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First published online June 10, 2009; 10.1104/pp.109.139253 Plant Physiology 150:1902-1915 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
ROOT UV-B SENSITIVE2 Acts with ROOT UV-B SENSITIVE1 in a Root Ultraviolet B-Sensing Pathway1,[C],[OA]Department of Biology, San Francisco State University, San Francisco, California 94132
Ultraviolet B light (UV-B; 280–320 nm) perception and signaling are well-known phenomena in plants, although no specific UV-B photoreceptors have yet been identified. We previously reported on the root UV-B sensitive1 (rus1) mutants in Arabidopsis (Arabidopsis thaliana), which display a block to development under very-low-fluence-rate UV-B (<0.1 µmol m–2 s–1) after the seedling emerges from the seed. Here, we report the analysis and cloning of the rus2-1 mutation in Arabidopsis. The phenotype of rus2-1 mutant seedlings is virtually indistinguishable from the phenotype of rus1 seedlings. A map-based approach was used to clone RUS2. RUS2 encodes a domain of unknown function (DUF647)-containing protein that is homologous to the RUS1 protein. rus1-2 rus2-1 double mutant seedlings have the same phenotype as both rus1 and rus2 single mutants, suggesting that the two genes work in the same pathway. RUS2-Green Fluorescent Protein shows a similar expression pattern as that of RUS1-Green Fluorescent Protein, and RUS1 and RUS2 proteins interact physically in yeast. This protein-protein interaction depends on the DUF647 domain, and site-directed mutagenesis identified specific residues in DUF647 that are required for both protein-protein interaction and physiological function. Six RUS genes are found in Arabidopsis, rice (Oryza sativa), and moss (Physcomitrella patens), and one RUS member, RUS3, is conserved in plants and animals. Our results demonstrate that RUS2 works with RUS1 in a root UV-B-sensing pathway that plays a vital role in Arabidopsis early seedling morphogenesis and development.
The ability to perceive and respond to light, both qualitatively and quantitatively, is critically important for the survival of plants (Whitelam and Halliday, 2007  ). Light is well known to be important to plants for photosynthesis, but it is also used as a developmental signal and can be a source of cellular damage that plants must cope with (Lao and Glazer, 1996; Ries et al., 2000). Ultraviolet B light radiation (UV-B; 280–320 nm) can cause severe deleterious effects in biological organisms, despite representing only a small amount of total solar radiation (Caldwell et al., 1998; McKenzie et al., 2003). UV-B can cause damage to DNA by creating cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone dimers, which can lead to breaks and point mutations if not correctly repaired (Garinis et al., 2005). Additionally, proteins, lipids, and cellular machinery, including photosynthetic machinery, can be damaged by UV-B light (Wilson et al., 1995; Ballare et al., 1996). The amount of UV-B that an organism is exposed to can vary greatly, depending on the position of the sun in the sky and atmospheric conditions. Organisms have evolved mechanisms to perceive and adapt to changing UV-B light conditions (e.g. production of UV-absorbing compounds; Frohnmeyer and Staiger, 2003). Plants are especially vulnerable to UV-B radiation, as they have no means by which to transport themselves away from the light source/sun. Extensive research has focused on the response of plants to damaging levels of UV-B. Interestingly, in addition to damaging biological organisms, UV-B is known to have some positive effects. Plants also have some well-documented photomorphogenic responses to UV-B light (Nogues et al., 1999; Suesslin and Frohnmeyer, 2003; Ulm and Nagy, 2005), although as yet no receptors have been identified in plants for UV-B wavelengths.
Although many genes have been found to be regulated by UV-B in plants, a specific receptor has yet to be identified (Ulm et al., 2004
In plants, UV-B is known to elicit certain photomorphogenic responses, including inhibition of hypocotyl elongation and root growth (Kim et al., 1998
The duplication and divergence of genes is an important phenomenon in the evolution of species (Taylor and Raes, 2004
We previously identified a mutant, root UV-B sensitive1 (rus1), that under LF to VLF UV-B light displayed a severe developmental arrest after germination (Tong et al., 2008
rus2-1 Mutant Seedling Development Is Blocked by UV-B Light rus2-1 mutant plants display a stall in development after the radicle and cotyledons have emerged from the seed (Fig. 1A). Cotyledons fail to green fully, the root does not elongate, and the development of true leaves is greatly delayed. Interestingly, rus2-1 plants recover when transplanted from Murashige and Skoog (MS) medium petri dishes into soil and also grow much better when sown directly onto soil. One major difference between the environments of the soil and the petri dish is the amount of light that the roots of the plants receive. We hypothesized that the light exposure difference between the soil and the MS medium plates affects the phenotype of the rus2-1 mutant plants. We observed that when the roots of rus2-1 mutants are covered, even on MS plates, the rus2 phenotype is partially alleviated and the plants develop more like wild-type plants (Fig. 1, B and C).
