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First published online August 11, 2006; 10.1104/pp.106.083519 Plant Physiology 142:651-661 (2006) © 2006 American Society of Plant Biologists Down-Regulation of the 26S Proteasome Subunit RPN9 Inhibits Viral Systemic Transport and Alters Plant Vascular DevelopmentCenter for Plant Cell Biology, Department of Plant Pathology, University of California, Riverside, California 92521
Plant viruses utilize the vascular system for systemic movement. The plant vascular network also transports water, photosynthates, and signaling molecules and is essential for plant growth. However, the molecular mechanisms governing vascular development and patterning are still largely unknown. From viral transport suppressor screening using virus-induced gene silencing, we identified a 26S proteasome subunit, RPN9, which is required for broad-spectrum viral systemic transport. Silencing of RPN9 in Nicotiana benthamiana inhibits systemic spread of two taxonomically distinct viruses, Tobacco mosaic virus and Turnip mosaic virus. The 26S proteasome is a highly conserved eukaryotic protease complex controlling many fundamental biochemical processes, but the functions of many 26S proteasome regulatory subunits, especially in plants, are still poorly understood. We demonstrate that the inhibition of viral systemic transport after RPN9 silencing is largely due to alterations in the vascular tissue. RPN9-silenced plants display extra leaf vein formation with increased xylem and decreased phloem. We further illustrate that RPN9 functions at least in part through regulation of auxin transport and brassinosteroid signaling, two processes that are crucial for vascular formation. We propose that RPN9 regulates vascular formation by targeting a subset of regulatory proteins for degradation. The brassinosteroid-signaling protein BZR1 is one of the targets.
Virus movement within the plant occurs via local cell-to-cell movement through plasmodesmata and long-distance transport through the vasculature, mainly the phloem (Crawford and Zambryski, 1999  ; Scholthof, 2005). Compared with work on virus cell-to-cell movement (Zambryski and Crawford, 2000; Zambryski, 2004), there have been relatively few studies on virus long-distance movement in the vascular system (Santa Cruz, 1999). Using a modified Tobacco mosaic virus (TMV) expressing green fluorescent protein (GFP), Cheng and colleagues studied the systemic vascular invasion routes of TMV in Nicotiana benthamiana and determined that TMV could enter minor, major, or transport veins directly from nonvascular cells to produce a systemic infection (Cheng et al., 2000). Only a few host genes have been identified to be important for virus systemic spread (Scholthof, 2005). A vascular-specific Gly-rich protein inhibits the long-distance movement of Turnip vein-clearing virus, probably by inducing callose accumulation in the phloem cell walls (Ueki and Citovsky, 2002). RTM1 and RTM2, which function in phloem, were found to restrict long-distance movement of Tobacco etch virus (Chisholm et al., 2000, 2001; Whitham et al., 2000). However, the proteins identified so far only affect the systemic transport of specific viruses. Host components required for broad-spectrum virus systemic transport have yet to be identified.
An integrated vascular network is not only required for plant viral systemic transport, but is also essential for transporting water, nutrients, and signaling molecules during plant growth. The vascular system consists of phloem, xylem, and meristematic cells—procambium (Ye, 2002
A major research impediment in this field has been the small number of vascular development mutants because knockout mutations in central regulatory components of vascular development are likely to be lethal. Notably, most of the genetic mutants studied so far, including monopteros (arf5; Hardtke and Berleth, 1998
RPN9 encodes a subunit of the 26S proteasome, a highly conserved eukaryotic protease responsible for intracellular protein degradation. The 26S proteasome consists of a 20S catalytic core particle (CP) and a 19S regulatory particle (RP), and controls many fundamental biochemical processes by programmed degradation of regulatory protein targets besides its role in removing damaged or misfolded proteins. The ubiquitin/26S proteasome pathway is implicated in numerous diseases, including cancer and neurodegenerative diseases (Mani and Gelmann, 2005
The capacity of protein selection for degradation by the proteasome is gained through the 19S RP, which recognizes, binds, and unfolds the target proteins, cleaves ubiquitin chains, and directs the target proteins into the lumen of the CP for degradation (Wolf and Hilt, 2004 Here we demonstrate that down-regulation of RPN9, but not two other 26S proteasome subunits, inhibits the systemic spread of two taxonomically distinct viruses. Inhibition of virus spread may be largely due to the alteration of vascular formation with reduced phloem and induced xylem. Our further analysis shows that RPN9 functions at least in part through regulation of auxin transport and brassinosteroid (BR) signaling. Given the established role of the 26S proteasome in programmed degradation of regulatory proteins, we propose that RPN9 regulates plant vascular development by targeting a subset of regulatory proteins for degradation. Our data suggest that the BR-signaling protein BZR1 is one such target.
