominant alleles of PHB and PHV, while the promoters remained unmethylated (Bao et al., 2004). The dominant alleles carry in the miRNA targeting motif, such that the encoded mRNAs are no longer susceptible to miRNA- guided cleavage.

Like PHB and PHV, the P. patens HD-ZIP homologs PpC3HDZIP1 and PpHB10 harbor an intron within their miRNA binding site (Figure S8), whereas the miRNA targeting motif in PpARF is not disrupted by an intron. Similar to the promoters,

PpC3HDZIP1 and PpARF sequences flanking the miRNA targeting motif as well as the

8 intron disrupting the miR166 binding site of PpC3HDZIP1 were CpG methylated in

ΔPpDCL1b mutants, but not in P. patens wild type (Figure 4E and 4F and Figure S7).

One scenario that can account for the findings presented so far is that

PpDCL1b normally acts in loading miRNAs into an RNA-cleavage competent RISC. In the absence of PpDCL1b, miRNAs might be loaded instead into an RNA-induced transcriptional silencing complex (RITS) directing DNA methylation of miRNA target loci. As the sequence of the miR166 binding site is disrupted by introns in two genes

(PpC3HDZIP1 and PpHB10), it is unlikely that their methylation in ΔPpDCL1b mutants is initiated by the formation of miRNA:DNA hybrids. Instead, the miRNA-loaded RITS complex might interact with the target mRNA, resulting in the formation of a stable miRNA:mRNA duplex. Subsequently, this duplex could guide the RITS complex to the corresponding genomic region, resulting in the initiation and spreading of DNA methylation.

If stable miRNA:mRNA duplexes are present in ΔPpDCL1b mutants, it should be possible to synthesize cDNA without added exogenous primers, which can subsequently be detected by conventional PCR. In support of such a scenario we obtained RT-PCR products for all miRNA targets examined, but not for a control locus in ΔPpDCL1b mutants. No such products were obtained with RNA from wild type plants

(Figure 4G). In addition, in the ΔPpDCL1b mutants no PCR products were obtained when using PCR primers located downstream of the miRNA targeting site. As a further control, we heated the RNA samples prior to cDNA synthesis. This should lead to denaturation of a miRNA:mRNA complex and hence eliminate priming; indeed, this procedure prevented the amplification of PCR products in the ΔPpDCL1b mutants

(Figure 4H). These results are compatible with base-paired miRNA:mRNA duplexes being present specifically in RNA samples from ΔPpDCL1b mutants.

9 To further scrutinize our hypotheses of transitivity and miRNA-dependent DNA methylation, we analyzed the ta-siRNA pathway in ΔPpDCL1b mutants. After miR390- mediated cleavage of TAS precursors, the RNA cleavage products are converted into dsRNA and further processed into ta-siRNAs (Axtell et al., 2006; Talmor-Neiman et al.,

2006). The mRNA encoding an EREBP/AP2 transcription factor is targeted by one of the ta-siRNAs derived from the TAS4 precursor (Talmor-Neiman et al., 2006). Hence, the production of ta-siRNAs presents an intermediate step in the miRNA-dependent control of mRNAs. TAS4 RNA cleavage products resulting from miRNA390-directed cleavage were detected by 5’ RACE-PCR in P. patens wild type, but not in the

ΔPpDCL1b mutants (Figure 5A), even though miR390 was present in equal amounts in

ΔPpDCL1b mutants and wild type (Figure 2C). Furthermore, ta-siRNAs of both sense and antisense orientation were present in wild type, but were undetectable in

ΔPpDCL1b mutants (Figure 5B), confirming that PpDCL1b is required to initiate the ta- siRNA pathway.

In agreement with our findings for other miRNA targets, TAS4 transcript levels were reduced in ΔPpDCL1b mutants (Figure 5C). Likewise, the TAS4 genomic locus was methylated only in ΔPpDCL1b mutants (Figure 5D). If, similar to miRNAs, ta- siRNA-mediated cleavage of target mRNAs also initiates the generation of transitive secondary siRNAs, the lack of ta-siRNAs in ΔPpDCL1b mutants should abolish both cleavage of EREBP/AP2 mRNA and transitive siRNAs. Consistent with this scenario, the EREBP/AP2 mRNA was cleaved in wild type, but not in ΔPpDCL1b mutants (Figure

5A), and only wild type produced EREBP/AP2 mRNA-derived siRNAs in sense and antisense orientation (Figure 5B). This observation indicates that the siRNA-dependent amplification of target RNA degradation that was initially triggered by ta-siRNA/miRNA- guided cleavage is a common mechanism in P. patens. In addition, we expected that, in contrast to direct miRNA targets, EREBP/AP2 mRNA levels should be elevated in

10 ΔPpDCL1b mutants, as stable ta-siRNA:mRNA duplexes that could guide DNA methylation at the corresponding genomic locus should be absent. Indeed, the

EREBP/AP2 RNA levels were increased in ΔPpDCL1b mutants (Figure 5C), and the genomic locus was methylated neither in wild type nor in ΔPpDCL1b mutants (Figure

5D).

Dependence of DNA methylation at miRNA target loci on miRNA expression levels

We propose that the formation of stable miRNA:target RNA duplexes leads to methylation of the corresponding genomic regions in ΔPpDCL1b mutants. Is this mechanism of epigenetic silencing also relevant in wild type P. patens? To investigate this question, we generated wild type and ΔPpDCL1b mutant plants expressing different levels of an artificial miRNA targeting our control gene PpGNT1.

Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA* sequence of endogenous miRNA precursor genes, while maintaining the general pattern of matches and mismatches in the foldback. We engineered an amiRNA against PpGNT1 into the A. thaliana miR319a precursor (Khraiwesh et al.,

2008) and expressed the hybrid construct in P. patens wild type and ΔPpDCL1b mutants (Figure 6A). RNA blots confirmed precise maturation of amiR-GNT1 in transformed lines, independent of expression level (Figure 6B). The expected PpGNT1 mRNA cleavage products were present in wild type, but not in ΔPpDCL1b mutants

(Figure 6C). Consequently, compared to P. patens wild type the PpGNT1 transcript levels were reduced in amiR-GNT1 plants (Figure 6D). Despite abolished amiRNA- directed cleavage of PpGNT1 mRNA, transcript levels were even lower in the

ΔPpDCL1b mutant background (Figure 6D).

11 Consistent with our model of miRNA-dependent epigenetic silencing, the

PpGNT1 promoter was methylated in the ΔPpDCL1b mutant background (Figure 6E and Figure S9). Importantly, the PpGNT1 promoter was also methylated in wild type lines, with strong expression of the amiR-GNT1, while it was unmethylated in lines with low levels of amiR-GNT1 (Figure 6E and Figure S9). Thus, specific methylation of miRNA target loci is not limited to ΔPpDCL1b mutants. Based on the observation that methylation in wild type is miRNA-dosage dependent, we hypothesized that the ratio of the miRNA and its target mRNA is crucial for the induction of DNA methylation at the target locus. If the miRNA concentration exceeds a certain threshold, the miRNA may either interact directly with its target and the duplex might then be recruited into a DNA methylation silencing complex, or the excess miRNA might be loaded immediately into an effector complex such as RITS triggering duplex formation that directs DNA methylation. We obtained supporting evidence for the expected amiR-GNT1:PpGNT1- mRNA duplexes by cDNA synthesis without exogenous primers and subsequent PCR in ΔPpDCL1b mutants. Importantly, we could amplify such products also in a wild type line with high levels of amiRNA expression, but not in a wild type line expressing only moderate amounts of amiR-GNT1 (Figure 6F).

Hormone-dependent DNA methylation of a miR1026 target locus

The analysis of amiRNA-GNT1 lines had shown that miRNA-directed epigenetic silencing occurs also wild type, but that it is dependent on miRNA levels. We therefore sought to identify endogenous miRNAs that might be induced to high levels in response to specific stimuli, which in turn should be reflected by downregulation of target mRNAs. In separate experiments we had found that treatment with the hormone abscisic acid (ABA) strongly represses expression of a basic helix-loop-helix (bHLH) transcription factor gene, PpbHLH, which has been predicted to be targeted by

12 miR1026 (Axtell et al., 2007). ABA is a well known signaling molecule in abiotic stress signaling pathways in plants including mosses (Frank et al., 2005b). RNA gel blots confirmed downregulation of PpbHLH in response to ABA (Figure 6G). This effect correlated well with an ABA-induced increase of miR1026 levels (Figure 6H), suggesting direct regulation of PpbHLH by miR1026. We confirmed miR1026-mediated cleavage of PpbHLH transcript by 5’ RACE (Figure 6I).

To evaluate transcriptional effects of miR1026, we analyzed DNA methylation of the PpbHLH gene, including the promoter and transcribed sequences. Upon ABA treatment, PpbHLH became methylated at specific CpG sites (Figure 6J and Figure

S10), consistent with the methylation patterns we had found before in ΔPpDCL1b mutants. The promoter of an unrelated gene, PpGNT1, was unmethylated regardless of ABA treatment (Figure 6J).

Our model posits that DNA methylation will be initiated if the miRNA target ratio exceeds a certain threshold. That DNA methylation of the PpbHLH locus is not quantitative, as deduced from the observation that unmethylation-specific primers allowed albeit inefficient PCR amplification in ABA-treated samples, may reflect cell type-specific differences in miRNA or target expression levels. Finally, we tried to obtain evidence for stable miR1026:PpbHLH-mRNA duplexes by unprimed RT-PCR.

Consistent with the DNA methylation status, such duplexes were only found in the

ABA-treated samples (Figure 6K).

We conclude that in P. patens, epigenetic silencing of miRNA target loci contributes to the control of target gene expression. Although we initially discovered this phenomenon in ΔPpDCL1b mutants, subsequent analyses of the miR1026/PpbHLH regulon confirmed that this type of miRNA-dependent control operates also in wild type.

13 Discussion

Our studies suggest that PpDCL1a is the functional ortholog of AtDCL1 required for miRNA and ta-siRNA biogenesis. Even though PpDCL1b shares a similar level of sequence identity with AtDCL1, we propose that it has a distinct function, since its inactivation does not affect miRNA biogenesis, but abolishes miRNA-directed target cleavage. It is unlikely that PpDCL1b directly cleaves target RNAs, as AGO proteins in

RISC are the catalytic enzymes in sRNA-dependent target cleavage (MacRae et al.,

2008). Biochemical analysis of AGO1 complexes immunoprecipitated from Arabidopsis dcl1-7, dcl2-1 and dcl3-1 mutants provided evidence for distinct functional properties.

An AGO1 complex extracted from dcl1-7 mutants was not able to cleave RNA targets due to the lack of ~21 nt small RNA accumulation in this mutant. In contrast, cleavage of RNA targets was not affected in AGO1 complexes from dcl2-1 and dcl3-1 mutants

(Qi et al., 2005). Furthermore, purification of Arabidopsis AGO1 revealed a ~160 kDa complex, most likely only consisting of AGO1 and associated sRNA (Baumberger and

Baulcombe, 2005). Thus, there is so far no evidence to support a function of plant DCL proteins in sRNA-mediated target cleavage. In contrast, studies in animals have shown that Dicer proteins are part of the RNA loading complex (RLC), which loads sRNAs into

RISC. Human RLC comprises the proteins Ago2, Dicer and TRBP and the purified protein components assemble spontaneously in vitro without requirement of any cofactors. The reconstituted RLC is fully functional and once Ago2 is loaded with a miRNA it tends to dissociate from the rest of the complex (MacRae et al., 2008).

Similarly, Dcr-2 from D. melanogaster, which produces siRNA, acts in the RISC assembly together with its partner R2D2 by loading one of the two siRNA strands into

RISC (Liu et al., 2003; Tomari et al., 2004). The C. elegans homolog of this protein,

RDE-4, was also found to interact with Dicer (Tabara et al., 2002). Given this particular function of animal Dicer proteins, we hypothesize that P. patens PpDCL1b may exhibit

14 an equivalent function in loading miRNAs into RISC, making it indispensable for miRNA-directed target cleavage.

Arabidopsis dcl1 and ago1 mutants, which are affected in miRNA biogenesis or miRNA-directed target cleavage, respectively, exhibit elevated transcript levels of miRNA targets (Ronemus et al., 2006). Likewise, miRNA target transcripts are increased in ΔPpDCL1a mutants due to the lack of miRNAs. In contrast, levels of miRNA target mRNAs are drastically reduced in ΔPpDCL1b mutants, in spite of abolished target RNA cleavage. We have shown that cytosine residues within the corresponding genomic loci are methylated in ΔPpDCL1b mutants, suggesting epigenetic control at the transcriptional level. Small RNAs initiate transcriptional silencing of homologous sequences by methylation of cytosine residues at CpG,

CpNpG, and CpHpH sequence motifs or by histone modifications (Bender, 2004; Cao and Jacobsen, 2002). In all genomic regions analyzed in the ΔPpDCL1b mutants, we only detected methylation at CpG dinucleotides, but cannot exclude that cytosine methylation may also occur at different sequence contexts in other regions. Moreover, we detected CpG methylation in large regions of the genomic loci encoding miRNA targets including introns, exons and promoter regions pointing to methylation that is able to spread over considerably long distances.

Although spreading of siRNA-directed DNA methylation into adjacent non repeated sequences is not common in A. thaliana, siRNA-mediated spreading of DNA methylation has been observed for the SUPPRESSOR OF drm1 drm2 cmt3 (SDC) locus, where methylation spreads beyond siRNA generating repeat regions present in the SDC promoter (Henderson and Jacobsen, 2008). In Arabidopsis, cytosine methylation can also spread in the PHV and PHB genes, which are targets of miR165/166 (Bao et al., 2004). In both genes, the miR165/166 complementary motif is disrupted by an intron and the coding sequence was found to be heavily methylated

15 downstream of the miRNA complementary site in differentiated, but not undifferentiated cells of wild type plants. Furthermore, methylation was reduced in phv-1d and phb-1d mutants, which have an altered miRNA recognition motif or a in the intron splice donor sequence, suggesting that miR165/166 needs to bind to nascent PHV and

PHB transcripts to trigger gene silencing (Bao et al., 2004).

Similarly, the P. patens HD-Zip genes PpC3HDZip1 and PpHB10 are targeted by miR166 and the miR166 binding sites are only reconstituted after splicing of an intron from the primary transcripts. These loci are hypermethylated in ∆PpDCL1b mutants, but not in wild type, suggesting that the initiation of CpG methylation upon defective target cleavage cannot be mediated by miR166, but involves binding of miR166 to the cognate target mRNAs. We have obtained evidence for the presence of stable duplexes of a miRNA and its target RNA in ∆PpDCL1b mutants. We propose that such duplexes guide a DNA modification complex.

In Arabidopsis, RNA-directed DNA methylation (RdRM) by siRNAs requires

RDR2, DCL3 and RNA PolIVa, which are all involved in siRNA biogenesis (Herr et al.,

2005; Kanno et al., 2005; Onodera et al., 2005; Pontier et al., 2005; Xie et al., 2004), whereas AGO4, DRM2, DRD1 and RNA PolIVb are indispensable for DNA methylation

(Cao and Jacobsen, 2002; Kanno et al., 2005; Zilberman et al., 2004). In fission yeast,

RNA-directed heterochromatic gene silencing at centromeres relies on two different complexes, the RITS complex comprising Ago1, Chp1 and TAS3, and the argonaute siRNA chaperone complex (ARC) comprising Ago1, Arb1 and Arb2. However, these complexes are required to direct histone H3 Lys9 methylation, but do not direct cytosine methylation. Nevertheless, it has been proposed that their action involves the recognition of nascent transcripts by RITS-bound siRNAs to promote recruitment of chromatin-modifying enzymes that implement silencing (Buker et al., 2007).

We also detected the specific silencing of miRNA target genes in P. patens wild type, where the expression of amiR-GNT1 caused methylation of the PpGNT1 genomic

16 locus. Moreover, we found that methylation of the locus is dependent on amiR-GNT1 abundance and only obtained evidence for amiR-GNT1:PpGNT1-mRNA duplexes in lines with high amiRNA levels, supporting the hypothesis that miRNA:target-RNA duplexes are required for DNA methylation. Finally, we have been able to show that the genomic region of the miR1026 target PpbHLH becomes methylated in response to

ABA, which upregulates miR1026 expression. Also in this case, DNA methylation was miR1026 dosage-dependent and appeared to correlate with the formation of stable miR1026:PpbHLH-mRNA duplexes. As ABA acts as a mediator of abiotic stress signaling, we assume that the miR1026-regulated silencing of PpbHLH is part of stress adaptation in P. patens.

In plants, epigenetic changes as a response to stress conditions have been previously shown to include DNA methylation, histone modifications and chromatin remodeling (Boyko and Kovalchuk, 2008; Dyachenko et al., 2006; Henderson and

Dean, 2004). Our analysis of the miR1026:PpbHLH regulon suggests that miRNAs may act in the epigenetic control of stress-responsive genes in plants.

Taken together, we propose that silencing of genomic loci can be triggered by stable duplexes of a miRNA and its target RNA, which can be either an mRNA or a primary TAS transcript. The epigenetic control of genes encoding miRNA target RNAs discovered in P. patens presents a new mechanism that affects the homeostasis of miRNA-regulated RNAs (Figure 7). The specific equilibrium of a cleavage-competent

RISC and a DNA-modifying RITS loaded with the same miRNA may determine the relative contribution of both pathways to miRNA-mediated downregulation of gene expression. In addition, siRNA-mediated transitivity as a major factor in amplifying the original miRNA- and ta-siRNA-directed cleavage signal appears to be more prevalent than in the flowering plant A. thaliana. It seems not unlikely that similar modifications and specializations of RNAi pathways will be common, which indicates that care needs

17 to be exercised when interpolating the results from single model organisms, in either plants or animals.

Experimental Procedures

Plant material

Culture of P. patens, protoplast transformation, and molecular analyses of transgenic plants were performed according to standard procedures (Frank et al., 2005a). Abscisic acid treatment was carried out by application of 10 µM (±)-cis-trans ABA to P. patens liquid cultures.

Generation of ΔPpDCL1a and ΔPpDCL1b mutant lines

An nptII selection marker cassette was cloned into single restriction sites present in

PpDCL1a and PpDCL1b, respectively. The gene disruption constructs were transfected into P. patens protoplasts and G418-resistant lines were analyzed by PCR to confirm precise integration events at the corresponding genomic loci. Loss of PpDCL1a and

PpDCL1b transcript, respectively, was confirmed by RT-PCR.

P. patens lines expressing amiR-GNT1

The generation of an amiRNA targeting PpGNT1 was described previously (Khraiwesh et al., 2008). The amiRNA expression construct was transfected into P. patens wild type and ΔPpDCL1b mutant lines.

RT-PCR of small RNAs

RT-PCR analyses of miRNAs and ta-siRNAs was carried out as described (Varkonyi-

Gasic et al., 2007). Oligonucleotides used for the cDNA synthesis and subsequent

PCR reactions are listed in Table S1.

18 DNA methylation analysis

DNA sequences were analyzed with the MethPrimer program (Li and Dahiya, 2002) to deduce methylation-specific (MSP) and unmethylation-specific primers (USP) (Figure

S6) for PCR analysis of bisulfite-treated DNA. Two µg of genomic DNA were used for sodium bisulfite treatment with the EpiTect Bisulfite Kit (Qiagen).

Detection of miRNA:mRNA duplexes by RT-PCR cDNA was synthesized from 4 µg total RNA with Superscript III (Invitrogen) without the addition of primers, with the exception of a primer specific for the PpEF1α transcript to monitor the efficiency of cDNA synthesis. RT-PCRs were carried out with gene-specific primers located upstream of miRNA binding sites (Table S1). Control experiments were performed by heating RNA samples to 95°C for 5 min prior to cDNA synthesis. PpEF1α control primers were added after cooling of the samples.

Supplemental Data

Supplemental Data include Figure S1-S10, Table S1 and Supplemental Experimental

Procedures and can be found with this article online

Acknowledgements

This work was supported by the Landesstiftung Baden-Württemberg (P-LS-RNS/40 to

D.W., W.F. and R.R.), the German Federal Ministry of Education and Research

(FRISYS: 0313921 to W.F. and R.R.), the Excellence Initiative of the German Federal and State Governments (EXC 294 to R.R.), the European Community FP6 IP

SIROCCO (contract LSHG-CT-2006-037900; D.W.), and the German Academic

Exchange Service (M.A.A.). We thank G. Gierga for assisting us in the small RNA blot

19 technique and T. Laux, W.R. Hess, R. Baumeister, and P. Beyer for comments on the manuscript.

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Figure Legends

Figure 1. Analysis of ΔPpDCL1a mutants

(A) Protonema filaments of identical density from wild type (WT) and two ΔPpDCL1a mutants grown for 28 days on solid medium. (B) Protonema filaments of plants grown in liquid cultures. (C) RT-PCR expression analysis of miR156, 160, 166, and 390. (D)

RT-PCR expression analysis of ta-siRNAs pptA013298 (processed from PpTAS3) and pptA079444 (processed from PpTAS1). (E) RT-PCR expression analysis of miRNA target genes in wild type and ΔPpDCL1a mutants. Error bars indicate standard errors with n=3.

Figure 2. Analysis of ΔPpDCL1b mutants (1-4)

(A) Regeneration of protoplasts from wild type (WT) and ΔPpDCL1b mutant 1 over indicated time points. (B) Scanning electron micrographs of gametophores. See

Supplementary Fig. 5 for phenotypes of other ΔPpDCL1b mutants. (C) Small RNA blots with 30 µg total RNA from protonema, probed for miR156, miR390, miR535, and miR538. An antisense probe for U6snRNA served as loading control. (D) Small RNA

25 blot with 80 µg total RNA from protonema treated with 5 µM auxin (NAA) for 8 hours, probed for miR160. (E) Small RNA blot with 80 µg total RNA from gametophores, probed for miR166. Ethidium bromide staining shown as loading control at the bottom for d and e. Size bars correspond to 100 µm in a, except for the 18 d and 8 week old plants, 500 µm.

Figure 3. RNA cleavage products, antisense transcripts, and transitive siRNAs of miRNA target genes

(A) 5’ RACE products of miRNA targets and a control transcript, PpGNT1, from wild type and ΔPpDCL1b mutants. Arrows indicate PCR fragments of the expected size for cleavage products. Numbers above miRNA:target alignments indicate sequenced

RACE products with the corresponding 5’ end. (B) Scheme for the generation of transitive siRNAs. Double stranded RNA is synthesized from cleaved miRNA targets by an RNA-dependent RNA polymerase (RdRP), processed into transitive siRNAs, which subsequently mediate cleavage of the miRNA target mRNA upstream and downstream of the miRNA recognition motif. Black line: mRNA; grey box: miRNA binding site; curved line: miRNA; arrows indicate oligonucleotide primers for RT-PCR, with grey indicating primers for cDNA synthesis from antisense strand, and black for sense strand. (C) RT-PCR products derived from antisense or sense-specific cDNAs from wild type and two ΔPpDCL1b mutants (KO1, KO2). (D) Detection of sense and antisense transitive siRNAs derived from PpARF and PpC3HDZIP1 RNAs, using hybridization probes targeting regions upstream and downstream of the miRNA binding sites. U6snRNA was used as control.

Figure 4. Expression of miRNA target genes, DNA methylation, and detection of miRNA:mRNA duplexes

26 (A) RT-PCR expression analysis of miRNA target genes and the control gene PpGNT1 in wild type and ΔPpDCL1b mutants (KO1-KO4). Bars indicate standard error (n=3). (B)

RNA blot analysis of miRNA target genes PpARF and PpC3HDZIP1 and two control genes, PpGNT1 and PpEF1α. (C) Specificity analysis of bisulfite PCR, using primers specific for unmodified sequences. PCR was performed with untreated and bisulfite- treated genomic DNA of wild type and two ΔPpDCL1b mutants (KO1, KO2). (D-F) PCR reactions with bisulfite-treated genomic DNA using methylation (MSP) and unmethylation specific primers (USP). (D) Bisulfite PCR for promoters of miRNA target genes and the PpGNT1 control. (E) Bisulfite PCR analysis of PpARF sequences surrounding the miR160 targeting motif. (F) Bisulfite PCR analysis of PpC3HDZIP1 sequences upstream of, the intron disrupting, and sequences downstream of the miR166 targeting motif. Arrows in d-f mark primer bands. (G) PCR products of miRNA target genes using cDNA synthesized from wild type and two ΔPpDCL1b mutants (1 and 2) without addition of exogenous primers. For the PpGNT1 control, no PCR products were detected in either wild type or ΔPpDCL1b mutants (not shown). A

PpEF1α primer specific for cDNA synthesis from the sense transcript was added as an internal control to all reactions, to monitor the efficiency of cDNA synthesis. (H) The same experiment performed with RNA samples that had been heated for 5 min to 95°C prior to cDNA synthesis. The control PpEF1α primer was added after cooling of the

RNA samples.

Figure 5. The ta-siRNA pathway in wild type and ΔPpDCL1b mutants

(A) 5’ RACE-PCRs from wild type and ΔPpDCL1b mutants (1-4) for the miR390 target

PpTAS4 and the ta-siRNA target PpEREBP/AP2. Arrows indicate products of the size expected for cleavage products. The number of sequenced RACE-PCR products with the corresponding 5’ end is indicated above the alignment. (B) ta-siRNAs derived from

27 PpTAS4 and transitive siRNAs derived from PpEREBP/AP2. U6snRNA served as control. (C) RNA blots for PpTAS4 and PpEREBP/AP2 transcripts. Ethidium bromide staining shown as loading control below. (D) Bisulfite PCR with methylation specific

(MSP) and unmethylation specific primers (USP) for PpTAS4 and PpEREBP/AP2. The arrow marks primer dimers.

Figure 6. Lines expressing amiR-GNT1 and analysis of miR1026 target PpbHLH

(A) PCR-based identification of two transgenic lines each harboring the PpGNT1- amiRNA expression construct in wild type (lines #1, #2), ΔPpDCL1b mutant 1 (lines #3,

#4), and ΔPpDCL1b mutant 2 (lines #5, #6) backgrounds. PpEF1α served as control.

(B) Detection of amiR-GNT1 on a small RNA blot loaded with 50 µg of total RNA.

U6snRNA served as control. (C) Cleavage mapping of PpGNT1 in amiR-GNT1 lines by

5’ RACE-PCR. The number of sequenced RACE-PCR products with the corresponding

5’ end is indicated above the alignment. (D) RNA blot of wild type and amiR-GNT1 lines, probed for PpGNT1. Hybridization signals were normalized to rRNA. Levels relative to wild type are indicated (E) Bisulfite PCR on genomic DNA from amiR-GNT1 lines using methylation (MSP) and unmethylation specific primers (USP) derived from the PpGNT1 promoter. (F) RT-PCR to detect amiR-GNT1:PpGNT1-mRNA duplexes, using cDNA synthesized without the addition of exogenous primers. PCR was carried out with a primer pair upstream of the amiR-GNT1 target motif. Amplification controls were as in Figure 4G and 4H. Arrows mark primer dimers. (G) RNA blots with 20 µg total RNA from untreated (Untr.) and ABA-treated wild type plants using probes for

PpbHLH, the loading control PpEF1α, and PpCOR47, a known ABA-induced gene.

PpbHLH levels were normalized to PpEF1α. Relative PpbHLH mRNA levels compared to wild type are given. (H) Small RNA blot with 50 µg total RNA from untreated (Untr.) and ABA-treated wild type. MiR1026 levels were normalized to the U6snRNA control.

28 Numbers indicate miR1026 levels relative to wild type. (I) 5’ RACE-PCR for PpbHLH using RNA from untreated (Untr.) and wild type treated for 4 h with ABA. Arrows indicate PCR fragments of the expected size for cleavage products. Numbers above miRNA:target alignments indicate sequenced RACE-PCR products with the corresponding 5’ end. (J) Bisulfite PCR reactions on DNA from untreated (Untr.) and

ABA-treated wild type using methylation (MSP) and unmethylation specific primers

(USP) targeting PpbHLH genomic sequences. PpGNT1 promoter served as control.

Arrows mark primer dimers. (K) RT-PCR to detect miR1026:PpbHLH-mRNA duplexes, using cDNA synthesized without the addition of exogenous primers. PCR was carried out with a primer pair upstream of the miR1026 binding site. Amplification controls were as in Figure 4G and 4H. Arrows mark primer dimers.

Figure 7. MiRNA expression levels determine post-transcriptional and transcriptional silencing of miRNA target genes in P. patens

At low miRNA:target-RNA ratios, miRNA targets are regulated primarily at the post- transcriptional level. The maturation of miRNAs from stem-loop precursors is catalyzed by PpDCL1a. PpDCL1b is required for loading miRNAs into cleavage competent RISC.

After loading of miRNAs into RISC, transient miRNA:target-RNA duplexes form based on sequence complementarity resulting in target RNA cleavage. In P. patens, the amplification of the miRNA signal by the generation of transitive siRNAs appears to be widespread. Elevated miRNA expression levels cause an increase in the miRNA:target

RNA ratio. In addition to the loading of miRNA into RISC (dotted arrow), miRNAs form stable duplexes with their cognate target RNAs. MiRNAs are either loaded into a RITS complex and subsequently interact with their target to form a duplex, or these duplexes are formed at first and then loaded into RITS. The miRNA:RNA duplexes bound by

RITS initiate DNA methylation at complementary genomic loci. The RITS complex is

29 able to act on adjacent regions (e. g., promoters) to complete CpG methylation of the entire genomic locus.

30 Figure 1

A DPpDCL1a DPpDCL1a WT mutant 1 mutant 2

0.5 cm 0.5 cm 0.5 cm

B DPpDCL1a DPpDCL1a WT mutant 1 mutant 2

100 µM 100 µM 100 µM

C DPpDCL1a D DPpDCL1a mutants mutants WT 1 2 WT 1 2 miR156 ta-siRNA pptA013298 miR160 ta-siRNA pptA079444 miR166 miR390 E

s 7 l e

v 6 e l

WT t 5 p i

r DPpDCL1a mutant 1 c 4 s n

a 3 DPpDCL1a mutant 2 r t

e 2 v i t a

l 1 e

R 0 PpSBP3 PpARF PpC3 PpHB10 PpTAS1 HDZIP1 Figure 2

A DPpDCL1b B DPpDCL1b WT mutant 1 WT mutant 1 100 µm

4 d

200 µm

C DPpDCL1b mutants WT 1 2 3 4 miR156

6 d miR390

miR535

miR538 8 d U6snRNA D miR160

18 d EtBr E miR166

EtBr 8 weeks Figure 3

A DPpDCL1b mutants WT 1 2 3 4

6/9 PpARF 5’ UGGCAUGCAGGGGGCCAGGCA 3’ miR160 3’ ACCGUAUGUCCCUCGGUCCGU 5’

CU U 7/9 PpC3HDZIP1 5’ GG AUGAAGCCUGGUCCGG 3’ miR166 3’ CC UACUUCGGACCAGGCU 5’ CC U

CU U 7/9 PpHB10 5’ GG AUGAAGCCUGGUCCGG 3’ miR166 3’ CC UACUUCGGACCAGGCU 5’ CC U

U 8/9 PpSBP3 5’ GUGCUC CUCUCUUCUGUCA 3’ miR156 3’ CACGAG GAGAGAAGACAGU 5’ U

PpGNT1 (control)

B 5’ 3’

5’ 3’ 5’ 3’

5’ 3’ 5’ 3’ RdRP RdRP

transitive siRNAs

5’ 3’ 5’ 3’

5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’

C PpARF PpEF1a PpARF PpEF1a KO KO KO KO KO KO KO KO WT 1 2 WT 1 2 WT 1 2 WT 1 2

antisense control sense control cDNA cDNA PpC3HD- PpC3HD- ZIP1 PpEF1a ZIP1 PpEF1a KO KO KO KO KO KO KO KO WT 1 2 WT 1 2 WT 1 2 WT 1 2

antisense control sense control cDNA cDNA PpGNT1 PpEF1a PpGNT1 PpEF1a KO KO KO KO KO KO KO KO WT 1 2 WT 1 2 WT 1 2 WT 1 2

antisense control sense control cDNA cDNA

D Downstream Upstream Antisense Sense Antisense Sense KO KO KO KO KO KO KO KO WT 1 2 WT 1 2 WT 1 2 WT 1 2 PpARF PpC3HDZIP1

U6 sRNA Figure 4

A B C 16 DPpDCL1b

s Untreated DNA l

e 14 WT 1 2 3 4 DNA + Bisulfite v WT KO1 KO2 KO3 KO4 e l

PpARF KO KO KO KO t 12 p

i WT 1 2 WT 1 2

r PpC3HDZIP1 c 10 PpARF s

n PpGNT1

a 8

r PpC3HDZIP1 t

PpEF1a e PpHB10 v 6 i t a l 4 PpSBP3 e R 2 PpGNT1 0 PpGNT1 PpC3 PpHB10 PpSBP3 PpARF HDZIP1 D E Exon Intron MSP USP KO KO KO KO MSP USP MSP USP WT 1 2 WT 1 2 KO KO KO KO KO KO KO KO WT 1 2 WT 1 2 WT 1 2 WT 1 2 PpARF upstream PpC3HDZIP1 of miR160 binding site PpHB10 downstream PpSBP3 of miR160 binding site PpGNT1

F Intron upstream Intron located in Exon downstream G H of miR166 miR166 of miR166 DPpDCL1b binding site binding site binding site PpC3HD- mutants PpEF1a PpARF ZIP1 PpHB10 PpSBP3 MSP USP MSP USP MSP USP WT 1 2 KO KO KO KO KO KO KO KO KO KO KO KO KOKO KO KO KO KO KO KO KO KO PpARF WT 1 2 WT 1 2 WT 1 2 WT 1 2 WT 1 2 WT 1 2 WT 1 2 WT 1 2 WT 1 2 WT 1 2 WT 1 2 PpC3HDZIP1 PpHB10 PpSBP3 PpEF1a Figure 5

A DPpDCL1b mutants WT 1 2 3 4 5/6 PpTAS4 5’ GGCGUUAUCCCUCUUGAGCUG 3’ miR390-5’ 3’ CCGCGAUAGGGAGGACUCGAA 5’ U A A 8/9 PpTAS4 5’ G UGU UAUC CUCCUGAGCUA 3’ miR390-3’ 3’ C GCG-AUAG GAGGACUCGAA 5’ C G C G 8/8 PpEREBP/AP2 5’ GAAGCA UCAUCACACCCUA 3’ ta-siRNA6(+)3’ CUUCGU AGUAGUGUGGGAU 5’ A G

B C Antisense Sense DPpDCL1b KO KO KO KO WT 1 2 3 4 WT 1 2 WT 1 2 PpTAS4 PpTAS4 PpEREBP/AP2 PpEREBP/AP2 rRNA U6 sRNA

D PpEREBP/AP2 PpTAS4 Coding Sequence Promoter MSP USP MSP USP MSP USP KOKO KOKO KOKO KOKO KOKO KOKO WT 1 2 WT 1 2 WT 1 2 WT 1 2 WT 1 2 WT 1 2 Figure 6 - A B C i

DPpDCL1b 1 m

DPpDCL1b #

amiRNA a

+ amiRNA A +

WT lines WT + KO1 + KO2 + N

KO1 KO2 T amiR amiR amiR R #1 #2 WT #3 #5 W WT background WT #1 #2 #3 #4 #5 #6 C 6/8 #3 #4 DPpDCL1b KO1 amiR- PpGNT1 5’ AA CGUCCUGAUUAUUUGGAG 3’ GNT1 #5 #6 amiRNA 3’ UU GCAGGACUAAUAAACCUU 5’ C DPpDCL1b KO2 U6 snRNA PpEF1a D E F DPpDCL1b WT + DPpDCL1b WT + DPpDCL1b MSP USP WT + KO1 + KO2 + amiRNA KO1 KO2 amiRNA KO1 KO2 DPpDCL1b DPpDCL1b amiRNA amiRNA amiRNA WT + WT + #1 #2 #3 #5 #1 #2 #3 #5 WT #1 #2 #3 #4 #5 #6 amiRNA KO1 KO2 amiRNA KO1 KO2 #1 #2 #3 #5 #1 #2 #3 #5 PpGNT1 PpGNT1 1.0 0.22 0.11 0.022 0.017 0.017 0.017 PpEF1a rRNA Duplex RT-PCR Heating control

G H I . .

r r ABA t ABA t ABA Untr. n n 4h

U 1h 2h 4h 6h 8h U 1h 2h 4h 6h 8h

PpbHLH miR1026 PpbHLH 1.0 0.5 0.3 0.3 0.4 0.4 1.0 2.9 2.5 3.3 3.0 3.0 5’ CC 8/10 C 3’ UCCUCUCAAGUCUUUCUC PpEF1a U6snRNA AGGAGAGUUCAGAAAGAG 3’ AC U 5’ PpCOR47 miR1026

J K . .

