RDR1 and SGS3, components of RNA-mediated gene silencing, are required for regulation of cuticular wax biosynthesis in developing inflorescence stems of Arabidopsis 1[W]

The cuticle is a protective layer that coats the primary aerial surfaces of land plants, and mediates plant interactions with the environment. It is synthesized by epidermal cells and is composed of a cutin polyester matrix that is embedded and covered with cuticular waxes. Recently, we have discovered a novel regulatory mechanism of cuticular wax biosynthesis which involves the CER7 ribonuclease, a core subunit of the exosome. We hypothesized that at the onset of wax production, the CER7 ribonuclease degrades an mRNA specifying a repressor of CER3, a wax biosynthetic gene whose protein product is required for wax formation via the decarbonylation pathway. In the absence of this repressor, CER3 is expressed, leading to wax production. To identify the putative repressor of CER3 and to unravel the mechanism of CER7 mediated regulation of wax production, we performed a screen for suppressors of the cer7 mutant. Our screen resulted in the isolation of components of the RNA silencing machinery, RDR1 and SGS3, implicating RNA silencing in the control of cuticular wax deposition during inflorescence stem development in Arabidopsis. the cer7 phenotype CER3 transcription, that the CER3 promoter sequence to CER7-mediated CER3 enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis.


INTRODUCTION
suppressor line (Fig. 3D). Here we report the cloning and characterization of genes disrupted in war3 and war4 mutants.

WAR3 encodes RNA-dependent RNA polymerase 1
Genetic analysis of the F 2 progeny from a backcross of war3-1 cer7-1 suppressor line to cer7-1 showed an approximately 3:1 segregation ratio of the glossy mutant to the waxy wild-type (620:232; χ 2 = 2.26; p>0.1), indicating that wax restoration was due to a recessive mutation in a single nuclear gene. To map the war3-1 mutation, war3-1 cer7-1 in the Landsberg erecta (Ler) background, was crossed to cer7-3 in the Columbia ecotype to create a mapping population.
Thirty-five F 2 plants exhibiting a waxy phenotype were used to establish linkage of war3-1 to markers F3F19 and F20D23 on chromosome 1 (Fig. 4A).
The map position of war3-1 was further delineated to a 150 kb genomic region between markers T5E21 and F10B6I-5 using a population of 232 waxy individuals (Fig. 4A). Sequencing of several candidate genes in this region revealed a point mutation in the third exon of At1g14790 at position 3171 (G to A transition), which is predicted to cause a premature stop codon in the war3-1 mutant. At1g14790 was also sequenced in war3-2 and war3-3, two additional alleles of war3 found in the suppressor screen, and in both cases missense mutations were detected (Fig. 4B deficiency. No other morphological phenotypes were detected in the war3 cer7 double mutants.
To verify that the mutation identified in war3 is responsible for the wax restoration of cer7-1, the genomic and promoter region encompassing At1g14790 was transformed into the war3-1 cer7-1 double mutant. Resulting transformants had a wax-deficient glossy stems confirming that WAR3 is RDR1 (Supplemental Fig. S2). Therefore, the war3 alleles described here will be subsequently referred to as rdr1 (Supplemental Table S1).

war4 contains a mutation in SUPPRESSOR OF GENE SILENCING 3
The unexpected finding that RDR1 is involved in regulation of stem wax deposition downstream of the CER7 ribonuclease prompted us to proceed with positional cloning of additional war suppressors to obtain more leads about the pathway involved. Genetic analysis of the F 2 progeny from a backcross of war4-1 cer7-1 suppressor line to cer7-3 showed an approximately 3:1 segregation ratio of the glossy mutant to the waxy wild type (1951:641; χ 2 = 0.101; p>0.7), indicating that wax restoration was due to a recessive mutation in a single nuclear gene. The approximate map position of war4 was determined using 22 F 2 progeny from a war4-1 cer7-1 (Ler ecotype) cross to cer 7-3 (Columbia ecotype) which localized the war4-1 mutation between markers CIW8 and NGA139 on chromosome 5 (Fig. 4C). Fine mapping was carried out using 641 F 2 plants, and allowed us to narrow down the war4-1 mutation to a 100 kb region flanked by the markers K19M13 and MQM1, which contained 22 genes. Sequencing of candidate genes in this region revealed a C to T point mutation at position 454 in the first exon of  Table S1).