To analyze the role of light in the mutant phenotype of rus2-1 plants, we cut the relative amount of light to these plants by the use of neutral density (ND) filters (Fig. 1A). Standard growth chamber fluorescent lights were used as the light source. MS medium plates were covered by the ND filters to reduce the total light intensity. The total photosynthetically active radiation (PAR) for each condition is shown in Table I. We previously analyzed the ND filters and found that they reduced the amount of light across all wavelengths (Tong et al., 2008 ). In this experiment, we grew rus2-1 seedlings alongside wild-type ecotype Columbia (Col) seedlings. We measured the amount of root growth for 7-day-old Col and rus2-1 seedlings under decreased total PAR conditions. As total PAR was reduced, the rus2-1 seedling root length increased (Table I). The lowered light intensities did not dramatically affect the wild-type control, although there was a slight increase in root length of the wild type as light intensities were reduced. Etiolated rus2-1 seedlings were very similar in this experiment to the wild-type, with an average root length 82.6% that of the wild type (Table I). This experiment shows that the severity of the rus2-1 phenotype is directly related to the intensity of light exposure.
rus2-1 mutant plants grown on MS medium plates with exposed roots recover considerably when transferred to soil, an environment where the roots are covered (see Fig. 4A below). Therefore, we hypothesized that the location of light perception responsible for the rus2-1 mutant phenotype is the root. To test this hypothesis, we covered the surface of MS medium plates with black metal foil with pinholes through which the roots could grow. The black surface reduces the amount of light that the roots are exposed to while allowing the apical portions of the plant to be exposed to the normal amount of light. rus2-1 plants grown horizontally on the uncovered control plate displayed a strong phenotype (Fig. 1B, top). rus2-1 seedlings grown horizontally on black metal foil-covered plates showed elongated roots and greener apical portions/cotyledons (Fig. 1C, top). This result suggests that the light environment of the root elicits not only the rus2-1 root phenotype but also the shoot phenotype.
Growth of rus2-1 plants on monochromatic light sources (blue light-emitting diode,  maximum emission of 470 nm; green light-emitting diode, maximum emission of 525 nm; red light-emitting diode, maximum emission of 633 nm) showed that none of the canonical light perception wavelengths (320–750 nm) is able to elicit the strong rus2-1 phenotypes (data not shown), a phenomenon found in rus1 (Tong et al., 2008). Additionally, rus2-1 double mutants with phototropin, phytochrome A, phytochrome B, and cryptochrome mutants (phot1, phot2, phot1phot2, nph3, phyA, phyB, phyAphyB, cry1, cry 2, cry1cry2) still displayed the same rus mutant phenotype (data not shown). Therefore, we hypothesized that UV-B light was responsible for the rus2 phenotype. To test this hypothesis, we covered plates in our growth chamber with either a UV shield or a sheet of non-UV-absorbing plastic as a control. Under the UV-B shield conditions, the rus2-1 mutant plants recovered considerably, reaching roughly half the root length of the wild-type plant (Fig. 1D). The non-UV-absorbing plastic-covered control plates did not show any recovery of the rus2-1 phenotype (Fig. 1E). This result suggests that UV-B is predominantly responsible for the mutant phenotypes in the rus2-1 mutant. We measured a UV-B fluence rate of 0.300 µmol m–2 s–1 in the normal light conditions in our growth chamber (Tong et al., 2008). Of the ND filters that we used, the one with the highest reduction in light intensity (ND 0.15) had a UV-B fluence rate of 0.025 µmol m–2 s–1. Even in the ND-0.15 light conditions, a partial rus2-1 phenotype was observed. Thus, we conclude from our data that LF and VLF UV-B can elicit the rus2-1 phenotype.