MSP USP r r t ABA t ABA n n . . r r U 4h 6h 8h U 4h 6h 8h t ABA t ABA n n

U 4h 6h 8h U 4h 6h 8h PpbHLH PpbHLH intragenic PpEF1a PpbHLH Duplex RT-PCR Heating control Promoter PpGNT1 Promoter Figure 7

Cytosol Low miRNA: RISC RISC target-RNA ratio

PpDCL1b AAAAA target-RNA target-RNA cleavage mRNA or (mRNA or TAS-RNA) + TAS-RNA AAAAA AAAAA cleavage Nucleus products RdRP

pre-miRNA double-stranded double-stranded mRNA TAS-RNA cleavage cleavage products products

transitive ta-siRNAs PpDCL1a siRNAs

Amplification of Targeting and cleavage the miRNA signal of ta-siRNA targets

miRNA transitive siRNAs

AAAAA High miRNA: target-RNA ratio RITS target-RNA gene (mRNA or TAS-RNA)

AAAAA CH3 CH3 CH3 RITS RITS AAAAA AAAAA Formation of elevated miRNA:target-RNA miRNA level AAAAA duplexes Formation of miRNA:target-RNA duplexes

Epigenetic Silencing Supplemental Data

Transcriptional control of gene expression by microRNAs

Basel Khraiwesh1, M. Asif Arif1, Gotelinde I. Seumel1, Stephan Ossowski2, Detlef

Weigel2, Ralf Reski1,3,4, Wolfgang Frank1,3*

1Plant Biotechnology, Faculty of Biology, University of Freiburg, Schänzlestraße 1, D-

79104 Freiburg, Germany

2Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-

72076 Tübingen, Germany

3Freiburg Initiative for Systems Biology (FRISYS), Faculty of Biology, University of

Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany

4Centre for Biological Signalling Studies (bioss), University of Freiburg, Schänzlestr. 1,

D-79104 Freiburg, Germany

Supplementary Figures

Figure S1. Neighbor-joining tree of DICER-LIKE proteins

Figure S2. Generation of ΔPpDCL1a mutants

Figure S3. Generation of ΔPpDCL1b mutants

Figure S4. Phenotypic analysis of ΔPpDCL1b mutants

Figure S5. Gene models of PpARF, PpC3HDZIP1, PpHB10, PpSBP3 and PpGNT1

Figure S6. Primer design for bisulfite PCR analyses

Figure S7. DNA methylation analysis of promoter and intragenic regions of the PpARF gene in P. patens wild type and two ΔPpDCL1b mutants

Figure S8. The miR166 binding sites of PpC3HDZIP1 and PpHB10 are disrupted by introns

Figure S9. DNA methylation analysis of the PpGNT1 promoter region in lines expressing the amiR-GNT1 Figure S10. DNA methylation analysis of promoter and intragenic regions of PpbHLH in untreated and ABA-treated P. patens wild type

Supplementary Tables

Table S1. Primers used in this study

Supplemental Experimental Procedures Figure S1

MmDCL1_NP_683750 HsDCL1_NP_803187 DmDCL1_AAF56056 CeDCL1_NP_498761 Animals DmDCL2_NM_079054 SpDCL1_Q09884 NcDCL1_XP_961898 PpDCL1b_DQ675601

PpDCL1a_EF670436 PtDCL1_Pt02g14226280 DCL1 MtDCL1_AC150443 AtDCL1_NP_171612 OsDCL1_NP_912466 PtDCL2a_Pt06g11470720 PtDCL2_Pt08g4686890 DCL2 AtDCL2_NP_566199 OsDCL2_XP_463595 Plants AtDCL3_NP_189978 PtDCL3_Pt10g16358340 OsDCL6_Os09g14610 DCL3 OsDCL5_Os03g38740 OsDCL3_NP_922059

PpDCL3_EF670437 PtDCL4_Pt18g3481550 AtDCL4_NP_197532 DCL4 OsDCL4_Os04g43050 PpDCL4_EF670438 CrDCL*

Figure S1. Neighbor-joining tree of DICER-LIKE proteins DICER-LIKE proteins from animals and plants are indicated by vertical lines. The four groups of DICER-LIKE proteins in plants are marked by coloured boxes. Species abbreviations are At (Arabidopsis thaliana), Ce (Caenorhabditis elegans), Cr (Chlamydomonas reinhardtii), Dm (Drosophila melanogaster), Hs (Homo sapiens), Mm (Mus musculus), Mt (Medicago truncatula), Nc (Neurospora crassa), Os (Oryza sativa), Pp (Physcomitrella patens), Pt (Populus trichocarpa), Sp (Schizosaccharomyces pombe). P. patens DCL proteins are highlighted in bold. * The sequence of DCL from Chlamydomonas reinhardtii can be retrieved at: http://genome.jgi-psf.org/chlre2.

Phylogenetic tree construction: The multiple sequence alignment was performed using the PROBCONS program. The phylogenetic tree was constructed by a neighbor-joining method using a WAG matrix model for amino acid substitution. Figure S2

A DEAD HEL DUF PAZ RNAse III dsrm

B nosP nptII nosT

EcoRV

PpDCL1aDCL1 cDNA gDNA

PpDCL1a nosP nptII nosT PpDCL1a

F2 F1 F3 PpDCL1a PpDCL1a nosP nptII nosT PpDCL1a PpDCL1a R2 R1 R3

C WT * * Genomic PCR-Screen

D DPpDCL1a E DPpDCL1a mutants mutants WT 1 2 1 2 WT PpDCL1a 5’ integration 3’ integration PpEF1a

Figure S2. Generation of DPpDCL1a mutants (A) Predicted domain structure of the P. patens DCL1a protein. DEAD: DEAD box helicase, HEL: helicase C, DUF: domain of unknown function, PAZ: PAZ domain, RNAseIII: ribonuclease III domain, dsrm: double- stranded RNA binding motif. (B) Scheme illustrating the generation of the DPpDCL1a mutants. The nptII cassette was cloned into a single EcoRV site of a PpDCL1a genomic DNA fragment (gDNA). Middle: Resulting PpDCL1a knockout construct. Bottom: Expected genomic structure of the PpDCL1a locus after integration of the PpDCL1a knockout construct by homologous recombination. Primers used for molecular analyses of the transgenic lines are indicated by arrows. White box: nptII cassette; grey boxes: PpDCL1a gDNA fragment; black boxes: genomic PpDCL1a locus. (C) PCR analysis of transgenic lines using genomic DNA performed with primers F1 and R1. Transgenic lines which failed to give rise to a PCR product are marked by asterisks. WT: wild type control; the remaining samples were derived from transgenic lines. (D) Analysis of PpDCL1a mRNA expression. Top: RT-PCR studies from two DPpDCL1a mutants and wild type (WT) with primers F1 and R1. Bottom: RT-PCR performed with PpEF1a control primers. (E) PCR analysis of DPpDCL1a mutants using genomic DNA to confirm 5’ and 3’ integration of the PpDCL1a knockout construct. PCR products obtained from PCR reactions with primers F2 and R2 (5’ integration), F3 and R3 (3’ integration). Sequences of primers used for the molecular analyses are listed in Experimental Procedures and Table S1. Figure S3

A DEAD HEL DUF PAZ RNAse III dsrm

B nosP nptII nosT

4550 Eco72I 5108

PpDCL1bDCL1 cDNA cDNA

PpDCL1b nosP nptII nosT PpDCL1b

F3 F2 F1 F4 PpDCL1b PpDCL1b nosP nptII nosT PpDCL1b PpDCL1b R3 R4 R1 R2

C WT * * * * Genomic PCR-Screen

D DPpDCL1b mutants E DPpDCL1b mutants 1 2 3 4 WT 1 2 3 4 WT PpDCL1b PpDCL1b PpEF1a PpEF1a

F DPpDCL1b mutants 1 2 3 4 5’ integration

3’ integration

Control PpEF1a

Figure S3. Generation of DPpDCL1b mutants (A) Predicted domain structure of the P. patens DCL1b protein. DEAD: DEAD box helicase, HEL: helicase C, DUF: domain of unknown function, PAZ: PAZ domain, RNAseIII: ribonuclease III domain, dsrm: double- stranded RNA binding motif. (B) Scheme illustrating the generation of the DPpDCL1b mutants. The nptII cassette was cloned into a single Eco72I site of a PpDCL1b cDNA fragment. Numbers indicate nucleotide positions in the PpDCL1b cDNA. Middle: Resulting PpDCL1b knockout construct. Bottom: Expected genomic structure of the PpDCL1b locus after integration of the PpDCL1b knockout construct by homologous recombination. Primers used for molecular analyses of the transgenic lines are indicated by arrows. White box: nptII cassette; grey boxes: PpDCL1b cDNA fragment; black boxes: genomic PpDCL1b locus. (C) PCR analysis of transgenic lines using genomic DNA performed with primers F1 and R1. Transgenic lines which failed to give rise to a PCR product are marked by asterisks. WT: wild type control; the remaining samples were derived from transgenic lines. (D) Analysis of PpDCL1b mRNA expression. Top: RT-PCR studies from four DPpDCL1b mutants and wild type (WT) with primers F2 and R2. Bottom: RT-PCR performed with PpEF1a control primers. (E) Top: RT-PCR analysis of PpDCL1b expression in DPpDCL1b mutants and wild type using primers F3 and R3 located upstream of the knockout construct integration site. Bottom: RT-PCR performed with PpEF1a control primers. (F) PCR analysis of DPpDCL1b mutants using genomic DNA to confirm 5’ and 3’ integration of the PpDCL1b knockout construct. PCR products obtained from PCR reactions with primers F2 and R4 (5’ integration), F4 and R2 (3’ integration) and PpEF1a control primers to confirm integrity of the used genomic DNA. Sequences of primers used for the molecular analyses are listed in Experimental Procedures and Table S1. Figure S4

A DPpDCL1b DPpDCL1b DPpDCL1b DPpDCL1b WT mutant 1 mutant 2 mutant 3 mutant 4

4 d

6 d

8 d

18 d

8 weeks

B

200 µm 100 µm 100 µm 90 µm 80 µm

Figure S4. Phenotypic analysis of DPpDCL1b mutants (A) Regeneration of protoplasts from wild type plants (WT) and DPpDCL1b mutants was monitored at the indicated time points. Size bars 4 d, 6 d, and 8 d: 100 µm; 18 d, 8 weeks: 500 µm.(B) Electron micrographs of gametophores from wild type and DPpDCL1b mutants. Figure S5

Start codon Intron 1 Intron 2 miRNA BS Intron 3 Stop codon 1 - 999 1315-1317 1893-2105 2731-2860 3115-3135 3749-3907 3959-3961 PpARF 1 Promoter Region 4194

1000 2000 3000 4000

I 2 I 4 I 6 I 8 I 10 I 12 I 14 I 16 3041-3247 3673-3984 4489-4641 5007-5135 5553-5735 6208-6446 7039-7300 7807-8039 Start codon Stop codon 1 - 1000 2329-2331 8181-8183 PpC3 1 Promoter Region 8183 HDZIP1 miRNA BS 2000 3662-3672 4000 6000 8000 I 1 I 3 3985-3894 I 5 I 7 I 9 I 11 I 13 I 15 2504-2839 3351-3581 4193-4402 4796-4929 5243-5363 5919-6130 6588-6726 7407-7658

Stop codon Start codon miRNA BS 4033-4035 1 - 999 1393-1395 2072-2092 PpHB10 1 Promoter Region 4590

1000 2000 3000 4000

Start codon miRNA BS Stop codon 1 - 1000 1300-1302 2687-2706 3280-3282 PpSBP3 1 Promoter Region 3282

1000 2000 3000

Start codon Stop codon 1 - 1000 1300-1302 2713-2715 PpGNT1 1 Promoter Region 2715

1000 2000

Figure S5. Gene models of PpARF, PpC3HDZIP1, PpHB10, PpSBP3 and PpGNT1 For PpARF and PpC3HDZIP1, complete gene models including intron sequences were predicted by comparing available cDNA sequences with the P. patens genomic trace files. For PpHB10, PpSBP3 and PpGNT1, promoter regions were analyzed following the same strategy. For PpHB10 the 5’ untranslated region was included based on the available full-length cDNA sequence. In the case of PpSBP3 and PpGNT1, cDNA sequences encompassing the open reading frame were available. Here, regions lying 300 nucleotides upstream of the start codon were used for promoter analysis. I 1 – I 16: Intron 1 to Intron 16; miRNA BS: miRNA binding site. The genomic nucleotide sequences were deposited in Genbank with the following accession numbers: BK006047 (PpHB10), BK006048 (PpC3HDZIP1), BK006049 (PpGNT1), BK006050 (PpSBP3) and BK006051 (PpARF). Figure S6

Promoter region of PpC3HDZIP1

1 CCTGCCTGCGCTGTCCTATCCTCCTCCTCTTCCTACTTCCCCTCACCTCCTCCGCCTCTG ::||::||++:|||::|||::|::|::|:||::||:||::::|:|::|::|:++::|:|| 1 TTTGTTTGCGTTGTTTTATTTTTTTTTTTTTTTTATTTTTTTTTATTTTTTTCGTTTTTG

61 CGCTCTGTGCACTGTCCCTTCCATGTCGTGCCAGGCTCTGCGGAGGGTGCGGCCAGGCAG ++:|:||||:|:|||:::||::||||++||::|||:|:||++|||||||++|::|||:|| 61 CGTTTTGTGTATTGTTTTTTTTATGTCGTGTTAGGTTTTGCGGAGGGTGCGGTTAGGTAG

WTfwd >>>>>>>>>>>>>>>>>>>>> 121 GCAGCTGTGATGGCGGTGTTGTACTGCCGCATGATTCTGGACCAACCGGGCCAGGGCGGG |:||:||||||||++||||||||:||:++:||||||:||||::||:++||::||||++|| 121 GTAGTTGTGATGGCGGTGTTGTATTGTCGTATGATTTTGGATTAATCGGGTTAGGGCGGG MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>

181 CGACTGTTACTGCAGACTGTTACTGACTCCTGAGGGCGCAGCAGGCTGGTGAGAGGGACG ++|:|||||:||:|||:|||||:|||:|::||||||++:||:|||:||||||||||||++ 181 CGATTGTTATTGTAGATTGTTATTGATTTTTGAGGGCGTAGTAGGTTGGTGAGAGGGACG

<<<< 241 GCCGCGGGTGGGGCGGGGCGAAGAAGAGAAAAAGGTGTGTAGGCGCGGGAGGCGTGCTTT |:++++|||||||++|||++|||||||||||||||||||||||++++|||||++||:||| 241 GTCGCGGGTGGGGCGGGGCGAAGAAGAGAAAAAGGTGTGTAGGCGCGGGAGGCGTGTTTT <<<<<<<< <<<<<<<<<< <<<<<<<<<<<<<<<<< WTrev 301 GTATGCGCACTACATGCCTTGAGCCTGGTGGTTGTTCATGACACTGTTCATCGCAGTATT |||||++:|:||:|||::|||||::|||||||||||:||||:|:||||:||++:|||||| 301 GTATGCGTATTATATGTTTTGAGTTTGGTGGTTGTTTATGATATTGTTTATCGTAGTATT <<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<< USPrev

361 CTACAACGACATCGTCCCCAGCTGGTAGTAGTATTGCAGTTGTATAAGTTGTCGCTGCAG :||:||++|:||++|::::||:||||||||||||||:|||||||||||||||++:||:|| 361 TTATAACGATATCGTTTTTAGTTGGTAGTAGTATTGTAGTTGTATAAGTTGTCGTTGTAG

421 CCAGGCGCCGCCAGTCAGCATCTTCTCGTTAGTGTTCAGTTAGTAGTTGAAGCGAGGGAG ::|||++:++::|||:||:||:||:|++||||||||:|||||||||||||||++|||||| 421 TTAGGCGTCGTTAGTTAGTATTTTTTCGTTAGTGTTTAGTTAGTAGTTGAAGCGAGGGAG

481 TATATTCCGCCTTCGATTTTTTGTTTCTCAGGGAGTCACAGCGCTGCGAATCAGAAGCCT ||||||:++::||++|||||||||||:|:|||||||:|:||++:||++|||:|||||::| 481 TATATTTCGTTTTCGATTTTTTGTTTTTTAGGGAGTTATAGCGTTGCGAATTAGAAGTTT

541 GTGAGAGCTTTGGGAACTGGTTTTCGTGTTTTAGAAAGCGAGGCCAACGAGAGAGCGAGA |||||||:||||||||:|||||||++||||||||||||++|||::||++||||||++||| 541 GTGAGAGTTTTGGGAATTGGTTTTCGTGTTTTAGAAAGCGAGGTTAACGAGAGAGCGAGA

601 TCGAGAGAGAGAGAGAGCGCGAGCGACAGCATGTCACGCATGAGAGGAGAGAAGAACAGA |++||||||||||||||++++||++|:||:||||:|++:|||||||||||||||||:||| 601 TCGAGAGAGAGAGAGAGCGCGAGCGATAGTATGTTACGTATGAGAGGAGAGAAGAATAGA

661 GGACGGAGCAGGGCTGGCCTATTGGTGTTACAGGAAGGGGGTTGCAGGAATTTGTAGGCG |||++|||:||||:|||::|||||||||||:|||||||||||||:|||||||||||||++ 661 GGACGGAGTAGGGTTGGTTTATTGGTGTTATAGGAAGGGGGTTGTAGGAATTTGTAGGCG

721 TGGCCGTCACTGTTTGGTTTTTGAAAGCTAGTGCTGCGACAAGAGATGCGGGTGGTCCTA |||:++|:|:|||||||||||||||||:|||||:||++|:||||||||++||||||::|| 721 TGGTCGTTATTGTTTGGTTTTTGAAAGTTAGTGTTGCGATAAGAGATGCGGGTGGTTTTA

781 GCTTGAGTACTTGTGCTAGGCGTCTGAGGCGTGAAGTTTCGGCTAGCTGATTGCAAATTC |:|||||||:|||||:||||++|:|||||++||||||||++|:|||:||||||:|||||: 781 GTTTGAGTATTTGTGTTAGGCGTTTGAGGCGTGAAGTTTCGGTTAGTTGATTGTAAATTT

841 AGTAAGATTGGAGAGGGCAATGGCTGACGGTCCGCATCCATTCGTACAAGAATGCCTTCT |||||||||||||||||:|||||:|||++||:++:||::|||++||:|||||||::||:| 841 AGTAAGATTGGAGAGGGTAATGGTTGACGGTTCGTATTTATTCGTATAAGAATGTTTTTT

901 TCTTGAAAAGCTGGTTGATCCTCGTCGTTGTAATCCGACGGTGCGGCTACGGAGCTAAAG |:||||||||:||||||||::|++|++|||||||:++|++|||++|:||++|||:||||| 901 TTTTGAAAAGTTGGTTGATTTTCGTCGTTGTAATTCGACGGTGCGGTTACGGAGTTAAAG

961 TTCAAACGCTTAGTCTCTTCTTTTCTGGTGTGAAGTAGGT ||:|||++:|||||:|:||:||||:||||||||||||||| 961 TTTAAACGTTTAGTTTTTTTTTTTTTGGTGTGAAGTAGGT

Used primers:

Forward MSP: 5’-ATTGTCGTATGATTTTGGATTAATC-3’ Reverse MSP: 5’-ACATATAATACGCATACAAAACACG-3’

Forward USP: 5’-GTTGTATGATTTTGGATTAATTGG-3’ Reverse USP: 5’-CATATAATACACATACAAAACACACC-3’

Forward WT: 5’-ACTGCCGCATGATTCTGGACC-3’ Reverse WT: 5’-GCATGTAGTGCGCATACAAAG-3’

Promoter region of PpHB10

1 AGGAGGTGGAGGAGGTGGAGGGTTCCAAGGTGAGGGAGCAAGCTGTCATACCGGTAGGAG ||||||||||||||||||||||||::||||||||||||:|||:|||:|||:++||||||| 1 AGGAGGTGGAGGAGGTGGAGGGTTTTAAGGTGAGGGAGTAAGTTGTTATATCGGTAGGAG

61 TCCGTAGAGGGAAATAGAGAGGAAGCAAGTCAGGAAGTGTTGGTGAAGGGGGAGAGAAAG |:++|||||||||||||||||||||:||||:||||||||||||||||||||||||||||| 61 TTCGTAGAGGGAAATAGAGAGGAAGTAAGTTAGGAAGTGTTGGTGAAGGGGGAGAGAAAG

121 AGAGCGAGAGGAGGAGGAGGAGTAGTAGAGGTGGTCGTGTCGATGATGGAAGAGATGATG ||||++|||||||||||||||||||||||||||||++|||++|||||||||||||||||| 121 AGAGCGAGAGGAGGAGGAGGAGTAGTAGAGGTGGTCGTGTCGATGATGGAAGAGATGATG 181 GTGTAGTTTTTGGTTGTATGTAGTAGTAGCCATGAAGGAGGGGTTGTTTTTACGGGTAAT |||||||||||||||||||||||||||||::|||||||||||||||||||||++|||||| 181 GTGTAGTTTTTGGTTGTATGTAGTAGTAGTTATGAAGGAGGGGTTGTTTTTACGGGTAAT

241 GGTTGTTGTTCGGAAGGTATGTACAAATGGAGAGGGCTATGTCGGGGATCAGCTGGAGTG ||||||||||++|||||||||||:||||||||||||:|||||++|||||:||:||||||| 241 GGTTGTTGTTCGGAAGGTATGTATAAATGGAGAGGGTTATGTCGGGGATTAGTTGGAGTG

301 ATGATTGATTGAGTGGAGAGGGAGTGGCGGTAGATATGGGGATGGAGTGGAATGGGGTTC |||||||||||||||||||||||||||++||||||||||||||||||||||||||||||+ 301 ATGATTGATTGAGTGGAGAGGGAGTGGCGGTAGATATGGGGATGGAGTGGAATGGGGTTC

361 GTATGTCATCATTAGAATCCAAGAGTGGAGAGTAGTTTACCTGGAGCAGCAGCGTTGTGC +|||||:||:||||||||::|||||||||||||||||||::|||||:||:||++|||||: 361 GTATGTTATTATTAGAATTTAAGAGTGGAGAGTAGTTTATTTGGAGTAGTAGCGTTGTGT

421 TCTTGCGCATCCTGGCGATGGACATTTGTGTTTGAGTAGTAGAGGTGGAGGCGTTGCTGT |:|||++:||::|||++|||||:||||||||||||||||||||||||||||++|||:||| 421 TTTTGCGTATTTTGGCGATGGATATTTGTGTTTGAGTAGTAGAGGTGGAGGCGTTGTTGT

481 TGTTGTTGTCGTGGTTGTTGTGTGGTAGTGGTAGTAGTGTGACTCTGTAGTGGCTATGGT |||||||||++|||||||||||||||||||||||||||||||:|:||||||||:|||||| 481 TGTTGTTGTCGTGGTTGTTGTGTGGTAGTGGTAGTAGTGTGATTTTGTAGTGGTTATGGT

541 GGGTCTATTGCTGATGGTTTTTGTGTTGCGTCAGGCCGGCGTCACGGTCGTGTAGCATCG ||||:|||||:|||||||||||||||||++|:|||:++|++|:|++||++|||||:||++ 541 GGGTTTATTGTTGATGGTTTTTGTGTTGCGTTAGGTCGGCGTTACGGTCGTGTAGTATCG

601 AGGGCGACGAAAGGTGAATGAACAAAGGGTGTGATTGTGTATAGGCATCCACATATTCTC ||||++|++|||||||||||||:||||||||||||||||||||||:||::|:|||||:|+ 601 AGGGCGACGAAAGGTGAATGAATAAAGGGTGTGATTGTGTATAGGTATTTATATATTTTC

WTfwd >>>>>>>>>>>>>>>>>>>>>>>> 661 GGCTGTGGAAGTTGGGAACAGGGATGCCTTGTGTGCGATTCAACTCGTGGTATAGAAGAA +|:|||||||||||||||:|||||||::|||||||++|||:||:|++||||||||||||| 661 GGTTGTGGAAGTTGGGAATAGGGATGTTTTGTGTGCGATTTAATTCGTGGTATAGAAGAA MSPfwd >>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>

721 GAAGAAGAGGAGCTTGAAGGTTGTCAAGAAAAGGGTAGGGTGTTGCTGCAGCAGCAGTAG ||||||||||||:|||||||||||:||||||||||||||||||||:||:||:||:||||| 721 GAAGAAGAGGAGTTTGAAGGTTGTTAAGAAAAGGGTAGGGTGTTGTTGTAGTAGTAGTAG

<<<<<<<<<<<<<<<<<<<<<<<<< WTrev 781 CAGCAGGAGCATCAGTAGCAGCTTGAGAGGACGAGGACCTAGGAGGAACAGAAGCTCTTG :||:|||||:||:|||||:||:|||||||||++||||::|||||||||:|||||:|:||| 781 TAGTAGGAGTATTAGTAGTAGTTTGAGAGGACGAGGATTTAGGAGGAATAGAAGTTTTTG <<<<<<<<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<<<<<<< USPrev

841 CGTGGTCTGTGAGGAGAATTCTTTGTTAGGGGTTGGAAGCTTCTAGGTTGGGCACGTAGT ++||||:|||||||||||||:||||||||||||||||||:||:|||||||||:|++|||| 841 CGTGGTTTGTGAGGAGAATTTTTTGTTAGGGGTTGGAAGTTTTTAGGTTGGGTACGTAGT

901 AGTGCGTTCTTTGTGTCTTGTCAACTGGGGTTTCAGTCGTATGAGTTGAACACGGGCTGT ||||++||:|||||||:||||:||:||||||||:|||++|||||||||||:|++||:||| 901 AGTGCGTTTTTTGTGTTTTGTTAATTGGGGTTTTAGTCGTATGAGTTGAATACGGGTTGT

961 CGTCACCAACCAGCAATTCGCAACCGGGCCTGCTCACGA ++|:|::||::||:||||++:||:++||::||:|:|++| 961 CGTTATTAATTAGTAATTCGTAATCGGGTTTGTTTACGA

Used primers:

Forward MSP: 5’-GATGTTTTGTGTGCGATTTAATTC-3’ Reverse MSP: 5’-ACTTCTATTCCTCCTAAATCCTCGT-3’

Forward USP: 5’-ATGTTTTGTGTGTGATTTAATTTGT-3’ Reverse USP: 5’-ACTTCTATTCCTCCTAAATCCTCATC-3’

Forward WT: 5’-GATGCCTTGTGTGCGATTCAACTC-3’ Reverse WT: 5’-GCTTCTGTTCCTCCTAGGTCCTCGT-3’

Promoter region of PpARF

1 AGGAGTGGTTTGTGATGCGAAGCTGGGAGGGTGACAGAAAGGACATCAGTGGATCTATGC |||||||||||||||||++|||:|||||||||||:||||||||:||:|||||||:||||: 1 AGGAGTGGTTTGTGATGCGAAGTTGGGAGGGTGATAGAAAGGATATTAGTGGATTTATGT

61 TCTTATTAGTCCTAGTATGGATTAGTATTCATTGATTATAGAGGCTGCGCGGGAGAAAAT |:||||||||::|||||||||||||||||:||||||||||||||:||++++||||||||| 61 TTTTATTAGTTTTAGTATGGATTAGTATTTATTGATTATAGAGGTTGCGCGGGAGAAAAT

121 GGAGAGACTAAGAAGATGAATTCTTCGTAGTTGTGACGAGATGGAAGGTTATTCAATTTA |||||||:||||||||||||||:||++|||||||||++|||||||||||||||:|||||| 121 GGAGAGATTAAGAAGATGAATTTTTCGTAGTTGTGACGAGATGGAAGGTTATTTAATTTA

181 TATTAGGGTACAATGGAAGGAATGCACTTAATTTTTGAAAGTTTTTCGCACGCCAGGATG ||||||||||:|||||||||||||:|:|||||||||||||||||||++:|++::|||||| 181 TATTAGGGTATAATGGAAGGAATGTATTTAATTTTTGAAAGTTTTTCGTACGTTAGGATG

241 GAACTCTTGATAATTGCGTATTATCTACGTATTGTTGAGTTTTCAATTTTCCCATACTGT |||:|:||||||||||++||||||:||++||||||||||||||:||||||:::|||:||| 241 GAATTTTTGATAATTGCGTATTATTTACGTATTGTTGAGTTTTTAATTTTTTTATATTGT

301 CTGTCTGGATTTGCTTCTCATGATACAGGAGTTGTCTGTGAATCTCATTGGATATTTCCG :|||:||||||||:||:|:||||||:|||||||||:|||||||:|:|||||||||||:++ 301 TTGTTTGGATTTGTTTTTTATGATATAGGAGTTGTTTGTGAATTTTATTGGATATTTTCG

361 GATGGTTATTAACCGGGTCCAGTTGATCGTCCAAGCTCCCTTGGCATTTGTGAGGGGTTC ||||||||||||:++|||::|||||||++|::|||:|:::||||:||||||||||||||: 361 GATGGTTATTAATCGGGTTTAGTTGATCGTTTAAGTTTTTTTGGTATTTGTGAGGGGTTT

421 TACTAGTTGTGTGAACTCAGCACACGTAAATTTATAGATTTCCTCTCAAGGCTCAAAGTA ||:||||||||||||:|:||:|:|++|||||||||||||||::|:|:||||:|:|||||| 421 TATTAGTTGTGTGAATTTAGTATACGTAAATTTATAGATTTTTTTTTAAGGTTTAAAGTA

WTfwd >>>>>>>>>>>>>>>>>>>>>> 481 CCAGACTTTTTATGCTAGGACAATCTTTGGATATGTGCCAGCGTATCTTGTGATCGTGGT ::|||:||||||||:|||||:|||:||||||||||||::||++|||:|||||||++|||| 481 TTAGATTTTTTATGTTAGGATAATTTTTGGATATGTGTTAGCGTATTTTGTGATCGTGGT MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>

541 TCTTAAGGGTCGAGTGCTTAGCTCCTCATCCTCATGCTTAGGTCTGGAAATATGTAAAAG |:||||||||++||||:||||:|::|:||::|:|||:||||||:|||||||||||||||| 541 TTTTAAGGGTCGAGTGTTTAGTTTTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAG

<<<<<<<<<<<<<<<<<<<<< WTrev 601 GGGACGTAATGACAACACGAAGCTTATAAAAACTCAAAGCTATATGATCATAGGGCTTTC ||||++||||||:||:|++|||:|||||||||:|:||||:||||||||:||||||:|||: 601 GGGACGTAATGATAATACGAAGTTTATAAAAATTTAAAGTTATATGATTATAGGGTTTTT <<<<<<<<<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<<<<<<< USPrev

661 ACGATGAGCGAGATATTTTCTCTCAAGCCTGTGAAGCATTTTGAACGTCTTTATTCTAGG |++|||||++|||||||||:|:|:|||::|||||||:||||||||++|:||||||:|||| 661 ACGATGAGCGAGATATTTTTTTTTAAGTTTGTGAAGTATTTTGAACGTTTTTATTTTAGG

721 AAGACGAGTTTGATGTTTATTGGTATTGAGTTTCGCTCTTTCAGAAGTATTTTCAGAAGT ||||++|||||||||||||||||||||||||||++:|:|||:|||||||||||:|||||| 721 AAGACGAGTTTGATGTTTATTGGTATTGAGTTTCGTTTTTTTAGAAGTATTTTTAGAAGT

781 TAGCAACGATTTCCTATGTTAGGTTCTGTTATTGGTTTTTTGCGTATGATTCGTGTCCTT |||:||++||||::|||||||||||:||||||||||||||||++|||||||++|||::|| 781 TAGTAACGATTTTTTATGTTAGGTTTTGTTATTGGTTTTTTGCGTATGATTCGTGTTTTT 841 CTGGTTGTAACCAAGCTGTACAAAAAAACGTGCAATTGATATCATTTGGTGGCGATTAGA :|||||||||::|||:||||:|||||||++||:|||||||||:|||||||||++|||||| 841 TTGGTTGTAATTAAGTTGTATAAAAAAACGTGTAATTGATATTATTTGGTGGCGATTAGA

901 CATTTTGGTGTCATTGACAAGTTCCAATGTACACTTCTCTTTAAGGTTTTTATTTAATTC :||||||||||:|||||:|||||::||||||:|:||:|:||||||||||||||||||||: 901 TATTTTGGTGTTATTGATAAGTTTTAATGTATATTTTTTTTTAAGGTTTTTATTTAATTT

961 CTAAGTATTGATATTTTATTTATTTATTTGTTGTGGTCA :||||||||||||||||||||||||||||||||||||:| 961 TTAAGTATTGATATTTTATTTATTTATTTGTTGTGGTTA

Used primers:

Forward MSP: 5’-GGATAATTTTTGGATATGTGTTAGC-3’ Reverse MSP: 5’-AACTTTAAATTTTTATAAACTTCGTA-3’

Forward USP: 5’-ATAATTTTTGGATATGTGTTAGTGT-3’ Reverse USP: 5’-AACTTTAAATTTTTATAAACTTCATA-3’

Forward WT: 5’-GGACAATCTTTGGATATGTGCC-3’ Reverse WT: 5’-AAGCTTCGTGTTGTCATTACG-3’

Promoter region of PpSBP3

1 ATAAAAGTCGTAAGGATCTCACTGGGTCCCTCTCACATTTCTCCCTGAAAAATGACGACG ||||||||++|||||||:|:|:|||||:::|:|:|:||||:|:::||||||||||++|++ 1 ATAAAAGTCGTAAGGATTTTATTGGGTTTTTTTTATATTTTTTTTTGAAAAATGACGACG

61 TCGTTTTCATGACGGTGATTCTCGGTTGTCCATTTGTGGCCTTGACGGAAATGTGTGGGC |++||||:||||++||||||:|++|||||::||||||||::||||++||||||||||||+ 61 TCGTTTTTATGACGGTGATTTTCGGTTGTTTATTTGTGGTTTTGACGGAAATGTGTGGGC

121 GATCTTTGATGGCCACTCTTTTTGTTTTGTTGCCAATCCTCCTCCTATATTTAGTGACTG +||:||||||||::|:|:||||||||||||||::|||::|::|::||||||||||||:|| 121 GATTTTTGATGGTTATTTTTTTTGTTTTGTTGTTAATTTTTTTTTTATATTTAGTGATTG

181 GAGGATCTTTGCTGTTGCTGATTTCCTGGCTTATCCTGGGCGCTGCTATAAGTTAGGCTT ||||||:||||:|||||:||||||::|||:||||::||||++:||:|||||||||||:|| 181 GAGGATTTTTGTTGTTGTTGATTTTTTGGTTTATTTTGGGCGTTGTTATAAGTTAGGTTT

241 TTCTTCATCCATTTTGAGGTGTCACAATATATTTATGGTCGTCGTAATTGTTTTTAATTT ||:||:||::||||||||||||:|:||||||||||||||++|++|||||||||||||||| 241 TTTTTTATTTATTTTGAGGTGTTATAATATATTTATGGTCGTCGTAATTGTTTTTAATTT

301 TACCTCCGTCGGGGTCTGCGCCACCATATGCTTGATAAATTGCAGATTTCAAAGCAGAAC ||::|:++|++||||:||++::|::|||||:|||||||||||:||||||:||||:||||+ 301 TATTTTCGTCGGGGTTTGCGTTATTATATGTTTGATAAATTGTAGATTTTAAAGTAGAAC

361 GTTTCGGTGATGCATGGTCACTTGTGCAGGTTTCTAGTTACCTGGTTGGTTATTTCTTTT +|||++||||||:|||||:|:|||||:||||||:||||||::|||||||||||||:|||| 361 GTTTCGGTGATGTATGGTTATTTGTGTAGGTTTTTAGTTATTTGGTTGGTTATTTTTTTT

421 TTGTTTATTTCTCGAGTTTGCGGGTAGTGGTGGAGTTATGGATGCTTAGAACGCTGCAAA ||||||||||:|++||||||++||||||||||||||||||||||:||||||++:||:||| 421 TTGTTTATTTTTCGAGTTTGCGGGTAGTGGTGGAGTTATGGATGTTTAGAACGTTGTAAA

481 TAGGCCAGTTTGGTGTTGGTGATGAGGATTGCGCTCCTTCCAGTCACGATTGTGTGCCTG ||||::|||||||||||||||||||||||||++:|::||::|||:|++||||||||::|| 481 TAGGTTAGTTTGGTGTTGGTGATGAGGATTGCGTTTTTTTTAGTTACGATTGTGTGTTTG

541 CATTCTGTGGAGTCTGTAATCCGCAGTTCAGTTTTTGTGTTTTAGCAAATTAGCGCATGC :|||:||||||||:||||||:++:||||:||||||||||||||||:|||||||++:|||: 541 TATTTTGTGGAGTTTGTAATTCGTAGTTTAGTTTTTGTGTTTTAGTAAATTAGCGTATGT

601 TTCGCAGTCTTACGTGCTTATGACGTTCCTATGGACGTCCTTCTATCGTTGCCCGAATTT ||++:|||:|||++||:||||||++||::||||||++|::||:|||++|||::++||||| 601 TTCGTAGTTTTACGTGTTTATGACGTTTTTATGGACGTTTTTTTATCGTTGTTCGAATTT

661 TCTGTGCTTCTTTCAAAGTCGCTGGCAATTGCAGACCTGGAAATTGGGTATTGTTTCCTC |:||||:||:|||:|||||++:|||:|||||:|||::|||||||||||||||||||::|: 661 TTTGTGTTTTTTTTAAAGTCGTTGGTAATTGTAGATTTGGAAATTGGGTATTGTTTTTTT

721 AGTTGCTTACTCTAAGTGCGAATACTACTTAGACGTGCTGTTGAGGGTAAACTTGCTTCT |||||:|||:|:||||||++||||:||:|||||++||:|||||||||||||:|||:||:| 721 AGTTGTTTATTTTAAGTGCGAATATTATTTAGACGTGTTGTTGAGGGTAAATTTGTTTTT

WTfwd >>>>>>>> 781 GAGGCTCTCCACAGTTTTAGAAGTTTGATTAATAAGATATAGAGGCTTTTCTCTGATCAC ||||:|:|::|:|||||||||||||||||||||||||||||||||:||||:|:||||:|: 781 GAGGTTTTTTATAGTTTTAGAAGTTTGATTAATAAGATATAGAGGTTTTTTTTTGATTAT MSPfwd >>>>>>>> USPfwd >>>>>>>>

>>>>>>>>>>>>>>>>>> 841 TTCAAATGGATGGTGATCGTGTTCTTTGATACTGCTGAAGCTTGGCGAGTTTTTTTGGTT ||:||||||||||||||++||||:|||||||:||:|||||:||||++||||||||||||| 841 TTTAAATGGATGGTGATCGTGTTTTTTGATATTGTTGAAGTTTGGCGAGTTTTTTTGGTT >>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>> <<<<<<<<<<< 901 CAAATCTCCGAAGCCTATGGACCATTCAGCAGCCTGAGCTTCCAATTTGGCCGTCAGTGT :||||:|:++|||::||||||::|||:||:||::||||:||::|||||||:++|:||||| 901 TAAATTTTCGAAGTTTATGGATTATTTAGTAGTTTGAGTTTTTAATTTGGTCGTTAGTGT <<<<<<<<<<< <<<<<<<<<<<

<<<<<<<<<<<<<<< WTrev 961 CGTATGTTACTCCTATGTTGAAGCTTGTGGGCTGGATCGC ++|||||||:|::||||||||||:|||||||:|||||++: 961 CGTATGTTATTTTTATGTTGAAGTTTGTGGGTTGGATCGT <<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<< USPrev

Used primers:

Forward MSP: 5’-TTGATTATTTTAAATGGATGGTGATC-3’ Reverse MSP: 5’-AAAAATAACATACGACACTAACGAC-3’

Forward USP: 5’-TTGATTATTTTAAATGGATGGTGATT-3’ Reverse USP: 5’-AAAAATAACATACAACACTAACAAC-3’

Forward WT: 5’-CTGATCACTTCAAATGGATGGTGATC-3’ Reverse WT: 5’-TAGGAGTAACATACGACACTGACGGC-3’

Promoter region of PpGNT1

1 CTGTTGATGTATCCTAGATATTGTTGCATAGTTCTTGTCTAGTTTATTAAAATAAGAATA :|||||||||||::||||||||||||:||||||:||||:||||||||||||||||||||| 1 TTGTTGATGTATTTTAGATATTGTTGTATAGTTTTTGTTTAGTTTATTAAAATAAGAATA