RDR1 and SGS3 are expressed throughout the plant
Quantitative RT-PCR was used to assess expression levels of RDR1 and SGS3 in various organs. Aerial tissues were harvested from 4-6 week old plants, whereas seedling and roots were collected from 14-day-old plants. RDR1 and SGS3 expression was detected in all tissues (Fig. 5), but at varying levels. Expression patterns for RDR1 and SGS3 were very similar, with high expression levels found in seedlings, cauline leaves, rosette leaves and flowers. Moderate levels were detected in the stem top and base. Low levels of RDR1 and SGS3 expression were detected in roots and siliques.

DISCUSSION
We previously proposed a novel mechanism of regulating cuticular wax biosynthesis in developing Arabidopsis inflorescence stems, which involves the CER7 exosomal ribonuclease (Hooker et al., 2007). We hypothesized that CER7 controls the transcription of CER3, a key wax biosynthetic gene, via degradation of an mRNA encoding a negative regulator of CER3. To test this model, we expressed the CER3 transgene in the cer7-3 mutant using the epidermis-specific CER6 promoter, which is not affected by the same negative regulator as CER3, and successfully complemented the cer7-3 stem wax phenotype.
To identify the proposed negative regulator and other factors required for CER7mediated control of CER3 expression, we performed a screen for suppressors of cer7-1, which restore cer7-related stem wax deficiency to wild-type wax levels. We isolated four classes of suppressors designated war1 to war4. In this study, we characterized war3 and war4, and the genes disrupted by these mutations. WAR3 encodes RNA-DEPENDENT RNA POLYMERASE 1 (RDR1), one of the six RDR proteins described in Arabidopsis. RDR proteins have been found in diverse eukaryotes, and are considered to be core members of the RNA silencing machinery.
They catalyze the conversion of a single-stranded RNA template into double-stranded RNA (dsRNA), which serves as a substrate for dicer-like enzymes in the production of a type of small The identification of RDR1 and SGS3 in our screen for the cer7-1 suppressors demonstrates that in addition to RDR6, RDR1 function also requires participation of SGS3.
Furthermore, even though RDR1 has not been reported to be involved in endogenous gene silencing, based on our results it seems reasonable to speculate that RDR1 and SGS3 are involved in the production of an as yet uncharacterized small RNA species that directly or indirectly mediates transcriptional gene silencing of CER3 to control wax deposition over the length of the stem. At the top of the stem where the stem is actively growing, wax biosynthetic genes are highly expressed (Suh et al., 2005). Conversely, at the base of the stem where growth has terminated, the expression of wax biosynthetic genes is reduced. As expected, in the wild type we found higher levels of the CER3 transcript in the stem top compared to the stem base ( Fig. 7). In the cer7-1 mutant, CER3 expression is significantly decreased, with CER3 transcript levels being similarly low in both the top and bottom of the stem, which results in the waxdeficient phenotype. In contrast to the cer7-1 mutant, CER3 transcript levels in rdr1-2 cer7-1 and sgs3-15 cer7-1 double mutants are considerably higher in both the top and the stem base than CER3 levels detected in the wild-type (Fig. 7), resulting in restoration of stem wax loads.
The simplest model that integrates all our findings is presented in Fig. 8. Small RNA precursors are known targets of the exosomal RNA ribonucleases (Chekanova et al., 2007). We hypothesize that in the wild-type stem tops where CER7 is highly expressed (Supplemental Fig.   S4), and the CER7 activity is presumably high, this exosomal ribonuclease degrades a precursor of a small RNA species that acts as a repressor of CER3 expression. This results in enhanced CER3 transcription and wax production via the decarbonylation pathway. CER7 expression progressively decreases from the top towards the base of the stem (Supplemental Fig. S4), causing a gradual increase in small RNA accumulation. This is associated with down-regulation of CER3 expression in the epidermal cells and cessation of wax production at the stem base. In the cer7 mutant, where the CER7 exosomal subunit is not functional, buildup of small RNA causes CER3 silencing and stem wax deficiency. The biogenesis of small RNA precursors involved in silencing of CER3 requires RDR1 and SGS3 activities. In the absence of RDR1 or SGS3 in the rdr1 cer7 or sgs3 cer7 double mutant, respectively, the small RNA species responsible for CER3 repression will not be generated, abolishing the need for CER7 in wax biosynthesis.