The rus2-1 mutation was identified from a screen for rus-like phenotypes using fast-neutron mutagenesis (Koornneef et al., 1982
To identify the nature of the rus2-1 mutation, we attempted to PCR amplify overlapping portions of At2g31190 for sequencing. Although we could amplify regions at the end of the gene, we failed to amplify internal portions of the gene in the rus2-1 background, despite being able to amplify these same fragments from the wild type. Additionally, we were unable to amplify across the entire length of the gene, something that should be possible with a simple deletion. These results suggested that the mutation was the result of a chromosomal rearrangement. We determined the exact nature of the rus2-1 mutation using thermal asymmetric interlaced PCR (Liu and Huang, 1998 ), working from both ends of the gene toward the middle. Thermal asymmetric interlaced PCR showed that the rus2-1 mutation is a disruption in the last intron and that the chromosome was broken there and reattached incorrectly to another portion of chromosome II (Fig. 2B). Our results indicate that two break points occurred in chromosome II and were repaired in a reverse orientation (Fig. 2C, red portion). The second break point is on BAC T1B3 after bp 15,812, in a region where no genes are predicted to occur (Swarbreck et al., 2008). At these two break point/repair sites, no genomic DNA was deleted, and a single T/A base pair was added at each site (Fig. 2, D and E, underlined base). Primers spanning either of the two break point sites yielded products only in the wild type (Fig. 2F). Recombining the same set of primers amplified mutant T1B3-RUS2 pieces as predicted (Fig. 2F). Reverse transcription (RT)-PCR analysis showed no expression of RUS2 after the break point (Fig. 2G). As the At2g31190 gene is disrupted in the rus2-1 background and capable of fully complementing the rus2-1 phenotype (Fig. 2G) in more than six independent lines, we conclude that RUS2 is At2g31190.
RUS2 encodes a 433-amino acid protein (48 kD) with no known function(s). A BLAST search with the RUS2 protein sequence uncovered the presence of an evolutionarily conserved domain called DUF647 (Pfam accession no. PF04884) in this protein. Additionally, the RUS2 protein is annotated by The Arabidopsis Information Resource (www.arabidopsis.org; Swarbreck et al., 2008
In order to ascertain the location of the RUS2 protein in vivo, we cloned the RUS2 gene, including promoter, in frame with the GFP coding sequence. This construct fully complements the rus2-1 phenotype (Figs. 1, D and E, and 2G). When visualized by confocal light scanning microscopy, we detected a low level of GFP fluorescence (Fig. 3E)
in the root tip that was consistently higher than the background autofluorescence of the wild-type controls (Fig. 3B). The GFP fluorescence in the root tip is strikingly similar to the localization we previously reported for the RUS1 protein (Tong et al., 2008
We analyzed the RUS2 protein sequence using the target signal prediction programs WoLF PSORT (www.wolfpsort.org; Horton et al., 2006 , 2007) and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP; Emanuelsson et al., 2000). Neither of these programs predicts a strong subcellular localization for RUS2. A previous well-conducted proteomic study found the RUS2 protein in purified plastid extracts (Ferro et al., 2003). This is consistent with our GFP fusion results, which show that RUS2 is not in the cell wall or membrane and is not localized to the nucleus. Additionally, WoLF PSORT and TargetP 1.1 predict that the RUS1 protein is localized to the plastid and that RUS1-GFP shows virtually the identical GFP fluorescence pattern as RUS2-GFP. Thus, our data here are consistent with the empirical results reported before, which show RUS2 to be in the chloroplast (Ferro et al., 2003).
We previously reported the characterization and cloning of the rus1 mutations and their effects on Arabidopsis seedlings grown under UV-B light (Tong et al., 2008 When grown vertically on MS medium plates under our normal growth chamber light conditions (71.2 µE, PAR), the rus1-2 rus2-1 double mutant appears indistinguishable from both the rus1-2 and rus2-1 single mutants (Fig. 4A). In the light, the double mutant germinates and arrests at the same time as either of the single mutants. On MS agar plates, the roots of 7-d-old light-grown rus1-2 rus2-1 double mutant seedlings were of a comparable length to either of the single mutants (Fig. 4B, Light). We next examined 7-d-old etiolated (dark-grown) seedlings for the wild type, rus1-2, and rus2-1 single mutants and the rus1-2 rus2-1 double mutant (Fig. 4A). The roots of rus1-2 rus2-1 double mutants greatly elongated in the dark, as compared with the roots of rus1-2 rus2-1 light-grown seedlings. The roots of the single and double mutants grew to about 75% that of the wild type in the dark. These results support a rejection of our initial hypothesis that the rus1-2 rus2-1 double mutant plants would be more severe than either single mutant alone. Instead, these data suggest that RUS1 and RUS2 are both part of the same genetic pathway and are likely to function in the same biochemical pathway. A loss of either RUS1 or RUS2 eliminates the function of this pathway, so that a further loss of the other gene has little to no effect.