61 ATAATAATAAATGTTTATATATTTAATATTAAAATAACCAATGTACAAAATATGTTAGAC |||||||||||||||||||||||||||||||||||||::||||||:|||||||||||||: 61 ATAATAATAAATGTTTATATATTTAATATTAAAATAATTAATGTATAAAATATGTTAGAT

121 ATTTTTGTATCAAATTCAAAAATATATTAAAAAAAGTACACAACATAGGTTACAATGGAT ||||||||||:|||||:|||||||||||||||||||||:|:||:||||||||:||||||| 121 ATTTTTGTATTAAATTTAAAAATATATTAAAAAAAGTATATAATATAGGTTATAATGGAT

181 CATAAATCATTAATTATTCTTGATATTATGTTAAAAAAGTTGAGAAACATCTACAATTAG :||||||:||||||||||:||||||||||||||||||||||||||||:||:||:|||||| 181 TATAAATTATTAATTATTTTTGATATTATGTTAAAAAAGTTGAGAAATATTTATAATTAG

241 TTAGAAACTTTCATATTGTTTAAAATCATTTTGTTATAAAAACAATACCATTTAATAAAG |||||||:|||:||||||||||||||:|||||||||||||||:||||::||||||||||| 241 TTAGAAATTTTTATATTGTTTAAAATTATTTTGTTATAAAAATAATATTATTTAATAAAG

301 ATGAATCTTATTAATAGGTAATTCTGTTGATATATTTCCTTGACACAGCAATATGGATAG ||||||:||||||||||||||||:|||||||||||||::||||:|:||:||||||||||| 301 ATGAATTTTATTAATAGGTAATTTTGTTGATATATTTTTTTGATATAGTAATATGGATAG

361 GAATCATAGTCTTAGATATAGTAGTTTTAAGGTGATTAATGTCAAAAGAACATAACTAGC ||||:|||||:|||||||||||||||||||||||||||||||:|||||||:||||:|||: 361 GAATTATAGTTTTAGATATAGTAGTTTTAAGGTGATTAATGTTAAAAGAATATAATTAGT

421 AAAAGAATTAAAGCATAGTCCACCAAACATATATTTTGAATAGCAAGATAATATAAATTA |||||||||||||:|||||::|::|||:|||||||||||||||:|||||||||||||||| 421 AAAAGAATTAAAGTATAGTTTATTAAATATATATTTTGAATAGTAAGATAATATAAATTA

481 CTTTAAAACAGAATATAATATAATATTAAATTTACTTTTATATTATTTTTAGATTAATGA :|||||||:|||||||||||||||||||||||||:||||||||||||||||||||||||| 481 TTTTAAAATAGAATATAATATAATATTAAATTTATTTTTATATTATTTTTAGATTAATGA

541 AACTTCACAATAATACATGAAAGAAATTTTTGTGACTTTGGCACCTTTTATTAGCAATGT ||:||:|:|||||||:|||||||||||||||||||:|||||:|::|||||||||:||||| 541 AATTTTATAATAATATATGAAAGAAATTTTTGTGATTTTGGTATTTTTTATTAGTAATGT

601 ATTACTCTTACTATGTAAAAGTATCAAATTTAACAAAAATTGAAAAAATATACATCCACT ||||:|:|||:|||||||||||||:||||||||:||||||||||||||||||:||::|:| 601 ATTATTTTTATTATGTAAAAGTATTAAATTTAATAAAAATTGAAAAAATATATATTTATT

661 TATACTATCAATTAATTAATTAAAATTTTATTTATTTTTTAATTTTTTGTTGACTTAAAA ||||:|||:||||||||||||||||||||||||||||||||||||||||||||:|||||| 661 TATATTATTAATTAATTAATTAAAATTTTATTTATTTTTTAATTTTTTGTTGATTTAAAA

721 TATATCTATATATATATATATATATATATATATGTATATTCACATTCTTGAACAAGAATT |||||:||||||||||||||||||||||||||||||||||:|:|||:|||||:||||||| 721 TATATTTATATATATATATATATATATATATATGTATATTTATATTTTTGAATAAGAATT

WTfwd >>>>>>>>>>>>>>>>>>>>>>> 781 TTGGATTCAAGGAGGTGAATGCTTTGCACAAAAAAAAGTTTTATCTCTAAATTCTTAGAC |||||||:|||||||||||||:||||:|:|||||||||||||||:|:||||||:|||||: 781 TTGGATTTAAGGAGGTGAATGTTTTGTATAAAAAAAAGTTTTATTTTTAAATTTTTAGAT MSPfwd >>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>

841 AACGTCATTCAAAATAAGTTTTAAAACAGCGACTAGTCATAAAATACGTATTTACACACT ||++|:|||:||||||||||||||||:||++|:||||:||||||||++||||||:|:|:| 841 AACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATACGTATTTATATATT >>>> >>>>>

901 TGTATATGATGTACCATAGACGGTAATCGTACATATTTGCCGACACCCTGCAATTAATAG |||||||||||||::|||||++|||||++||:|||||||:++|:|:::||:||||||||| 901 TGTATATGATGTATTATAGACGGTAATCGTATATATTTGTCGATATTTTGTAATTAATAG

<<<<<<<<<<<<<<<<<<<<<<< WTrev 961 AGTTCGAATATCCCCGCCGCGTTCAAGTCGCCTCGTGCAA ||||++|||||:::++:++++||:||||++::|++||:|| 961 AGTTCGAATATTTTCGTCGCGTTTAAGTCGTTTCGTGTAA <<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<< USPrev

Used primers:

Forward MSP: 5’-TTTATTTTTAAATTTTTAGATAACG-3’ Reverse MSP: 5’-AACGACTTAAACGCGACGA-3’

Forward USP: 5’-TTATTTTTAAATTTTTAGATAATGT-3’ Reverse USP: 5’-AAACAACTTAAACACAACAAA-3’

Forward WT: 5’-GTTTTATCTCTAAATTCTTAGAC-3’ Reverse WT: 5’-GGCGACTTGAACGCGGCGGGGAT-3’

Intron 4 within the miRNA166 binding site of PpC3HDZIP1

1 GGTATGAAGGTATGGATGCCATGCCTTCCTACGGCACGTTCTACAGTGTATTGTGGAGTA ||||||||||||||||||::|||::||::||++|:|++||:||:|||||||||||||||| 1 GGTATGAAGGTATGGATGTTATGTTTTTTTACGGTACGTTTTATAGTGTATTGTGGAGTA MSPfwd >>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>

61 GCGAGCCTCACCTGTAACTCTTGATCTATAGATTCCATTATCAGAGATATGATCGCACGA |++||::|:|::|||||:|:|||||:||||||||::|||||:|||||||||||++:|++| 61 GCGAGTTTTATTTGTAATTTTTGATTTATAGATTTTATTATTAGAGATATGATCGTACGA <<<<<<< <<<<<<<

121 AATAACTCTTTGTTCCAACCTTTTGTAAAATAAGTATTAGCGGAGTCATGGTACTGGAGC |||||:|:||||||::||::||||||||||||||||||||++||||:||||||:|||||: 121 AATAATTTTTTGTTTTAATTTTTTGTAAAATAAGTATTAGCGGAGTTATGGTATTGGAGT <<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<< USPrev

181 AAAGTCAAACAAATTAATTTGACTCAAAACACGACTTCGAATTAATTTAGGAGCTAACAA |||||:|||:||||||||||||:|:||||:|++|:||++||||||||||||||:|||:|| 181 AAAGTTAAATAAATTAATTTGATTTAAAATACGATTTCGAATTAATTTAGGAGTTAATAA

241 GGTAATGATATTGATTCTTTAATTCAAATTAAAGTGGTTGATTGCAAATGCCATTGCTGA ||||||||||||||||:|||||||:|||||||||||||||||||:|||||::||||:||| 241 GGTAATGATATTGATTTTTTAATTTAAATTAAAGTGGTTGATTGTAAATGTTATTGTTGA

301 TACGTCACTAGT ||++|:|:|||| 301 TACGTTATTAGT

Used primers:

Forward MSP: 5’-GTTATGTTTTTTTACGGTACGT-3’ Reverse MSP: 5’-AAACAAAAAATTATTTCGTACG-3’

Forward USP: 5’-TGTTATGTTTTTTTATGGTATGT-3’ Reverse USP: 5’-TTAAAACAAAAAATTATTTCATACA-3’

Intron 1 upstream of the miRNA166 binding site of PpC3HDZIP1

1 GGTATGTATCCGTGTCCTTCGCCAGATTCTAGGAGAGGAAAATTGTTTGGGCATAAACCT |||||||||:++|||::||++::|||||:||||||||||||||||||||||:|||||::| 1 GGTATGTATTCGTGTTTTTCGTTAGATTTTAGGAGAGGAAAATTGTTTGGGTATAAATTT

61 CGTATAGAAGTTCGTTCGATCTTGAAATCTTTCTCAAACGGAGATTCTGGATGACATGCA ++||||||||||++||++||:|||||||:|||:|:|||++||||||:|||||||:|||:| 61 CGTATAGAAGTTCGTTCGATTTTGAAATTTTTTTTAAACGGAGATTTTGGATGATATGTA MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>

121 CGTGAAGTTGATTCACTATATTATGTCGCCTCGAATTTCACTTCGTATTGAGCCATCTCA ++|||||||||||:|:||||||||||++::|++|||||:|:||++|||||||::||:|:| 121 CGTGAAGTTGATTTATTATATTATGTCGTTTCGAATTTTATTTCGTATTGAGTTATTTTA

181 TGTTTTATTCACTTCTGTCTTGAGATTGTCATACGTGCCGCTCCGTGATGTGCGGATAGG |||||||||:|:||:|||:||||||||||:|||++||:++:|:++|||||||++|||||| 181 TGTTTTATTTATTTTTGTTTTGAGATTGTTATACGTGTCGTTTCGTGATGTGCGGATAGG <<<<<<<< <<<<<<<<<<

241 TGGACAGGTGCACAACATGATAATCCATGTTGTGGTCGAGGGGTAGGGGGGTGGTACACG ||||:|||||:|:||:||||||||::||||||||||++||||||||||||||||||:|++ 241 TGGATAGGTGTATAATATGATAATTTATGTTGTGGTCGAGGGGTAGGGGGGTGGTATACG <<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<< USPrev

301 ACACAATTAATTGAAATGAGTGGAGAGTGTATTGCAG |:|:||||||||||||||||||||||||||||||:|| 301 ATATAATTAATTGAAATGAGTGGAGAGTGTATTGTAG

Used primers:

Forward MSP: 5’-TTCGATTTTGAAATTTTTTTTAAAC-3’ Reverse MSP: 5’-TATTATACACCTATCCACCTATCCG-3’

Forward USP: 5’-TGATTTTGAAATTTTTTTTAAATGG-3’ Reverse USP: 5’-ATTATACACCTATCCACCTATCCACA-3’

Exon 14 downstream of the miRNA166 binding site of PpC3HDZIP1

1 GGACGGAATCGGGCTGAATCGTACACTTGATCTGGCTTCCACACTTGAAGATCACGAGGC |||++||||++||:|||||++||:|:|||||:|||:||::|:|:||||||||:|++|||: 1 GGACGGAATCGGGTTGAATCGTATATTTGATTTGGTTTTTATATTTGAAGATTACGAGGT MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>

61 AGGATTGAATGGAGAGAGCAAGTCTAATGGCAGCTCTAGCCAAGTGCGATCCGTTCTGAC ||||||||||||||||||:||||:||||||:||:|:|||::|||||++||:++||:|||: 61 AGGATTGAATGGAGAGAGTAAGTTTAATGGTAGTTTTAGTTAAGTGCGATTCGTTTTGAT

121 AATAGCTTTTCAGTTTGCGTATGAAGTTCATACACGCGAAACATGCGCAGTGATGGCCCG |||||:||||:||||||++|||||||||:|||:|++++|||:|||++:||||||||::++ 121 AATAGTTTTTTAGTTTGCGTATGAAGTTTATATACGCGAAATATGCGTAGTGATGGTTCG <<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<

181 CCAGTATGTTCGCACAGTGGTTGCATCCGTGCAGCGGGTTGCCATGGCTTTGGCACCGTC ::||||||||++:|:||||||||:||:++||:||++|||||::||||:|||||:|:++|: 181 TTAGTATGTTCGTATAGTGGTTGTATTCGTGTAGCGGGTTGTTATGGTTTTGGTATCGTT <<<<<<<<<< MSPrev <<<<<<<< USPrev

241 CCGTGGTCAGCCCCGTCCAGCACTGGGCAACTCAGATGCCATCAGTTTGGCGCGTCACAT :++||||:||:::++|::||:|:||||:||:|:|||||::||:|||||||++++|:|:|| 241 TCGTGGTTAGTTTCGTTTAGTATTGGGTAATTTAGATGTTATTAGTTTGGCGCGTTATAT

301 CCTGAGCAGCTACAG ::||||:||:||:|| 301 TTTGAGTAGTTATAG

Used primers:

Forward MSP: 5’-TGGTTTTTATATTTGAAGATTACGA-3’ Reverse MSP: 5’-AACATACTAACGAACCATCACTACG-3’

Forward USP: 5’-TGGTTTTTATATTTGAAGATTATGA-3’ Reverse USP: 5’-CATACTAACAAACCATCACTACACA-3’

Exon 1 upstream of the miRNA160 binding site of PpARF

1 GGTATCGATCTGGAGCCCGTTGCAAACTCAATGGTGTATTTTATAGGGCAAAAGTCTGAT |||||++||:|||||::++|||:|||:|:|||||||||||||||||||:||||||:|||| 1 GGTATCGATTTGGAGTTCGTTGTAAATTTAATGGTGTATTTTATAGGGTAAAAGTTTGAT

61 CTATATGGAATGCATCCTCTCAGAGTTGCAAATCATGGACTGCATGTCACTCTGGGTTAT :|||||||||||:||::|:|:|||||||:||||:|||||:||:||||:|:|:|||||||| 61 TTATATGGAATGTATTTTTTTAGAGTTGTAAATTATGGATTGTATGTTATTTTGGGTTAT

121 TCTCGATCACCTAGCTTTGCTGGAGTTCAAATTGGTGAGTACGAGTATTATGAGTGATCT |:|++||:|::|||:||||:|||||||:|||||||||||||++|||||||||||||||:| 121 TTTCGATTATTTAGTTTTGTTGGAGTTTAAATTGGTGAGTACGAGTATTATGAGTGATTT

181 CGAGTTTATGGTCCCCTTCTTTCATGATCAAGGGTAATTTATATCAAGGGTGTATATGAG ++||||||||||::::||:|||:|||||:|||||||||||||||:||||||||||||||| 181 CGAGTTTATGGTTTTTTTTTTTTATGATTAAGGGTAATTTATATTAAGGGTGTATATGAG

241 AGATACGCACTTATTGAGTGGACCTTTTCTCATACTGCATTTACACCCCTGTCAGTTGCA |||||++:|:||||||||||||::||||:|:|||:||:|||||:|::::|||:|||||:| 241 AGATACGTATTTATTGAGTGGATTTTTTTTTATATTGTATTTATATTTTTGTTAGTTGTA

301 GCATCCTGGTTTGGAATGCCGGGTCCAGTCCCTCTATTATCCATGAGTGTAAAATCGGAG |:||::||||||||||||:++|||::|||:::|:||||||::|||||||||||||++||| 301 GTATTTTGGTTTGGAATGTCGGGTTTAGTTTTTTTATTATTTATGAGTGTAAAATCGGAG

361 AGTCTCGATGACATTGGAGGTCACGAGAAAAAATCTGTAACTGGGTCGGAAGTGGGTGGC |||:|++||||:|||||||||:|++|||||||||:|||||:|||||++|||||||||||: 361 AGTTTCGATGATATTGGAGGTTACGAGAAAAAATTTGTAATTGGGTCGGAAGTGGGTGGT

421 CTCGATGCTCAGCTGTGGCATGCCTGTGCTGGGGGTATGGTTCAACTGCCTCATGTGGGT :|++|||:|:||:|||||:|||::||||:|||||||||||||:||:||::|:|||||||| 421 TTCGATGTTTAGTTGTGGTATGTTTGTGTTGGGGGTATGGTTTAATTGTTTTATGTGGGT

481 GCTAAGGTTGTCTATTTTCCCCAAGGCCATGGCGAACAAGCTGCTTCAACTCCCGAGTTC |:|||||||||:||||||::::||||::||||++||:|||:||:||:||:|::++||||: 481 GTTAAGGTTGTTTATTTTTTTTAAGGTTATGGCGAATAAGTTGTTTTAATTTTCGAGTTT

541 CCCCGCACTTTGGTTCCAAATGGAAGTGTTCCCTGCCGAGTTGTGTCAGTTAACTTTCTG :::++:|:|||||||::|||||||||||||:::||:++||||||||:||||||:|||:|| 541 TTTCGTATTTTGGTTTTAAATGGAAGTGTTTTTTGTCGAGTTGTGTTAGTTAATTTTTTG MSPfwd >>>>> USPfwd >>>>>

601 GCTGATACAGAAACAGACGAGGTATTTGCTCGTATTTGCCTGCAGCCTGAGATTGGCTCC |:|||||:|||||:|||++|||||||||:|++||||||::||:||::|||||||||:|:: 601 GTTGATATAGAAATAGACGAGGTATTTGTTCGTATTTGTTTGTAGTTTGAGATTGGTTTT >>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>

661 TCCGCTCAGGATTTAACAGATGATTCTCTTGCGTCTCCGCCTCTAGAGAAACCAGCTTCA |:++:|:|||||||||:||||||||:|:|||++|:|:++::|:||||||||::||:||:| 661 TTCGTTTAGGATTTAATAGATGATTTTTTTGCGTTTTCGTTTTTAGAGAAATTAGTTTTA

721 TTTGCCAAAACGCTCACTCAAAGTGATGCAAACAACGGTGGAGGCTTTTCAATACCTCGT ||||::||||++:|:|:|:|||||||||:|||:||++|||||||:||||:||||::|++| 721 TTTGTTAAAACGTTTATTTAAAGTGATGTAAATAACGGTGGAGGTTTTTTAATATTTCGT <<<<<<<<<<<<<<<<<<<<<<<<

841 GTTCTTGCAAAAGATGTCCATGGAGAGGTGTGGAAATTTCGTCACATTTACAGG |||:|||:|||||||||::||||||||||||||||||||++|:|:|||||:||| 841 GTTTTTGTAAAAGATGTTTATGGAGAGGTGTGGAAATTTCGTTATATTTATAGG

Used primers:

Forward MSP: 5’-TTTTGGTTGATATAGAAATAGACGA-3’ Reverse MSP: 5’-GAAATATTAAAAAACCTCCACCGTT-3’

Forward USP: 5’-TTTTGGTTGATATAGAAATAGATGA-3’ Reverse USP: 5’-AAAATATTAAAAAACCTCCACCATT-3’

Exon 4 downstream of the miRNA160 binding site of PpARF

1 AGGAATTCCATGGAGACAGTCAGACGCCTCATACTCCTGCATCTGGTAGCCAATGAGGCT |||||||::|||||||:|||:|||++::|:|||:|::||:||:||||||::|||||||:| 1 AGGAATTTTATGGAGATAGTTAGACGTTTTATATTTTTGTATTTGGTAGTTAATGAGGTT

61 AAAGCTTGATCATAGCTCATAACCCTCTCACAGGACGTAATGGGGGTGACAACATGCTAA ||||:|||||:||||:|:||||:::|:|:|:||||++||||||||||||:||:|||:||| 61 AAAGTTTGATTATAGTTTATAATTTTTTTATAGGACGTAATGGGGGTGATAATATGTTAA MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>>

121 CAGAATTGCACGGTAAAGGAAAACTGTACTAGGCATGTTATATGGGAATTCGGATCGCTT :|||||||:|++|||||||||||:||||:||||:||||||||||||||||++|||++:|| 121 TAGAATTGTACGGTAAAGGAAAATTGTATTAGGTATGTTATATGGGAATTCGGATCGTTT

181 CTTGCAATTAAACACGCTAGCGCCGTTTGGTGCCAATGTTATTCTGGCATTTGTTTTGTT :|||:|||||||:|++:|||++:++|||||||::|||||||||:|||:|||||||||||| 181 TTTGTAATTAAATACGTTAGCGTCGTTTGGTGTTAATGTTATTTTGGTATTTGTTTTGTT <<<<<<<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<<<<<<< USPrev

241 TCCTTTGGAAACAAATTGCTATATTTCAAAGTCCTTTGGAGGAGCTCGC |::||||||||:||||||:|||||||:|||||::||||||||||:|++: 241 TTTTTTGGAAATAAATTGTTATATTTTAAAGTTTTTTGGAGGAGTTCGT

Used primers:

Forward MSP: 5’-AGTTTATAATTTTTTTATAGGACGT-3’ Reverse MSP: 5’-AAAATAACATTAACACCAAACGAC-3’

Forward USP: 5’-TAGTTTATAATTTTTTTATAGGATGT-3’ Reverse USP: 5’-CCAAAATAACATTAACACCAAACAAC-3’

Intron 2 upstream of the miRNA160 binding site of PpARF

1 AGTTCTCCATGGCGGGTTCTGCAGGTTAGCTTTTTGTTTGTCTAATCAAAGCAATCAATG ||||:|::||||++||||:||:|||||||:|||||||||||:||||:||||:|||:|||| 1 AGTTTTTTATGGCGGGTTTTGTAGGTTAGTTTTTTGTTTGTTTAATTAAAGTAATTAATG MSPfwd >>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>

61 TGCTAATGAACGTGCTGCCTGCATTGTCTGCAATGACTATGTACTGCGTAGTCAGTGGAA ||:|||||||++||:||::||:|||||:||:|||||:||||||:||++||||:||||||| 61 TGTTAATGAACGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGCGTAGTTAGTGGAA >>>>>>>>>>>>> >>>>>>>>>>>>>

121 ATAGGTGGTTGATTATGAGATTTTGGTTGTGCAGGTCACTTGGGATGAGCCGGACCTATT |||||||||||||||||||||||||||||||:||||:|:||||||||||:++||::|||| 121 ATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTCGGATTTATT

181 GCAGGGAGTGAATCGTGTAAGCCCATGGCAGTTAGAGCTTGTGGCGACACTTCCTATGCA |:|||||||||||++||||||:::||||:||||||||:||||||++|:|:||::||||:| 181 GTAGGGAGTGAATCGTGTAAGTTTATGGTAGTTAGAGTTTGTGGCGATATTTTTTATGTA <<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<

241 GCTGCCCCCTGTCTCTCTTCCCAAAAAGAAACTGCG |:||:::::|||:|:|:||:::|||||||||:||++ 241 GTTGTTTTTTGTTTTTTTTTTTAAAAAGAAATTGCG <<<<<<< MSPrev <<<<<<< USPrev

Used primers:

Forward MSP: 5’-TAAAGTAATTAATGTGTTAATGAACGT-3’ Reverse MSP: 5’-AAACAACTACATAAAAAATATCGCC-3’

Forward USP: 5’-TTAAAGTAATTAATGTGTTAATGAATGT-3’ Reverse USP: 5’-AAACAACTACATAAAAAATATCACC-3’

Intron 3 downstream of the miRNA160 binding site of PpARF

1 AGGATCAGGTTTGTATCAACAAATCTCTAGTTCGTTTTGGCATGGAGTTCACAATGTGCT |||||:||||||||||:||:||||:|:|||||++||||||:||||||||:|:||||||:| 1 AGGATTAGGTTTGTATTAATAAATTTTTAGTTCGTTTTGGTATGGAGTTTATAATGTGTT MSPfwd >>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>

61 CTTCAAGCTTCGTTGTATGCTAAACTCTACTGCAATACTGCTACGGCGTGCCTTTTCTTT :||:|||:||++|||||||:||||:|:||:||:||||:||:||++|++||::||||:||| 61 TTTTAAGTTTCGTTGTATGTTAAATTTTATTGTAATATTGTTACGGCGTGTTTTTTTTTT >>>>>>>>>>>>> >>>>>>>>>>>>>

121 TTTCGAAGTATGATATGATGACCTAATGGTCTTTTTGAATACGACAGGAATTCCATGGAG |||++||||||||||||||||::|||||||:||||||||||++|:|||||||::|||||| 121 TTTCGAAGTATGATATGATGATTTAATGGTTTTTTTGAATACGATAGGAATTTTATGGAG <<<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<<<

181 ACAGTCAGACGCCTCATACTCCTGCATCTGGTAGCCAATGAGGCTAAAGCTTGATC |:|||:|||++::|:|||:|::||:||:||||||::|||||||:|||||:|||||: 181 ATAGTTAGACGTTTTATATTTTTGTATTTGGTAGTTAATGAGGTTAAAGTTTGATT <<<< MSPrev <<<< USPrev

Used primers:

Forward MSP: 5’-TTTATAATGTGTTTTTTAAGTTTCGT-3’ Reverse MSP: 5’-CTATCTCCATAAAATTCCTATCGTA-3’

Forward USP: 5’-TTTATAATGTGTTTTTTAAGTTTTGT-3’ Reverse USP: 5’-CTATCTCCATAAAATTCCTATCATA-3’

Sequence of PpTAS4

1 ACCAAAGTAGATTGAATCAATCCGTGCATCGATTCACAGGAGCACTGACCTGATCTATGC |::||||||||||||||:|||:++||:||++|||:|:|||||:|:|||::||||:||||+ 1 ATTAAAGTAGATTGAATTAATTCGTGTATCGATTTATAGGAGTATTGATTTGATTTATGC

61 GACGGTGCGAGAAAAATCATCCCAGCGTGGTGCTACGCTAGTCACCTAGTCATCAGCATC +|++|||++||||||||:||:::||++|||||:||++:||||:|::||||:||:||:||+ 61 GACGGTGCGAGAAAAATTATTTTAGCGTGGTGTTACGTTAGTTATTTAGTTATTAGTATC MSPfwd >>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>

121 GGTGCTACGTAAACTTATGGCAATGGTGCGTTCCTATCAGTGCGTCACTTCAAGGAAGCC +|||:||++||||:||||||:|||||||++||::|||:||||++|:|:||:|||||||:: 121 GGTGTTACGTAAATTTATGGTAATGGTGCGTTTTTATTAGTGCGTTATTTTAAGGAAGTT <<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<<

181 CTTCCCAAGTCTTCATCCGGCCCGCTACCTTTGCGTAGCTGCTTCACTGGAGGCCTGGGT :||:::||||:||:||:++|::++:||::||||++|||:||:||:|:||||||::||||| 181 TTTTTTAAGTTTTTATTCGGTTCGTTATTTTTGCGTAGTTGTTTTATTGGAGGTTTGGGT <<<<< MSPrev <<<<< USPrev

241 GGAGACTGGCGGACAGCTTTGCGATGTTACGGTTGTAGCCAATTCTTGTTGCACTTAGAT |||||:|||++||:||:||||++||||||++|||||||::||||:||||||:|:|||||| 241 GGAGATTGGCGGATAGTTTTGCGATGTTACGGTTGTAGTTAATTTTTGTTGTATTTAGAT

301 TTCCACTGGGCGTTATCCCTCTTGAGCTGAGAAGACAAGGGCTCCCTCCTAGGGGGCGAA ||::|:||||++||||:::|:|||||:||||||||:|||||:|:::|::|||||||++|| 301 TTTTATTGGGCGTTATTTTTTTTGAGTTGAGAAGATAAGGGTTTTTTTTTAGGGGGCGAA

361 AATAGGTGAGCTGGGGTCACCTTGTTAGCGGGGTGTTAAGCATTTGAATGCAACACTCCT ||||||||||:||||||:|::|||||||++||||||||||:|||||||||:||:|:|::| 361 AATAGGTGAGTTGGGGTTATTTTGTTAGCGGGGTGTTAAGTATTTGAATGTAATATTTTT

421 ACGCAAGACCCTAGCTATGGCTCCATAGGGTGTGATGAGTGCTTCATCCGGTGCTCTTCT |++:||||:::|||:|||||:|::|||||||||||||||||:||:||:++|||:|:||:| 421 ACGTAAGATTTTAGTTATGGTTTTATAGGGTGTGATGAGTGTTTTATTCGGTGTTTTTTT

481 ACTGCCTTGCCCACCTACCCTTGTGATATGGGCCGCGCGTGTCTGCGTGTCTCCTGTATC |:||::|||:::|::||:::||||||||||||:++++++|||:||++|||:|::|||||+ 481 ATTGTTTTGTTTATTTATTTTTGTGATATGGGTCGCGCGTGTTTGCGTGTTTTTTGTATC

541 GGTTGTATATCACTCCTGAGCTACGGGTGTGCAATTCCCATGTCTTTTGGGAATAGGCGT +|||||||||:|:|::||||:||++||||||:||||:::||||:|||||||||||||++| 541 GGTTGTATATTATTTTTGAGTTACGGGTGTGTAATTTTTATGTTTTTTGGGAATAGGCGT

601 CAAGACTAGAGGTAGTTTTGTTGTCTTAGCCGGCCACAGGCGGCGGTGATAAAACCTGCA :||||:||||||||||||||||||:||||:++|::|:|||++|++|||||||||::||:| 601 TAAGATTAGAGGTAGTTTTGTTGTTTTAGTCGGTTATAGGCGGCGGTGATAAAATTTGTA

661 GTTGATGTAATGGAGTCACATACTGAATCCACTTGACTGGCTGTGGCTGAAATAAAAACA ||||||||||||||||:|:|||:|||||::|:||||:|||:|||||:|||||||||||:| 661 GTTGATGTAATGGAGTTATATATTGAATTTATTTGATTGGTTGTGGTTGAAATAAAAATA

721 TTTTCCAC ||||::|: 721 TTTTTTAT

Used primers: Forward MSP: 5’-GGTGCGAGAAAAATTATTTTAGC-3’ Reverse MSP: 5’-AAAAAAACTTCCTTAAAATAACGCA-3’

Forward USP: 5’-GTGTGAGAAAAATTATTTTAGTGT-3’ Reverse USP: 5’-AAAAAAACTTCCTTAAAATAACACA-3’

Coding Sequence of PpEREBP/AP2

1 NATGTCTGGTAGCGGAAGCATAGGCACTTCCGGAGTGGACTCATGGGTTGAGCAGAGCTA |||||:||||||++||||:|||||:|:||:++|||||||:|:||||||||||:||||:|| 1 NATGTTTGGTAGCGGAAGTATAGGTATTTTCGGAGTGGATTTATGGGTTGAGTAGAGTTA MSPfwd >>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>

61 ACTGACATGCTTTGCGGGGGAATTACTCACTGATTCAGCAGCACTGGCAGATCTGTGTGA |:|||:|||:||||++|||||||||:|:|:|||||:||:||:|:|||:||||:||||||| 61 ATTGATATGTTTTGCGGGGGAATTATTTATTGATTTAGTAGTATTGGTAGATTTGTGTGA

121 GGCGTTGAAGGTTTTCTACGAGTCGCTGCTTCCCCAGCTAATCCAAAGAGATGCGGATAG ||++|||||||||||:||++|||++:||:||::::||:||||::|||||||||++||||| 121 GGCGTTGAAGGTTTTTTACGAGTCGTTGTTTTTTTAGTTAATTTAAAGAGATGCGGATAG <<<<<<<<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<<<<<< USPrev 181 AGATTCCGCAACATCATGCTGAAGTGGAAGAAAGTGTTGCGAGTGTGGGTTTATAGATTC |||||:++:||:||:|||:||||||||||||||||||||++||||||||||||||||||+ 181 AGATTTCGTAATATTATGTTGAAGTGGAAGAAAGTGTTGCGAGTGTGGGTTTATAGATTC

241 GCGCTTTATTGACGATTGAGGTGAAGCAGAGGGTGGGGTTTTCGGAAAGCTGCAGCGTCA +++:||||||||++||||||||||||:|||||||||||||||++|||||:||:||++|:| 241 GCGTTTTATTGACGATTGAGGTGAAGTAGAGGGTGGGGTTTTCGGAAAGTTGTAGCGTTA

301 CTCGAGTGCAGAGGGTGTGCCAGGTTGTAGTGGTTTTCGAATTTCAGCGGACGAGGTAAG :|++||||:||||||||||::||||||||||||||||++|||||:||++||++||||||| 301 TTCGAGTGTAGAGGGTGTGTTAGGTTGTAGTGGTTTTCGAATTTTAGCGGACGAGGTAAG

361 GAAGCCTAGCAGACGAGAACGTGAGTAAGTCAGCGCAAGATGGTAGGCAAAGTGCAGGCG ||||::|||:|||++||||++|||||||||:||++:|||||||||||:||||||:|||++ 361 GAAGTTTAGTAGACGAGAACGTGAGTAAGTTAGCGTAAGATGGTAGGTAAAGTGTAGGCG

421 TCCCTAGCTGGTGCCCATGGCAAGCAGTCTACTCATCATGCCATGGTTCGAAGCAGTCAT |:::|||:|||||:::||||:|||:|||:||:|:||:|||::||||||++|||:|||:|| 421 TTTTTAGTTGGTGTTTATGGTAAGTAGTTTATTTATTATGTTATGGTTCGAAGTAGTTAT

481 CACACCCTAGTTCCGGAGATCATGGGTCGCTCGCGACCTGTTTCACAAAAGCTGAAAGCC :|:|:::|||||:++|||||:||||||++:|++++|::|||||:|:|||||:||||||:+ 481 TATATTTTAGTTTCGGAGATTATGGGTCGTTCGCGATTTGTTTTATAAAAGTTGAAAGTC

541 GCCAGCATCAAGAGGGCCAAAAAGGTTCAAGAGGGGAGGTACAGAGGGGTGCGGCAGCGG +::||:||:|||||||::|||||||||:|||||||||||||:|||||||||++|:||++| 541 GTTAGTATTAAGAGGGTTAAAAAGGTTTAAGAGGGGAGGTATAGAGGGGTGCGGTAGCGG

601 CCGTGGGGGCGATTTGCGGCGGAGATTAGAGACCCCAATACTAAGGAACGGAAGTGGCTA :++||||||++|||||++|++|||||||||||::::||||:|||||||++|||||||:|| 601 TCGTGGGGGCGATTTGCGGCGGAGATTAGAGATTTTAATATTAAGGAACGGAAGTGGTTA

661 GGCACTTTTGACACTGCTGAGGATGCAGCTCTCGCTTACGACACTGGTAAGAATATCAAC ||:|:||||||:|:||:||||||||:||:|:|++:|||++|:|:||||||||||||:||: 661 GGTATTTTTGATATTGTTGAGGATGTAGTTTTCGTTTACGATATTGGTAAGAATATTAAT

721 TTCTCCATTGCGCATTTGGTTACAAGGTGGCGATGACGATCACGTATCTCTATCCCTGAT ||:|::||||++:|||||||||:|||||||++||||++||:|++|||:|:|||:::|||| 721 TTTTTTATTGCGTATTTGGTTATAAGGTGGCGATGACGATTACGTATTTTTATTTTTGAT

781 AGAGCTAACTCTATCCGCCTTCCGTTTCTTGTAGCGGCAAGATCTATGAGAGGACCTAAG ||||:|||:|:|||:++::||:++|||:||||||++|:|||||:||||||||||::|||| 781 AGAGTTAATTTTATTCGTTTTTCGTTTTTTGTAGCGGTAAGATTTATGAGAGGATTTAAG

841 GCACGTACCAACTTTGTGTACCCTACGCATGAGACCTGTCTTCTTTCCGCTGCAGCGGCA |:|++||::||:||||||||:::||++:||||||::|||:||:|||:++:||:||++|:| 841 GTACGTATTAATTTTGTGTATTTTACGTATGAGATTTGTTTTTTTTTCGTTGTAGCGGTA

901 CTGGCGGCGCCAAATGGTAATTCGCAGCATCACCAGGTGGGTCTAATCGCTCAGAAGACC :|||++|++::|||||||||||++:||:||:|::||||||||:||||++:|:||||||:: 901 TTGGCGGCGTTAAATGGTAATTCGTAGTATTATTAGGTGGGTTTAATCGTTTAGAAGATT

961 TTGGGAAGTGCTGCTGCTCTCAGCAGCAGTACCGGCTTATTGCACNNAACCCTNNNNNNN ||||||||||:||:||:|:|:||:||:||||:++|:||||||:|:||||:::|||||||| 961 TTGGGAAGTGTTGTTGTTTTTAGTAGTAGTATCGGTTTATTGTATNNAATTTTNNNNNNN

1021 GGGGA ||||| 1021 GGGGA

Used primers:

Forward MSP: 5’-GGTAGCGGAAGTATAGGTATTTTC-3’ Reverse MSP: 5’-TTAACTAAAAAAACAACGACTCGTA-3’

Forward USP: 5’-GTAGTGGAAGTATAGGTATTTTTGG-3’ Reverse USP: 5’-TTAACTAAAAAAACAACAACTCATA-3’

Promoter region of PpEREBP/AP2

1 GCGTGACATCGTAGATATTGAGGATGAAGATTCGTCTGAGAATGGAACTTGCGTGGATAG |++|||:||++|||||||||||||||||||||++|:|||||||||||:|||++||||||| 1 GCGTGATATCGTAGATATTGAGGATGAAGATTCGTTTGAGAATGGAATTTGCGTGGATAG

61 CAGACATTTTCTGGGCTCGATGACTCCAAGCTCTGAACCTGTGTCTTCGAAATTTACGAT :|||:|||||:||||:|++||||:|::|||:|:||||::|||||:||++|||||||++|| 61 TAGATATTTTTTGGGTTCGATGATTTTAAGTTTTGAATTTGTGTTTTCGAAATTTACGAT

121 CACGCTGGAGTCCACGTACTTCGACACTACTTGGTCCAGTGTGCCACTTATAATTTTAGA :|++:||||||::|++||:||++|:|:||:|||||::||||||::|:||||||||||||| 121 TACGTTGGAGTTTACGTATTTCGATATTATTTGGTTTAGTGTGTTATTTATAATTTTAGA

181 CTCAAACTGCCACTGCATTAGTGCTTGCGAAGGAGAAGTTACTGTAGAAGCTTTTTTGTA :|:|||:||::|:||:|||||||:|||++||||||||||||:||||||||:||||||||| 181 TTTAAATTGTTATTGTATTAGTGTTTGCGAAGGAGAAGTTATTGTAGAAGTTTTTTTGTA

241 AAGCATAATCGAATCGCTTGGTGGTGAATCGTTTTCGGAGGAAACTGAGATATCATCGCT |||:|||||++|||++:||||||||||||++||||++|||||||:||||||||:||++:| 241 AAGTATAATCGAATCGTTTGGTGGTGAATCGTTTTCGGAGGAAATTGAGATATTATCGTT