RPN9-Silenced Plants Inhibit Systemic Transport of Two Taxonomically Distinct Viruses
The RPN9 gene was identified in a functional high-throughput VIGS screen to identify genes necessary for viral infection and systemic spread in N. benthamiana, one of the best hosts for plant virus studies. A cDNA library was enriched for TMV-induced genes by suppression subtractive hybridization between cDNAs prepared from TMV-challenged and untreated N. benthamiana plants. The cDNA library was cloned into Potato virus X (PVX)-derived silencing vector pGr106 and introduced into Agrobacterium for high-throughput silencing in wild-type N. benthamiana. GFP-labeled TMV was inoculated on the silenced leaves (start from the third or fourth leaf above the VIGS agroinfiltration sites) 2 weeks after Agrobacterium inoculation for visualization of the viral infection and systemic spread in the silenced plants (Jin et al., 2002 The onset of viral infection foci was clearly visible on the inoculated leaves of both RPN9-silenced plants (RPN9–) and pGr106 empty vector-treated controls at 4 days postinoculation (4 DPI) of TMV-GFP (Fig. 1A ). The infection foci enlarged to a similar size in both RPN9– plants and control leaves, which indicates that cell-to-cell movement of TMV-GFP was not affected. However, the virus failed to achieve systemic spread in RPN9-silenced plants because no GFP fluorescence was detected on the upper uninoculated leaves at 10 DPI (Fig. 1A). In contrast, the virus invaded the vascular tissue of control plants and spread systemically to upper uninoculated leaves (Fig. 1A, 10 DPI). Real-time reverse transcription (RT)-PCR analysis showed that RPN9 mRNA was reduced by 80% to 95% in the silenced leaves beginning at 8 days postsilencing induction (DPS) relative to the empty vector-treated control (Fig. 1B). VIGS induces RNA degradation based on nucleotide sequence identity. Southern analysis under stringent conditions showed at most two copies of RPN9, which might share greater than 80% identity in the N. benthamiana genome (data not shown). Database searches revealed that two RPN9 genes sharing more than 87% identity are present in Arabidopsis and tomato (Lycopersicon esculentum). VIGS of RPN9 in N. benthamiana very likely silences both homologs because the silencing insertion is about 1 kb and covers the conserved region.
Previous studies have identified host components that only affect the systemic transport of specific viruses. To determine whether RPN9-silenced plants inhibit systemic transport of other viruses, we tested Turnip mosaic virus (TuMV), a potyvirus that is taxonomically very different from TMV, a tobamovirus. The systemic transport of GFP-tagged TuMV was also attenuated after RPN9 silencing (Fig. 1C); no virus was visualized in the upper uninoculated leaves. Therefore, RPN9 is required for systemic transport of both TMV and TuMV.