As noted earlier, rus1 and rus2 single mutants have virtually identical phenotypes and the double rus1 rus2 mutant has a phenotype very similar to either of the single mutants alone. Therefore, rus1 and rus2 mutants genetically interact in a way that strongly suggests that RUS1 and RUS2 are working in the same pathway. Additionally, the RUS2-GFP fluorescence pattern is very similar to the RUS1-GFP fluorescence pattern. As the RUS1 and RUS2 proteins share a similar domain, we hypothesized that these proteins interact physically. In our experiences, RUS1 and RUS2 proteins are highly unstable in vitro and rapidly degrade, even in the presence of SDS, protease inhibitors, and a reducing agent (data not shown). This characteristic of the RUS1 and RUS2 proteins made our in vitro analyses of these proteins unsuccessful. Therefore, we utilized a yeast-two-hybrid system to test for interactions between these proteins in an in vivo assay using various combinations of the RUS1 and RUS2 proteins to test our hypothesis. We used the pGADT7 and pGBKT7 vectors to create C-terminal fusions with the Gal4 activating domain (AD) and Gal4 DNA-binding domain (BD), respectively. Neither the RUS1-BD nor the RUS2-BD construct was self-activating when cotransformed with an empty AD vector (Fig. 5C ; data not shown). We did not observe any self-interaction for RUS1 or RUS2 in our system (Fig. 5C). In support of our original hypothesis, we did observe interaction between RUS1 and RUS2 (Fig. 5C).
Specific Residues in the DUF647 Domain Are Required for RUS1-RUS2 Physical Interactions and Functionalities We next hypothesized that the interaction between RUS1 and RUS2 was via the DUF647 domain. We created constructs for RUS1 that contained either the DUF647 domain (R1DUF) or the region of the protein preceding the DUF647 domain (R1preDUF). The R1DUF corresponds to amino acids 182 to 608, and the R1preDUF corresponds to amino acids 1 to 181. R1DUF was able to interact with RUS2, and R1preDUF647 did not show any interaction with RUS2 (Fig. 5C; data not shown). The RUS2 protein has only about 50 amino acids before its DUF647 domain; thus, RUS2 is almost entirely a DUF647 domain already. Therefore, we obtained strong evidence that the DUF647 domain has a protein-protein interaction function and is required for the interaction of RUS1 with the RUS2 protein. Next, we used site-directed mutagenesis on the RUS1-AD constructs to alter four selected conserved (100% in DUF647 domains from various species) amino acids in the DUF647 domain (Fig. 5B). Two of these mutant rus1 proteins (K281G and K349G) failed to interact with RUS2 in our yeast two-hybrid system (Fig. 5C). The other two mutant rus1 proteins (E298G and N342G) interacted with RUS2, but the observed interaction was much weaker than that with the wild type (Fig. 5C). We created pRUS1::RUS1 constructs with these same point mutations and transformed them into rus1-1 mutant plants. We observed at least partial rescue of the rus1-1 phenotype with all of these mutant constructs (Fig. 5D). Interestingly, the amount of rescue was greater in the two mutants that showed partial interaction via yeast two-hybrid assay (Fig. 5D). The fact that these mutant proteins can at least partially suppress the rus1-1 phenotype in vivo is evidence that the mutant versions are being produced. The ability of the non-yeast two-hybrid-interacting mutants to partially suppress the rus1-1 phenotype suggests that there are additional factors in the plant that support the function of the RUS1/RUS2 complex. Thus, these data strongly support the hypothesis that RUS1 and RUS2 physically interact via the DUF647 domain and that this interaction is important for their function in vivo.