301 GTCAAAGACCTGCCACAAGGATTTGAAAAAGTGGTGAACTCCAGTAAGGTGGGATATCTC ||:|||||::||::|:||||||||||||||||||||||:|::|||||||||||||||:|: 301 GTTAAAGATTTGTTATAAGGATTTGAAAAAGTGGTGAATTTTAGTAAGGTGGGATATTTT

361 CAAGGATGAAATGCATTTAGCAGAGCTTATTCCAATAACAACTGCACCATCACTACGTAA :||||||||||||:||||||:||||:|||||::|||||:||:||:|::||:|:||++||| 361 TAAGGATGAAATGTATTTAGTAGAGTTTATTTTAATAATAATTGTATTATTATTACGTAA

421 TGACCAAATAAACTAGTTATAAACAAAATGCTACGAGCATCTTCATAATGCGAAACATAA |||::|||||||:||||||||||:||||||:||++||:||:||:||||||++|||:|||| 421 TGATTAAATAAATTAGTTATAAATAAAATGTTACGAGTATTTTTATAATGCGAAATATAA

481 AGCCTGCTTCAAACACACCTTGAAACAGGTGTGAGTATTCACGTTGCTGGTTCACAAGAC ||::||:||:|||:|:|::||||||:|||||||||||||:|++|||:|||||:|:||||: 481 AGTTTGTTTTAAATATATTTTGAAATAGGTGTGAGTATTTACGTTGTTGGTTTATAAGAT

541 TCCGGAAAAAGTAATAAGTTTCTGGATCGAGTAGTGAAAGAGAATTACCTGAAATGGTTG |:++|||||||||||||||||:|||||++||||||||||||||||||::||||||||||| 541 TTCGGAAAAAGTAATAAGTTTTTGGATCGAGTAGTGAAAGAGAATTATTTGAAATGGTTG

601 GGATCCTGCTCCGCAATAGCGTGACACAAACAGCTCGGAACTGACAGTTGGACTCCGTTT ||||::||:|:++:|||||++|||:|:|||:||:|++|||:|||:|||||||:|:++||| 601 GGATTTTGTTTCGTAATAGCGTGATATAAATAGTTCGGAATTGATAGTTGGATTTCGTTT MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>> 661 GAATTGGGTCAGACGAAATTGTTGGTCTTCGTCAGAGACGCAATGCCATTGCTCTTGCTT |||||||||:|||++|||||||||||:||++|:|||||++:||||::||||:|:|||:|| 661 GAATTGGGTTAGACGAAATTGTTGGTTTTCGTTAGAGACGTAATGTTATTGTTTTTGTTT

721 CCCCGGACTTACTTTAAAACCTTCTACCTCAACCTTGTTGATCGAGGGACTTTTCGGCAT :::++||:|||:|||||||::||:||::|:||::||||||||++|||||:||||++|:|| 721 TTTCGGATTTATTTTAAAATTTTTTATTTTAATTTTGTTGATCGAGGGATTTTTCGGTAT <<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<< 781 CACCAATCTTTCATCCTGCGAAGGGCTGTGGAGAGCGTTGGGAGGAGGCATCTGTTGAGA :|::|||:|||:||::||++|||||:|||||||||++|||||||||||:||:|||||||| 781 TATTAATTTTTTATTTTGCGAAGGGTTGTGGAGAGCGTTGGGAGGAGGTATTTGTTGAGA

<<<<<<< MSPrev <<<<< USPrev 841 AGCTGGTTGGATCTTTCTGTCTACGTACCTACACCCATACCCAGGCAACCTGTATCCTTC ||:|||||||||:|||:|||:||++||::||:|:::|||:::|||:||::|||||::||: 841 AGTTGGTTGGATTTTTTTGTTTACGTATTTATATTTATATTTAGGTAATTTGTATTTTTT

901 TACCAGTTGCTCCTGTACTGCTGGTGCATTGAACATATTGTTGGAATGTTCATCATAATT ||::|||||:|::||||:||:|||||:||||||:||||||||||||||||:||:|||||| 901 TATTAGTTGTTTTTGTATTGTTGGTGTATTGAATATATTGTTGGAATGTTTATTATAATT

961 CAGCTGAGTATTTTTCCAGTTGCAGTTGCGAGCATTGTTT :||:|||||||||||::|||||:|||||++||:||||||| 961 TAGTTGAGTATTTTTTTAGTTGTAGTTGCGAGTATTGTTT

Used primers:

Forward MSP: 5’-TAATAGCGTGATATAAATAGTTCGG-3’ Reverse MSP: 5’-ATTAATAATACCGAAAAATCCCTCG-3’

Forward USP: 5’-GTAATAGTGTGATATAAATAGTTTGG-3’ Reverse USP: 5’-TAATAATACCAAAAAATCCCTCAAT-3’

Promoter region of PpbHLH

1 GAACAAGGGTTTAAAGCATTGCAGGCAGGTGATTGCATTTGTATTAACCGAGTAGTACAA |||:||||||||||||:||||:|||:|||||||||:|||||||||||:++|||||||:|| 1 GAATAAGGGTTTAAAGTATTGTAGGTAGGTGATTGTATTTGTATTAATCGAGTAGTATAA 61 TTCGAGTTTGTGTGTCATTTCGCAGAATATTGGTGGTTGGGGTTCCATGATATTGTTCAC ||++|||||||||||:||||++:|||||||||||||||||||||::|||||||||||:|: 61 TTCGAGTTTGTGTGTTATTTCGTAGAATATTGGTGGTTGGGGTTTTATGATATTGTTTAT

121 TGCTTTGATGTTTTTATTTGTGTGATTGTGATTTTATCATGATCAAACGCAAACAAAAGT ||:||||||||||||||||||||||||||||||||||:|||||:|||++:|||:|||||| 121 TGTTTTGATGTTTTTATTTGTGTGATTGTGATTTTATTATGATTAAACGTAAATAAAAGT

181 ATTCTTCTGTTGCTGCTGTATCACGTTTTACTGTGGGTTGAAGAATGTTGCAGTCTAACA |||:||:|||||:||:|||||:|++|||||:|||||||||||||||||||:|||:|||:| 181 ATTTTTTTGTTGTTGTTGTATTACGTTTTATTGTGGGTTGAAGAATGTTGTAGTTTAATA

241 ATGTGGTTCTCTAGAAGGACTGTCTAAGGCGACGGAATATTTCAGGCCTCTGTTGGGCTG ||||||||:|:||||||||:|||:|||||++|++||||||||:|||::|:|||||||:|| 241 ATGTGGTTTTTTAGAAGGATTGTTTAAGGCGACGGAATATTTTAGGTTTTTGTTGGGTTG

301 TGTTTATTATTTCTTTTTTGTTTCTTCTTCTTCTTCTTCTTCTTCTTCTTTTACCTCATT ||||||||||||:||||||||||:||:||:||:||:||:||:||:||:|||||::|:||| 301 TGTTTATTATTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTATT

361 CATATATATATATATATATATATATATATATATGTGTATGGATTTGTGAATGATATGAAT :||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 361 TATATATATATATATATATATATATATATATATGTGTATGGATTTGTGAATGATATGAAT

421 TCACAGTTTTAATCATAACCTTGGATGCTGGGGGATACTCTGAATGAAATAGTTTTCCCC |:|:|||||||||:||||::|||||||:|||||||||:|:||||||||||||||||:::: 421 TTATAGTTTTAATTATAATTTTGGATGTTGGGGGATATTTTGAATGAAATAGTTTTTTTT

481 TACAGCAGTTATTCACGAAGTTGCTTTGAGCAATACCCGATATTACCATGGCTCAAGCTT ||:||:|||||||:|++||||||:||||||:||||::++||||||::||||:|:|||:|| 481 TATAGTAGTTATTTACGAAGTTGTTTTGAGTAATATTCGATATTATTATGGTTTAAGTTT

541 ATTGAATCTTTCAAATTCCGCTTCCCTCGCACATGACTAAATCTAACATAATTTCTAAAC |||||||:|||:|||||:++:||:::|++:|:||||:|||||:|||:|||||||:||||: 541 ATTGAATTTTTTAAATTTCGTTTTTTTCGTATATGATTAAATTTAATATAATTTTTAAAT MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>> 601 TGCTGATTTTGTCCCATCGGTGTGTCAGGAAAGACTTCGACTCGTCAGCTGTAACTTCAG ||:|||||||||:::||++||||||:||||||||:||++|:|++|:||:|||||:||:|| 601 TGTTGATTTTGTTTTATCGGTGTGTTAGGAAAGATTTCGATTCGTTAGTTGTAATTTTAG

661 TTTCGAAATCCCAGCTTGGACAGAACTTCGTTTTATCTAGACGGAGGTCACCAGGACTGG |||++||||:::||:|||||:||||:||++||||||:||||++|||||:|::||||:||| 661 TTTCGAAATTTTAGTTTGGATAGAATTTCGTTTTATTTAGACGGAGGTTATTAGGATTGG <<<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<<< 721 TAACACGTCAAGAATTGGCATCGGCTCTTCAATTAGTACTATTGAACTTTCCGAGGACGT |||:|++|:|||||||||:||++|:|:||:||||||||:|||||||:|||:++||||++| 721 TAATACGTTAAGAATTGGTATCGGTTTTTTAATTAGTATTATTGAATTTTTCGAGGACGT <<<< MSPrev <<<< USPrev 781 GCTATGGCGTCGCAATCCAATTCCGACATGTGGATGGGCGCTGCATCATCAGAAATCAGG |:|||||++|++:|||::||||:++|:|||||||||||++:||:||:||:||||||:||| 781 GTTATGGCGTCGTAATTTAATTTCGATATGTGGATGGGCGTTGTATTATTAGAAATTAGG 841 TTCAGTTCATGATCACCCCTGCATTTCCTTCCAACAACCATTCTCATTACGAACAGGAGT ||:||||:|||||:|::::||:||||::||::||:||::|||:|:||||++||:|||||| 841 TTTAGTTTATGATTATTTTTGTATTTTTTTTTAATAATTATTTTTATTACGAATAGGAGT

901 TCTGCTGGTTTCTGTGATTGATAAAGACTCGGTTCGGAGTTAAATCACTTGGAATAAATC |:||:||||||:|||||||||||||||:|++|||++|||||||||:|:|||||||||||: 901 TTTGTTGGTTTTTGTGATTGATAAAGATTCGGTTCGGAGTTAAATTATTTGGAATAAATT

961 CATGAATGTGTTTTTTTTATTTTTTATTTTATTTTCCCATAGTTTGCCT :||||||||||||||||||||||||||||||||||:::||||||||::| 961 TATGAATGTGTTTTTTTTATTTTTTATTTTATTTTTTTATAGTTTGTTT

Used primers:

Forward MSP: 5’- ATTTTTTAAATTTCGTTTTTTTCGT-3’ Reverse MSP: 5’- ATTACCAATCCTAATAACCTCCGTC-3’

Forward USP: 5’- ATTTTTTAAATTTTGTTTTTTTTGT-3’ Reverse USP: 5’- ATTACCAATCCTAATAACCTCCATC-3’

Gene model of PpbHLH (Phypa1_209063)

1 ATTACCAAATCAAGTTGATCCATCGATTGCTAATTTGCAGACTGGAGTGCAGGAAAATGT ||||::||||:||||||||::||++||||:|||||||:|||:|||||||:|||||||||| 1 ATTATTAAATTAAGTTGATTTATCGATTGTTAATTTGTAGATTGGAGTGTAGGAAAATGT

61 AGGAACACCAAGTTTCAGCAAGGGCGTGCTGGACGAGGAGTGGTACACGCCCGAGACCTC |||||:|::||||||:||:|||||++||:||||++||||||||||:|++::++|||::|: 61 AGGAATATTAAGTTTTAGTAAGGGCGTGTTGGACGAGGAGTGGTATACGTTCGAGATTTT

121 CTTAATGGAGCTCTCTTACTCATTACCATATGGGATATCCGATACTCGCACAGGCTTTGG :|||||||||:|:|:|||:|:||||::|||||||||||:++|||:|++:|:|||:||||| 121 TTTAATGGAGTTTTTTTATTTATTATTATATGGGATATTCGATATTCGTATAGGTTTTGG

181 AATGCTCGAGTCGTCGCTGAATTTTGACAGCAGCAGCAACCTCATGTCTAGCTTCCGCCC ||||:|++|||++|++:||||||||||:||:||:||:||::|:||||:|||:||:++::: 181 AATGTTCGAGTCGTCGTTGAATTTTGATAGTAGTAGTAATTTTATGTTTAGTTTTCGTTT

241 TGCTCCCTCAGCCTTGAGCATGGGCCTTGAGAGCAACCGCAGTCTGGAGGATCTCGTTTG ||:|:::|:||::|||||:|||||::|||||||:||:++:|||:||||||||:|++|||| 241 TGTTTTTTTAGTTTTGAGTATGGGTTTTGAGAGTAATCGTAGTTTGGAGGATTTCGTTTG

301 CACTGGTCAGGGCTCGAGCAACGTTGGCCTCCTCTCAAGTCTTTCTCCAGGTCTTGTGGT :|:||||:||||:|++||:||++||||::|::|:|:||||:|||:|::||||:||||||| 301 TATTGGTTAGGGTTCGAGTAACGTTGGTTTTTTTTTAAGTTTTTTTTTAGGTTTTGTGGT

361 CTTGTCCCATTTTTCAGCAATTGTACACTTGTGATATCCGTTTCTCAACACATACACCGC :||||:::||||||:||:|||||||:|:|||||||||:++|||:|:||:|:|||:|:++: 361 TTTGTTTTATTTTTTAGTAATTGTATATTTGTGATATTCGTTTTTTAATATATATATCGT

421 AGCATTTTATAAAATTCATCTCAACAATGGATGAGAACCATGGTACCTGCTCAACTTACA ||:|||||||||||||:||:|:||:||||||||||||::||||||::||:|:||:|||:| 421 AGTATTTTATAAAATTTATTTTAATAATGGATGAGAATTATGGTATTTGTTTAATTTATA

481 AGGTCCTCAAGTAGGGAATTGAAGGATATCCTGTTGGTTGTGATTGCAGGCGGTCAACTC ||||::|:|||||||||||||||||||||::|||||||||||||||:|||++||:||:|+ 481 AGGTTTTTAAGTAGGGAATTGAAGGATATTTTGTTGGTTGTGATTGTAGGCGGTTAATTC

541 GGCCGGAGCACCGTAATGGAAAGCTTCAGCTCAGGTCTGCCAACAAGCTTCAACCAAGGA +|:++|||:|:++||||||||||:||:||:|:||||:||::||:|||:||:||::||||| 541 GGTCGGAGTATCGTAATGGAAAGTTTTAGTTTAGGTTTGTTAATAAGTTTTAATTAAGGA

601 ATCATCAACGCTGGTGGAAGCATAACCAACATCACCAGTAGCAACATAAATAACGTCCGC ||:||:||++:|||||||||:||||::||:||:|::|||||:||:||||||||++|:++: 601 ATTATTAACGTTGGTGGAAGTATAATTAATATTATTAGTAGTAATATAAATAACGTTCGT

661 TCTAACTTCCCCCTCATGGCCTCACCTTCGAACTTTTCCGATGCGTACCGCGCTCGATCA |:|||:||:::::|:||||::|:|::||++||:||||:++|||++||:++++:|++||:| 661 TTTAATTTTTTTTTTATGGTTTTATTTTCGAATTTTTTCGATGCGTATCGCGTTCGATTA

721 GTGAGCGAAGACAAGTCTGGGAAAGTTGTTGGCTCTGGCGGCCCACGGAATGAACTTGTG |||||++||||:||||:|||||||||||||||:|:|||++|:::|++|||||||:||||| 721 GTGAGCGAAGATAAGTTTGGGAAAGTTGTTGGTTTTGGCGGTTTACGGAATGAATTTGTG

781 CCATATCACAGAAACAAGGGGGCGGAAACTCGGAGCCATGGTCAGGGTCAGCAAACTCTA ::||||:|:|||||:|||||||++||||:|++|||::|||||:|||||:||:|||:|:|| 781 TTATATTATAGAAATAAGGGGGCGGAAATTCGGAGTTATGGTTAGGGTTAGTAAATTTTA

841 TTCTTGAAGCGCGCAGCTTCTCGCCGTTGTGCGGGGTCTAGTGGGACTGTCTCTCCAGTG ||:||||||++++:||:||:|++:++|||||++||||:||||||||:|||:|:|::|||| 841 TTTTTGAAGCGCGTAGTTTTTCGTCGTTGTGCGGGGTTTAGTGGGATTGTTTTTTTAGTG

901 AGCAAGTCACCCCCACGCGTGGTCACTAGTGCCTCCAACGATTCTTCTGTGGACACGCCG ||:||||:|:::::|++++||||:|:|||||::|::||++|||:||:||||||:|++:++ 901 AGTAAGTTATTTTTACGCGTGGTTATTAGTGTTTTTAACGATTTTTTTGTGGATACGTCG

961 GATAAAGACAGCCCTCATCCGCGTAATGCTCACTTGCAGAGCGCATCTGGAAGATTAAAT ||||||||:||:::|:||:++++|||||:|:|:|||:||||++:||:||||||||||||| 961 GATAAAGATAGTTTTTATTCGCGTAATGTTTATTTGTAGAGCGTATTTGGAAGATTAAAT

1021 ATCAACTCCGGATCGGATGATCCCAACGACATGGGATTAGATGGTGACGACTACGATGCC ||:||:|:++|||++||||||:::||++|:|||||||||||||||||++|:||++|||:: 1021 ATTAATTTCGGATCGGATGATTTTAACGATATGGGATTAGATGGTGACGATTACGATGTT

1081 AAAGACGACGATGATTTGGATGAGAGTGGTGACGGCTCAGGGGGGCCCTACGAGGTGGAA |||||++|++||||||||||||||||||||||++|:|:|||||||:::||++|||||||| 1081 AAAGACGACGATGATTTGGATGAGAGTGGTGACGGTTTAGGGGGGTTTTACGAGGTGGAA

1141 GAAGGCGCAGGCAATGGAGCAGATCAAAGCATTGGAAAGGGAAACGGCAAAGGGAAACGA |||||++:|||:|||||||:||||:||||:||||||||||||||++|:|||||||||++| 1141 GAAGGCGTAGGTAATGGAGTAGATTAAAGTATTGGAAAGGGAAACGGTAAAGGGAAACGA

1201 GGACTTCCTGCGAAAAACCTCATGGCTGAGCGCAGGCGCCGCAAAAAACTCAACGATCGC |||:||::||++|||||::|:||||:||||++:|||++:++:||||||:|:||++||++: 1201 GGATTTTTTGCGAAAAATTTTATGGTTGAGCGTAGGCGTCGTAAAAAATTTAACGATCGT MSPfwd >>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>> 1261 CTGTACACGCTACGGTCTGTAGTTCCTAAGATTACAAAGGTGCTTCCAAACTCTATCTTT :||||:|++:||++||:|||||||::||||||||:|||||||:||::|||:|:|||:||| 1261 TTGTATACGTTACGGTTTGTAGTTTTTAAGATTATAAAGGTGTTTTTAAATTTTATTTTT

1321 GAACATGTTGCCCGCCTCGATTGCTGAATTGCACATCATGTATGTTGAGATGTCCACTTA |||:||||||::++::|++||||:|||||||:|:||:||||||||||||||||::|:||| 1321 GAATATGTTGTTCGTTTCGATTGTTGAATTGTATATTATGTATGTTGAGATGTTTATTTA

1381 CGAATCAGTGGGGTGTGGAGTACAGATGGATAGAGCCTCCATATTGGGGGATGCGATTGA ++|||:||||||||||||||||:||||||||||||::|::|||||||||||||++||||| 1381 CGAATTAGTGGGGTGTGGAGTATAGATGGATAGAGTTTTTATATTGGGGGATGCGATTGA

1441 GTACCTAAAGGAGCTCCTGCAACGCATCAATGAAATCCATAACGAACTGGAAGCAGCAAA |||::||||||||:|::||:||++:||:||||||||::||||++||:||||||:||:||| 1441 GTATTTAAAGGAGTTTTTGTAACGTATTAATGAAATTTATAACGAATTGGAAGTAGTAAA <<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<< 1501 GCTGGAGCAGTCGCGGTCGATGCCGTCTAGCCCCACTCCACGATCCACCCAAGGTTATCC |:|||||:|||++++||++|||:++|:|||::::|:|::|++||::|:::||||||||:: 1501 GTTGGAGTAGTCGCGGTCGATGTCGTTTAGTTTTATTTTACGATTTATTTAAGGTTATTT <<<<<< MSPrev <<<<< USPrev 1561 AGCTACAGTTAAAGAAGAATGCCCCGTCTTGCCGAATCCTGAATCCCAGCCTCCTCGAGT ||:||:|||||||||||||||:::++|:|||:++|||::|||||:::||::|::|++||| 1561 AGTTATAGTTAAAGAAGAATGTTTCGTTTTGTCGAATTTTGAATTTTAGTTTTTTCGAGT

1621 ATGTTGTTTATAATTTCTCACCTTCTTGGAATTGCATCTCAGTACTTATTTCGCAATGCC ||||||||||||||||:|:|::||:|||||||||:||:|:||||:||||||++:||||:: 1621 ATGTTGTTTATAATTTTTTATTTTTTTGGAATTGTATTTTAGTATTTATTTCGTAATGTT

1681 AACGACGTTCTGAAATGTCTACACTTTGCACTGTTCTGAAGTTCTGGAATGCTGAACATA ||++|++||:||||||||:||:|:||||:|:||||:|||||||:|||||||:||||:||| 1681 AACGACGTTTTGAAATGTTTATATTTTGTATTGTTTTGAAGTTTTGGAATGTTGAATATA

1741 GTTTACTTTGCACATTGTTTCATAGGTGGAAGTGAGGAAAAGAGAAGGTCAGGCCCTCAA |||||:||||:|:|||||||:||||||||||||||||||||||||||||:|||:::|:|| 1741 GTTTATTTTGTATATTGTTTTATAGGTGGAAGTGAGGAAAAGAGAAGGTTAGGTTTTTAA

1801 CATTCATATGTTCTGTGCCCGCCGGCCTGGACTCCTCCTCTCTACTGTGAAGGCGCTGGA :|||:|||||||:||||::++:++|::||||:|::|::|:|:||:||||||||++:|||| 1801 TATTTATATGTTTTGTGTTCGTCGGTTTGGATTTTTTTTTTTTATTGTGAAGGCGTTGGA

1861 CGCCCTTGGCTTGGATGTACAACAGGCTGTCATCAGCTGCTTCAATGGTTTCGCCCTTGA ++:::||||:|||||||||:||:|||:|||:||:||:||:||:||||||||++:::|||| 1861 CGTTTTTGGTTTGGATGTATAATAGGTTGTTATTAGTTGTTTTAATGGTTTCGTTTTTGA

1921 CCTCTTCCGTGCAGAGGTAAGAGTCTTTCGCCTCAAGAATTCGATGTGATTGCAACTAAT ::|:||:++||:||||||||||||:|||++::|:|||||||++|||||||||:||:|||| 1921 TTTTTTTCGTGTAGAGGTAAGAGTTTTTCGTTTTAAGAATTCGATGTGATTGTAATTAAT

1981 AGAGTTTGTGCGTTGACATGGGCGGGAAATGTACATCGGTTGTCTTATTTAGCTGTGTTA ||||||||||++||||:|||||++|||||||||:||++|||||:||||||||:||||||| 1981 AGAGTTTGTGCGTTGATATGGGCGGGAAATGTATATCGGTTGTTTTATTTAGTTGTGTTA

2041 GGCCTCAAGCTGAAGATCACCTTGGGTGTGGGTTATTGCTGTGGGCAGGCCAAAGATGTG ||::|:|||:|||||||:|::|||||||||||||||||:||||||:|||::||||||||| 2041 GGTTTTAAGTTGAAGATTATTTTGGGTGTGGGTTATTGTTGTGGGTAGGTTAAAGATGTG

2101 GACGTTGGACCAGAAGAAATAAAGGCCGTTCTGCTGCTCACTGCGGGATGTGATTTGCAC ||++|||||::||||||||||||||:++||:||:||:|:|:||++||||||||||||:|: 2101 GACGTTGGATTAGAAGAAATAAAGGTCGTTTTGTTGTTTATTGCGGGATGTGATTTGTAT

2161 TCTTTGCAGTAGATCCCACAATGCAGACGGACAAGTTGAATGAATTCTCTTCTTTTCTGC |:||||:|||||||:::|:||||:|||++||:||||||||||||||:|:||:||||:||: 2161 TTTTTGTAGTAGATTTTATAATGTAGACGGATAAGTTGAATGAATTTTTTTTTTTTTTGT

2221 ATGGGAAACAAAACACAAATTGATACGAGTGTGGTTTCAAAGTCTCTCCTCACCAGGCAG ||||||||:||||:|:|||||||||++||||||||||:|||||:|:|::|:|::|||:|| 2221 ATGGGAAATAAAATATAAATTGATACGAGTGTGGTTTTAAAGTTTTTTTTTATTAGGTAG

2281 TGTTCTTCAATTTTCACCATACAAGCTAAAAAATTTGACGCTACCTAAATTCAGTGGTTT ||||:||:||||||:|::|||:|||:||||||||||||++:||::||||||:|||||||| 2281 TGTTTTTTAATTTTTATTATATAAGTTAAAAAATTTGACGTTATTTAAATTTAGTGGTTT

2341 GAACCTGACATTGTTGTAGGCTGTACTCAGCTTTTTCTGTTTTTGTATAACTTAGTATAC |||::|||:|||||||||||:||||:|:||:|||||:|||||||||||||:||||||||+ 2341 GAATTTGATATTGTTGTAGGTTGTATTTAGTTTTTTTTGTTTTTGTATAATTTAGTATAC

2401 GGTAAAGGCCCACAGAGCAGAGAACGGTGGGACCTTCACGGTACTGCCACTAGACCAAGC +|||||||:::|:||||:||||||++||||||::||:|++|||:||::|:||||::|||: 2401 GGTAAAGGTTTATAGAGTAGAGAACGGTGGGATTTTTACGGTATTGTTATTAGATTAAGT

2461 CTTGAAAACGTTATGCAGGTGAAATGTTTCTGTACATCATCTTCAAGACTTGTGTGCTGT :|||||||++|||||:|||||||||||||:||||:||:||:||:||||:|||||||:||| 2461 TTTGAAAACGTTATGTAGGTGAAATGTTTTTGTATATTATTTTTAAGATTTGTGTGTTGT

2521 GTGTGCCTCTATGTGTTTGCCTATGTAACCAAGTTGTCCGTTGCTATCCAACACCTGCCA |||||::|:||||||||||::|||||||::|||||||:++|||:|||::||:|::||::| 2521 GTGTGTTTTTATGTGTTTGTTTATGTAATTAAGTTGTTCGTTGTTATTTAATATTTGTTA

Used primers:

Forward MSP: 5’-GCGAAAAATTTTATGGTTGAGC-3’ Reverse MSP: 5’-TCCAACTTTACTACTTCCAATTCGT-3’

Forward USP: 5’-TGTGAAAAATTTTATGGTTGAGTGT-3’ Reverse USP: 5’-CCAACTTTACTACTTCCAATTCATT-3’

Figure S6. Primer design for bisulfite PCR analyses

Primer design for promoter, exon and intron regions of miRNA target genes

(PpC3HDZIP1, PpHB10, PpSBP3, PpARF, PpbHLH, PpTAS4), the ta-siRNA target gene PpEREBP/AP2, and the PpGNT1 control gene as described in the manuscript. In the gene model sequence of PpbHLH start and stop codons are highlighted in grey and intron sequences are marked in blue. The upper strand of each sequence depicts the wild type sequence, the lower strand indicates the expected cytosine to thymine conversions after the bisulfite treatment. Upright lines mark unaltered nucleotides, double points mark cytosine to thymine conversions. CpG dinucleotides are marked by plus signs (+).

Figure S7

A

WT GGACAATCTTTGGATATGTGCCAGCGTATCTTGTGATCGTGGTTCTTAAGGGTCGAGTGCTTAGCT WT BT GGATAATTTTTGGATATGTGTTAGTGTATTTTGTGATTGTGGTTTTTAAGGGTTGAGTGTTTAGTT KO1 BT GGATAATTTTTGGATATGTGTTAGCGTATTTTGTGATTGTGGTTTTTAAGGGTCGAGTGTTTAGTT KO2 BT GGATAATTTTTGGATATGTGTTAGCGTATTTTGTGATTGTGGTTTTTAAGGGTCGAGTGTTTAGTT

WT CCTCATCCTCATGCTTAGGTCTGGAAATATGTAAAAGGGGACGTAATGACAACACGAAGCTTATAA WT BT TTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAGGGGATGTAATGATAATATGAAGTTTATAA KO1 BT TTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAGGGGACGTAATGATAATACGAAGTTTATAA KO2 BT TTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAGGGGACGTAATGATAATACGAAGTTTATAA

WT AAACTCAAAGCT WT BT AAATTTAAAGTT KO1 BT AAATTTAAAGTT KO2 BT AAATTTAAAGTT

B

WT TTTCTGGCTGATACAGAAACAGACGAGGTATTTGCTCGTATTTGCCTGCAGCCTGAGATTGGCTCC WT BT TTTTTGGTTGATATAGAAATAGATGAGGTATTTGTTTGTATTTGTTTGTAGTTTGAGATTGGTTTT KO1 BT TTTTTGGTTGATATAGAAATAGACGAGGTATTTGTTTGTATTTGTTTGTAGTTTGAGATTGGTTTT KO2 BT TTTTTGGTTGATATAGAAATAGACGAGGTATTTGTTTGTATTTGTTTGTAGTTTGAGATTGGTTTT

WT TCCGCTCAGGATTTAACAGATGATTCTCTTGCGTCTCCGCCTCTAGAGAAACCAGCTTCATTTGCC WT BT TTTGTTTAGGATTTAATAGATGATTTTTTTGTGTTTTTGTTTTTAGAGAAATTAGTTTTATTTGTT KO1 BT TTCGTTTAGGATTTAATAGATGATTTTTTTGCGTTTTTGTTTTTAGAGAAATTAGTTTTATTTGTT KO2 BT TTCGTTTAGGATTTAATAGATGATTTTTTTGCGTTTTTGTTTTTAGAGAAATTAGTTTTATTTGTT

WT AAAACGCTCACTCAAAGTGATGCAAACAACGGTGGAGGCTTTTCAATACCTC WT BT AAAATGTTTATTTAAAGTGATGTAAATAATGGTGGAGGTTTTTTAATATTTT KO1 BT AAAATGTTTATTTAAAGTGATGTAAATAACGGTGGAGGTTTTTTAATATTTC KO2 BT AAAATGTTTATTTAAAGTGATGTAAATAACGGTGGAGGTTTTTTAATATTTC

C

WT TAGCTCATAACCCTCTCACAGGACGTAATGGGGGTGACAACATGCTAACAGAATTGCACGGTAAAG WT BT TAGTTTATAATTTTTTTATAGGATGTAATGGGGGTGATAATATGTTAATAGAATTGTATGGTAAAG KO1 BT TAGTTTATAATTTTTTTATAGGACGTAATGGGGGTGATAATATGTTAATAGAATTGTACGGTAAAG KO2 BT TAGTTTATAATTTTTTTATAGGACGTAATGGGGGTGATAATATGTTAATAGAATTGTACGGTAAAG

WT GAAAACTGTACTAGGCATGTTATATGGGAATTCGGATCGCTTCTTGCAATTAAACACGCTAGCGCC WT BT GAAAATTGTATTAGGTATGTTATATGGGAATTTGGATTGTTTTTTGTAATTAAATATGTTAGTGTT KO1 BT GAAAATTGTATTAGGTATGTTATATGGGAATTTGGATCGTTTTTTGTAATTAAATACGTTAGTGTC KO2 BT GAAAATTGTATTAGGTATGTTATATGGGAATTTGGATCGTTTTTTGTAATTAAATACGTTAGTGTC

WT GTTTGGTGCCAATGTTATTCTGG WT BT GTTTGGTGTTAATGTTATTTTGG KO1 BT GTTTGGTGTTAATGTTATTTTGG KO2 BT GTTTGGTGTTAATGTTATTTTGG

D

WT TCAAAGCAATCAATGTGCTAATGAACGTGCTGCCTGCATTGTCTGCAATGACTATGTACTGCGTAG WT BT TTAAAGTAATTAATGTGTTAATGAATGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGTGTAG KO1 BT TTAAAGTAATTAATGTGTTAATGAACGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGCGTAG KO2 BT TTAAAGTAATTAATGTGTTAATGAACGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGCGTAG

WT TCAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGCAGGTCACTTGGGATGAGCCGGACCT WT BT TTAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTTGGATTT KO1 BT TTAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTCGGATTT KO2 BT TTAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTCGGATTT

WT ATTGCAGGGAGTGAATCGTGTAAGCCCATGGCAGTTAGAGCTTGTGGCGACACTTCCTATGCAGCT WT BT ATTGTAGGGAGTGAATTGTGTAAGTTTATGGTAGTTAGAGTTTGTGGTGATATTTTTTATGTAGTT KO1 BT ATTGTAGGGAGTGAATTGTGTAAGTTTATGGTAGTTAGAGTTTGTGGCGATATTTTTTATGTAGTT KO2 BT ATTGTAGGGAGTGAATTGTGTAAGTTTATGGTAGTTAGAGTTTGTGGCGATATTTTTTATGTAGTT

WT GCCC WT BT GTTT KO1 BT GTTT KO2 BT GTTT

E

WT TTCACAATGTGCTCTTCAAGCTTCGTTGTATGCTAAACTCTACTGCAATACTGCTACGGCGTGCC WT BT TTTATAATGTGTTTTTTAAGTTTTGTTGTATGTTAAATTTTATTGTAATATTGTTATGGTGTGTT KO1 BT TTTATAATGTGTTTTTTAAGTTTCGTTGTATGTTAAATTTTATTGTAATATTGTTACGGCGTGTT KO2 BT TTTATAATGTGTTTTTTAAGTTTCGTTGTATGTTAAATTTTATTGTAATATTGTTACGGCGTGTT

WT TTTTCTTTTTTCGAAGTATGATATGATGACCTAATGGTCTTTTTGAATACGACAGGAATTCCATG WT BT TTTTTTTTTTTTGAAGTATGATATGATGATTTAATGGTTTTTTTGAATATGATAGGAATTTTATG KO1 BT TTTTTTTTTTTTGAAGTATGATATGATGATTTAATGGTTTTTTTGAATACGATAGGAATTTTATG KO2 BT TTTTTTTTTTTTGAAGTATGATATGATGATTTAATGGTTTTTTTGAATACGATAGGAATTTTATG

WT GAGACAG WT BT GAGATAG KO1 BT GAGATAG KO2 BT GAGATAG

Figure S7. DNA methylation analysis of promoter and intragenic regions of the

PpARF gene in P. patens wild type and two ΔPpDCL1b mutants

Nucleotide sequences of PCR products obtained from methylation-specific PCRs are aligned. (A) Promoter region. (B) Exon 1 upstream of the miR160 binding site. (C) Exon

4 downstream of the miR160 binding site. (D) Intron 2 upstream of the miR160 binding site. (E) Intron 3 downstream of the miR160 binding site. WT: Wild type nucleotide sequence of the analyzed region; CpG residues are highlighted in green. WT BT:

Sequences of PCR products obtained with USP primers from bisulfite-treated DNA from wild type. KO1+2 BT: Sequences of PCR products obtained with MSP primers from bisulfite-treated DNA from two ΔPpDCL1b mutants. Cytosine residues of CpG dinucleotides which are methlyated in the ΔPpDCL1b mutants are indicated in red.

Cytosine residues of CpG dinucleotides which are not methylated are highlighted in yellow. Cytosine to thymine conversions are highlighted in bold and are underlined.

Five independent clones from each PCR product were sequenced.

Figure S8

A PpC3HDZIP1 mRNA GACGGATTTCCTGGCGAAGGCAACGGGAACCGCAGTGGATTGGATACAGT |||||||||||||||||||||||||||||||||||||||||||||||||| PpC3HDZIP1 genomic gacggatttcctggcgaaggcaacgggaaccgcagtggattggatacagt

PpC3HDZIP1 mRNA TACCTGGTATGAAG------|||||||||||||| PpC3HDZIP1 genomic tacctggtatgaagGTatggatgccatgccttcctacggcacgttctaca

PpC3HDZIP1 mRNA ------

PpC3HDZIP1 genomic gtgtattgtggagtagcgagcctcacctgtaactcttgatctatagattc

PpC3HDZIP1 mRNA ------

PpC3HDZIP1 genomic cattatcagagatatgatcgcacgaaataactctttgttccaaccttttg

PpC3HDZIP1 mRNA ------

PpC3HDZIP1 genomic taaaataagtattagcggagtcatggtactggagcaaagtcaaacaaatt

PpC3HDZIP1 mRNA ------

PpC3HDZIP1 genomic aatttgactcaaaacacgacttcgaattaatttaggagctaacaaggtaa

PpC3HDZIP1 mRNA ------

PpC3HDZIP1 genomic tgatattgattctttaattcaaattaaagtggttgattgcaaatgccatt

PpC3HDZIP1 mRNA ------CCTGGTCCGGATGCCATTGGCATC |||||||||||||||||||||||| PpC3HDZIP1 genomic gctgatacgtcactagtgcaatgcAGcctggtccggatgccattggcatc

PpC3HDZIP1 mRNA ATTGCTATATCCCATGGTTGCGTGGGCATAGCAGCTCGAGCGTGCGGCCT |||||||||||||||||||||||||||||||||||||||||||||||||| PpC3HDZIP1 genomic attgctatatcccatggttgcgtgggcatagcagctcgagcgtgcggcct

B PpHB10 mRNA AGCTACCGCTGAGGAGACGCTGACAGAATTCCTGGCTAAAGCCACAGGAA |||||||||||||||||||||||||||||||||||||||||||||||||| PpHB10 genomic agctaccgctgaggagacgctgacagaattcctggctaaagccacaggaa

PpHB10 mRNA CGGCGGTGGATTGGATTCAGTTACCTGGTATGAAG------||||||||||||||||||||||||||||||||||| PpHB10 genomic cggcggtggattggattcagttacctggtatgaagGTatgtcatctctcc

PpHB10 mRNA ------

PpHB10 genomic gcgatggtgatgagtgattcaccgcatcacctcttaccgtaatctgagtg

PpHB10 mRNA ------

PpHB10 genomic atttgaagtaattgcaatgctctgaaattgaaatctgaagttctgaaagg

PpHB10 mRNA ------

PpHB10 genomic gcggtttggctgaatttgttaactggtgagactattgctgttgcactaat

PpHB10 mRNA ------CCTGGTCCGGATGCCATTGGCATTATTGCT |||||||||||||||||||||||||||||| PpHB10 genomic agtaagatgtttatttgcAGcctggtccggatgccattggcattattgct

Figure S8. The miR166 binding sites of PpC3HDZIP1 and PpHB10 are disrupted by introns

(A) PpC3HDZIP1; (B) PpHB10. The miR166 binding sites are indicated in red and are underlined. Intron borders (GT and AG) are marked in bold.