The inhibition of systemic spread of two taxonomically distinct viruses suggested that a common structure required for viral systemic transport, such as the vascular system, might be altered in RPN9– plants. Leaf-clearing experiments did not reveal obvious differences in vein patterning between RPN9– and control leaves. However, veins were slightly denser and clearly thicker in RPN9– leaves than the same order of veins in control leaves (Fig. 2A
). Scanning electron microscopy (SEM) revealed that, at 14 DPS, double- and even triple-twisted veins were present on the abaxial surface of RPN9– leaves (Fig. 2B). Interestingly, the additional veins emerged adjacent to the existing veins (Fig. 2B, arrows), including both midveins and high-order veins. In wild-type plants, vascular bundles with normal auxin transport suppress the formation of new vascular strands in their immediate vicinity (Sachs, 1991
Increased Xylem and Reduced Phloem Were Observed in RPN9– Plants Increased vein formation might be expected to aid viral systemic spread. However, long-distance movement of viruses was inhibited in RPN9– plants. To further explore this unexpected finding, we characterized the fine structures of the veins and stems of RPN9– plants. Compared to control plants that formed veins with a single vascular bundle, two or three vascular bundles were observed in RPN9– plants as early as 12 DPS (Fig. 3A , black circles). These vascular bundles contained increased xylem and decreased phloem in both veins (Fig. 3A) and stems (Fig. 3B). The phenotype became more severe at later stages of silencing (16 DPS or later) and the highly organized bicollateral vascular bundle patterning was disrupted (Fig. 3, A and B). Lignin deposition was greatly increased in RPN9– plants, which confirms that RPN9 silencing promotes xylem formation (Fig. 3C). These data indicate that RPN9 silencing suppresses phloem and promotes xylem, which may be the major cause of viral transport inhibition.
The Function of RPN9 in Vascular Development Is Specific
RPN9 is a subunit of the 19S RP lid, which is a part of the 26S proteasome. To determine whether the phenotype observed is specific to RPN9 silencing or is a common phenomenon of silencing any of the 26S proteasome subunits, genes encoding two other subunits, the
Auxin Transport Is Reduced in RPN9– Plants
Various phytohormones and steroids have been implicated in vascular development and vein patterning (Fukuda, 2004
To test this hypothesis, we first analyzed the gene expression level of PIN1 and PINOID, two major players in auxin transport. PIN1 mRNA levels were similar in control and RPN9– plants (data not shown). In contrast, PINOID mRNA levels were reduced in RPN9– plants compared to the control (Fig. 5A
). PINOID is preferentially expressed in young vascular tissues and controls the polar localization of PIN proteins for auxin transport (Christensen et al., 2000
To confirm that the inhibition of auxin transport really induces vein formation in N. benthamiana, we germinated seeds in Murashisge and Skoog medium containing the auxin transport inhibitor NPA. The presence of 25 µM NPA induced thick vein formation (Fig. 5D), mimicking the phenotype of extra vein formation in RPN9– plants (Fig. 3C). Plants germinated on NPA arrested and could not be used for the viral infection assay. To examine the effect of extra vein formation on viral systemic transport, we sprayed 3-week-old N. benthamiana with 150 µM of NPA as described in Nemhauser et al. (2000) . Leaves became crinkly and extra veins were observed in these leaves 1 week after NPA treatment, especially along the leaf margins (Fig. 5E). Systemic transport of TMV-GFP was clearly inhibited in plants that had been sprayed with NPA, but was not completely blocked (Fig. 5E). Virus could enter the vascular system and could occasionally be transported to the upper uninfected leaves (data not shown). These results suggest that inhibition of auxin transport indeed induces extra vein formation, which may partially inhibit viral systemic transport.