We searched the completed Arabidopsis genome and found a total of six DUF647-encoding genes (RUS genes; Arabidopsis Genome Initiative, 2000
We next looked for RUS genes in the completed animal genomes by BLAST search with the RUS1 protein. We found a single RUS gene in each of the human, mouse, Drosophila, zebrafish, pufferfish, horse, and cow genomes. Interestingly, we were unable to identify a RUS homolog in the chicken genome (Gallus gallus), despite using multiple search methods. The Mouse Genome Database lists MmRUS (BC017158) cDNAs from various tissues, including heart, spleen, bone marrow, dendritic cells, salivary gland, melanoma, and mammary gland tumors (www.informatics.jax.org; Eppig et al., 2007 Various fungal species have RUS proteins, but the relationship to plant or animal RUS proteins is not clear and some fungi, such as Saccharomyces cerevisiae do not have any RUS proteins. This lack shows that it is possible for some eukaryotes to live without RUS protein(s), although the vast majority of eukaryotic genomes that we searched have at least one. A RUS protein from the fungal species Coprinopsis cinerea okayama7#130 has a predicted protein with an oxidoreductase enzyme fused in front of a DUF647 domain (XP_001838896). This is interesting because, in the over 50 RUS proteins we looked at from various species, it is the only example that has an additional predicted domain besides DUF647. This has perhaps been misannotated, as previously a mevalonate diphosphate decarboxylase-DUF647-fused protein was predicted in a Neurospora species, only later to be annotated as two separate proteins. Therefore, the overwhelming bulk of sequence data suggest that RUS proteins are made up primarily of the DUF647 domain. Additional parts of RUS proteins are typically small and nonconserved. To analyze the phylogenetic relationships between RUS proteins, we performed a ClustalW alignment, which we then used to create a phylogenetic tree using maximum-likelihood point-accepted mutation with neighbor joining. For our analysis, we used protein sequences from Arabidopsis, rice, moss, the green algae Ostreococcus lucimarinus, human, mouse, zebrafish (zgc:162613), and Drosophila (CG10338). Interestingly, the RUS protein in animals clusters with AtRUS3 (At1g13770) from Arabidopsis, and the phylogeny constructed for RUS3 proteins mimics the phylogenetic relationship of these species. These data suggest that the plant RUS3 proteins are true orthologs of the RUS proteins in animal lineages. RUS1, RUS2, and RUS6 also have highly supported clades between the moss, rice, and Arabidopsis lineages, strongly supporting the orthology of these genes between these species. The RUS3 cluster is a monophyletic grouping within the larger RUS phylogeny, which suggests that there were several RUS genes in the ancestor of animals and plants and that only the RUS3 ortholog has been maintained in animals. However, the base of the RUS family tree is only weakly defined, with very short branch lengths between groupings. Therefore, we cannot rule out that the additional RUS genes in plants are the result of early duplications in the plant kingdom. It is very likely, however, that the common ancestor of moss and higher plants had multiple RUS genes, as there is a clear orthological relationship between the moss RUS proteins and the rice and Arabidopsis RUS proteins. The O. lucimarinus genome has two RUS genes, one that clusters with RUS4 and another that does not clearly cluster with any of the RUS proteins from plants. Thus, RUS proteins exist in most plants and animals and in some fungi, but the basal relationships are not clearly definable. We conclude that the animal RUS protein has an ortholog in plants and that that ortholog is RUS3.
We have isolated and analyzed the RUS2 gene, a second gene in addition to RUS1 that when mutated gives a root UV-B-sensitive phenotype (Tong et al., 2008 ). Under our standard growth conditions, the rus1 and rus2 mutant plants display a virtually indistinguishable development-block phenotype that occurs after the seedling has emerged from the seed. Rather than being more severe, the double rus1 rus2 mutant has the same phenotype as either of the single mutants alone. Additionally, we have shown that the RUS1 and RUS2 proteins interact in a yeast two-hybrid assay via the DUF647 domain, a conserved domain in eukaryotes that both RUS1 and RUS2 share. RUS1 and RUS2 also share similar gene expression and GFP fluorescence patterns, supporting the hypothesis that they function together. RUS1 is predicted to be localized to plastids, and RUS2 has been empirically proposed to be in plastids in a previous study (Ferro et al., 2003). Interestingly, RUS2 does not have a clear subcellular signaling sequence, which suggests that RUS2 may be localized to the plastid as a consequence of its interaction with RUS1.