Figure S9

WT TTTATCTCTAAATTCTTAGACAACGTCATTCAAAATAAGTTTTAAAACAGCGACTAGTCATAAAATA WT1+amiRNA BT TTTATTTTTAAATTTTTAGATAATGTTATTTAAAATAAGTTTTAAAATAGTGATTAGTTATAAAATA WT2+amiRNA BT TTTATTTTTAAATTTTTAGATAACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATA KO1+amiRNA BT TTTATTTTTAAATTTTTAGATAACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATA KO2+amiRNA BT TTTATTTTTAAATTTTTAGATAACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATA

WT CGTATTTACACACTTGTATATGATGTACCATAGACGGTAACCGTACATATTTGCCGACACCCTGCAA WT1+amiRNA BT TGTATTTATATATTTGTATATGATGTATTATAGATGGTAATTGTATATATTTGTTGACATTTTGTAA WT2+amiRNA BT CGTATTTATATATTTGTATATGATGTATTATAGACGGTAATTGTATATATTTGTTGACATTTTGTAA KO1+amiRNA BT CGTATTTATATATTTGTATATGATGTATTATAGACGGTAATTGTATATATTTGTTGACATTTTGTAA KO2+amiRNA BT CGTATTTATATATTTGTATATGATGTATTATAGACGGTAATTGTATATATTTGTCGACATTTTGTAA

WT TTAATAGAGTTCGAATATCCCCGCCGCGTTCAAGTCGCCT WT1+amiRNA BT TTAATAGAGTTTGAATATTTTTGTTGTGTTTAAGTTGTTT WT2+amiRNA BT TTAATAGAGTTCGAATATTTTCGTCGCGTTTAAGTCGTTT KO1+amiRNA BT TTAATAGAGTTTGAATATTTTCGTCGCGTTTAAGTCGTTT KO2+amiRNA BT TTAATAGAGTTTGAATATTTTCGTCGCGTTTAAGTCGTTT

Figure S9. DNA methylation analysis of the PpGNT1 promoter region in lines expressing the amiR-GNT1

Nucleotide sequences of PCR products obtained from methylation-specific PCRs are aligned. WT: Wild type nucleotide sequence of the analyzed region; CpG residues are highlighted in green. WT1 + amiRNA BT: Sequences of PCR products obtained with

USP primers from bisulfite-treated DNA from wild type line 1 expressing the amiR-

GNT1 at low levels. WT2 + amiRNA BT: Sequences of PCR products obtained with

MSP primers from bisulfite-treated DNA from wild type line 2 expressing the amiR-

GNT1 at high levels. KO1 + amiRNA BT and KO2 + amiRNA BT: Sequences of PCR products obtained with MSP primers from bisulfite-treated DNA from ΔPpDCL1b mutants expressing the amiR-GNT1. Cytosine residues of CpG dinucleotides which are methlyated are indicated in red. Cytosine residues of CpG dinucleotides which are not methylated are highlighted in yellow. Cytosine to thymine conversions are highlighted in bold and are underlined. Five independent clones from each PCR product were sequenced.

Figure S10 A WT ATCTTTCAAATTCCGCTTCCCTCGCACATGACTAAATCTAACATAATTTCTAAACTGCTGATTTTG WT/Con. BT ATTTTTTAAATTTTGTTTTTTTTGTATATGATTAAATTTAATATAATTTTTAAATTGTTGATTTTG WT/ABA BT ATTTTTTAAATTTCGTTTTTTTCGTATATGATTAAATTTAATATAATTTTTAAATTGTTGATTTTG

WT TCCCATCGGTGTGTCAGGAAAGACTTCGACTCGTCAGCTGTAACTTCAGTTTCGAAATCCCAGCTT WT/Con. BT TTTTATTGGTGTGTTAGGAAAGATTTTGATTTGTTAGTTGTAATTTTAGTTTTGAAATTTTAGTTT WT/ABA BT TTTTATTGGTGTGTTAGGAAAGATTTCGATTCGTTAGTTGTAATTTTAGTTTTGAAATTTTAGTTT

WT GGACAGAACTTCGTTTTATCTAGACGGAGGTCACCAGGACTGGTAAC WT/Con. BT GGATAGAATTTTGTTTTATTTAGATGGAGGTTATTAGGATTGGTAAT WT/ABA BT GGATAGAATTTCGTTTTATTTAGACGGAGGTTATTAGGATTGGTAAT

B WT GCGAAAAACCTCATGGCTGAGCGCAGGCGCCGCAAAAAACTCAACGATCGCCTGTACACGCTACGG WT/Con. BT GTGAAAAATTTTATGGTTGAGTGTAGGTGTTGTAAAAAATTTAATGATTGTTTGTATATGTTATGG WT/ABA BT GCGAAAAATTTTATGGTTGAGCGTAGGTGTCGTAAAAAATTTAATGATCGTTTGTATACGTTACGG

WT TCTGTAGTTCCTAAGATTACAAAGGTGCTTCCAAACTCTATCTTTGAACATGTTGCCCGCCTCGAT WT/Con. BT TTTGTAGTTTTTAAGATTATAAAGGTGTTTTTAAATTTTATTTTTGAATATGTTGTTTGTTTTGAT WT/ABA BT TTTGTAGTTTTTAAGATTATAAAGGTGTTTTTAAATTTTATTTTTGAATATGTTGTTTGTTTCGAT

WT TGCTGAATTGCACATCATGTATGTTGAGATGTCCACTTACGAATCAGTGGGGTGTGGAGTACAGAT WT/Con. BT TGTTGAATTGTATATTATGTATGTTGAGATGTTTATTTATGAATTAGTGGGGTGTGGAGTATAGAT WT/ABA BT TGTTGAATTGTATATTATGTATGTTGAGATGTTTATTTACGAATTAGTGGGGTGTGGAGTATAGAT

WT GGATAGAGCCTCCATATTGGGGGATGCGATTGAGTACCTAAAGGAGCTCCTGCAACGCATCAATGA WT/Con. BT GGATAGAGTTTTTATATTGGGGGATGTGATTGAGTATTTAAAGGAGTTTTTGTAATGTATTAATGA WT/ABA BT GGATAGAGTTTTTATATTGGGGGATGTGATTGAGTATTTAAAGGAGTTTTTGTAACGTATTAATGA

WT AATCCATAACGAACTGGAAGCAGCAAAGCTGGA WT/Con. BT AATTTATAATGAATTGGAAGTAGTAAAGTTGGA WT/ABA BT AATTTATAACGAATTGGAAGTAGTAAAGTTGGA

Figure S10. DNA methylation analysis of promoter and intragenic regions of

PpbHLH in untreated and ABA-treated P. patens wild type

Nucleotide sequences of PCR products obtained from methylation-specific PCRs are aligned. (A) Promoter region of PpbHLH. (B) Coding Sequence of PpbHLH (intron sequences are marked in blue). WT: Wild type nucleotide sequence of the analyzed region; CpG residues are highlighted in green. WT/Con. BT: Sequences of PCR products obtained with USP primers from bisulfite-treated DNA from untreated wild type. WT/ABA BT: Sequences of PCR products obtained with MSP primers from bisulfite-treated DNA from ABA-treated wild type. Cytosine residues of CpG dinucleotides which are methlyated in the ABA-treated plants are indicated in red.

Cytosine residues of CpG dinucleotides which are not methylated are highlighted in

yellow. Cytosine to thymine conversions are highlighted in bold and are underlined.

Five independent clones from each PCR product were sequenced.

Table S1. Primers used in this study

Primer sequence (5’ Ö 3’) Description of experiment

TGGCATACAGGGAGCCAGGCA Antisense oligonucleotide of miR160.

GGGGAATGAAGCCTGGTCCGA Antisense oligonucleotide of miR166.

GGCGCTATCCCTCCTGAGCTT Antisense oligonucleotide o f miR390.

GTGCTCACTCTC TTCTGTCA Antisense oligonucleotide of miR156.

GCGTGCTCTCTCTCGTTGTCA Antisense oligonucleotide of miR535.

TCCAGACATAGACTCCATGCAA Antisense oligonucleotide of miR538.

TGTCCTCTCAAGTCTTTCTCA Antisense oligonucleotide of miR1026.

AAGCGTCCTGATTATTTGGAA Antisense oligonucleotide of amiR-GNT1.

GGGGCCATGCTAATCTTCTCTG Antisense oligonucleotide of U6snRNA.

GGGTGTACAAGAGCTCTATAGTGCCACCG 5’ RACE primer to detect the cleavage product of PpC3HDZIP1.

GCCACCGTTTCCTGTCGGGAGAGTTCC 5’ RACE nested primer to detect the cleavage product of PpC3HDZIP1.

AACCGCCGCCATCACACGGCCGGATC 5’ RACE primer to detect the cleavage product of PpHB10.

CACACGGCCGGATCAGGTAACCACTTG 5’ RACE nested primer to detect the cleavage product of PpHB10.

CGCAGATCGGTGAACCCGCGGTGCTCAC 5’ RACE primer to detect t he cleavage product of PpSBP3.

CCCGCGGTGCTCAC CAACTGAGACCGGA 5’ RACE nested primer to detect the cleavage product of PpSBP3.

ATGAGGGCTGTCTTTATCCGGCGTGT 5’ RACE primer to detect the cleavage product of PpbHLH.

ACTTTGGAGCAAGTTCTTCCCAGGTGGA 5’ RACE primer to detect t he cleavage product of PpGNT1 in PpGNT1-amiRNA expressing lines.

CGGTGAGAAATACACGCTTTTGACCCT 5’ RACE primer of the control gene PpGNT1. Primer sequence (5’ Ö 3’) Description of experiment

GATGCTTACCATCCCCAGCAACGGA 5’ RACE primer to detect the cleavage product of PpARF. PpARF reverse primer to detect sense and antisense transcript downstream of the miR160 binding site by RT-PCR. Primer used for the synthesis of the PpARF sense transcript derived cDNA.

CAAGATCATCAAGTCTTCCATCCT PpARF forward primer to detect sense and antisense transcript downstream of the miR160 binding site by RT-PCR. Primer used for the synthesis of the PpARF antisense transcript derived cDNA.

CAAAGAGTGTCCAATCCTGGC PpC3HDZIP1 forward primer to detect sense and antisense transcript upstream of the miR166 binding site by RT-PCR. Primer used for the synthesis of the PpC3HDZIP1 antisense transcript derived cDNA. PpC3HDZIP1 forward primer to detect the miRNA: mRNA duplexes by RT-PCR.

TTGAAGCCACACCAGCCTGAC PpC3HDZIP1 reverse primer to detect sense and antisense transcript ups tream of the miR166 binding site by RT-PCR. Primer used for the synthesis of the PpC3HDZIP1 sense transcript derived cDNA. PpC3HDZIP1 reverse primer to detect the miRNA: mRNA duplexes by RT-PCR.

CAAAGATGTGGCGGAGAAAT PpGNT1 forward primer to detect sense and antisense transcript by RT-PCR. Primer used for the synthesis of the PpGNT1 antisense transcript derived cDNA. PpGNT1 forward primer fo r expression analysis by RT-PCR. PpGNT1 forward primer to detect the miRNA: mRNA duplexes by RT-PCR. Forward primer used for the amplification of the PpGNT1 hybridisation probe from cDNA. Primer sequence (5’ Ö 3’) Description of experiment

ATAACCTGGCGACCTTTCCT PpGNT1 reverse primer to detect sense and antisense transcript by RT-PCR. Primer used for the synthesis of the PpGNT1 sense transcript derived cDNA. PpGNT1 reverse primer for expression analysis by RT-PCR. PpGNT1 reverse primer to detect the miRNA: mRNA duplexes by RT-PCR. Reverse primer used for the amplification of the PpGNT1 hybridisation probe from cDNA.

CCCCTGTCAGTTGCAGCATCC PpARF forward primer to detect the miRNA: mRNA duplexes by RT-PCR.

CTAGAGGCGGCGACGCAAGAG PpARF reverse primer to detect the miRNA: mRNA duplexes by RT-PCR.

GGAAAGAAGCAACAAGGTTGG PpHB10 forward primer to detect the miRNA: mRNA duplexes by RT-PCR.

ATCCCGCAGGACTGGAATCGC PpHB10 reverse prime r to detec t the miRNA: mRNA duplexes by RT-PCR.

GTGCAGGGTTGTGATGCCGAC PpSBP3 forward primer to detect the miRNA: mRNA duplexes by RT-PCR.

ATGCAAGAAACTGGACTGCTTC PpSBP3 reverse primer to detect the miRNA: mRNA duplexes by RT-PCR.

TTGATCCATCGATTGCTAATTT PpbHLH forward primer to detect the miRNA: mRNA duplexes by RT-PC R.

AGCCCTGACCAGTGCAAAC PpbHLH reverse primer to detect the miRNA: mRNA duplexes by RT-PCR.

AGCGTGGTATCACAATTGAC PpEF1α forward primer for expression analysis by RT-PCR and PCRs from genomic DNA. Forward primer used for the amplification of the PpEF1α hybridisation probe from cDNA. Primer sequence (5’ Ö 3’) Description of experiment

GATCGCTCGATCATGTTATC PpEF1α reverse primer for for expression analysis by RT-PCR and PCRs from genomic DNA. Primer used for the synthesis of PpEF1α sense transcript derived cDNA. Reverse primer used for the amplification of the PpEF1α hybridisation probe from cDNA.

CAGGCTTTCGCGTAATTCCCGTTG Forward primer used for the amplification of the PpC3HDZIP1 hybridisation probe from cDNA.

AGTGCCTCCAACTTCGGGCCTAAC Reverse primer used for the amplification of the PpC3HDZIP1 hybridisation probe from cDNA.

TTCTGCTGTCACTGGTGGACTT PpC3HDZIP1 forward primer for expression analysis by RT-PCR.

AGAGTTCCAAGAACCTCCATGC PpC3HDZIP1 reverse primer f or expression analysis by RT-PCR.

GATTCTGCTGTCACTGGTGGTC PpHB10 forward primer for expression analysis by RT-PCR.

GTCTTGTAACCAACGTGGACGA PpHB10 reverse primer for expression analysis by RT-PCR.

GGCTATCACTTCCTGGATGGAC PpSBP3 forward primer for expression analysis by RT-PCR.

ACAAGGAAGTTGCAGATGGTGA PpSBP3 reverse primer for expression analysis by RT-PCR.

TGGTTCTCGGTTCAAGATGAAA PpARF forward primer for expression analysis by RT-PCR.

CAACTTGTTGGACTGCTGAGGA PpARF reverse primer for expression analysis by RT-PCR.

GGTGAACGTTTTGAGGTTGTG PpARF forward primer for the amplification of the PpARF hybridisation probe.

CAAAGGAAACAAAACAAATGCC PpARF reverse primer for the amplification of the PpARF hybridisation probe.

GGACGTTGGACCAGAAGAAA PpbHLH forward primer for the amplification of the PpbHLH hybridisation probe. Primer sequence (5’ Ö 3’) Description of experiment

CGCTTTATTCAGCCTCCTCA PpbHLH reverse primer for the amplification of the PpbHLH hybridisation probe.

GGTTGGTCATGGGTTGCG PpCOR47 forward primer for the amplification of the PpCOR47 hybridisation probe.

GAGGTCAACTGT CTCGCC PpCOR47 reverse primer for the amplification of the PpCOR47 hybridisation probe.

CAGCCACAGCCAGTCAAGTGGATTCAGT 5’ RACE primer to detect the cleavage product of PpTAS4.

ATGTGACTCCATTACATCAACTGCAGGT 5’ RACE nested primer to detect the cleavage product of PpTAS4.

GACCCACCTGGTGATGCTGCGAATTACC 5’ RACE primer to detect the cleavage product of PpEREBP/AP2.

ATTTGGCGCCGCCAGTGCCGCTGCAGCG 5’ RACE nested primer to detect the cleavage product of PpTAP2.

GCACTTAGATTTCCACTGGGCG PpTAS4 forward primer for expression analysis by RT-PCR. Forward primer used for the amplification of the PpTAS4 hybridisation probe from cDNA.

AAGACATGGGAATTGCACACCC PpTAS4 reverse primer for expression analysis by RT-PCR. Reverse primer used for the amplification of the PpTAS4 hybridisation probe from cDNA.

TTTGCGATGTTACGGTTGTAGC PpTAS1 forward primer for expression analysis by RT-PCR.

ACAGCCAGTCAAGTGGATTCAG PpTAS1 reverse primer for expression analysis by RT-PCR.

CCGCAACATCATGCTGAAGTGG PpEREBP/AP2 forward primer for expression analysis by RT-PCR. Forward primer used for the amplification of the PpEREBP/AP2 hybridisation probe from cDNA.

GATGCTGGCGGCTTTCAGCTTT PpEREBP/AP2 reverse primer for expression analysis by RT-PCR. Reverse primer used for the amplification of the PpEREBP/AP2 hybridisation probe from cDNA. Primer sequence (5’ Ö 3’) Description of experiment

GAAGGAAGCAACGAGGCTGGTGGCGTGAAT Oligonucleotide to detect PpC3HDZIP1 sense GCTAAGCTGACAGCC siRNA upstream of the miR166 binding site.

GTTCTTGGAACTCTCCCGACAGGAAACGGTG Oligonucleotide to detect PpC3HDZIP1 sense GCACTATAGAGCTCTT siRNA downstream of the miR166 binding site.

GGCTGTCAGCTTAGCATTCACGCTCACCAGC Oligonucleotide to detect PpC3HDZIP1 antisense CTCGTTGCTTCCTTC siRNA upstream of the miR166 binding site.

AAGAGCTCTATAGTGCCACCGTTTCCTGTCG Oligonucleotide to detect PpC3HDZIP1 antisense GGAGAGTTCCAAGAAC siRNA downstream of the miR166 binding site.

TACACGATTCACTCCCTGCAATAGGTCCGGC Oligonucleotide to detect PpARF sense siRNA TCACTCCAAGTGAC upstream of the miR160 binding site.

CTCCAGCTGTGAGCTTCCAGAAGCAGACATG Oligonucleotide to detect PpARF sense siRNA AAGGCTAAGGGCTG downstream of the miR160 binding site.

GTCACTTGGGATGAGCCGGACCTATTGCAGG Oligonucleotide to detect PpARF antisense siRNA GAGTGAATCGTGTA upstream of the miR160 binding site.

CAGCCCTTAGCCTTCATGTCTGCTTCTGGAA Oligonucleotide to detect PpARF antisense siRNA GCTCACAGCTGGAG downstream of the miR160 binding site.

TGAAGCACTCATCACACCCTATGGAGCCATA Oligonucleotide to detect PpTAS4 sense ta- GCTAGGGTCTTGCG siRNA.

TGGCTCCATAGGGTGTGATGATGTCTTCATC Oligonucleotide to dete ct PpTAS4 antisense ta- CGGTGCTCTTCTACTGCCTT siRNA.

GGCAAAGTGCAGGCGTCCCTAGCTGGTGCC Oligonucleotide to detect PpEREBP/AP2 sense CATGGCAAGCAGTCT siRNA.

AGACTGCTTGCCATGGGCACCAGCTAGGGA Oligonucleotide to detect PpEREBP/AP2 CGCCTGCACTTTGCC antisense siRNA.

GTCGTATCCAGTGCAGGGTCCGAGGTATTCG Oligonucleotide for miR156-specific cDNA CACTGGATACGACGTGCTC synthesis.

GTCGTATCCAGTGCAGGGTCCGAGGTATTCG Oligonucleotide for miR160-specific cDNA CACTGGATACGACTGGCAT synthesis.

GTCGTATCCAGTGCAGGGTCCGAGGTATTCG Oligonucleotide for miR166-specific cDNA CACTGGATACGACGGGGAA synthesis.

GTCGTATCCAGTGCAGGGTCCGAGGTATTCG Oligonucleotide for miR390-specific cDNA CACTGGATACGACGGCGCT synthesis. Primer sequence (5’ Ö 3’) Description of experiment

GTCGTATCCAGTGCAGGGTCCGAGGTATTCG Oligonucleotide for ta-siRNA (pptA013298)- CACTGGATACGACGCAGAG specific cDNA synthesis.

GTCGTATCCAGTGCAGGGTCCGAGGTATTCG Oligonucleotide for ta-siRNA (pptA079444)- CACTGGATACGACTTGCCC specific cDNA synthesis.

GCGGCGGTGACAGAAGAGAGT Forward primer for miR156 RT-PCR.

CCTCCCGTGCCTGGCTCCCTGT Forward primer for miR160 RT-PCR.

GCGGCGGTCGGACCAGGCTTCA Forward primer for miR 166 RT-PCR.

GCGGCGGAAGCTCAGGAGGGAT Forward primer for miR390 RT-PCR.

GCGGCGGGTGATTGCACTGCAG Forward primer for ta-siRNA (pptA013298) RT- PCR.

GCGGCGGATCACAAGGGTAGGT Forward primer for ta-siR NA (pptA079444) RT- PCR.

GTGCAGGGTCCGAGGTAT Universal reverse primer for miRNA and ta-siRNA RT-PCR.

Supplemental Experimental Procedures

Isolation of PpDCL full-length cDNAs

Partial cDNA sequences of P. patens DCL genes were initially identified by tblastn searches in P. patens EST sequences (Rensing et al., 2002) using A. thaliana DCL1-4 protein sequences (accession numbers P84634, Q9SP32, NP_189978, NP_566199) as queries. Corresponding cDNA clones were sequenced and used to obtain the full-length sequences. The cloning of full-length cDNA sequences was performed by 5’RACE-

PCRs and RT-PCRs using primers derived from available P. patens genomic sequence data. To confirm that the amplicons were derived from the same cDNA all PCR and 5’

RACE primers were selected to produce overlapping PCR fragments of already known sequence stretches.

Generation and molecular analysis of PpDCL1a and PpDCL1b knockout lines

For the generation of PpDCL1a and PpDCL1b knockout constructs we amplified a

PpDCL1a genomic region with the primers 5’-CCAGTTGCGCATAAAGTTGA-3’ and 5’-

TCCAAGGCATCCAGAGAGTC-3’ and a PpDCL1b cDNA region using the primers 5’-

GCATTCCTGTGGAGTTTGATG-3’ and 5’-ACCTTCCACACTTGGTGTGTG-3’. An nptII selection marker cassette was cloned into a single Eco72I restriction site present in the

PpDCL1b cDNA fragment and into a single EcoRV restriction site of the PpDCL1a genomic fragment. The complete knockout cassettes were released from the vector prior to transformation. Primers used to identify ΔPpDCL1a transgenic lines were: 5’-

TTATGTGGATTCAGTGCGCTTC-3’ and 5’-CCATCGACTTAGCCAAACCAGT-3’. To confirm a precise 5’ and 3’ integration of the PpDCL1a knockout construct we used the primers 5’-TTTGCAGTTGACTGACCTCAAGA-3’ and 5’-

GCGGCTGAGTGGCTCCTTCA-3’ (5’ integration) and 5’-

CCAAGGATCCCGGAAGAGGA-3’ and 5’-AAATTATCGCGCGCGGTGTC-3’ (3’ integration). To confirm the loss of PpDCL1a transcript by RT-PCR the primers 5’-

TTGGTCCGTTGGAATACACA-3’ and 5’- AATCTTTGTGCGCCTCTCAC-3’ were used.

Primers used for the screening of transgenic ΔPpDCL1b lines were: 5’-

GCATTCCTGTGGAGTTTGATG-3’ and 5’-ACCTTCCACACTTGGTGTGTG-3’. The same primers were used to confirm the loss of PpDCL1b transcript by RT-PCR. A second primer pair upstream of the integration site was used for RT-PCR: 5’-

AGGATTGTTACTGCGGTGCA-3’ and 5’-AAGCTCTGCACGCTCATAGC-3’. To confirm a precise 5’ and 3’ integration of the PpDCL1b knockout construct we used the primers

5'-TGCTACTCACTTCATGAACTG-'3 and 5'-ACGTGACTCCCTTAATTCTCC-'3 (5’ integration) and 5'-CCCGCAATTATACATTTAATACG-'3 and 5'-

GCACCATGGCTGCAACAAAG-'3 (3’ integration). RT-PCR control primers for the

PpEF1α control gene are listed in Table S1.

P. patens lines expressing an artificial miRNA (amiRNA) targeting PpGNT1

The amiR-GNT1 sequence was introduced into the A. thaliana miRNA319a precursor by overlapping PCR as described (Khraiwesh et al., 2008). The resulting construct was used for transfections of P. patens wild type and ΔPpDCL1b mutant lines. To identify transgenic lines harboring the amiR-GNT1 expression construct PCR was performed using the primers 5’-TGATATCTCCACTGACGAAAGGG-3’ and 5’-

GGATCCCCCCATGGCGATGCCTTAAAT-3’.

Detection of RNA cleavage products

Synthesis of 5’ RACE-ready cDNAs was carried out according to Zhu et al. (Zhu et al.,

2001) with the BD Smart RACE cDNA Amplification Kit (Clontech). PCR reactions were performed using the UPM Primer-Mix in combination with gene-specific primers derived from target RNAs (Table S1). Cleavage products were excised from the gel, cloned and sequenced.

Small RNA Blots

Total RNA was separated in a 12% denaturing polyacrylamide gel containing 8.3 M urea in TBE buffer. The RNA was electroblotted onto nylon membranes for 1 h at 400 mA.

Radiolabeled probes were generated by end-labeling of DNA oligonucleotides complementary to miRNA, siRNA and ta-siRNA sequences and the U6snRNA control

(Table S1) with γ32P-ATP using T4 polynucleotide kinase. Blot hybridization was carried out in 0.05 M sodium phosphate (pH 7.2), 1 mM EDTA, 6 x SSC, 1 x Denhardt’s, 5%

SDS. Blots were washed 2-3 times with 2 x SSC, 0.2% SDS and one time with 1 x SSC,

0.1% SDS. Blots were hybridized and washed at temperatures 5°C below the Tm of the oligonucleotide. The sequences of the oligonucleotides used for the detection of small

RNAs are listed in Table S1.

Detection of small RNAs by RT-PCR

The RT-PCR analyses of the miR156, 160, 166, 390, and the ta-siRNAs pptA079444

(processed from the PpTAS1 gene) and pptA013298 (processed from the PpTAS3 gene) were carried out as described (Varkonyi-Gasic et al., 2007). The sequences of oligonucleotides used for the cDNA synthesis and subsequent PCR reactions are listed in Table S1.

Expression analysis by RT-PCR and RNA gel blots

RT-PCRs were performed for PpEF1α, PpGNT1, PpC3HDZIP1, PpHB10, PpSBP3,

PpARF, and PpTAS1 from three independent biological replicates with gene-specific primers (Table S1). PCR products were quantified with the Quantity One Software (Bio-

Rad). The relative amounts of the transcripts were normalized to the constitutive control

PpEF1α. 20 µg of total RNA isolated from wild type, ΔPpDCL1b mutants and transgenic lines expressing the amiR-GNT1, respectively, were separated in denaturing agarose gels and blotted onto nylon membranes. Hybridization probes for PpARF, PpC3HDZIP1,

PpGNT1, PpEF1α, PpTAS4 PpEREBP/AP2, PpbHLH, and PpCOR47 were amplified from wild type cDNA (primers listed in Table S1). The ABA-responsive gene PpCOR47

(Frank et al., 2005) was used to control the efficiency of the ABA treatments.

DNA methylation analysis

The cDNA sequences of PpC3HDZIP1 (DQ385516), PpHB10 (AB032182), PpARF

(AR452951), PpSBP3 (AJ968318) and PpGNT1 (AJ429143) were used for BLASTN searches to identify corresponding genomic sequences from the P. patens whole- genome-shotgun (WGS) traces (accessible via www.ncbi.nlm.nih.gov/Traces/trace.cgi).

The identified genomic sequences were clustered and assembled using the Paracel

Transcript Assembler to determine the genomic exon/intron structure (Figure S5). The parameters for clustering threshold, overlap length and overlap identity were 100 nt, 80 nt and 90%, respectively. Primers to analyse the PpTAS4 genomic locus were derived from the reported PpTAS4 sequence (Talmor-Neiman et al., 2006). Primers for the analysis of the PpEREBP/AP2 and PpbHLH gene were derived from the corresponding gene model of the available P. patens genomic sequence (http://genome.jgi- psf.org/Phypa1_1/Phypa1_1.home.html; gene model accession numbers

Phypa1_129196 [PpEREBP/AP2] and Phypa1_209063 [PpbHLH]). The derived promoter, exon and intron regions were analyzed with the MethPrimer program (Li and Dahiya, 2002) to deduce methylation-specific (MSP) and unmethylation-specific primers

(USP) (Figure S6) for PCR analysis of bisulfite-treated DNA.

Detection of sense and antisense transcripts

cDNA from wild type plants and ΔPpDCL1b mutants was synthesized from 4 µg total

RNA with Superscript III (Invitrogen) using primers specific for sense and antisense transcripts, respectively (Table S1). To monitor the efficiency of cDNA synthesis, primers specific for the PpEF1α sense transcript were added to each cDNA synthesis reaction.

RT-PCRs were carried out with gene-specific primers (Table S1).

Supplemental Experimental Procedures References

Frank, W., Ratnadewi, D., and Reski, R. (2005). Physcomitrella patens is highly tolerant against drought, salt and osmotic stress. Planta 220, 384-394.

Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., and Frank, W. (2008). Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockouts. Plant Physiol.

Li, L. C., and Dahiya, R. (2002). MethPrimer: designing primers for methylation PCRs.

Bioinformatics 18, 1427-1431.

Rensing, S. A., Rombauts, S., Van de Peer, Y., and Reski, R. (2002). Moss transcriptome and beyond. Trends Plant Sci 7, 535-538.

Talmor-Neiman, M., Stav, R., Klipcan, L., Buxdorf, K., Baulcombe, D. C., and Arazi, T.

(2006). Identification of trans-acting siRNAs in moss and an RNA-dependent RNA polymerase required for their biogenesis. Plant J 48, 511-521. Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F., and Hellens, R. P. (2007).

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3, 12.

Zhu, Y. Y., Machleder, E. M., Chenchik, A., Li, R., and Siebert, P. D. (2001). Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30, 892-897.

Chapter III Artificial microRNAs in Physcomitrella patens

3 Chapter III: Publication 1 Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockout

Plant Physiology, October 2008, Vol. 148, pp. 684–693 Received August 13, 2008 Accepted August 22, 2008

Own contribution:

Carried out all experimental work reported in the publication and contributed to prepare the manuscript (drafting the manuscript and preparing the figures). The work was supervised by W. Frank.

121 Breakthrough Technologies

Specific Gene Silencing by Artificial MicroRNAs in Physcomitrella patens: An Alternative to Targeted Gene Knockouts1[C][W][OA]

Basel Khraiwesh, Stephan Ossowski, Detlef Weigel, Ralf Reski, and Wolfgang Frank* Plant Biotechnology, Faculty of Biology (B.K., R.R., W.F.), Freiburg Initiative for Systems Biology (R.R., W.F.), and Centre for Biological Signaling Studies (R.R.), University of Freiburg, 79104 Freiburg, Germany; and Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (S.O., D.W.)

MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNAs processed from nuclear-encoded transcripts, which include a characteristic hairpin-like structure. MiRNAs control the expression of target transcripts by binding to reverse complementary sequences directing cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern of matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of interest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism, which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genome necessitates the development of alternative techniques to speed up reverse genetics analyses and to allow for more flexible inactivation of genes. To prove the adaptability of amiRNA expression in Physcomitrella, we designed two amiRNAs, targeting the gene PpFtsZ2-1, which is indispensable for chloroplast division, and the gene PpGNT1 encoding an N-acetylglucosami- nyltransferase. Both amiRNAs were expressed from the Arabidopsis (Arabidopsis thaliana) miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of the amiRNAs and an efficient knock down of the cognate target mRNAs. Furthermore, chloroplast division was impeded in PpFtsZ2-1-amiRNA lines that phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of the initial amiRNA signal by secondary transitive small interfering RNAs, although these small interfering RNAs do not seem to have a major effect on sequence-related mRNAs, confirming specificity of the amiRNA approach.

During the last decade, small nonprotein-coding classes of small RNAs include microRNAs (miRNAs) RNAs (20–24 nucleotides [nt] in size) have been dem- and small interfering RNAs (siRNAs), which differ with onstrated to be involved in RNA-mediated phenomena respect to their biogenesis (Bartel, 2004; Chapman and such as RNA interference (RNAi), cosuppression, gene Carrington, 2007). MiRNAs are approximately 21-nt silencing, and quelling (Matzke et al., 1989; Napoli et al., RNAs that are encoded by endogenous MIR genes. 1990; de Carvalho et al., 1992; Romano and Macino, 1992; Their primary transcripts form precursor RNAs exhib- Lee et al., 1993; Hamilton and Baulcombe, 1999). Major iting a partially double-stranded stem-loop structure that is processed by DICER-LIKE proteins to release mature miRNAs (Bartel, 2004). MiRNAs are recruited 1 This work was supported by the Landesstiftung Baden- to the RNA-induced silencing complex (RISC), where Wu¨ rttemberg (grant no. P–LS–RNS/40 to W.F., R.R., and D.W.), the they become activated by unwinding of the double Federal Ministry of Education and Research (Freiburg Initiative for strand and subsequently bind to complementary mRNA Systems Biology grant no. 0313921 to R.R. and W.F.), the Excellence Initiative of the German Federal State Governments (Biological sequences resulting in either direct cleavage of the Signaling Studies grant no. EXC294 to R.R.), and European Com- mRNA or repression of their translation by RISC munity FP6 IP SIROCCO (contract no. LSHG–CT–2006–037900 to (Bartel, 2004; Kurihara and Watanabe, 2004; Brodersen D.W.). et al., 2008). Recently, miRNAs have been identified as * Corresponding author; e-mail wolfgang.frank@biologie. important regulators of gene expression in both plants uni-freiburg.de. and animals (Jones-Rhoades et al., 2006), and particular The author responsible for distribution of materials integral to the miRNA families were shown to be highly conserved in findings presented in this article in accordance with the policy evolution (Jones-Rhoades et al., 2006; Axtell et al., 2007; described in the Instructions for Authors (www.plantphysiol.org) is: Fahlgren et al., 2007; Fattash et al., 2007; Axtell and Wolfgang Frank ([email protected]). Bowman, 2008). In contrast, precursors of siRNAs [C] Some figures in this article are displayed in color online but in black and white in the print edition. form perfectly complementary double-stranded RNA [W] The online version of this article contains Web-only data. (dsRNA) molecules (Myers et al., 2003). They originate [OA] Open Access articles can be viewed online without a sub- from transgenes, viruses, and transposons and may scription. require RNA-dependent RNA polymerases for dsRNA www.plantphysiol.org/cgi/doi/10.1104/pp.108.128025 formation (Waterhouse et al., 2001; Aravin et al., 2003).