RPN9– plants had curled crinkly leaves with increased xylem and reduced phloem in the vascular bundles. The silenced leaves were dark green and underwent early senescence initiated from the veins (Fig. 4B). These phenotypes were not observed in NPA-treated plants and cannot all be explained simply by reduced auxin transport, but point to the likelihood of the involvement of the BR-signaling pathway in RPN9-mediated regulation. BRs are involved in xylem cell differentiation by regulating secondary wall formation and the programmed cell death that occurs during TE differentiation (Yamamoto et al., 1997
BRs are perceived by the receptor-like kinases BRI1, BRL1, and BRL3, which then inactivate the negative regulator BIN2 and stabilize its downstream transcription factors BZR1 and BES1 to promote BR-regulated gene expression (Cano-Delgado et al., 2004 ; Nemhauser and Chory, 2004; Vert and Chory, 2006). BRL1 and BRL3 are specifically expressed in vascular cells and a loss-of-function mutation in BRL1 causes reduced xylem and increased phloem differentiation (Cano-Delgado et al., 2004). Conversely, overexpression of BRI1 results in increased procambial activity leading to increased xylem differentiation. These results suggest the existence of a vascular cell-specific BR perception system. The N. benthamiana ortholog of the vascular-localized BRL1 was clearly induced in RPN9– leaves (Fig. 6B), whereas the mRNA levels of NbBRI1 and NbBIN2 remained unchanged in RPN9– plants (data not shown). Gene expression analyses were carried out on samples collected 8 DPS, a time when the expression of RPN9 was clearly suppressed (Fig. 1B), but the plants appeared morphologically normal. Thus, the observed changes in gene expression are likely to reflect direct effects of RPN9 silencing rather than secondary effects from morphological alterations. Our results indicate a possible increase of BR perception in vascular tissues in RPN9– plants. Loss of function in BRL1 causes increased phloem and reduced xylem differentiation (Cano-Delgado et al., 2004), which correlates perfectly with our data that RPN9– plants have elevated BRL1 expression and display increased secondary xylem and reduced phloem.
As a subunit of the 26S proteasome, RPN9 is likely to function through protein degradation. To determine whether RPN9 regulates degradation of many proteins or only a small number of targets, we analyzed total polyubiquinated proteins. If RPN9 affects global protein degradation, we would expect to see an accumulation of polyubiquinated proteins in RPN9– plants, as was observed in the rpn10 mutant in Arabidopsis (Smalle et al., 2003
Previous studies have shown that a downstream transcription factor of the BR-signaling pathway, BZR1, is regulated by 26S proteasome-mediated degradation (Wang et al., 2002
To determine whether RPN9 silencing affects other proteins regulated by 26S proteasome degradation, we examined the protein level of ICE1 (Chinnusamy et al., 2003
Many plant viruses rely on vascular structure, primarily the phloem, for their systemic transport. The vascular system is also essential for plant development. However, the molecular regulatory mechanisms of plant vascular development are still poorly understood. From a viral transport suppressor screen using VIGS in N. benthamiana, we have identified RPN9, a regulatory subunit of the 26S proteasome. Silencing RPN9 inhibits the systemic transport of two taxonomically distinct viruses, which leads to the finding that RPN9 regulates plant vascular development. Silencing of RPN9 induced extra vein formation with increased xylem and reduced phloem, which may largely contribute to the inhibition of broad-spectrum viral systemic transport. This genetic evidence indicates that plant vascular development is regulated at the proteolysis level via a specific 26S proteasome subunit. RPN9 is a subunit of the 19S RP lid. Limited studies suggest that different subunits of 19S RP have well-differentiated functions (Glickman et al., 1999 ). On the basis of recent studies on RPN12a (Smalle et al., 2002), RPN10 (Smalle et al., 2003), and RPN1 (Brukhin et al., 2005) in plants that function specifically in cytokinin-, ABA-, and embryogenesis-signaling pathways, respectively, we hypothesize that RPN9 regulates vascular development via selective degradation of specific target proteins.