We have shown that like rus1 mutants, rus2 mutant plants are hypersensitive to light in the UV-B range (280–320 nm). rus2-1 mutant roots are approximately 80% the length of wild-type plants when grown in the dark. As light increases, the roots become progressively shorter until they essentially fail to elongate at all in the full growth chamber light. We interpret these results to suggest that the RUS1/RUS2 complex acts as a negative modulator of a unique UV-B perception pathway in Arabidopsis. Without either RUS1 or RUS2, the small amount of UV-B present in the growth chamber lights is perceived by the plant as a much larger amount. It is unlikely that RUS1/RUS2 is involved in a UV-B-defensive pathway for several reasons. First, rus1 and/or rus2 mutant plants do not die in the light; they are merely blocked from developing, and they recover when the root is covered. If such a small amount of UV-B were causing severe damage to rus mutant plants, then it is logical to conclude that these plants would die after a few days. Second, the amount of UV-B needed to induce a strong rus phenotype is very low (<0.1 µmol m–2 s–1) and is considered to be nondamaging at that level (Ulm and Nagy, 2005
Previous reports have focused on the role of UVR8, HY5, and HYH genes in regulating the response to UV-B in Arabidopsis (Ulm et al., 2004
Based on the similar phenotypes of rus1 and rus2 mutants, we hypothesized that the proteins might interact physically in the same complex. RUS1 and RUS2 proteins did indeed interact in our yeast two-hybrid analysis. Additionally, the DUF647 domain of RUS1 was necessary and sufficient for this interaction. The region of RUS1 preceding the DUF647 domain did not interact with RUS2, and it is yet to be determined whether this region is required for RUS1 function. In vivo, these two proteins have indistinguishable localizations when analyzed via GFP fusion constructs. RUS1 is strongly predicted to be plastid localized, and RUS2, although not predicted to be plastid localized, has been previously found in a study that isolated plastid proteins (Ferro et al., 2003
DUF647 proteins (RUS proteins) exist in many eukaryotic species, including all of the plants (Goff et al., 2002 Our results strongly support a model where the RUS1 and RUS2 genes work together in the same genetic and biochemical pathway(s). In our model, RUS1 and RUS2 physically interact and are necessary to modulate a signal from a UV-B receptor negatively (Fig. 7A). This signal positively regulates a proposed "developmental block" that prevents further development after germination (Fig. 7A). The RUS1/RUS2 complex is required to dampen or diminish this signal under normal UV-B light quantities. In the dark or in a UV-B-free light environment, the receptor is not activated; thus, the action of the RUS1/RUS2 complex is not required (Fig. 7B). Without RUS1 or RUS2, the signal from the UV-B receptor is not properly dampened and is thus large enough to activate the postgermination developmental block (Fig. 7C). This model best represents our current understanding of our rus mutant data. rus mutant plants exhibit a hypersensitive response to LF or VLF UV-B, and our model represents this by placing the RUS1/RUS2 complex in a position to modulate the signal in this response.
Our model predicts that high levels of UV-B light will elicit a rus-like phenotype in wild-type plants. This is virtually impossible to test, however, as UV-B light at high fluence is very damaging, making it impossible to distinguish signaling responses from damage responses. This model also predicts that a loss of the receptor or of key signaling components would restore a wild-type phenotype to rus plants. Since there are currently no known photoreceptors for UV-B in plants, the rus1 and rus2 mutant phenotypes represent an opportunity for identifying the photoreceptor(s) responsible for perceiving low-level, nondamaging UV-B. Currently, we are focusing on identifying suppressors of rus mutants in Arabidopsis. Having a strong UV-B-induced phenotype under VLF UV-B should be ideal for identifying additional members of this UV-B perception pathway.
Plant Material and Growth Conditions
Arabidopsis (Arabidopsis thaliana Col-0) plants were grown as described before (Lally et al., 2001
The rus2 mutation segregates as a typical recessive allele. The ratio of wild type to rus2 from the F1 parents is 3:1. rus1 rus2 double mutants were created by crossing rus2-1 to the three available rus1 alleles (rus1-1, rus1-2, and rus1-3), and the double mutation was confirmed by known established markers. Genotyping analysis and a map-based approach were followed as described previously (Tong et al., 2008
Seven-day-old seedlings were used for GFP detection. GFP fluorescence was excited by a blue argon laser (10 mW, 488-nm blue excitation) and detected at 515- to 530-nm wavelengths in a Nikon C1 Confocal E600FN microscope. Whole roots were directly mounted in water and observed with water objectives (203 and 603). Wild-type seedlings were used as negative controls. Images were processed and arranged by Adobe Photoshop version CS3.