684 Plant Physiology, October 2008, Vol. 148, pp. 684–693, www.plantphysiol.org Ó 2008 American Society of Plant Biologists Artificial MicroRNAs in Physcomitrella patens

Unlike miRNAs, the diced siRNA products derived from Alternative approaches to analyze gene functions the long complementary precursors are not uniform in in Physcomitrella were recently reported, which were sequence, but correspond to different regions of their based on the expression of classical inverted repeat precursor. Whereas miRNAs mainly mediate posttran- sequences resulting in the formation of dsRNA mol- scriptional control of endogenous transcripts, siRNAs ecules, which give rise to siRNAs and consequently have been implicated in transcriptional silencing of silence the target transcript (Bezanilla et al., 2003, 2005; transposable elements as well as posttranscriptional Vidali et al., 2007). One drawback to applying classical control of endogenous and exogenous RNAs, for exam- RNAi constructs is the production of a diverse set ple, viral transcripts (Waterhouse et al., 2001; Aravin of siRNAs from the complete dsRNA, which may af- et al., 2003; Myers et al., 2003; Ossowski et al., 2008). fect off-target transcripts. Furthermore, in some cases, Previous reports demonstrated that the alteration of gene silencing triggered by the expression of inverted several nucleotides within the miRNA sequence does repeat sequences was found to be unstable in Phys- not affect its biogenesis as long as the positions of comitrella (Bezanilla et al., 2005). Additional differ- matches and mismatches within the precursor stem ences in the action of siRNAs and amiRNAs may loop remain unaffected (Vaucheret et al., 2004). This result from their varying mobility within the plant. raises the possibility of modifying miRNA sequences Recent studies have shown that transgene-derived or and creating artificial miRNAs (amiRNA) directed viral-induced siRNAs are able to move from cell to against any gene of interest resulting in posttran- cell, whereas miRNAs are not mobile and act cell scriptional silencing of the corresponding transcript autonomously (Tretter et al., 2008). (Zeng et al., 2002; Parizotto et al., 2004; Alvarez et al., Independent studies on small RNAs in Physcomitrella 2006; Niu et al., 2006; Schwab et al., 2006; Warthmann revealed the existence of a diverse miRNA repertoire, et al., 2008). In addition, genome-wide expression including highly conserved miRNA families (Arazi analyses in Arabidopsis (Arabidopsis thaliana) have et al., 2005; Axtell et al., 2006, 2007; Talmor-Neiman shown that plant amiRNAs exhibit high specificity et al., 2006; Fattash et al., 2007). Furthermore, their similar to natural miRNAs (Schwab et al., 2005, 2006), corresponding precursor transcripts share the charac- such that their sequences can easily be optimized to teristic hairpin-like structure known from seed plants. knock down the expression of a single gene or several Thus, the design and expression of amiRNAs for the highly conserved genes without affecting the expres- specific knock down of genes in Physcomitrella should sion of other genes. be feasible. To test amiRNA function in Physcomitrella, The moss Physcomitrella patens has become a recog- we targeted the gene PpFtsZ2-1,whichisrequired nized model system to study diverse processes in for chloroplast division (Strepp et al., 1998), and the plant biology, which was mainly based on the unique PpGNT1 gene encoding an N-acetylglucosaminyltrans- ability to efficiently integrate DNA into its nuclear ferase (Koprivova et al., 2003). PpFtsZ2-1 null mutants genome by means of homologous recombination en- form macrochloroplasts, presenting an obvious pheno- abling the generation of targeted gene knockout lines type, which enables direct evaluation of the efficiency of (Schaefer, 2002). Furthermore, based on the predomi- the intended amiRNA approach. nant haploid phase of Physcomitrella’s life cycle, the frequency of phenotypic deviations caused by the disruption of a single gene is higher compared to seed plants (Egener et al., 2002). Nevertheless, the RESULTS generation of targeted knockout mutants in Physcomi- Expression and Detection of PpFtsZ2-1-amiRNA and trella has limitations. For example, despite the haploid PpGNT1-amiRNA in Physcomitrella genome, homologs might still compensate for each other and one cannot recover knockouts of genes with The use of amiRNAs for efficient gene silencing has essential functions. Furthermore, the targeted knock- been reported in various seed plants (Alvarez et al., out of a single gene requires several cloning steps, 2006; Niu et al., 2006; Schwab et al., 2006; Warthmann repetitive selection of transgenic lines, and detailed et al., 2008), but it has not been tested in nonseed molecular analysis of putative knockout candidates. plants such as the bryophyte P. patens. This is an issue The recently published Physcomitrella genome (Rensing because functional studies of essential members of the et al., 2008) now opens the way for medium- to large- RNAi machinery in Physcomitrella, such as Dicer and scale analysis of gene functions in a postgenomic era Argonaute proteins, are still missing (Axtell et al., (Quatrano et al., 2007) requiring the development of 2007). Furthermore, the complement of essential RNAi- new techniques. The posttranscriptional silencing of related proteins in Physcomitrella differsfromthatin genes by amiRNAs may serve as an appropriate tool seed plants (Axtell et al., 2007; Rensing et al., 2008). to speed up such analyses because they can be de- We designed two amiRNAs that were predicted to signed to target several genes (as long they contain at target the genes PpFtsZ2-1 and PpGNT1, respectively, least one conserved sequence stretch), amiRNAs can using the amiRNA designer interface WMD (Schwab be expressed from inducible promoters, and amiRNA et al., 2006; Ossowski et al., 2008). The designed constructs can easily be generated using a standard- amiRNAs contain a uridine residue at position 1 and ized cloning procedure (Schwab et al., 2006). an adenine residue at position 10, both of which are

Plant Physiol. Vol. 148, 2008 685 Khraiwesh et al. overrepresented among natural plant miRNAs and AmiRNA-Directed Cleavage of PpFtsZ2-1 and increase the efficiency of miRNA-mediated target cleav- PpGNT1 mRNAs age (Schwab et al., 2005). Furthermore, we also pre- ferred that the amiRNAs exhibit 5# instability relative The expression of the PpFtsZ2-1-amiRNA and to the miRNA*, which positively affects separation of PpGNT1-amiRNA should cause cleavage of the cog- nate mRNAs within the region complementary to the both strands during RISC loading (Fig. 1A; Mallory # et al., 2004; Schwab et al., 2005). In previous studies, the amiRNA sequences. To prove this, we performed 5 RACE-PCRs to detect specific PpFtsZ2-1 and PpGNT1 Arabidopsis miR319a precursor was used to introduce # specific nucleotide changes within the miRNA/miRNA* mRNA cleavage products. Using 5 RACE-ready stem-loop region. Based on the conservation of the cDNA prepared from one PpFtsZ2-1-amiRNA, one miR319 family among land plants (Jones-Rhoades et al., PpGNT1-amiRNA overexpression line, and wild type, 2006; Fattash et al., 2007; Axtell and Bowman, 2008; cleavage products of the expected size were only amplified from the amiRNA lines. Conversely, in Warthmann et al., 2008) and similar secondary struc- # tures of miR319 precursor transcripts from Arabidopsis wild type, the 5 RACE-PCRs yielded exclusively and Physcomitrella (Fig. 1B; Supplemental Fig. S1), fragments derived from the full-length transcripts we hypothesized that the PpFtsZ2-1-amiRNA and (Fig. 1E). The PCR products corresponding to the PpGNT1-amiRNA will be correctly processed from the expected size of the PpFtsZ2-1 and PpGNT1 mRNA Arabidopsis miR319a precursor. The PpFtsZ2-1-amiRNA cleavage products in the amiRNA lines were cloned and PpGNT1-amiRNA and the corresponding miRNA* and sequenced to determine the precise mRNA cleav- sequences were introduced into the miR319a precur- age sites. In 12 of 18 clones analyzed, PpFtsZ2-1 mRNA sor by overlapping PCR using primers harboring the cleavage occurred between nucleotide positions 11 respective amiRNA and miRNA* sequences, cloned and 12 with respect to the PpFtsZ2-1-amiRNA se- into the plant expression vector pPCV downstream quence, whereas the remaining six clones resulted of a double cauliflower mosaic virus 35S promoter from cleavage of the PpFtsZ2-1 mRNA between nu- (Fig. 1A) and used for transfection of Physcomitrella cleotides 12 and 13 (Fig. 1E). Normally, in plants, protoplasts. After selection of regenerating plants, cleavage within a target transcript that is mediated by genomic DNA of individual lines was analyzed by a 21-nt miRNA occurs between positions 10 and 11 PCR with primers flanking the amiRNA sequence with respect to the miRNA sequence (Llave et al., present in the expression constructs to identify trans- 2002), suggesting that the actual amiRNAs produced genic lines that had integrated the PpFtsZ2-1-amiRNA from the PpFtsZ2-1-amiRNA construct were shifted by and PpGNT1-amiRNA constructs, respectively. Eight 1 or 2 nt. However, the sequencing of six independent of 12 regenerated lines derived from the transforma- clones of PpGNT1 mRNA cleavage products revealed tion with the PpFtsZ2-1-amiRNA construct and seven that cleavage occurred between positions 10 and 11 of 12 regenerated lines derived from the transforma- with respect to the PpGNT1-amiRNA sequence (Fig. tion with the PpGNT1-amiRNA construct produced 1E), indicating precise processing of the PpGNT1- the expected PCR amplicon. Thus, we cannot ex- amiRNA from the precursor construct. clude that some lines survived the antibiotic selection Target sites in plant mRNAs normally share high without the integration of the DNA constructs. From sequence complementarity to the respective miRNA the lines harboring an overexpression construct, three (Schwab et al., 2005). To prove the specificity of the PpFtsZ2-1-amiRNA lines and two PpGNT1-amiRNA expressed PpFtsZ2-1-amiRNA, we analyzed whether lines were selected for further analysis (Fig. 1C). As the the mRNA of PpFtsZ2-2, the closest homolog of generated amiRNA overexpression constructs do not PpFtsZ2-1, is targeted by the PpFtsZ2-1-amiRNA. contain homologous sequences of the Physcomitrella ge- Compared to the PpFtsZ2-1-amiRNA recognition site nome, the constructs are expected to integrate into the in PpFtsZ2-1, the corresponding region within the PpFtsZ2-2 sequence contains two mismatches at posi- Physcomitrella genome by an illegitimate recombina- # tion event. To prove the correct maturation of the tions 12 and 16. 5 RACE-PCRs were performed using a PpFtsZ2-1-amiRNA and PpGNT1-amiRNA from the PpFtsZ2-2 gene-specific primer. PCR products indicat- ing amiRNA-guided cleavage products were not ob- Arabidopsis miR319a precursor and its accumulation # in the transgenic lines, we performed small RNA gel- tained. Instead, the 5 RACE-PCR yielded exclusively blot analyses with antisense probes for both amiRNAs. fragments corresponding to the PpFtsZ2-2 full-length Accumulation of the mature PpFtsZ2-1-amiRNA and transcript (Fig. 1E). Thus, the PpFtsZ2-1-amiRNA ex- PpGNT1-amiRNA was detected in all lines analyzed, hibits high specificity, comparable to natural miRNAs. demonstrating that the amiRNAs are efficiently pro- cessed from the Arabidopsis miR319a precursor in AmiRNAs Efficiently Down-Regulate PpFtsZ2-1 and Physcomitrella (Fig. 1D). However, normalization of PpGNT1 mRNA Levels the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA hybri- dization signals to the U6snRNA controls revealed As we detected amiRNA-directed cleavage of the amiRNA expression levels that differed up to 8-fold PpFtsZ2-1 and PpGNT1 target mRNAs, we next ana- for the PpFtsZ2-1-amiRNA and up to 5-fold for the lyzed the target transcript levels by RNA gel blots. PpGNT1-amiRNAbetweentheindividuallines(Fig.1D). Compared to wild type, we detected strongly reduced

686 Plant Physiol. Vol. 148, 2008 Artificial MicroRNAs in Physcomitrella patens

Figure 1. Analysis of Physcomitrella lines expressing PpFtsZ2-1-amiRNA and PpGNT1-amiRNA. A, Scheme illustrating the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA overexpression constructs. The modified ath-miRNA319a precursor DNA fragments were cloned into the SmaI and BamHI sites of the pPCV plant expression vector containing a double 35S promoter, nos terminator, and hpt selection marker cassette. Primers that were used for molecular analyses of the transgenic lines are indicated

Plant Physiol. Vol. 148, 2008 687 Khraiwesh et al. steady-state levels of PpFtsZ2-1 and PpGNT1 mRNAs in from PpFtsZ2-1 and PpGNT1 mRNA regions down- the respective amiRNA overexpression lines (Fig. 2A). stream of the amiRNA recognition site for RNA gel- However, PpFtsZ2-1 transcript levels were reduced blot analysis. Sense and antisense siRNAs were only to 1% to 2% in PpFtsZ2-1-amiRNA lines, whereas detected in PpFtsZ2-1-amiRNA and PpGNT1-amiRNA PpGNT1 mRNA levels dropped to 10% to 20% in lines, respectively, but not in wild type (Fig. 2C). We PpGNT1-amiRNA lines when compared to wild-type conclude that amiRNAs allow for efficient down- plants. Furthermore, the efficiency of posttranscrip- regulation of mRNAs in Physcomitrella and the genera- tional silencing of PpFtsZ2-1 was similar in all three tion of transitive siRNAs from mRNA cleavage products amiRNA overexpression lines, even though they dif- may amplify the initial amiRNA trigger. However, fered with respect to the PpFtsZ2-1-amiRNA accumu- the transitive effects are apparently not sufficient to lation (Fig. 1D), whereas the reduction of PpGNT1 have a major impact on sequence-related genes, as the transcript levels correlated with the PpGNT1-amiRNA PpFtsZ2-2 steady-state RNA levels were unaffected in expression levels. From these results, we conclude that PpFtsZ2-1-amiRNA overexpression lines (Fig. 2A). amiRNAs confer efficient down-regulation of their target mRNAs in Physcomitrella. As a control, we also PpFtsZ2-1-amiRNA Overexpressors Phenocopy PpFtsZ2-1 analyzed the steady-state levels of the sequence- Null Mutants related PpFtsZ2-2 mRNA in PpFtsZ2-1-amiRNA over- expression lines. In agreement with the absence of In this study, we have chosen two genes to evaluate amiRNA-induced mRNA cleavage products, PpFtsZ2-2 the use of an amiRNA expression system in Physco- transcript levels were similar in wild-type and the three mitrella. The targeted deletion of PpGNT1 that is in- PpFtsZ2-1-amiRNA lines (Fig. 2A). volved in the N-glycosylation of proteins did not cause The 5# RACE-PCR experiments performed with any phenotypic deviations (Koprivova et al., 2003). In one of the PpFtsZ2-1-amiRNA lines yielded additional agreement with this previous study, the two charac- fragments that differed substantially in size from the terized PpGNT1-amiRNA lines were indistinguish- expected cleavage products (Fig. 1E). After amiRNA- able from Physcomitrella wild-type plants. In contrast, mediated cleavage of the mRNA, the cleavage prod- PpFtsZ2-1 null mutants, which were generated by tar- ucts may serve as templates for synthesizing cRNA geted gene disruption and lack expression of PpFtsZ2-1 by RNA-dependent RNA polymerase (Vaistij et al., mRNA, are impeded in chloroplast division leading 2002) leading to the formation of dsRNA. Subse- to the formation of macrochloroplasts (Strepp et al., quently, the dsRNA may be processed into secondary 1998). In our study, the expression of PpFtsZ2-1- siRNAs, resulting in spreading of the initial amiRNA amiRNA led to strongly reduced PpFtsZ2-1 mRNA signal (Fig. 2B). This mechanism, known as transitiv- levels. To compare knockout and amiRNA lines, we ity, usually is initiated by dsRNA triggers. In plants, investigated the phenotypes of the three PpFtsZ2-1- the transitivity occurs in both directions of the initial amiRNA lines. In all lines, the accumulation of the dsRNA trigger (Moissiard et al., 2007), whereas in amiRNA targeting PpFtsZ2-1 resulted in impaired animals, spreading of the initial signal occurs only chloroplast division and the formation of macrochlo- upstream of the trigger (Pak and Fire, 2007). However, roplasts that phenocopied the PpFtsZ2-1 null mutants the onset of transitivity is a rare event after miRNA- (Fig. 3; Supplemental Fig. S2). The formation of macro- mediated target cleavage (Howell et al., 2007; Moissiard in the PpFtsZ2-1-amiRNA lines was ob- et al., 2007) and is normally not observed after served in all tissues and cells analyzed indicating amiRNA-mediated target cleavage (Schwab et al., an efficient production of mature amiRNAs from con- 2006). To investigate the possibility of transitivity, we stitutively expressed precursor transcripts. Further- used sense and antisense oligonucleotides derived more, we did not observe any particular phenotypic

Figure 1. (Continued.) by arrows. B, Secondary structures of foldbacks of the P. patens miR319d precursor (ppt-MIR319d) and Arabidopsis miR319a precursor (ath-MIR319a). The mature miRNA is highlighted in green with uppercase letters. C, PCR screen to identify transgenic lines harboring the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression constructs. WT, Wild type; amiRNA lines, 1, 2, and 3 for PpFtsZ2-1-amiRNA; 1 and 2 for PpGNT1-amiRNA; PpEF1a, control PCRs. D, Expression analysis of PpFtsZ2-1-amiRNA and PpGNT1-amiRNA in Physcomitrella wild type (WT), and lines harboring the PpFtsZ2-1-amiRNA or PpGNT1-amiRNA expression constructs. Fifty micrograms of each RNA was blotted and hybridized with a PpFtsZ2-1-amiRNA and PpGNT1- amiRNA antisense probe, respectively. Hybridization with an antisense probe for U6snRNA served as control. PpFtsZ2-1- amiRNA and PpGNT1-amiRNA expression levels were normalized to the U6snRNA control hybridization. Numbers indicate the relative PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression levels. E, Top, 5# RACE-PCRs for the genes PpFtsZ2-1 and PpFtsZ2-2 from wild type (WT) and line 1 expressing the PpFtsZ2-1-amiRNA; bottom, 5# RACE-PCR for the gene PpGNT from wild type (WT) and line 1 expressing the PpGNT1-amiRNA. The arrows mark PCR fragments corresponding to the expected size of the cleavage products that were isolated, cloned, and sequenced. The right images show the sequence complementarity of PpFtsZ2-1, PpFtsZ2-2, and PpGNT1 to the amiRNA sequences. The determined cleavage sites within the PpFtsZ2-1 and PpGNT1 mRNAs are marked by vertical arrows and numbers above indicate the number of sequenced products cleaved at this site. [See online article for color version of this figure.]

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Figure 2. Expression analysis of PpFtsZ2-1, PpFtsZ2-2, and PpGNT1, and detection of transitive siRNAs. A, Left, RNA gel blots (20 mg each) from wild type (WT) and PpFtsZ2-1-amiRNA overexpression lines (1–3) hybridized with PpFtsZ2-1 and PpFtsZ2-2 probes; right, RNA gel blots (20 mg each) from wild type (WT) and PpGNT1-amiRNA overexpression lines (1 and 2) hybridized with a PpGNT1 probe. The ethidium bromide-stained gels below indicate equal loading. The hybridization signals were normalized to the rRNA bands, and the PpFtsZ2-1, PpFtsZ2-2,andPpGNT1 expression levels in wild type were set to 1. Numbers indicate the relative PpFtsZ2-1, PpFtsZ2-2,and PpGNT1 mRNA levels. B, Scheme illustrating the generation of transitive siRNAs from amiRNA target cleavage products requiring an RNA-dependent RNA polymerase (RdRP) to generate dsRNA, which is subsequently processed into siRNAs. Black line, mRNA; gray box, amiRNA binding site; curved line, amiRNA. C, Detection of sense and antisense transitive siRNAs produced from PpFtsZ2-1 (left) and PpGNT1 (right) mRNA cleavage products by RNA gel blots hybridized with oligonucleotides derived from regions downstream of the amiRNA binding sites. Hybridiza- tion with an antisense probe for U6snRNA served as control.

differences among the transgenic lines expressing the pre-miRNAs within seed plants (Alvarez et al., 2006). PpFtsZ2-1-amiRNA, which is consistent with the sim- In our study, we tested the application of amiRNAs for ilar degree of PpFtsZ2-1 mRNA reduction. Our results the specific silencing of genes in the bryophyte Phys- demonstrate that the expression of amiRNAs in Phys- comitrella making use of an amiRNA expression sys- comitrella leads to efficient silencing of their target tem, where the Arabidopsis miR319a precursor serves mRNAs comparable to the effects of targeted gene as the backbone for amiRNA expression and subse- knockouts. quent maturation and was developed to control gene expression in Arabidopsis (Schwab et al., 2006). The miR319 family belongs to the highly conserved amiRNA DISCUSSION families, even over large evolutionary distances, and was also found in Physcomitrella (Arazi et al., 2005; The successful use of amiRNAs for the specific Jones-Rhoades et al., 2006; Fattash et al., 2007). Nota- down-regulation of genes was shown for the dicoty- bly, miR319 stands out in that there is also consider- ledonous plants Arabidopsis, tomato (Solanum lyco- able sequence conservation in the foldback, not only persicum), and tobacco (Nicotiana tabacum), and for the in the miRNA itself. Our comparison of the Arabidop- monocot rice (Oryza sativa; Parizotto et al., 2004; sis miR319a precursor and the Physcomitrella miR319 Alvarez et al., 2006; Niu et al., 2006; Schwab et al., precursor sequences confirmed nucleotide sequence 2006; Qu et al., 2007; Ossowski et al., 2008; Warthmann conservation outside the miRNA/miRNA* region, et al., 2008). In most cases, the amiRNA was expressed implying similar foldback structures of the Arabidop- from endogenous miRNA precursors. However, high sis and Physcomitrella miR319 pre-miRNAs. Indeed, expression rates of amiRNAs were achieved in tobacco we detected the correct processing of a mature 21-nt and tomato using the Arabidopsis miR164b precursor PpFtsZ2-1-amiRNA and PpGNT1-amiRNA, respec- sequence indicating correct processing of conserved tively, from the Arabidopsis miR319a precursor in

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Figure 3. Impeded plastid division and formation of macrochloroplasts in PpFtsZ2-1-amiRNA overexpressors. A, Light microscopy from protonema and leaves of wild type (WT) and one PpFtsZ2-1-amiRNA overexpression line (size bars, 100 mm). B, Confocal laser- scanning microscopy from protonema and leaves of wild type (WT) and one PpFtsZ2-1-amiRNA overexpression line (size bars, 50 mm). Red, Chlorophyll auto- fluorescence in plastids. See Supplemental Figure S2 for phenotypes of the other two PpFtsZ2-1-amiRNA lines.

transgenic Physcomitrella lines, indicating that the re- et al., 2005). Furthermore, cleavage of the PpFtsZ2-1 constructed miR319a pre-miRNA contains the essen- mRNA within the amiRNA recognition site indicates tial recognition and processing information to enter correct amiRNA/amiRNA* duplex recognition and the Physcomitrella miRNA biogenesis pathway. The amiRNA loading into the RISC complex. PpFtsZ2-1 mRNA cleavage products were, however, Previous studies have shown that the transcript offset by 1 and 2 nt relative to the expected products levels of amiRNA targets are in most cases anticorre- (Llave et al., 2002), suggesting that the PpFtsZ2-1- lated with corresponding amiRNA levels (Schwab amiRNA was shifted by 1 or 2 nt, respectively, relative et al., 2006). Among PpFtsZ2-1-amiRNA and PpGNT1- to the intended amiRNA. A similar effect has been amiRNA lines analyzed, the amiRNA expression levels observed for some Arabidopsis amiRNAs (Schwab varied 8-fold and 5-fold, respectively. Nevertheless, the et al., 2006). Because the originally designed amiRNAs amiRNA expression caused a similar reduction of were perfectly complementary, the shifted amiRNAs PpFtsZ2-1 and PpGNT1 mRNA levels to 1% to 2% and should still adhere to the targeting rules for miRNAs. 10% to 20%, respectively, compared to transcript levels The observation of shifted cleavage products suggested in wild type. This suggests that the amount of amiRNAs that release of the PpFtsZ2-1-amiRNA/miRNA* du- is not limiting in any of the lines. Instead, it is plex from the precursor was not always precise, likely that the competition of natural miRNAs and consistent with observations on endogenous miRNAs amiRNAs in RISC loading determines the efficiency (Rajagopalan et al., 2006). Nevertheless, the Arabidop- of posttranscriptional silencing of the PpFtsZ2-1 and sis miR319a precursor can be used routinely for the PpGNT1 transcripts. expression of amiRNAs in Physcomitrella as the appar- The formation of macrochloroplasts in the PpFtsZ2-1- ent shift by 1 nt during the maturation of the amiRNA amiRNA lines indicated impeded plastid division may result in a mismatch at the 3# end of the miRNA, and resembled the phenotype of PpFtsZ2-1 knockout which is not affecting target mRNA cleavage (Schwab lines, which completely lack a functional transcript

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(Strepp et al., 1998). We therefore conclude that the useful for targeting groups of closely related genes remaining PpFtsZ2-1 transcripts in the amiRNA lines (Alvarez et al., 2006; Schwab et al., 2006). (3) AmiRNAs are not able to generate sufficient PpFtsZ2-1 protein to can be expressed from inducible or tissue-specific support proper plastid division. In addition, amiRNA promoters (Schwab et al., 2006) enabling the analysis expression in the transgenic lines seems to be stable of genes with essential functions that cannot be ana- over long time periods as we did not observe any lyzed by targeted gene disruption. Provided that other phenotypic reversion to wild-type plastids in the amiRNAs have a similar effect on the knock down of PpFtsZ2-1-amiRNA overexpression lines after 1 year of their cognate target genes in Physcomitrella as observed subculture. We anticipate that the described amiRNA in this study, they can be considered as an efficient expression system will result in similar silencing alternative tool to the targeted gene knockout approach efficiencies of any target gene and thus can be rou- for reverse genetics studies in Physcomitrella. tinely used as an alternative to the generation of knockout mutants in Physcomitrella. The efficient silencing of PpFtsZ2-1 and PpGNT1 by MATERIALS AND METHODS amiRNAs might be enhanced by the generation of transitive siRNAs, as we detected such siRNAs from Plant Material and Growth Conditions # the 3 cleavage products of the PpFtsZ2-1 and PpGNT1 Physcomitrella patens plants were cultured in modified liquid Knop me- mRNAs. Usually, transitive siRNAs are produced 21 21 21 dium containing 250 mg L KH2PO4, 250 mg L KCl, 250 mg L 21 21 from exogenous RNA sequences such as viruses or MgSO4·7H2O, 1,000 mg L Ca(NO3)2, and 12.5 mg L FeSO4·7H2O (pH 5.8) sense transgene transcripts (Baulcombe, 2004), but the or on solid Knop plates. Erlenmeyer flasks containing 400 mL of suspension culture were agitated on a rotary shaker at 120 rpm at 25°C under a 16-h-light/ formation of transitive siRNAs from miRNA-guided 22 21 8-h-dark regime (Philips TLD 25; 50 mM m s ). Liquid cultures were cleavage products appears to be the exception (Howell mechanically disrupted every week to maintain the plants in the protonema et al., 2007; Moissiard et al., 2007). Furthermore, tran- stage. Gametophore development was induced by transferring protonema sitivity was suggested not to be a major factor con- tissue to solidified Knop medium. tributing to amiRNA efficacy in previous studies, although this was inferred only indirectly from the Transformation of Physcomitrella Protoplasts lack of effects on sequence-related transcripts (Schwab Polyethylene glycol-mediated transformation of Physcomitrella protoplasts et al., 2006; Warthmann et al., 2008). Although we was performed according to standard procedures (Frank et al., 2005). Briefly, cannot exclude that siRNAs, which are produced from transformation was carried out using 25 mg of linearized plasmid DNA. amiRNA-mediated mRNA cleavage products, can af- Transformed protoplasts were cultivated for 24 h under standard conditions fect other genes not targeted by the original amiRNA, in the dark and were then transferred to light. After 10 d, the protoplasts were the PpFtsZ2-1 homolog PpFtsZ2-2, which shares high transferred to solid Knop medium. Three days later, regenerating plants were transferred to Knop medium supplemented with hygromycin (Promega). The identity in sequence stretches of the coding region selection lasted 2 weeks and was followed by a 2-week release period on Knop (Supplemental Fig. S3), seemed unaffected in PpFtsZ2-1- medium without antibiotic followed by another round of selection and amiRNA lines. We detected neither cleavage products release. Plants surviving the second round of selection were screened by by 5# RACE-PCR, indicating siRNA-mediated cleav- PCR to confirm integration of the DNA construct. age of PpFtsZ2-2 transcripts, nor reduced PpFtsZ2-2 steady-state mRNA levels, pointing to a posttran- Generation of Physcomitrella Lines Expressing scriptional silencing of this gene. Thus, even though AmiRNAs Targeting PpFtsZ2-1 and PpGNT1 transitivity might be more common in Physcomitrella, AmiRNAs targeting PpFtsZ2-1 (accession no. AJ001586; amiRNA, the specificity of posttranscriptional silencing is ap- 5#-TTCGTAATTAACGTGTCCGCG-3#)andPpGNT1 (accession no. AJ429143; parently sufficient to silence single members of highly amiRNA, 5#-TTCCAAATAATCAGGACGCTT-3#) were designed using the conserved gene families. Moreover, it might be pref- amiRNA designer interface WMD (Schwab et al., 2006; Ossowski et al., 2008). erable to design amiRNAs lacking perfect sequence The PpFtsZ2-1-amiRNA and PpGNT1-amiRNA sequences were introduced into # the Arabidopsis (Arabidopsis thaliana) miR319a precursor by overlapping PCR us- complementarity at the 3 end, as this reduces tran- ing the following primers. PpFtsZ2-1-amiRNA, miRNA-sense, 5#-GATTCGTA- sitivity (Moissiard et al., 2007). ATTAACGTGTCCGCGTCTCTCTTTTGTATTCC-3#;miRNA-antisense,5#-GAC- GCGGACACGTTAATTACGAATCAAAGAGAATCAATGA-3#;miRNA*- sense, 5#-GACGAGGACACGTTATTTACGATTCACAGGTCGTGATATG-3#; miRNA*-antisense, 5#-GAATCGTAAATAACGTGTCCTCGTCTACATATATAT- CONCLUSION TCCT-3#;primerA,5#-CCCGGGTGCAGCCCCAAACACACGCTC-3#;primer B, 5#-GGATCCCCCCATGGCGATGCCTTAAAT-3#. PpGNT1-amiRNA, miRNA- Compared to the conventional targeted gene knock- sense, 5#-GATTCCAAATAATCAGGACGCTTTCTCTCTTTTGTATTCC-3#; out approach in Physcomitrella, the expression of miRNA-antisense, 5#-GAAAGCGTCCTGATTATTTGGAATCAAAGAGAATC- # # amiRNA provides several advantages. (1) The gener- AATGA-3 ; miRNA*-sense, 5 -GAAAACGTCCTGATTTTTTGGATTCACAGG- TCGTGATATG-3#; miRNA*-antisense, 5#-GAATCCAAAAAATCAGGACGT- ation and molecular analysis of amiRNA overexpres- TTTCTACATATATATTCCT-3#; same primers A and B as described above. The sion lines is sped up as each regenerated transgenic plasmid pRS300 harboring the Arabidopsis miR319a precursor was used as line harboring an amiRNA expression construct and PCR template (Schwab et al., 2006). The resulting precursor fragments were should produce the desired mature amiRNA. (2) cloned into the pJET1.2 cloning vector (Fermentas) and sequenced. The modified ath-miRNA319a precursor DNA fragments were cloned into SmaIandBamHI Instead of the generation of multigene knockout lines, sites of the plant expression vector pPCV (Koncz et al., 1989) containing the which is experimentally difficult, but feasible (Hohe cauliflower mosaic virus 35S promoter, nos terminator, and hpt selection marker et al., 2004), amiRNAs are likely to be particularly cassette. Transgenic lines were analyzed by PCR to identify lines that had

Plant Physiol. Vol. 148, 2008 691 Khraiwesh et al.

integrated the amiRNA overexpression constructs using the primers 5#-TGA- Sequence data from this article can be found in the GenBank/EMBL data TATCTCCACTGACGAAAGGG-3# and 5#-GGATCCCCCCATGGCGATGCCT- libraries under accession numbers AJ001586 (PpFtsZ2-1), XM_001766723 TAAAT-3#. PCR primers for the amplification of the Physcomitrella control gene (PpFtsZ2-2), and AJ429143 (PpGNT1). EF1a were 5#-AGCGTGGTATCACAATTGAC-3# and 5#-GATCGCTCGAT- CATGTTATC-3#. The one-step isolation of genomic DNA was performed ac- cording to the method of Schween et al. (2002). Supplemental Data

The following materials are available in the online version of this article. Small RNA Blots Supplemental Figure S1. Analysis of Physcomitrella and Arabidopsis miR319 precursors. Total RNA was isolated from protonema using TRIzol reagent (Invitrogen) and separated in a 12% denaturing polyacrylamide gel containing 8.3 M urea Supplemental Figure S2. Impeded plastid division and formation of in Tris-borate/EDTA buffer. The RNA was electroblotted onto nylon mem- macrochloroplasts in PpFtsZ2-1-amiRNA overexpressors. branes for 1 h at 400 mA. Radiolabeled probes were generated by end labeling of DNA oligonucleotides with [g-32P]ATP using T4 polynucleotide kinase. Supplemental Figure S3. Nucleotide sequence alignment of PpFtsZ2-1 and The following probes were used. Antisense probe for PpFtsZ2-1-amiRNA, PpFtsZ2-2 coding regions. 5#-CGCGGACACGTTAATTACGAA-3#; antisense probe for PpGNT1-amiRNA, 5#-AAGCGTCCTGATTATTTGGAA-3#; detection of sense transitive PpFtsZ2-1 siRNAs, 5#-CCCCAGTGACGGAAGCGTTCAATCTTGCAGACGACATCCTT- CGGC-3#; detection of antisense transitive PpFtsZ2-1 siRNAs, 5#-GCCGAAG- ACKNOWLEDGMENTS GATGTCGTCTGCAAGATTGAACGCTTCCGTCACTGGGG-3#; detection of sense transitive PpGNT1 siRNAs, 5#-GTGAATTTCCTGCAGCATTTAG- We thank Andras Viczian for providing the pPCV expression vector, Enas ATGAAAATCCTCCCAAGACAAGG-3#; detection of antisense transitive Qudeimat for assisting with confocal laser-scanning microscopy, and Bjo¨rn PpGNT1 siRNAs, 5#-CCTTGTCTTGGGAGGATTTTCATCTAAATGCTGCAG- Voß and Isam Fattash for advice on miR319 precursor sequence analysis. GAAATTCAC-3#; detection of the U6snRNA control, 5#-GGGGCCATGC- Received August 13, 2008; accepted August 22, 2008; published August TAATCTTCTCTG-3#. Blot hybridization was carried out in 0.05 M sodium 27, 2008. phosphate (pH 7.2), 1 mM EDTA, 63 SSC, 13 Denhardt’s, 5% SDS. Blots were washed three times with 23 SSC, 0.2% SDS, and one time with 13 SSC, 0.1% SDS. Blots were hybridized and washed at temperatures 5°Cbelowthemelting temperature of the oligonucleotide. LITERATURE CITED

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Plant Physiol. Vol. 148, 2008 693 Supplemental Figure S1

A

5

’ 3

’ ppt-MIR319a

5

’

3 ’ ppt-MIR319b

5

’ 3 ’ ppt-MIR319c

5

’ 3

’ ppt-MIR319d

5

’ 3

’ ath-MIR319a

B miRNA*

miRNA

Supplemental Figure S1. Analysis of Arabidopsis and Physcomitrella miRNA319 precursors. A, Secondary structures of foldbacks of Physcomitrella patens miR319a-d precursors (ppt-MIR319a, b, c, d) and Arabidopsis thaliana miR319a precursor (ath-MIR319a). The mature miRNA is highlighted in green with uppercase letters.B, Multiple sequence alignment of Physcomitrella patens miR319a-d precursors (ppt-MIR319a, b, c, d) and the Arabidopsis thaliana miR319a precursor (ath-MIR319a). Black boxes with underlined sequences indicate miRNA/miRNA* sequences. Supplemental Figure S2

A PpFtsZ2-1-amiRNA lines

WT 1 2 3

Protonema - - - -100µm

Leaves - - - - B

Protonema -- - -

Leaves - -- -50-µm Supplemental Figure S2. Impeded plastid divison and formation of macrochloroplasts in PpFtsZ2-1-amiRNA overexpressors. A, Light microscopy from protonema and leaves of wild type (WT) and three PpFtsZ2-1-amiRNA overexpression lines (1-3) (Size bars: 100 µm). B, Confocal laser scanning microscopy from protonema and leaves of wild type (WT) and three PpFtsZ2-1- amiRNA overexpression lines (1-3) (Size bars: 50 µm). Red: chlorophyll autofluorescence in plastids. Supplemental Figure S3

FtsZ2-1 1 ATGGCGTTGTTTAGTGGGTGCTCGGGATGGGCGGGGCTCAAGGTGTCATC 50 |||||| |||| | | ||||||| | || ||| |||| |||| || FtsZ2-2 1 ATGGCGCTGTTAGGCAGTCGCTCGGGCTTGGTGGGCCTCAGGGTGAGCTC 50

FtsZ2-1 51 GCGAGTGGGTGGGGAGGCTTGCAGAA-----CCCCCCCCGTT-GTTCACT 94 ||||||||| |||||| | |||| ||| | || | || || FtsZ2-2 51 GCGAGTGGGCGGGGAGAGCAGTAGAATAGTGCCCGCGACGAGAGATCGCT 100

FtsZ2-1 95 GCAGCATGCATTCTAGGTCAAGCGTTCGAGCTCTACGCCGAATCGACCGA 144 | || |||| | ||| | ||| ||| || | || || | || | FtsZ2-2 101 TCTGCGTGCACTTGAGGCCGAGCACTCGGGCGCATCGTCGTCTGGATAGG 150

FtsZ2-1 145 GCTTTGAGTAATGGGGGTCTTTGCAATTTTGGAGAGAGGGACTTGTTGGC 194 || | | |||| | ||||||||| | | |||||||||| |||| FtsZ2-2 151 ACTGTAGGGAATGAGAGTCTTTGCACTCCCCGGGAGAGGGACT---TGGC 197

FtsZ2-1 195 TTTGGAAGCAAAATC---GCCTTTGCGATGTGAACCCCCCTCGA------235 | ||| | |||| || | |||| || | | || FtsZ2-2 198 TGCGGAGCCTAAATTCTTGCACACGGGATGGGAGTCTTCTTCTTCTTCTT 247

FtsZ2-1 236 ------GCGTGATGCGGAATCCTGTCATGGCATTTGAAGGAAGC 273 ||| || ||| || |||| |||||||| ||| | FtsZ2-2 248 CTTCTTCTTCTTGCGAGACTGGGATACCCGTCACGGCATTTGGAGGTAAT 297

FtsZ2-1 274 GGAGACGACACTGGAAGTTATAACGAAGCGAAAATTAAAGTAATAGGGGT 323 |||||||| || |||| || || |||||||||||||| ||||| || FtsZ2-2 298 GGAGACGAATATGAGAGTTCCAATGAGGCGAAAATTAAAGTGATAGGCGT 347

FtsZ2-1 324 CGGAGGTGGGGGTTCCAACGCCGTAAACCGAATGCTTGAGAGCGAGATGC 373 || || ||||||||||||||||| |||||||||||||||||||| |||| FtsZ2-2 348 GGGGGGCGGGGGTTCCAACGCCGTCAACCGAATGCTTGAGAGCGAAATGC 397

FtsZ2-1 374 AAGGGGTAGAATTTTGGATCGTGAATACTGATGCGCAGGCTATGGCCTTG 423 |||| || ||||| ||||| |||||||| ||||| ||||| ||||| ||| FtsZ2- 398 AAGGTGTGGAATTCTGGATTGTGAATACGGATGCTCAGGCAATGGCGTTG 447

FtsZ2-1 424 TCCCCTGTTCCGGCTCAGAATCGTCTGCAGATTGGGCAAAAATTGACGAG 473 || || ||||||||||||||||||||||||||||| || ||||||||| | FtsZ2-2 448 TCTCCGGTTCCGGCTCAGAATCGTCTGCAGATTGGTCAGAAATTGACGCG 497

FtsZ2-1 474 AGGTCTGGGGGCGGGCGGGAATCCAGAAATAGGGTGTAGTGCTGCGGAAG 523 ||||||||||||||| || ||||| ||||||||||||||||| ||||||| FtsZ2-2 498 AGGTCTGGGGGCGGGTGGTAATCCGGAAATAGGGTGTAGTGCCGCGGAAG 547

FtsZ2-1 524 AGAGCAAAGCTATGGTGGAAGAAGCCCTACGCGGAGCTGACATGGTTTTC 573 |||||||||||||||||||||||||| ||||||||||||||||||||||| FtsZ2-2 548 AGAGCAAAGCTATGGTGGAAGAAGCCTTACGCGGAGCTGACATGGTTTTC 597

FtsZ2-1 574 GTAACGGCGGGTATGGGTGGCGGCACTGGCAGCGGTGCAGCACCAATAAT 623 || || ||||| |||||||| ||||||||||||||||| |||||||| || FtsZ2-2 598 GTTACAGCGGGCATGGGTGGTGGCACTGGCAGCGGTGCTGCACCAATCAT 647

FtsZ2-1 624 TGCGGGTGTGGCGAAGCAGTTGGGAATTCTTACTGTAGGAATAGTTACTA 673 ||| ||||| |||||||| |||||||||||||| || |||||||| |||| FtsZ2-2 648 TGCTGGTGTAGCGAAGCAATTGGGAATTCTTACCGTGGGAATAGTAACTA 697