Quantitative RT-PCR
RNA was extracted from plant tissue using TRIzol (Invitrogen) as per manufacturer's protocol.
RNA quantification was performed using a NanoDrop 8000 (Thermo Scientific). 500ng of total RNA was treated with DNaseI (Fermentas) and then used for first strand cDNA synthesis using iScript RT supermix (Bio-Rad). Quantitative RT-PCR was performed using gene-specific primer sets from Supplemental Table S3, in 20µL reactions using iQ SYBR green supermix (Bio-Rad) and run on the iQ5 real-time PCR detection system (Bio-Rad). Data were analyzed using the Pfaffl method (Pfaffl, 2001), and control samples were normalized to 1. Statistical significance was measured with a student's T-test.

Positional cloning of suppressor lines
To map the position of suppressor lines, each suppressor line was crossed to cer7-3 and grown to the F 2 generation. DNA from leaves was collected on FTA cards (Whatman), and 30-40 plants with the wildtype waxy stem phenotype (plants homozygous for the suppressor mutation) were subjected to PCR using simple sequence length polymorphism (SSLP) markers to determine linkage. To further pinpoint the location of each suppressor loci, over 1000 plants were screened with SSLP markers until a narrow interval was found.

Molecular complementation of suppressor lines and subcellular localization of RDR1 and SGS3
A 5252bp DNA fragment containing 1754bp of the upstream region of RDR1 and the coding region minus the STOP codon was amplified from WT Col plants with primers RDR1p-attB1 and RDR1-attB2_noSTOP using Phusion polymerase (Finnzymes). Gateway adapters were added using the adapter protocol (Invitrogen). This 5252bp fragment was cloned into pDONR221 using BP Clonase II (Invitrogen) to create pDONR221:ProRDR1:RDR1ΔSTOP and was sequenced to confirm that no mutations were introduced during PCR. The fragment was then recombined into the destination vector pGWB4 (Nakagawa et al., 2007) using LR Clonase II (Invitrogen) to generate pGWB4:ProRDR1:RDR1:GFP.
To generate SGS3:YFP for sub-cellular localization analysis, the coding sequence of SGS3 (At5g23570) was obtained from leaf cDNA using primers SGS3-attB1 and SGS3-attB2_noSTOP with Phusion polymerase (Finnzymes). The PCR product was introduced into the pDONR207 entry vector using BP Clonase II (Invitrogen). Sequencing was performed to confirm error free inserts which were then transferred to the binary vectors pEarleyGate104 (Earley et al., 2006) using LR Clonase II (Invitrogen).
Spinning disk confocal microscopy was performed on a Perkin Elmer Ultraview VoX Spinning Disk Confocal mounted on a Leica DMI6000 inverted microscope. GFP and YFP were detected using a 488nm laser and 528/38-nm emission filters. For ER staining, stems and leaves of transgenic sgs3-15 cer7-1 plants expressing SGS3-YFP were immersed in hexyl rhodamine B solution (1.6 µM) for 10 to 30 min. Hexyl rhodamine B was excited with a 561nm laser line and a 600-nm long-pass emission filter. Acquired images were processed using Volocity (Improvision) and ImageJ.

RDR1 and SGS3 promoter:GUS fusions and GUS Activity Assay
To generate ProRDR1:GUS, a 1754bp region upstream of the RDR1 initiation codon was amplified from genomic DNA using the primers RDR1pro_EcoRI-F and RDR1_XbaI-R with Phusion polymerase (Finnzymes). The PCR product was digested with EcoRI and XbaI and cloned into the corresponding restriction enzyme sites of pBluescriptIISK(+) (Stratagene). After confirmation that no errors were induced from PCR, the ProRDR1 region was excised using SalI and BamHI and cloned into the corresponding sites of pBI101 (Clontech) to generate pBI101:ProRDR1:GUS. To generate ProSGS3p:GUS, a 2177-bp long region containing 2141 bps immediately upstream of the SGS3 translation start site and 36 bps downstream of the SGS3 translation start site was amplified from genomic DNA using gene specific primers SGS3pro-attB1 and SGS3pro-attB2 with Phusion polymerase (Finnzymes). The obtained fragment was introduced to pDONR207 entry vector, sequenced to confirm accuracy and transferred into the pMDC163 destination vector.