Vertically grown plates were photographed, and the images were analyzed using the ImageJ program (Rasband et al., 1997–2008
RNA was extracted from plant tissues using the RNeasy Mini Kit (Qiagen catalog no. 74106) and quantified spectrophotometrically. Reverse transcriptase reactions were carried out using the OneStep RT-PCR Kit (Qiagen catalog no. 210210). The reactions were scaled down from 50 to 15 µL with all reagents kept at the same final concentrations. For each reaction, 100 ng of total extracted RNA was used. Reverse transcriptase reactions were done for 50 min at 50°C, followed by a 2-min 95°C step to activate the HotStarTaq DNA polymerase. The PCRs were done for 25 to 35 cycles, depending on the gene. The PCR temperatures and times were as follows: 30 s of denaturing at 94°C; 30 s of annealing at 52°C; and 1 min of extension at 72°C. The reaction products were run on 2% agarose gels and imaged using a Kodak 4000R Image Station. Products were sequenced to confirm identity. Primer sequences and cycle numbers are available upon request.
Yeast two-hybrid analysis was performed using the vectors from the Matchmaker Two-Hybrid Library and Construction Kit (Clontech catalog no. 630445). cDNAs were cloned into either pGADT7 or pGBKT7 and transformed into yeast to create yeast containing a single vector. Yeast were mated together to create yeast with two vectors for two-hybrid analysis. Confirmation of the presence of both vectors was performed by growing the yeast on medium lacking Trp and Leu. Experimental protein-protein interaction was determined by growth on plates lacking Trp, Leu, and His and containing 5-Bromo-4-chloro-3-indolyl-
Proteins were aligned using the ClustalW program. The BLOSUM30 matrix was used for pair-wise alignment with an open gap penalty of 10 and an extend gap penalty of 0.1. Multiple alignment was performed using the BLOSUM Series with an open gap penalty of 10, an extend gap penalty of 0.05, and a delay divergent of 40%. Trees were constructed using the PHYLIP phylogenetic analysis programs. Trees were constructed using Protdist with point-accepted mutation settings, followed by the Neighbor program for neighbor-joining analysis. For bootstrap analysis, the Seqboot program was used prior to the Protdist and Neighbor programs, followed by the Consensus program to create a consensus tree. A total of 1,000 samples were used for bootstrapping. Trees were drawn with the Drawtree program. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers NP_609897.2 (DmRUS), NP_001103923.1 (DrRUS), Q91W34.1 (MmRUS), NP_073581.1 (HsRUS), XP_001419164.1 (OlRUSA), XP_001418386.1 (OlRUSB), XP_001755448.1 (PpRUS1), XP_001766030.1 (PpRUS2), XP_001764017.1 (PpRUS3), XP_001759421.1 (PpRUS4), XP_001762143.1 (PpRUS6A), XP_001764974.1 (PpRUS6B), CAE02373.2 (OsRUS1), NP_001053319.1 (OsRUS2), ABF94623.1 (OsRUS3), NP_001041984.1 (OsRUS5), BAD82242.1 (OsRUS6A), NP_190175.2 (AtRUS1), NP_565718.1 (AtRUS2), NP_172832.3 (AtRUS3), NP_179928.2 (AtRUS4), NP_195771.2 (AtRUS5), and NP_568713.1 (AtRUS6/EMB1879).
We are very grateful to Winslow Briggs (Carnegie Institute of Washington, Stanford, CA) for critically reading the manuscript and for helpful discussions. We thank members of the He laboratory for discussions and Annette Chan (Cell Molecular Imaging Center, San Francisco State University, San Francisco) for help with the confocal microscopy. Received March 30, 2009; accepted June 6, 2009; published June 10, 2009.
1 This work was supported by the National Science Foundation (Faculty Early Career Development [CAREER] Program award no. MCB9985185) and the National Institutes of Health (Support of Competitive Research [SCORE] Institutional Development award no. S06 GM52588) to Z.-H.H. 
2 These authors contributed equally to the article.
3 Present address: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.
4 Present address: Laboratory of Molecular Plant Physiology, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Zheng-Hui He (zhe{at}sfsu.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.139253 * Corresponding author; e-mail zhe{at}sfsu.edu.
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was used as a control. Bar = 1 cm. [See online article for color version of this figure.]