FtsZ2-1 674 CTCCTTTCGCCTTTGAAGGGCGGAGACGAGCTGTCCAAGCCCACGAGGGT 723 | ||||| ||||||||||||||||||||| | || ||||| ||||| || FtsZ2-2 698 CGCCTTTTGCCTTTGAAGGGCGGAGACGATCCGTTCAAGCTCACGAAGGC 747

FtsZ2-1 724 ATTGCAGCTCTCAAAAATAACGTGGACACGTTAATTACGATTCCAAACAA 773 || || |||||||||||||| || ||||| ||||||||||| |||||||| FtsZ2-2 748 ATCGCGGCTCTCAAAAATAATGTTGACACTTTAATTACGATACCAAACAA 797

FtsZ2-1 824 CAAACTTTTGACTGCAGTTGCGCAGTCTACCCCAGTGACGGAAGCGTTCA 823 ||| ||||||||||||||||||||||||||||| ||||||||||| |||| FtsZ2-2 798 CAAGCTTTTGACTGCAGTTGCGCAGTCTACCCCCGTGACGGAAGCATTCA 847

FtsZ2-1 824 ATCTTGCAGACGACATCCTTCGGCAGGGAGTGCGGGGTATTTCAGATATT 873 ||||||| || ||||||||||||||||||||||||||||||||||||||| FtsZ2-2 848 ATCTTGCCGATGACATCCTTCGGCAGGGAGTGCGGGGTATTTCAGATATT 897

FtsZ2-1 874 ATCACGGTCCCTGGGCTGGTTAACGTAGATTTTGCCGACGTGCGGGCGAT 923 ||||| || ||||| || |||||||| || ||||| || ||||||||||| FtsZ2-2 898 ATCACTGTTCCTGGTCTCGTTAACGTGGACTTTGCGGATGTGCGGGCGAT 947

FtsZ2-1 924 CATGGCTAATGCAGGATCATCTTTGATGGGCATAGGGACCGCCACAGGTA 973 |||||| ||||||||||||||||||||||| || || ||||| ||||| | FtsZ2-2 948 CATGGCCAATGCAGGATCATCTTTGATGGGAATTGGAACCGCTACAGGGA 997

FtsZ2-1 974 AGTCAAGAGCTAGAGAAGCAGCATTGAGCGCAATCCAATCTCCTCTATTG 1023 |||||| ||||||||| ||||||||||| || || || ||||| | ||| FtsZ2-2 998 AGTCAAAAGCTAGAGAGGCAGCATTGAGTGCCATTCAGTCTCCATTGTTG 1047

FtsZ2-1 1024 GATGTGGGTATTGAGCGAGCCACAGGGATAGTCTGGAATATCACTGGGGG 1073 ||||||||||||||||||||||||||||| || |||||||| |||||||| FtsZ2-2 1048 GATGTGGGTATTGAGCGAGCCACAGGGATCGTTTGGAATATTACTGGGGG 1097

FtsZ2-1 1074 AAGCGACATGACTCTCTTTGAGGTAAATGCTGCAGCAGAGGTGATTTATG 1123 |||||||||||| |||||||| || ||||||||||||||||| || |||| FtsZ2-2 1098 AAGCGACATGACCCTCTTTGAAGTCAATGCTGCAGCAGAGGTAATCTATG 1147

FtsZ2-1 1124 ATTTGGTCGATCCCAACGCAAATCTTATTTTTGGAGCCGTAGTAGACGAA 1173 ||||||| ||||| ||||||||||||||||| |||||||||||||||||| FtsZ2-2 1148 ATTTGGTGGATCCTAACGCAAATCTTATTTTCGGAGCCGTAGTAGACGAA 1197

FtsZ2-1 1174 GCACTTCATGGCCAAGTTAGTATAACTTTGATAGCAACAGGATTTAGTTC 1223 |||||||||| |||| |||| ||||| || ||||||||||| |||||||| FtsZ2-2 1198 GCACTTCATGACCAAATTAGCATAACCTTAATAGCAACAGGGTTTAGTTC 1247

FtsZ2-1 1224 TCAAGATGAACCTGATGCGCGTAGTATGCAAAATGTGAGTCGTATTTTGG 1273 ||||||||| |||||||| || |||||||| ||| ||||| | |||| FtsZ2-2 1248 TCAAGATGATCCTGATGCACGGAGTATGCAGTATGCAAGTCGCGTATTGG 1297

FtsZ2-1 1274 ATGGACAAGCTGGTCGATCACCGACAGGTTTATCTCAAGGCAGCAATGGC 1323 | || ||||||||||||||| ||| | | ||| | ||| ||||| || FtsZ2-2 1298 AGGGTCAAGCTGGTCGATCATCGATGGCCTCATCCCGAGGTGGCAATAGC 1347

FtsZ2-1 1324 TCTGCGATCAATATACCAAGTTTCTTAAGGAAGCGAGGCCAGACACGTCA 1373 ||| |||| || ||||||| ||||||| | |||||||| || | FtsZ2-2 1348 TCTACGATTAACATACCAAATTTCTTACGAAAGCGAGGGCAAAGG----- 1392

FtsZ2-1 1374 TTAA 1377 || FtsZ2-2 1393 –TAG 1395

Supplemental Figure S3. Nucleotide sequence alignment of PpFtsZ2-1 and PpFtsZ2-2 coding regions. The highly conserved central region with 89% sequence identity is highlighted in yellow. The PpFtsZ2-1-amiRNA target site is highlighted in green. Chapter IV Appendices

4 Chapter IV: Appendices 4.1 Flow cytometric measurements (FCM) For ploidy level determination 10-20 mL of protonema liquid culture were used. The plant material was harvested four to seven days after the last sub-culturing, resuspended with 2 mL of DAPI buffer, and chopped up with a razor blade in a . The solution was filtered through a sieve of 30 μm pore size prior to measuring the fluorescence intensity with a PAS cell analyser (Partec, Münster) using a 100 W high-pressure mercury lamp for detection. The ploidy level was derived from the resulting histograms. For Physcomitrella, a prominent peak at a fluorescence intensity of about 200 indicates a haploid genotype, whereas signals at fluorescence intensities of about 400 represent a diploid plant.

Figure 1: Flow cytometric analysis. Flow cytometric histograms of protonema from WT and ΔPpDCL1b mutants (1-4) grown in parallel in Knop medium. The abscissa represents the channel numbers corresponding to the relative fluorescence intensities of analyzed particles (linear mode), while the ordinate indicates the number of events counted.

136 Chapter IV Appendices

4.2 Physcomitrella patens DCL1b (PpDCL1b) mRNA

LOCUS DQ675601 6052 bp mRNA linear PLN 03-JUL-2008 DEFINITION Physcomitrella patens Dicer-like 1b protein (DCL1b) mRNA, complete cds. ACCESSION DQ675601 VERSION DQ675601.1 GI: 110520366 KEYWORDS SOURCE Physcomitrella patens ORGANISM Physcomitrella patens Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Bryophyta; Moss Superclass V; Bryopsida; Funariidae; Funariales; Funariaceae; Physcomitrella REFERENCE 1 (bases 1 to 6052)

AUTHORS Khraiwesh,U B.U , Seumel, G.I., Reski, R. and Frank, W. TITLE Direct genetic evidence for the involvement of a Dicer-like gene in microRNA mediated target cleavage in plants. JOURNAL Unpublished REFERENCE 2 (bases 1 to 6052)

AUTHORS Khraiwesh,U B.,U Seumel, G.I., Reski,R. and Frank,W. TITLE Direct Submission JOURNAL Submitted (06-JUN-2006) Plant Biotechnology, University of Freiburg, Schaenzlestrasse 1, Freiburg 79104, Germany FEATURES Location/Qualifiers Source 1..6052 /organism="Physcomitrella patens" /mol_type="mRNA" /db_xref="taxon: 3218" Gene 1..6052 /gene="DCL1b" CDS 607..5694 /gene="DCL1b" /codon_start=1 /product="Dicer-like 1b protein" /protein_id="ABG74922.1" /db_xref="GI:110520367" /translation="MRRARSVRLENGLNGKDEGEEKARTYQLEVLAQAKV KITVAFLDTGAGKTLIAILLMKHKHQVLREYDKRMLALFLVPKVPLVYQQADVIRNGTKFSVGHYCGEMGSRFWD ARGWQREFDTKDVFVMTAQILLNILRHSIVKMEAIHLLILDECHHAVKKHPYSLVMSEFYLMTPKDKRPCVFGMI ASPVNLKGVSNQEDCAIEIRNLESKLDSIVCTIRDRKELEKHVPLPSETMILYDKPALLFSLRKERKQMEATVEK AANASVRRSKWKCMGARDAGAKEELQLVYSVAERTESDGAASLSQKLRAITYALDELGQWCAYKVSLGYLTSLHN DERVNHQLDVKFQKLYLKKVCTLLRCSLREGAAGWEVPAEIGESEGDKAQDPMDVEEGSFLTLVSVGEHLDEILG AAVADGKVTPKVQSLIKVLIGYQHTDDFRAIIFVERVWVGVTLCRSLQSCPSLKFVKCASLIGHNNNQDMPTRQM QETISKFRDGRVTLLVATSVAAEGLDIRQCNVVIRFDLAKTVLAYIQSRGRARKPGSDYILMLERGNLQHEAFFR NAKNSEETLRKEAIERTDLGEKRENAILASIDIGEGEIYQVPATGAVVSMNSAVGLIHFYCSQLPSDRYSLLRPE FIMNKIEDQRGAIRYSCRLQLPCHAPFEAVEGPECNSMRGAQQSCVLEGLQKMHEMGAFTDMLLSNKGSREEAAK LEGSEEGESLPGTSRHREYYPEGIADILKGDRIVAEKDSDTKEGSKVLVFMYTVKCENVGFSRDSLLTETSDFTL LVGQQLHDQVLTMTINLFVANPTLLITMSWKKRDLDCSNSQLTELKSFHVRLMSIVLDVNVEPATTRWDPAKAYL FAPVLHKDASDPKDLVDWVVMRRTIETDSWSNPLQRASPDVNLGTDERALGGDRREYGFGKLRCSLAFGQGAHPT YGARGAKAQFDVVKATGLLPTSDMVEETTVQEVPPEGKLLIVDGFVEVEVLVGRIVTAVHSGKRLYVDSVRFDMT ADSSFPQKDGYLGPLEYTSYADYYKQKYGVELVCKKQPLLRGRGVSHCKNLLSPRFETSGDSLDALDKTYYVMLP PELCLIHPLPGSLVRGAQRLPSVMRRVESMLLAIQLKHQIDYPIAASKVALTAASGQETSYERAELLSDAYLEWV VSHRLFLKFPSKHEGQLTRMRQKIVSNSVLYQHALEKGLQSYIQADRFAPSRWAAPGVPPAFDEDLRDGDDSDKE SKPEVEREVVEIVGEEGEIVKELNTESENMEDGEIEGDSGSYRVLSSKTLADVVEVFMGMYYVEGGGEAATHFMN WVGIPVEDDVETDLATGGCQVPETVMRSIDFSSLQKNVGHEFRERSLLVEAITHASRPSLGVPCYQRLEFVGDAV LDHLITRYLFFKYTNLPPGRLTDLRAAAVNNENFARVAVKHSYHLHLRHGSTALETQIRNFVNDIHSELDKPGVN SFGLGDFKAPKVLGDIFESIAGALFLDARLDTHQVWKVFEPLLQPMVSPETLPIHPVRGLQERCQQEAEGLEYKV SRAESVATVEVYVDGVQIGSTQSAQKKMAQKLGARNALVKLKDKEVIKVKAEAENGDLNAGKSSKNGHTNFTRKT INDLCLKRQWPMPQYKCVLESGPAHAKKFTLSVRVLTTTDGWTEECVGEPMASVKKAKDSAALVLLATLRRSYPL RNNIIDC"

137 Chapter IV Appendices

ORIGIN

1 aacgcgggga ggtggagaag tggctctttt tctagcacta tccctctcga ggagcggagg 61 tgaattgtca agtaaggaac gattcaatta gagcgccgcg aattgattga attacgagtg 121 gtttgatgga gcaggtgggg agcggagggg tgaaatgcgc aagcaagggg tgtacatgac 181 gaggctcgtg atcgagggaa gaggagccgg gagagcgacg ggagagttgg cgaggctgga 241 atgaagggta gaggtggtgg tggtgtgaga ggattcgtca ggaggagcag ggagaggcgg 301 tctgtgtcga ggtctaggtc agagagaggc ctaaagagag ggcggagatg acgggagaca 361 gcagggaggg gaggaagcga aggtcggctt tcgaatatgc ctttgatgat aggcgcgatg 421 agaagagggg gcggcatttc catgaattgg gagattatcg agattaccac ggttccatag 481 tgacgcgcga cagaccttgg attggcagag gggattgcga ccggcgatca aggatggagg 541 ctcgggagcg atttgttgcg tcgtctcgtg atcgggagag agagtgggag cggtcgcgtg 601 tttgaaatga gaagggcgcg aagcgtgaga ctggagaatg ggctgaatgg gaaggatgag 661 ggtgaggaga aggcgcggac ttatcagctt gaagtgctgg ctcaggcgaa ggtgaagatt 721 acggttgcat ttctagacac gggcgctggg aagaccctaa ttgcgattct gttgatgaag 781 cataagcacc aggtgttgcg ggagtatgac aagcgtatgc tcgctctgtt cctcgtccct 841 aaagtaccgc tcgtctacca gcaagcagat gtgattcgca acggcacaaa gtttagtgtt 901 ggtcactact gcggagagat gggatcaaga ttttgggacg cccgagggtg gcagcgagaa 961 tttgatacca aagatgtttt tgtaatgacc gcacagattc ttttgaacat ccttaggcat 1021 agcattgtaa aaatggaagc cattcatcta cttattctcg atgagtgcca ccatgccgtg 1081 aagaaacatc cctattcttt ggtgatgtct gaattctatc ttatgacacc taaagataag 1141 cgaccgtgtg tctttgggat gatagcatcg cctgtgaacc tcaaaggggt atcaaaccag 1201 gaagattgtg caatagagat tcgaaattta gaaagcaagt tggactcgat agtgtgtaca 1261 atcagggatc ggaaagagct cgaaaagcac gtgcctttgc cgtcagagac aatgattctg 1321 tacgataagc cggccttgct tttctcgttg cggaaagaga gaaaacagat ggaggccact 1381 gtagaaaagg ctgctaatgc aagtgtcaga cgcagcaaat ggaaatgcat gggcgctcgg 1441 gatgcgggtg ctaaagagga actgcaactt gtgtacagtg tcgcggagag aacggaaagc 1501 gatggcgcag ctagtctttc tcaaaagctt agagccatta cctatgcact tgatgaatta 1561 ggtcaatggt gtgcttacaa ggtctcgctg ggatatctga caagtcttca taatgatgaa 1621 agggttaatc atcagttaga cgtgaagttt caaaagttgt acttgaagaa ggtttgtact 1681 cttctgcgat gcagtctacg tgaaggtgct gcagggtggg aggtacctgc tgaaattgga 1741 gagtctgagg gcgataaagc acaagatcca atggatgtgg aagaaggaag cttcctgact 1801 ctcgtatcag tgggtgaaca tttggatgag attcttgggg ccgctgtagc agatggaaaa 1861 gtgactccga aggtgcagtc tttaattaag gttttaatag gttatcagca tacggatgat 1921 ttccgagcta ttatatttgt ggagcgagtc tgggtgggtg ttacgctttg caggtctttg 1981 cagagttgcc cttcattgaa gtttgtgaaa tgtgccagtc tgatagggca caacaataac 2041 caagacatgc cgacacggca gatgcaggag actatttcca agtttcgaga tggacgggtg 2101 acgttgctgg tggctacaag cgtggccgca gagggattag atattcgcca atgtaatgtg 2161 gtcatccgtt ttgatcttgc taaaaccgtg ttagcctaca tccagtctcg tggtcgtgct 2221 cggaagcctg gttcagatta tattttaatg cttgagagag gaaatctgca acatgaggcg 2281 ttttttcgga atgcaaaaaa tagtgaggag actttacgga aggaggctat tgaaagaact 2341 gatctgggtg aaaaacggga gaatgcgatt ctggcctcca ttgacattgg ggaaggggag 2401 atttaccagg tgccagccac tggggcagtc gtgagcatga actcagctgt aggtcttatt 2461 cacttctact gctctcagct tcccagtgac aggtattctc tcttgcgtcc tgagttcatt 2521 atgaacaaaa ttgaggatca aagaggtgct ataagatact cgtgcagact gcagttgcct 2581 tgccatgctc cgtttgaagc tgtggaaggc ccagaatgta attctatgcg aggagcgcag 2641 cagtcctgtg tgcttgaagg cttgcaaaaa atgcacgaaa tgggggcatt cacggacatg 2701 ctattatcta ataaaggaag tagggaagaa gctgctaagt tggagggtag tgaagaggga 2761 gagtctcttc ctggcacatc ccgtcatcga gaatattatc cagaggggat tgcagatatt 2821 ctgaagggcg atcggatagt ggctgagaaa gattcggata caaaggaagg cagcaaggtg 2881 ctcgtattca tgtacacggt gaagtgtgaa aatgttggct tctcgagaga cagccttttg 2941 accgagacat cagactttac cttacttgtc ggccaacagc ttcatgacca ggtgttaacc 3001 atgacaataa atctttttgt cgcaaacccg actttactga tcacgatgag ctggaaaaag 3061 agggatttgg attgctctaa ttcccagttg actgagctca agagttttca tgtgaggctt 3121 atgagcattg ttttagacgt aaatgtcgag ccggcaacaa ctcgttggga tcccgccaag 3181 gcgtatctct ttgctccagt tctgcataag gatgcctccg atcctaaaga cttggtggac 3241 tgggtcgtta tgagaaggac gatcgagact gattcatgga gtaatcccct ccagcgcgca 3301 tcacctgatg tgaacttggg gactgacgaa cgtgctcttg gtggggatcg tagagagtac 3361 gggtttggaa aactgcgatg tagtctggcc tttgggcagg gagcgcatcc aacgtatggt 3421 gctcgtggcg ctaaagctca atttgatgtt gtgaaagcca caggtctact tcctacctca 3481 gacatggtgg aggagacaac tgtgcaggaa gtacctcccg agggtaagct gttgatagtg

138 Chapter IV Appendices

3541 gatggttttg ttgaagttga agtattggtg ggaaggattg ttactgcggt gcattctggg 3601 aagaggcttt atgtggattc ggtgcgcttt gacatgacag ccgacagctc ttttcctcaa 3661 aaggatggat accttggtcc actggaatac acatcgtatg cggattatta caaacaaaag 3721 tacggtgtcg agttggtttg caagaaacag cctctgttga ggggtcgtgg ggtttctcat 3781 tgcaaaaatt tattgtcgcc acgttttgag acctctggcg actctctgga tgccttggat 3841 aagacgtatt atgtgatgct gccacctgag ctttgcctta tacatcctct tccgggatcc 3901 ttggtgagag gcgcacaaag attgccatcg gtcatgagac gtgtagagag catgttgctt 3961 gccatacaac taaagcacca aatcgattac cctattgctg cttcgaaggt agcgttgacg 4021 gctgcgtctg gtcaagagac attcagctat gagcgtgcag agcttttaag cgacgcgtac 4081 ctcgaatggg ttgttagtca tcgattgttc ctgaagttcc ctagtaaaca tgaggggcag 4141 cttacacgta tgagacagaa aattgtcagc aattccgttc tgtatcaaca tgccctagag 4201 aaaggtcttc agagttacat tcaggccgac cgctttgcac cgtcccggtg ggccgcaccg 4261 ggagtgcctc ctgcattcga tgaggacttg agagatggcg atgattcgga taaggagtcg 4321 aaacctgaag ttgaaagaga agtagtggag attgtcggtg aggaaggtga aattgttaag 4381 gaactaaata cagaaagtga aaatatggaa gacggtgaaa ttgaaggtga ttccggttcc 4441 tatcgagtgc tttcgagtaa aaccttggca gacgtggtag aggtattcat gggaatgtat 4501 tatgtggagg ggggggggga ggctgctact cacttcatga actgggtagg cattcctgtg 4561 gagtttgatg acgtggagac agacttagcc acaggtggct gccaagttcc tgaaaccgtt 4621 atgcggagca tagacttttc atcattacaa aaaaacgttg gccatgaatt tcgtgaacga 4681 agtttattgg tagaggccat cacgcacgcg tctcgaccat cgttgggagt tccttgctac 4741 caaaggctgg agtttgtggg ggatgccgtg ttggaccatc tgattacacg ttatctattc 4801 tttaaatata ctaatttgcc cccaggtagg ttgaccgatt tgcgagctgc tgcagtgaat 4861 aacgaaaatt tcgcacgtgt tgctgtgaag cactcgtatc atcttcattt gcggcatggt 4921 tcaaccgctt tagaaactca gattcgcaat ttcgtgaatg atatacactc ggagttagac 4981 aagcctggag tgaactcttt tggactaggg gattttaagg cccctaaagt gctgggtgat 5041 attttcgaat ccattgcagg cgctctattc ctggacgctc gtcttgacac acaccaagtg 5101 tggaaggttt ttgagccttt gttgcagccc atggtgtccc cagagacatt gccgatccat 5161 ccagtacgag ggttgcagga gcgttgtcaa caagaagctg aaggtctgga gtacaaagtg 5221 tctcgtgcag agagtgttgc gaccgtggag gtgtatgtag acggtgtaca gataggttct 5281 acgcaaagtg ctcagaagaa aatggcccaa aaattaggtg ctcgtaatgc gttggtcaaa 5341 ttgaaggata aggaggtgat caaagtgaaa gctgaggcag agaatggtga cttgaacgct 5401 ggaaaatcga gcaagaacgg tcacactaac ttcactcgca aaacaattaa cgacctttgt 5461 cttaagagac agtggccgat gccacagtac aaatgcgttc tggagagcgg accagcgcat 5521 gctaagaagt ttacgctctc tgtacgggtt ctgaccacca ctgatggatg gaccgaagaa 5581 tgtgttgggg agcctatggc gagtgtgaag aaagctaagg actctgcagc tcttgtactt 5641 ttggctactt tgagacgatc atatcctttg cgtaataata ttatagactg ctaaaatgac 5701 ccaaattgat cagaaaacat acagaactac ataccggcct gtgggtgctt caggttcaac 5761 atatccgtgc cccgtcaaca taaatttgtg aatgcacaaa tcacaaggtt tggatagcac 5821 tagccagcgc cagttcgttc aaggagctgc aggccagctc agccctcgtt ttataccttt 5881 catgatatgt tttgtctttt tggatcaaat tgtgagagac agagcacagg tcagtatacc 5941 gttcaaagaa agtagtgagt ttttgtaccg taagacagct gcgtctctcc ctcttaattt 6001 tgtctttcat ttttcagttt ttcagagaaa aaaaaaaaaa aaaaaaaaaa aa

139 Chapter IV Appendices

4.3 DNA vectors

Origin of DNA vectors that were used for cloning and transformations Name Backbone Insert Reference Invitrogen, pCR4-TOPO pCR4-TOPO PpDCL1b cDNA region; nptII Karlsruhe PpFtsZ2-1-amiRNA and PpGNT1- pPCV ---- amiRNA sequences with A. Koncz, C. et al., (Figure 2) thaliana miRNA319a Precursor 1989

Figure 2: pPCV plant overexpression vector containing a double 35S promoter, nos terminator and hpt selection marker cassette.

140 Chapter IV Appendices

4.4 Genes downregulated in ΔPpDCL1b mutants

Sequence ID Cosmoss Annotation Fold change EST BJ165956 **: Homolog of OSJNBa0003O19.20|putative MYC transcription factor -1.46 BJ172212 ***: Q7XN04 OSJNBb0038F03.7 protein. -1.49 BJ200093 **: Homolog of Similar to phytochrome and flowering time 1 protein -1.59 BJ200754 ***: Q8RYB8 Aldehyde dehydrogenase Aldh21A1. -1.82 BJ579811 **: Homolog of Roc1-related|o_sativa|chr_8|P0020B10|4196 -1.59 BJ580674 **: Homolog of (AB032182) protein PpHB10 [Physcomitrella patens] -1.80 BJ583348 **: Homolog of aldehyde dehydrogenase, putative|o_sativa|chr_11|OSJNBa0052O08|5272 -3.15 BJ583460 **: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0406G08|2927 -2.21 BJ587267 **: Homolog of Helicase conserved C-terminal domain, -1.60 BJ589432 ***: Q84WK0 At4g33880. -1.66 BJ601044 **: Homolog of (68417.m02544 vernalization 2 protein VRN2) -1.43 PP_10059_C1 ***: Q8SA80 Disease-resistent-related protein. -1.34 PP_10115_C2 **: Homolog of 68416.m05958 protein kinase family protein contains eukaryotic protein -1.50 kinase domain PP_10382_C1 ***: PSAG_ARATH Photosystem I reaction center subunit V, chloroplast precursor -3.18 PP_10385_C1 ***: Q6Z5T6 Putative intensifier. -1.58 PP_10479_C1 ***: Q8S1X3 Putative SUVH4. -1.90 PP_10658_C1 **: Homolog of AP2 domain, putative|o_sativa|chr_6|P0021C04|3631 -1.50 PP_1073_C1 **: Homolog of AT3g02790/F13E7_27|o_sativa|chr_6|P0621D05|1946 -1.70 PP_10745_C1 **: Homolog of (AB084898) mitochondrial aldehyde dehydrogenase [Sorghum bicolor] -1.45 PP_10817_C1 not annotated Physcomitrella patens -1.39 PP_11302_C1 ***: Q8X1E7 Histidine kinase. -2.11 PP_11331_C1 ***: Q9M551 Polyubiquitin. -1.86 PP_11112_C1 ***: Q948P1 Peroxisomal ascorbate peroxidase. -2.22 PP_11390_C1 ***: Q41067 Polyubiquitin. -1.66 PP_1146_C3 ***: Q9LV44 Similarity to signal peptidase. -1.60 PP_11513_C1 ***: Q9M077 Putative serine/threonine protein kinase. -1.87 PP_11795_C1 ***: Q93V58 Putative serine threonine-protein kinase. -1.35 PP020018263R **: Homolog of 68418.m05603 YEATS family protein contains Pfam domain -1.50 PP_12005_C1 **: Homolog of Neutral/alkaline nonlysosomal ceramidase|o_sativa|chr_1|P0501G01|2708 -1.58 PP_12101_C1 ***: Q9ARE4 ZF-HD homeobox protein. -1.65 PP_12167_C1 **: Homolog of (AY034888) aldehyde dehydrogenase Aldh21A1 [Tortula ruralis] -4.19 PP_12301_C1 contains: (COIL:coil) -2.40 PP_12301_C1 contains: (COIL:coil) -2.06 PP_12365_C2 ***: Q9FH40 Similarity to unknown protein (TAF14b) (Hypothetical protein At5g45600). -1.35 PP_12367_C1 ***: Q8W314 Putative dehydratase/deaminase. -1.71 PP_125_C1 **: Homolog of (68415.m03211 plectin-related contains -1.25 PP_12576_C1 **: Homolog of SNF2 family N-terminal domain -1.70 PP_12599_C1 **: Homolog of expressed protein|o_sativa|chr_4|OSJNBa0013K16|5362 -2.34 PP_12681_C1 ***: Q7XPK1 OSJNBa0087O24.9 protein. -2.51 PP_12858_C1 **: Homolog of 68417.m05252 expressed protein contains Pfam profile PF04784 -1.74 PP_12940_C1 ***: Q7Y1Z3 Putative small nuclear ribonucleoprotein Prp4p. -1.42 PP_13101_C1 ***: Q43303 Histone H3 (Fragment). -1.43 PP_13149_C1 ***: Q9ZPN6 Transcription factor MYC7E (Fragment). -1.79 PP_13160_C1 ***: RL71_ARATH 60S ribosomal protein L7-1. -1.38 PP_13170_C1 ***: Q9SII9 Putative ubiquitin protein. -1.42 PP_13256_C1 **: Homolog of expressed protein|o_sativa|chr_2|P0506A08|3865 -1.85 PP_13508_C1 contains: Protein kinase-like(InterPro:IPR011009,SUPERFAMILY:SSF56112) -1.58

141 Chapter IV Appendices

PP_13734_C1 **: Homolog of 68415.m02009 ARID/BRIGHT DNA-binding domain-containing protein -2.23 contains PP_13750_C1 **: Homolog of (AB015183) transcription factor Vp1 [Mesembryanthemum crystallinum] -1.91 PP_13846_C1 ***: Q8LB52 Scarecrow-like protein. -1.37 PP_1387_C1 ***: Q7X9V3 Nuclear shuttle interacting protein. -1.51 PP_14366_C1 **: Homolog of (Transcription factor E2F/dimerisation partner TDP) -1.74 PP_14581_C1 **: Homolog of (AY077758) WRKY transcription factor 1 [Physcomitrella patens] -1.75 PP_14609_C1 ***: Q39183 Serine/threonine protein kinase (Protein kinase (EC 2.7.1.37) 5) (AT5g47750/MCA23_7). -1.56 PP_14674_C1 "**: Homolog of (68417.m04184 acid phosphatase class B family protein similar to acid -1.58 phosphatase PP_1468_C1 not annotated Physcomitrella patens -1.92 PP_15181_C1 ***: O82527 Polyubiquitin (Fragment). -1.46 PP_1665_C2 ***: RL71_ARATH 60S ribosomal protein L7-1. -2.11 PP_17120_C1 **: Homolog of 68417.m02122 myb family transcription factor contains Pfam profile -1.55 PP_1761_C1 contains: Nascent polypeptide-associated complex -1.89 NAC(InterPro:IPR002715,PFAM:PF01849) PP_18023_C1 **: Homolog of (68415.m03211 plectin-related contains -2.11 PP_18168_C1 ***: SU91_HUMAN Histone-lysine N-methyltransferase, H3 lysine-9 specific 1 -1.63 PP_11285_C4 **: Homolog of (AB112672) auxin response factor 2 [Cucumis sativus] -1.80 PP_18237_C1 ***: ARP_ARATH Apurinic endonuclease-redox protein (DNA-(apurinic or apyrimidinic site) -1.73 PP_18520_C1 "**: Homolog of ((AP004849) putative CCR4-NOT transcription complex -1.56 PP_2007_C1 ***: Q7XU22 OSJNBb0034G17.2 protein (Transcription factor DREB). -1.95 PP_2015_C1 ***: Q8LKS8 Early drought induced protein. -1.38 PP_2094_C3 ***: Q8RXD3 ABI3-interacting protein 2. -1.71 PP_2103_C2 ***: Q8GRK2 Somatic embryogenesis receptor kinase 1. -1.43 PP_2104_C1 **: Homolog of Similar to chloroplast DNA-binding protein PD3|o_sativa|chr_2|P0017C12|3841 -1.74 PP_214_C9 ***: AHM7_ARATH Potential copper-transporting ATPase 3 (EC 3.6.3.4). -2.08 PP_224_C1 **: Homolog of 68417.m02632 lil3 protein identical to Lil3 protein [Arabidopsis thaliana] -1.34 PP_2272_C1 ***: O81077 Putative cytochrome P450. -40.91 PP_2294_C1 **: Homolog of expressed protein|o_sativa|chr_6|OSJNBa0019F11|6897 -1.59 PP_2113_C3 ***: Q9FIX3 Gb|AAD30619.1. -2.07 PP_2320_C1 ***: Q852K5 Putative zinc finger protein (Putative zinc finger transcription factor ZFP38). -1.65 PP_2360_C1 ***: AHM1_ARATH Potential cadmium/zinc-transporting ATPase HMA1 (EC 3.6.3.3) (EC -1.64 3.6.3.5). PP_233_C4 ***: Q943L1 Putative Ubiquitin carrier protein UBC7. -1.96 PP_2537_C1 ***: Q9ZS93 T4B21.6 protein. -2.16 PP_2633_C1 contains: Transcription factor, MADS-box -1.67 PP_2646_C2 contains: GCN5-related N-acetyltransferase -1.66 PP_2738_C1 ***: O23310 CCAAT-binding transcription factor subunit A(CBF-A) -1.82 PP_276_C1 **: Homolog of (AB106274) SCARECROW-like protein [Lilium longiflorum] -1.50 PP_2920_C1 **: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0406G08|2927 -1.45 PP_2921_C1 ***: T2AG_ARATH Transcription initiation factor IIA gamma chain (TFIIA-gamma). -1.56 PP_285_C2 **: Homolog of AT3g02790/F13E7_27|o_sativa|chr_6|P0621D05|1946 -1.31 PP_2980_C1 ***: Q8H1G0 Putative flowering protein CONSTANS (GATA-type zinc finger protein). -1.37 PP_2817_C1 contains: Protein of unknown function DUF296(InterPro:IPR005175,PFAM:PF03479) -1.56 PP_6215_C1 ***: Q8S9V3 Putative zinc finger protein. -2.49 PP_3091_C1 ***: Q9SNA4 Receptor-like protein kinase homolog. -1.53 PP_323_C1 ***: PSAG_ARATH Photosystem I reaction center subunit V, chloroplast precursor (PSI-G). -1.59 PP_3684_C1 "**: Homolog of (X58577) DNA-binding protein; bZIP type [Petroselinum crispum] -1.70 PP_3846_C1 **: Homolog of aldehyde dehydrogenase, -1.85 PP_3876_C1 **: Homolog of (68414.m05059 Ras-related GTP-binding protein -1.36 PP_3950_C1 **: Homolog of (AB028078) homeobox protein PpHB7 [Physcomitrella patens] -1.58 PP_3864_C1 **: Homolog of (68416.m02515 basic helix-loop-helix bHLH) family protein -2.01 PP_4087_C4 "**: Homolog of (68416.m01203 aspartate/glutamate/uridylate kinase family protein -1.50

142 Chapter IV Appendices

PP_4109_C1 **: Homolog of (M62985) protein kinase [Zea mays] -1.86 PP_4163_C1 **: Homolog of (AF029984) COP1 homolog [Lycopersicon esculentum] -1.68 PP_4175_C1 **: Homolog of (68414.m07887 basic helix-loop-helix bHLH) family protein -2.79 PP_4238_C1 **: Homolog of (68414.m01246 eukaryotic translation initiation factor 3 subunit 3 / eIF-3 -1.61 PP_4183_C1 **: Homolog of (AY077758) WRKY transcription factor 1 [Physcomitrella patens] -1.50 PP_4368_C1 **: Homolog of (68418.m04281 histidine kinase AHK2) identical to histidine kinase -2.10 PP_438_C1 **: Homolog of 68418.m05840 myb family transcription factor contains Pfam profile -1.42 PP_4383_C1 **: Homolog of (68415.m02898 basic helix-loop-helix bHLH) family protein -1.56 PP_4394_C1 **: Homolog of Protein kinase domain, putative|o_sativa|chr_1|OJ1529_G03|4777 -3.02 PP_4227_C2 **: Homolog of (transcription initiation factor iib general transcription factor tfiib). -1.39 PP_4501_C1 **: Homolog of OSJNBa0003O19.1|putative AT-Hook DNA-binding protein -1.43 PP_4570_C3 "**: Homolog of (68418.m08464 F-box family protein similar to unknown protein -1.63 (dbj|BAA78736.1) PP_4710_C1 **: Homolog of 68416.m05166 Dof-type zinc finger domain-containing protein [Arabidopsis -1.53 thaliana] PP_4819_C1 **: Homolog of Similar to histidine kinase-like protein|o_sativa|chr_6|P0709F06|1935 -2.13 PP_4820_C1 **: Homolog of (AF378125) GAI-like protein 1 [Vitis vinifera] -4.01 PP_4988_C1 **: Homolog of (68415.m05233 basic helix-loop-helix bHLH) family protein -1.80 PP_5004_C1 "**: Homolog of (68418.m01242 sensory transduction histidine kinase-related -1.70 PP_5046_C1 "**: Homolog of (68415.m04408 zinc finger (C3HC4-type RING finger) family protein -3.59 PP_5138_C1 **: Homolog of (AF439278) ethylene-responsive transciptional coactivator-like protein -3.39 [Retama raetam] PP_5262_C1 **: Homolog of 68415.m04505 PHD finger transcription factor, putative -1.92 PP_5396_C1 **: Homolog of (AF311224) C2H2 zinc-finger protein [Zea mays] -1.73 PP_5526_C1 **: Homolog of 68418.m04803 Dof-type zinc finger domain-containing protein [Arabidopsis -1.31 thaliana] PP_5624_C1 **: Homolog of TAZ zinc finger, putative|o_sativa|chr_1|P0696G06|5647 -1.62 PP_5836_C2 **: Homolog of 68416.m05063 myb family transcription factor -27.29 PP_5905_C1 **: Homolog of OSJNBa0053C23.4|putative serine/threonine protein kinase -1.50 PP_6171_C1 ***: Q84XK6 Peroxisomal targeting signal type 2 receptor. -1.29 PP_6223_C1 contains: Helix-turn-helix, Fis- -3.32 type(InterPro:IPR002197,GO:0003700,GO:0006355,PRINTS:PR01590) PP_6242_C1 not annotated Physcomitrella patens -2.62 PP_6285_C1 **: Homolog of (Y10685) G/HBF-1 [Glycine max] -1.94 PP_6114_C1 **: Homolog of F-box domain, putative|o_sativa|chr_4|OSJNBa0043A12|8129 -1.91 PP_6587_C1 ***: O82064 Putative beta-subunit of K+ channels. -3.86 PP_646_C2 **: Homolog of Dof domain, zinc finger, putative|o_sativa|chr_1|P0453A06|2679 -2.29 PP_6731_C1 not annotated Physcomitrella patens -1.89 PP_6766_C1 ***: Q8W2B8 Serine acetyltransferase (Hypothetical protein At4g35640). -2.19 PP_683_C1 ***: YPT6_CHLRE Ras-related protein YPTC6. -1.97 PP_6888_C1 contains: Ubiquitin-conjugating enzymes -1.65 PP_6969_C1 **: Homolog of (68415.m03211 plectin-related contains -9.85 PP_6973_C1 "***: Q9C8A0 Serine/arginine-rich protein, putative; 48931-50251 (TAF7) (At1g55300)." -1.39 PP_6875_C1 contains: U2 snRNP auxilliary factor -1.60 PP_7128_C1 **: Homolog of (AF467900) hypothetical transcription factor [Prunus persica] -2.40 PP_7321_C2 ***: Q9FJC9 26S proteasome regulatory particle chain RPT6-like protein -1.71 (AT5g53540/MNC6_8). PP_11287_C4 ***: RL5_ARATH 60S ribosomal protein L5. -1.55 PP_7371_C1 contains: Tubby(InterPro:IPR000007,PFAM:PF01167) -2.04 PP_7586_C1 ***: DR1D_ARATH Dehydration responsive element binding protein -1.54 PP_7694_C1 ***: Q39031 Protein kinase. -2.02 PP_7708_C1 ***: Q7X976 Putative AT-Hook DNA-binding protein. -1.43 PP_775_C1 ***: Q9SQ79 Helix-loop-helix protein 1A. -1.79 PP_7994_C1 **: Homolog of (AY077758) WRKY transcription factor 1 [Physcomitrella patens] -1.50 PP_8047_C1 **: Homolog of 68417.m01380 KOW domain-containing transcription factor family protein -1.45 chromatin PP_8107_C1 **: Homolog of Similar to TINY-related|o_sativa|chr_2|OJ1711_D06|4260 -2.55