The extra vein formation in the immediate vicinity of the existing veins led us to the finding that auxin transport is reduced in RPN9– plants. The down-regulation of PINOID may contribute at least in part to the reduction of auxin transport in RPN9– plants. The roles of EMB30/GN and VAN3 in auxin transport and auxin signaling strongly indicate the involvement of vesicle transport in leaf vein formation (Geldner et al., 2003
BRs have been shown to regulate plant vascular development. Up-regulation of the vascular-specific BR receptor BRL1 and the distinctive phenotype resulting from RPN9 silencing strongly indicate the involvement of BR signaling in RPN9 regulation. BRL1 is specifically expressed in vascular tissues and the brl1 knockout mutant displays increased phloem and reduced xylem (Cano-Delgado et al., 2004
Several recent studies address cross talk and overlap between BR and auxin responses and indicate a complex interdependency between the two phytohormones (Mussig et al., 2002
Cytokinin also plays an important role in vascular development by regulating the formation of procambial cells (Mahonen et al., 2000
Within plant, fungal, and animal kingdoms, RPN9 is the most divergent subunit of the 26S proteasome (Yang et al., 2004
Plant Growth Conditions Nicotiana benthamiana plants and Arabidopsis (Arabidopsis thaliana) plants were grown in a plant growth room at 22°C under a 16-h light/8-h dark cycle. For BL and auxin treatments, seedlings were grown on Murashige and Skoog medium with 100 nM BL or 10 µM IAA.
A subtraction cDNA library was made by suppression subtractive hybridization between cDNAs prepared from TMV-challenged N. benthamiana (mixture of 1-, 2-, 4-, 8-, 24-, and 48-h postinoculation) and unchallenged plants with a PCR-select cDNA subtraction kit (BD Biosciences). The cDNA library was cloned into the PVX-derived silencing vector pGr106 (Takken et al., 2000
Leaf and stem tissues were fixed and dehydrated for SEM analysis as described (Dong et al., 2005
For light microscopy, leaf and stem tissues were fixed, dehydrated, embedded in Technovit 7100 resin (Kulzer), and sectioned at 4-µm thickness as described (Dong et al., 2005
Samples were collected at 8 DPI and subjected to RNA extraction and cDNA synthesis as described (Jin et al., 2002
IAA transport activity was measured as described (Okada et al., 1991
For seedling assays, seeds were germinated on Murashige and Skoog medium with 25 µM NPA (Uniroyal Chemical). Leaves were taken after 10 to 14 d for leaf-clearing analysis. For the viral infection assay, 3-week-old plants were sprayed with a heavy mist of 100 or 200 µM NPA with 0.01% Silwet L-77 (a surfactant; Lehle Seeds) as described in Nemhauser et al. (2000)
Agrobacterium GV2260 (OD600 = 1.0) carrying various GFP fusion constructs (BRI-GFP, BZR1-CFP, or GFP-ICE1) was infiltrated into RPN9– and control leaves at 9 DPI. Leaf discs were collected at 66 h postinoculation and subjected to western analysis with GFP antibody sc-8334 (Santa Cruz), which recognizes both GFP and cyan fluorescent protein fusions. For detecting GFP-ICE1 fusion, the total protein extract was immunoprecipitated with GFP antibody prior to western-blot analysis.
Sequences of the mRNA fragments used for silencing RPN9 (DQ226994), RPN8 (DQ226995), and the 20S
We thank Dr. Elizabeth Lord for advice on histological analysis and access to the microscopy facility in her lab; Dr. Barbara Baker for the TMV-GFP construct; Dr. Darleen DeMason for [14C]IAA, NPA, and helpful discussions; Dr. Zhiyong Wang for the BZR1-CFP construct; Dr. Joanne Chory for the BRI1-GFP construct; Dr. Jian-Kang Zhu for the GFP-ICE1 construct and helpful discussions; Dr. James Borneman for access to a Bio-Rad real-time iCycler in his lab; and Dr. Xuemei Chen, Dr. Patty Springer, and Dr. Zhenbiao Yang for critical reading of this manuscript. Received May 12, 2006; accepted August 2, 2006; published August 18, 2006.
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: Hailing Jin (hailingj{at}ucr.edu). www.plantphysiol.org/cgi/doi/10.1104/pp.106.083519 * Corresponding author; e-mail hailingj{at}ucr.edu; fax 951–827–4294.
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ABSTRACT
RESULTS
4-subunit of the 20S CP and RPN8 of the 19S lid complex, were silenced. Silencing the 