143 Chapter IV Appendices

PP_8413_C1 contains: Zn-finger, RING(InterPro:IPR001841,PFAM:PF00097) -1.47 PP_8463_C1 ***: Q86AZ8 Similar to Anabaena sp. (Strain PCC 7120). Hypothetical WD-repeat protein -1.76 alr2800. PP_8293_C1 contains: (COIL:coil) -2.52 PP_8547_C1 **: Homolog of 68414.m01463 hypothetical protein -1.84 PP_86_C1 **: Homolog of (AY566696) unknown [Xerophyta humilis] -1.83 PP020016117R **: Homolog of (AB032182) homeobox protein PpHB10 [Physcomitrella patens] -1.93 PP_723_C1 ***: Q8LST6 Mitochondrial aldehyde dehydrogenase. -1.51 PP_9108_C3 ***: FTH2_ARATH Cell division protein ftsH homolog 2, chloroplast precursor -1.70 PP_12500_C1 **: Homolog of (68414.m01494 basic helix-loop-helix bHLH) family protein / F-box family -3.17 protein PP_9253_C1 ***: Q9C1Q7 Putative two-component histidine kinase Fos-1. -1.41 PP_9264_C1 "***: Q9SRM4 Putative nucleic acid binding protein (At3g11200/F11B9.12) -1.33 PP_9369_C1 ***: Q9FJ00 Gb|AAF24948.1. -1.59 PP_9394_C1 ***: O94094 Histidine kinase FIK. -1.81 PP_9399_C1 ***: Q8X1E7 Histidine kinase. -1.67 PP_9419_C1 **: Homolog of 68417.m04480 WRKY family transcription factor contains Pfam profile -2.93 PP_9627_C1 ***: Q7WZ30 MmoS. -3.06 PP_9785_C1 ***: Q8LPA5 MADS-box protein PpMADS1. -1.73 PP_9953_C1 ***: Q852U6 At1g49850. -1.59 PP_9960_C1 contains: Nascent polypeptide-associated complex NAC -2.92 (InterPro:IPR002715,PFAM:PF01849) PP_6995_C2 ***: Q9ZNX9 Sigma-like factor precursor (RNA polymerase sigma subunit SigE). -1.77 PP020064243R ***: Q9LWW0 Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 6, -1.40 clone:P0425F02. PP_SD_251_C1 **: Homolog of (AB115546) phototropin 2 [Adiantum capillus-veneris] -1.33 PP_SD_60_C1 ****: Physcomitrella patens mRNA for homeobox protein PpHB9, complete cds. -1.87 PP_SD_88_C1 not annotated Physcomitrella patens -3.49 PP_SD_92_C1 ****: Physcomitrella patens PpHB10 mRNA for homeobox protein PpHB10, complete cds. -2.00 PP_SD_245_C1 **: Homolog of (68418.m05453 disease resistance protein TIR-NBS-LRR class) -1.66 PP_5766_C1 **: Homolog of (68418.m02952 zinc finger B-box type) family protein similar to -1.47 CONSTANS-like protein ****: Physcomitrella patens subsp. patens mRNA for MADS-box protein PpMADS1, -1.96 PP_SD_0_C1 complete cds. PP001001061F **: Homolog of (X98744) chloroplast DNA-binding protein PD3 [Pisum sativum] -2.13 PP001008059F **: PP_CL_6374.Singlet Homolog of (AB046872) PpSIG2 [Physcomitrella patens] -1.52 PP001009093F **: Homolog of OSJNBa0010C11.3|putative transcription regulatory protein -1.35 PP001019006F **: PP_CL_8395.Singlet Homolog of (68416.m00477 RNA recognition motif (RRM) -1.54 PP001030028F **: Homolog of (AC005315) putative non-LTR retroelement reverse transcriptase -1.96 PP001030033F "**: Homolog of 68414.m08061 paired amphipathic helix repeat-containing protein -1.47 PP001068061R **: PP_CL_18208.Singlet Homolog of 68415.m05104 expressed protein -2.18 PP001085009R **: PP_CL_6389.Singlet Homolog of (AY324646) katanin [Gossypium hirsutum] -1.60 BJ165389 ***: Q41067 Polyubiquitin. -1.41 ****: Physcomitrella patens subsp. patens mRNA for MADS-box protein PpMADS1, -1.48 PP004021140R complete cds. PP004058038R ***: O49977 Ubiquitin (Fragment). -1.31 PP004067310R **: PP_CL_7066.Singlet Homolog of (AB026657) unnamed protein product -1.60 PP004086076R ***: Q41067 Polyubiquitin. -1.58 PP004087236R ****: Physcomitrella patens subsp. patens mRNA for MADS-box protein PpMADS1, -1.52 complete cds. BI488008 ***: O82527 Polyubiquitin (Fragment). -1.59 PP004095105R ***: O82527 Polyubiquitin (Fragment). -1.59 PP013015004R **: Homolog of OSJNBb0070O09.2|unknown protein -1.51 PP015004308R ***: Q8LNW1 Putative transcription factor. -1.70 PP015011331R **: Homolog of (68414.m08326 GCN5-related N-acetyltransferase GNAT) family protein -1.43 PP015020123R **: Homolog of (AB032182) homeobox protein PpHB10 [Physcomitrella patens] -1.47 PP015029288R "**: PP_CL_5054.Singlet Homolog of (68418.m08510 TAZ zinc finger family protein -1.99

144 Chapter IV Appendices

PP015033075R **: Homolog of (AB121445) histidine kinase 3 [Zea mays] -4.00 PP015037157R **: Homolog of Helicase conserved C-terminal domain, -1.73 putative|o_sativa|chr_8|OJ1034_C08|7210 **: PP_CL_1670.Singlet Homolog of (AJ419328) putative MADS-domain transcription -1.50 PP015044155R factor **: Homolog of Helix-loop-helix DNA-binding domain, -1.53 PP015054317R putative|o_sativa|chr_1|OSJNBa0093F16|4658 PP015071162R ***: AGO1_ARATH Argonaute protein. -1.40 ****: Physcomitrella patens subsp. patens PPLFY1 mRNA for FLORICAULA/LEAFY -1.40 PP020027384R homolog PP020032226R **: PP_CL_18682.Singlet Homolog of (AJ011828) NDX1 homeobox protein [Lotus -1.59 corniculatus PP020034124R **: PP_CL_4373.Singlet Homolog of (AB115546) phototropin 2 [Adiantum capillus-veneris] -1.65 PP020036015R **: PP_CL_9604.Singlet Homolog of OJ1365_D05.10|putative response regulator protein -2.19 PP020036307R ***: Q9FT60 Histidine kinase-like protein. -1.44 PP020044335R "**: Homolog of 68416.m03380 oligopeptide transporter OPT family protein -1.94 PP020051260R ***: Q9SXL4 Histidine kinase 1. -2.12 PP020062195R **: Homolog of (AJ419328) putative MADS-domain transcription factor [Physcomitrella -1.82 patens] PP020065285R **: PP_CL_10589.Singlet Homolog of (AL670011) related to regulatory protein SET1 -1.49 PP020069226R ***: Q9LRI1 Homeobox protein PpHB10. -1.60 PP030015063R ***: O82527 Polyubiquitin (Fragment). -1.55 AY123146 PP_SD_72.Singlet not annotated Physcomitrella patens -1.76

145 Chapter IV Appendices

4.5 Genes upregulated in ΔPpDCL1b mutants

Sequence ID Cosmoss Annotation Fold change EST AW561368 "**: Homolog of (68417.m01135 F-box family protein (FBL8) FBL24) contains 1.32 BJ160934 **: Homolog of (AF004165) 2-isopropylmalate synthase [Lycopersicon pennellii] 2.24 BJ163321 ***: Q8S0L3 Ankyrin-kinase-like protein. 1.94 BJ181458 ***: Q9AV93 Response regulator 8. 1.97 BJ187175 ***: Q84VL6 Putative polyubiquitin (Fragment). 1.81 BJ189887 **: PP_CL_6916.Singlet Homolog of ((AP003273) histone H1-like protein (Oryza sativa ) 1.30 BJ609923 ***: EFTM_ARATH Elongation factor Tu, mitochondrial precursor. 1.43 BJ610672 **: Homolog of expressed protein|o_sativa|chr_4|OSJNBa0013K16|5362 1.70 BU051751 ***: Q7X864 OSJNBa0093F12.4 protein. 1.55 PP_10130_C2 ***: Q8H9A2 Dehydratiion responsive element binding protein 1 like protein. 1.76 PP_10143_C1 ***: Q8YMN1 All4902 protein. 1.87 PP_10308_C1 **: Homolog of OSJNBb0015I11.23|putative ubiquitin 1.62 protein|o_sativa|chr_10|OSJNBb0015I11|31 PP_1034_C1 ***: Q9SEK4 Putative succinic semialdehyde dehydrogenase 1.53 PP_10379_C1 "**: Homolog of (68417.m00335 AAA-type ATPase family protein 1.95 PP_10320_C1 ***: Q96327 Putative nuclear DNA-binding protein G2p (Nuclear DNA-binding protein) 1.75 PP_10567_C1 not annotated Physcomitrella patens 1.57 PP_10875_C1 ***: Q94H06 Putative zinc finger protein. 2.11 PP_10880_C1 ***: Q8GYN7 Putative SCARECROW gene regulator. 1.67 PP_10919_C1 ***: Q8S8P6 Putative salt-inducible protein. 1.69 PP_11080_C1 ***: Q9SA69 F10O3.17. 1.39 PP_1112_C1 ***: Q9ZVG0 Putative ATP-dependent DNA helicase RECG. 1.41 PP_11139_C1 **: Homolog of 68414.m03428 expressed protein 1.38 PP_12499_C1 ***: PRS7_ARATH 26S protease regulatory subunit 7 (26S proteasome subunit 7) 1.43 PP_13554_C1 ***: Q9LKG4 Putative DNA binding protein. 1.30 PP_11359_C1 **: Homolog of 68416.m01881 DNA-binding protein-related 2.03 PP032001093R **: PP_CL_11223.Singlet Homolog of 68414.m05053 expressed protein 1.58 PP_18394_C1 **: Homolog of RNA polymerase I specific transcription initiation factor 2.11 PP_1179_C1 **: Homolog of Similar to probable zinc finger protein [imported] - Arabidopsis thaliana 2.07 PP_1012_C1 **: Homolog of 68418.m05158 kelch repeat-containing F-box family protein contains Pfam 2.43 profiles PP_12120_C1 ***: Q7X6J0 RNA binding protein Rp120. 1.73 PP_12140_C1 **: Homolog of (AF470350) WD40 [Tortula ruralis] 1.41 PP_12145_C1 **: Homolog of hypothetical protein|o_sativa|chr_1|P0470A12|2880 1.78 PP001005057F ***: O82527 Polyubiquitin (Fragment). 1.65 PP_12587_C1 **: Homolog of AP2-related transcription factor, 2.46 putative|o_sativa|chr_4|OSJNBa0079A21|8302 ***: Q9GZS3 Homo sapiens cDNA: FLJ21101 fis, clone CAS04682 (G protein beta 1.56 PP_12713_C2 subunit) PP_12802_C1 ***: Q7XXN2 Putative serine/threonine-protein kinase ctr1. 2.19 PP_1303_C1 contains: Dihydrodipicolinate synthetase(InterPro:IPR002220,PFAM:PF00701) 1.86 PP_13105_C1 ***: Q84QC2 Putative AP2 domain transcription factor. 1.72 PP_13136_C1 ***: Q9SGP0 F3M18.14. 1.96 PP_13592_C1 ***: Q94DZ5 Putative helicase-like transcription factor. 2.17 PP_13985_C1 ***: Q9FHJ4 Arabidopsis thaliana genomic DNA, chromosome 5, P1 clone: MFC19. 1.39 PP_14045_C1 ***: O65567 Puative protein. 1.81 PP_14113_C1 ***: IF35_ARATH Eukaryotic translation initiation factor 3 subunit 5 (eIF-3 epsilon) (eIF3 1.47 p32 subunit) PP_1440_C1 contains: RNA-binding region RNP-1 (RNA recognition motif) 1.69 PP_14547_C1 **: Homolog of (68414.m00475 zinc finger (C3HC4-type RING finger) family protein 1.32

146 Chapter IV Appendices

PP_14811_C1 **: Homolog of (AF098674) lateral suppressor protein [Lycopersicon esculentum] 1.40 PP_15007_C1 ***: Q9ZV05 Expressed protein. 1.60 PP_15083_C1 **: Homolog of zinc finger protein, putative|o_sativa|chr_6|P0550B04|1978 1.69 PP_15177_C2 ***: Q9SJR0 Putative AP2 domain transcription factor (Putative AP2/EREBP transcription 10.73 factor). PP_15255_C1 ***: Q9FHA7 Emb|CAB62312.1 (Putative bHLH transcription factor). 1.41 PP_15299_C1 ***: Q7XJM3 Putative mitochondrial translation elongation factor G. 2.15 PP_15344_C1 ***: O81763 Protein kinase-like protein. 1.84 PP_15382_C1 **: Homolog of 68416.m05464 phototropic-responsive protein 1.59 PP_15384_C1 **: Homolog of 68417.m00399 elongation factor Tu, putative / EF-Tu, putative 1.50 PP_15546_C1 ***: Q9LW84 Gb|AAF26996.1. 1.65 PP_15582_C1 **: Homolog of Similar to DNA helicase-like|o_sativa|chr_2|P0724B10|6621 1.42 PP_15610_C1 ***: MAT1_MOUSE CDK-activating kinase assembly factor MAT1 (RING finger protein 1.44 MAT1) PP_15633_C1 ***: Q9SJW0 Transfactor-like protein. 2.38 PP_15636_C1 ***: Q8VZG7 AT5g07350/T2I1_60. 2.24 PP_15695_C1 **: Homolog of expressed protein|o_sativa|chr_4|OSJNBa0013K16|5362 24.51 PP_1585_C2 contains: Zn-binding protein, LIM(InterPro:IPR001781,PFAM:PF00412) 1.53 PP_15995_C1 ***: EFTM_ARATH Elongation factor Tu, mitochondrial precursor. 1.96 PP_15997_C1 ***: Q9SI75 F23N19.11 (Hypothetical protein At1g62750). 2.72 PP_16050_C1 ***: Q851S7 Pescadillo-like protein. 4.28 PP_1618_C1 ***: Q9LVF7 Gb|AAD14441.1. 1.44 PP_16284_C1 "**: Homolog of (68414.m09356 coatomer protein complex, subunit beta 2 (beta prime), 3.16 PP_1662_C1 ***: Q8S3E7 Putative bHLH transcription factor. 1.84 PP_1663_C1 ***: Q9LSQ8 Arabidopsis thaliana genomic DNA, chromosome 5, BAC clone: F24B18. 1.98 PP_16437_C1 ***: EFGM_ARATH Probable elongation factor G, mitochondrial precursor (mEF-G). 2.48 PP_1496_C1 contains: Pathogenesis-related transcriptional factor and ERF 3.69 BJ181914 ***: O82527 Polyubiquitin (Fragment). 1.61 PP_16865_C1 **: Homolog of auxin response factor 1 [imported] - Arabidopsis 2.62 thaliana|o_sativa|chr_2|P0506A08|3865 PP_1691_C1 ***: Q9LS31 Homeobox protein Pphb7 short form. 1.91 PP_16923_C1 **: Homolog of OSJNBa0003O19.20|putative MYC transcription factor 1.52 PP_1738_C3 contains: Zn-finger, Dof type(InterPro:IPR003851,GO:0003677,PFAM:PF02701) 2.99 PP_17440_C1 ***: Q8W3M3 AP2 domain containing protein (Putative AP2/EREBP transcription factor). 20.48 PP_17575_C1 ***: Q9SAK5 T8K14.15 protein. 1.34 PP_17900_C1 **: Homolog of (AJ131113) VP1/ABI3-like protein [Chamaecyparis nootkatensis] 1.43 PP_17924_C1 ***: Q7XSB1 OJ991113_30.18 protein. 1.89 PP_1804_C1 ***: Q9SSF9 F25A4.28 protein. 1.76 PP_18357_C1 contains: (COIL:coil) 1.54 PP_18393_C1 not annotated Physcomitrella patens 2.06 PP_18403_C1 **: Homolog of RNA polymerase I specific transcription initiation factor 2.84 PP_1844_C1 ***: O65567 Puative protein. 1.42 PP_18489_C1 "**: Homolog of (68417.m02830 nucleoside phosphatase family protein / GDA1/CD39 1.67 family protein PP_18663_C1 **: Homolog of Kelch motif, putative|o_sativa|chr_6|OJ1378_E04|3445 1.72 PP_18676_C1 not annotated Physcomitrella patens 2.63 PP_1999_C1 ***: Q8SB10 Putative crp1 protein. 1.70 PP_2158_C1 **: Homolog of EREBP-type transcription factor, putative|o_sativa 2.02 PP_2271_C5 ***: Q9ZVU6 T5A14.12 protein. 1.40 PP_2334_C1 ***: Q8GZ22 Putative ankyrin (At2g03430). 1.52 PP_2362_C1 ***: Q84TU4 Arm repeat-containing protein. 1.50 PP_7120_C2 contains: (SUPERFAMILY:SSF54171) 2.14 PP_2324_C1 ***: Q94ID6 ERF domain protein12 (Ethylene responsive element binding factor, putative). 1.58 PP_2520_C1 ***: Q9M8Z0 T6K12.4 protein. 2.89 PP_2344_C1 ***: Q9FWR5 F14P1.8 protein. 1.35

147 Chapter IV Appendices

PP_2372_C1 ***: Q9SAI2 F23A5.13 protein (Putative CCR4-associated factor). 1.39 BQ041789 ***: Q9LMP8 F7H2.20 protein (At1g15870/F7H2_19). 1.55 PP_10621_C1 ***: Q9FPV8 Putative methionine aminopeptidase. 1.33 PP_3086_C1 ***: Q949D4 Putative AP2-related transcription factor. 3.09 PP_3132_C1 "**: Homolog of (68415.m02229 expressed protein contains Pfam profiles 1.49 PP_319_C1 ***: Q9LQ28 F14M2.12 protein (Putative AP2/EREBP transcription factor). 1.55 PP_3479_C1 "**: Homolog of 68416.m01095 KOW domain-containing transcription factor family protein 1.51 PP_3728_C1 **: Homolog of 68417.m00223 WRKY family transcription factor 1.79 PP_3738_C1 "**: Homolog of 68418.m07197 protein kinase family protein similar to protein kinase 1.67 [Glycine max] PP_4015_C1 **: Homolog of DRE-binding protein 1A|o_sativa|chr_8|OJ1323_A06|7300 5.29 PP_4112_C6 "**: Homolog of (AC016529) putative AP2 domain transcription factor 6.24 PP_4166_C1 **: Homolog of OSJNBb0033N16.2|putative RNA 1.31 PP_4300_C1 **: Homolog of (68414.m05749 basic helix-loop-helix bHLH) family protein contains Pfam 1.51 profile PP_4459_C1 **: Homolog of myb-like DNA-binding domain, SHAQKYF class 1.77 PP_4595_C1 "**: Homolog of (68414.m00292 GCN5-related N-acetyltransferase (GNAT) family protein 1.35 PP_4414_C1 not annotated Physcomitrella patens 2.27 PP_4697_C1 **: Homolog of (AB067689) MADS-box protein PpMADS2 [Physcomitrella patens] 2.22 PP_4719_C1 **: Homolog of 68414.m01735 expressed protein 22.24 PP_4825_C1 **: Homolog of OSJNBa0053C23.4|putative serine/threonine protein kinase 1.53 PP_4986_C1 "**: Homolog of (68415.m03129 transducin family protein / WD-40 repeat family protein 1.45 PP_5002_C1 **: Homolog of (68415.m04914 eukaryotic translation initiation factor 3 subunit 5 / eIF-3 1.54 epsilon PP_5288_C1 **: Homolog of (AY346455) histone deacetylase [Solanum chacoense] 1.67 **: Homolog of ((AP002092) unnamed protein product [Oryza sativa japonica cultivar 1.46 PP_5296_C1 group)] PP_543_C1 **: Homolog of 68416.m05511 expressed protein 2.02 PP_4563_C1 **: Homolog of (AF184886) LIM domain protein WLIM2 [Nicotiana tabacum] 1.37 PP_5681_C1 **: Homolog of (AB111943) hypothetical protein [Nicotiana benthamiana] 1.51 PP_5719_C1 **: Homolog of expressed protein|o_sativa|chr_5|OSJNBb0015A05|5000 1.70 PP_573_C2 ***: Q852S5 Nucleoside diphosphate kinase. 1.54 PP_5803_C2 **: Homolog of (AB042267) response regulator 5 [Zea mays] 2.33 PP_584_C1 **: Homolog of (68414.m05651 scarecrow-like transcription factor 3 (SCL3) 2.15 PP_560_C1 "**: Homolog of (68418.m07708 no apical meristem (NAM) family protein 1.75 PP_5870_C1 **: Homolog of 68418.m01679 expressed protein 2.76 PP_5912_C2 **: Homolog of (AF506028) CTV.22 [Poncirus trifoliata] 1.37 PP_12254_C1 **: Homolog of (AF098674) lateral suppressor protein [Lycopersicon esculentum] 1.70 PP_6275_C1 contains: Pathogenesis-related transcriptional factor and ERF 1.43 PP_63_C1 ***: Q7Y1U0 Kinesin-like calmodulin binding protein. 1.81 PP_6340_C1 ***: Q9SR03 Ankyrin-like protein. 1.52 PP_6354_C2 ***: Q9SAU3 CAO. 1.58 PP_6368_C4 ***: Q762A0 BRI1-KD interacting protein 114 (Fragment). 1.33 PP_6455_C1 ***: Q9SZM7 Protein kinase like protein. 2.29 PP_648_C2 **: Homolog of (L76926) putative zinc finger protein [Arabidopsis thaliana] 1.79 PP_18379_C1 not annotated Physcomitrella patens 1.78 PP_6564_C1 "**: Homolog of 68414.m01006 protein kinase 1.64 PP_6651_C1 ***: O22826 Putative splicing factor (At2g43770). 1.64 PP_6682_C1 ***: Q7XMI6 OSJNBb0006N15.13 protein. 1.37 PP_6732_C1 not annotated Physcomitrella patens 1.66 PP_7252_C1 **: Homolog of ((AP005190) putative p53 binding protein [Oryza sativa japonica cultivar- 1.65 group)] PP_729_C1 **: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0466H10|5982 1.66 PP_753_C1 ***: Q94D32 P0712E02.24 protein (P0700A11.5 protein). 1.88 PP_7668_C1 ***: PCNA_TOBAC Proliferating cell nuclear antigen (PCNA). 1.40 PP_10278_C1 ***: Q9FP06 P0038C05.18 protein. 2.75

148 Chapter IV Appendices

PP_793_C4 **: Homolog of (68418.m08455 basic helix-loop-helix bHLH) family protein 1.56 PP_7999_C1 **: Homolog of 68415.m03352 DC1 domain-containing protein contains Pfam profile 1.37 PP_8_C1 contains: Protein synthesis factor, GTP-binding 1.62 PP_8013_C1 ***: RM21_ARATH 50S ribosomal protein L21, mitochondrial precursor. 1.51 PP_14769_C1 **: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0406G08|2927 1.32 PP_8021_C1 **: Homolog of putative AP2 domain transcription 8.04 factor|o_sativa|chr_4|OSJNBb0034G17|5454 PP_8274_C1 not annotated Physcomitrella patens 1.66 PP_8314_C1 **: Homolog of Similar to Lil3 protein|o_sativa|chr_2|P0018H03|4896 1.61 PP_8332_C1 **: Homolog of AP2 domain, putative|o_sativa|chr_6|P0638H11|5506 7.71 PP_8337_C1 ***: Q7XNE0 OSJNBa0088A01.11 protein. 1.79 PP_8348_C1 ***: Q9ZV05 Expressed protein. 1.68 PP_8372_C1 "***: Q9CAN3 Transcription factor SCARECROW, putative; 52594-50618." 2.32 PP_8392_C1 ***: Q8RYF8 P0592G05.19 protein. 2.63 PP_8343_C1 "***: Q9C550 2-isopropylmalate synthase 2.61 PP_8584_C1 ***: Q39216 RNA polymerase subunit (Isoform B). 2.05 PP_8642_C1 **: Homolog of (AY192369) ethylene response factor 3 [Lycopersicon esculentum] 3.41 PP_8479_C1 contains: Basic helix-loop-helix dimerisation region bHLH 1.63 PP_8784_C1 contains: Protein kinase-like(InterPro:IPR011009,SUPERFAMILY:SSF56112) 1.67 PP_8794_C1 contains: Pathogenesis-related transcriptional factor 1.87 PP_8838_C1 ***: Q7XU78 OSJNBa0029H02.4 protein. 2.11 PP_8990_C3 **: Homolog of (GDA1/CD39 nucleoside phosphatase) family, putative 1.59 PP_900_C1 ***: O49591 Putative zinc finger protein. 2.44 PP_8906_C1 ***: Q9M551 Polyubiquitin. 1.42 PP_9142_C1 "**: Homolog of 68418.m03534 bZIP transcription factor family protein 1.55 PP_9252_C1 ***: Q84QD7 Avr9/Cf-9 rapidly elicited protein 276. 1.49 PP_765_C1 ***: O80582 Expressed protein (At2g44130/F6E13.26). 2.39 PP_9420_C1 ***: Q7XU22 OSJNBb0034G17.2 protein (Transcription factor DREB). 3.06 PP_9498_C1 **: Homolog of 68416.m01360 PHD finger family protein contains Pfam domain 1.63 PP_9599_C1 **: Homolog of similar to CH6 and COP9 complex subunit 1.35 6|o_sativa|chr_8|OJ1118_A06|3036 PP_9374_C1 **: Homolog of SelR domain|o_sativa|chr_3|OSJNBa0048D11|3659 1.91 PP_9607_C1 ***: O65639 Glycine-rich protein. 1.33 PP_9750_C1 ***: Q8LBL6 Cell division protein FtsH-like protein. 1.28 PP_976_C1 ***: Q6ZHJ5 Pentatricopeptide (PPR) repeat-containing protein-like. 1.74 PP_17043_C1 **: Homolog of ((AP004068) GCN5-related N-acetyltransferase protein-like (Oryza sativa 1.79 japonica ) PP_9949_C1 **: Homolog of Myb-like DNA-binding domain, putative|o_sativa|chr_1|P0038F12|2733 1.71 PP_SD_12_C1 ****: Physcomitrella patens mRNA for RNA polymerase alpha subunit, complete cds. 2.09 PP_SD_17_C1 ****: Physcomitrella patens WRKY transcription factor 1 (WRKY1) gene, complete cds. 1.55 PP_SD_252_C1 **: Homolog of 68418.m05899 protein kinase, putative similar to protein kinase G11A 1.49 [Oryza sativa] PP_SD_46_C1 ****: Physcomitrella patens mRNA for homeobox protein PpHB7, complete cds. 3.51 PP_SD_67_C1 ****: Physcomitrella patens MADS-domain protein PPM1 (ppm1) mRNA, complete cds. 1.37 PP_SD_90_C1 not annotated Physcomitrella patens 2.32 PP_10747_C1 ***: O81763 Protein kinase-like protein. 1.54 **: PP_CL_18202.Singlet Homolog of (AC026238) Hypothetical protein [Arabidopsis PP001063096R thaliana] 1.89 PP001072036R **: PP_CL_15170.Singlet Homolog of 68418.m06538 myb family transcription factor 1.32 PP001077051R ***: O49459 Predicted protein. 1.89 PP001090095R **: PP_CL_15183.Singlet Homolog of Similar to Z97341 apetala2 domain TINY like protein 1.59 PP002015081R **: PP_CL_5597.Singlet Homolog of (AC006072) putative tubby protein [Arabidopsis thaliana] 1.56 PP002023001R ***: O24460 Calmodulin-like domain protein kinase. 4.72 PP004003286R "**: PP_CL_18294.Singlet Homolog of (68414.m07536 gibberellin regulatory protein 2.15 RGL1) PP004006023R ****: Physcomitrella patens phytochrome (phy2) gene, complete cds. 1.44

149 Chapter IV Appendices

PP004007192R ***: Q9FJ91 Dbj|BAA78737.1 (AT5g52010/MSG15_9). 2.04 PP004009367R **: Homolog of (AF004165) 2-isopropylmalate synthase [Lycopersicon pennellii] 1.77 PP004012159R **: Homolog of 68414.m06061 mechanosensitive ion channel domain 1.79 PP004015085R **: Homolog of OSJNBa0010C11.3|putative transcription regulatory protein 1.64 PP004020107R **: PP_CL_11223.Singlet Homolog of 68414.m05053 expressed protein 1.92 PP004024038R ***: Q8VYE7 Putative calcium-dependent protein kinase. 2.59 PP004029122R ***: RPOA_PSINU DNA-directed RNA polymerase alpha chain (EC 2.7.7.6) (PEP) 1.56 PP004030378R **: Homolog of Similar to DRE binding factor 1|o_sativa|chr_6|P0516A04|3745 1.59 PP004032225R **: Homolog of (AB164647) vascular plant one zinc finger protein [Physcomitrella patens] 1.57 PP004040295R **: PP_CL_18294.Singlet Homolog of (AY269087) GAI-like protein [Lycopersicon 2.13 esculentum] PP004043210R ***: Q9SGT9 T6H22.8.2 protein. 4.14 PP004046136R **: Homolog of VIP2 protein|o_sativa|chr_2|OJ1311_D08|4248 1.43 PP004054012R **: PP_CL_15491.Singlet Homolog of (68418.m00567 zinc finger (C3HC4-type RING 1.39 finger) PP004054209R PP_SD_13.Singlet not annotated Physcomitrella patens 1.92 PP004057142R ***: Q94C56 Putative FtsH protease (Fragment). 2.33 PP004062128R **: Homolog of 68418.m06038 phototropic-responsive NPH3 family protein 1.34 PP004075103R ***: Q7XPK1 OSJNBa0087O24.9 protein. 1.52 BQ827548 ***: Q8H386 Casein kinase II alpha subunit. 1.47 PP004034373R ****: Physcomitrella patens phytochrome (phy2) gene, complete cds. 1.58 PP004082282R **: Homolog of (AJ579910) NIN-like protein 1 [Lotus corniculatus var. japonicus] 2.23 PP004083344R ***: Q40164 Ubiquitin. 1.67 PP004088245R ***: Q9M1K4 Leucine zipper-containing protein AT103. 1.43 PP004092140R **: PP_CL_18318.Singlet Homolog of (AB028621) unnamed protein product [Arabidopsis 2.09 thaliana] PP004094219R ***: Q8LST4 Mitochondrial aldehyde dehydrogenase. 1.33 PP004095066R **: Homolog of NF-X1 type zinc finger, putative|o_sativa|chr_1|P0041E11|2738 1.48 PP004095253R **: Homolog of (AF328842) homeodomain protein HB2 [Picea abies] 1.44 PP004096225R ****: PP_SD_29.Singlet Physcomitrella patens mRNA for putative P-type II calcium 2.55 PP004097116R **: Homolog of ((AP005243) VP1/ABI3 family regulatory protein-like [Oryza sativa] 1.60 PP004103024R ***: O99018 Chloroplast protease precursor. 1.72 PP004105269R **: Homolog of 68416.m01747 scarecrow transcription factor family protein 1.60 PP006002067R ***: Q9M378 TATA box binding protein (TBP) associated factor (TAF)-like protein. 2.78 PP010001010R ***: Q8GV68 Phytochrome. 1.60 PP010002038R **: Homolog of (AC068602) F14D16.2 [Arabidopsis thaliana] 1.64 PP010008086R **: Homolog of (AL163912) putative protein [Arabidopsis thaliana] 1.94 PP011003080R "**: PP_CL_17423.Singlet Homolog of (68418.m02981 transducin family protein 1.52 PP011005059R **: Homolog of ATP-dependent metalloprotease FtsH, 2.28 putative|o_sativa|chr_5|OJ1362D02|3955 PP011006015R **: Homolog of (AY514604) gibberelin response modulator dwarf 8 Zea mays 3.63 PP015001075R ****: PP_SD_46.Singlet Physcomitrella patens gene for homeobox protein 3.50 PP015006184R **: PP_CL_2250.Singlet Homolog of Cyclin, N-terminal domain, putative 2.35 PP015015236R ****: PP_SD_46.Singlet Physcomitrella patens mRNA for homeobox protein PpHB7, 3.32 complete cds. PP015024237R **: PP_CL_3101.Singlet Homolog of (68414.m03658 DNA-directed RNA polymerase 1.58 family protein PP015028003R **: Homolog of (68414.m07827 zinc finger B-box type) family protein 3.56 PP015028194R **: Homolog of 2-isopropylmalate synthase|o_sativa|chr_12|OSJNBb0034E23|7059 2.35 PP015030306R "**: PP_CL_3039.Singlet Homolog of (68417.m04350 translation initiation factor 3 IF-3) 1.66 PP015033189R **: Homolog of (68414.m08377 CCAAT-box-binding transcription factor-related 2.41 PP015040077R **: Homolog of (AF098674) lateral suppressor protein [Lycopersicon esculentum] 1.54 PP015041065R **: Homolog of Similar to RSSG8|o_sativa|chr_12|OJ1396_C02|7247 1.75 PP015041271R **: Homolog of Helix-loop-helix DNA-binding domain, 2.00 putative|o_sativa|chr_11|P0410D09|7510 PP015042038R **: PP_CL_6025.Singlet Homolog of expressed protein|o_sativa|chr_1|B1064G04|2860 1.72 PP015044222R **: PP_CL_18339.Singlet Homolog of (AF328786) EIL3 [Lycopersicon esculentum] 1.32

150 Chapter IV Appendices

PP015050353R ***: Q9S786 Calcium-dependent protein kinase. 1.50 PP015058275R ***: Q9FGR7 Similarity to salt-inducible protein. 1.66 PP015060167R ***: Q8W3N8 26S proteasome regulatory particle triple-A ATPase subunit4b (Fragment). 1.39 PP020009267R ***: Q9SI82 F23N19.4. 1.42 PP020016269R **: Homolog of OSJNBb0033N16.3|putative protein kinase|o sativa|chr 1.63 3|OSJNBb0033N16|761 PP020019294R **: PP_CL_15.Singlet Homolog of (AF534891) type-B response regulator [Catharanthus 1.34 roseus] PP020024305R ***: O04235 Transcription factor. 1.72 PP020026165R contains: Response regulator receiver 1.90 PP020029315R "**: Homolog of (68418.m00739 transcription factor jumonji (jmjC) domain-containing 1.56 protein PP020031042R ***: Q9LPC6 F22M8.8 protein. 2.58 PP020031185R **: Homolog of expressed protein|o_sativa|chr_5|P0683B12|6231 1.64 "**: Homolog of (68416.m05316 bacterial transferase hexapeptide repeat-containing 1.54 PP020032141R protein PP020039216R **: Homolog of A67797 unnamed protein product-related|o sativa|chr 8|OSJNBa0054L03|4675 1.41 PP020041086R ***: Q9LTD4 Similarity to unknown protein. 1.96 PP020043294R ***: Q6K7E2 Mitochondrial transcription termination factor-like. 3.27 PP020053142R ***: Q9S729 GlsA. 1.78 PP020054111R "**: PP_CL_10402.Singlet Homolog of (68414.m01147 NF-X1 type zinc finger family protein 1.85 PP020054231R ***: Q9FP06 P0038C05.18 protein. 1.50 PP020058260R ***: Q9FLM7 Gb|AAC33480.1 (MYB transcription factor). 1.51 PP020060244R **: PP_CL_10622.Singlet Homolog of (AB107691) AG-motif binding protein-3 [Nicotiana 1.77 tabacum] PP020063226R ***: Q9LV30 Emb|CAB40755.1. 1.48 PP020063256R **: Homolog of Protein kinase domain, putative|o_sativa|chr_1|P0695A04|2742 1.46 PP020070127R PP_SD_242.Singlet not annotated Physcomitrella patens sporophyte 1.81 PP030007003R ***: Q8S9J9 At1g14000/F7A19_9. 1.93 PP030013070R ***: O82527 Polyubiquitin (Fragment). 1.34 PP032009070R ***: Q8GV68 Phytochrome. 2.22

151 Acknowledgment

4.6 Acknowledgments

First of all I would like to thank Prof. Dr. Ralf Reski for giving me the opportunity of doing my PhD in his research group and for his support and encouragement along the way. I am also indebted to him for his guidance

I would like to thank PD Dr. Wolfgang Frank for his invaluable supervision, great efforts in guidance, encouragement throughout the research work.

Great appreciation is also due to my family for their encouragement and support. Special thanks to my wife Enas for valuable help, encouragement and support.

I would like to say thanks to: Dr. Volker Speth and Dr. Claudia Gack for sample preparation and valuable help at the scanning electron micrographs Andras Viczian for providing the pPCV expression vector Björn Voß for advice on miR319 precursor sequence analysis Gregor Gierga for assisting in the small RNA blots technique Richard Haas for practical support in the lab Anne Katrin Prowse for proofreading my thesis.

Special thanks are expressed to my dearest colleagues; Fattash, I., Arif, M. A., Rödel, P., Tomek, M.

Finally, I would like to say thanks to all members from the Reski group for the wonderful working ambience.

Thank you all…

152 Erklärung

4.7 Erklärung

Hiermit erkläre ich, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quellen gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von Vermittlungs, beziehungsweise Beratungsdiensten (Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen. Die Arbeit wurde bisher weder im In noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt. Die Bestimmungen der Promotionsordnung der Fakultät für Biologie der Universität Freiburg sind mir bekannt; insbesondere weiß ich, dass ich vor Vollzug der Promotion zur Führung des Doktortitels nicht berechtigt bin.

Basel Khraiwesh

März, 2009 Freiburg,

153