Running Head: Regulator of viral systemic transport and vascular formation

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 a viral transport suppressor screening using virus-induced gene silencing (VIGS), we identified a 26S proteasome subunit, RPN9, which is required for broad-spectrum viral systemic transport. Silencing of RPN9 in N. benthamiana inhibits systemic spread of two taxonomically distinct viruses, tobacco mosaic virus (TMV) and turnip mosaic virus (TuMV). 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. RPN9silenced 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 (BR) 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 BR signaling protein BZR1 is one of the targets. 3 www.plantphysiol.org on September 4, 2017 Published by Downloaded from Copyright © 2006 American Society of Plant Biologists. All rights reserved. Introduction Virus movement within the plant occurs via local cell-to-cell movement through plasmodesmata and long-distance transport through vasculature, mainly the phloem (Crawford, 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 N. benthamiana and determined that TMV could enter minor, major or transport veins directly from non-vascular 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 glycine-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; Whitham et al., 2000; Chisholm et al., 2001). However, these 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). The phloem forms a macromolecular trafficking network for transport of nutrients and signaling molecules that regulate physiological and developmental events at the whole-plant level (Ruiz-Medrano et al., 2001). The xylem consists of vessels and tracheary elements (TE), which are essential for plant mechanical support and water transport. However, the molecular regulatory mechanisms of plant vascular development are still poorly understood. 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), axr6 (Hobbie et al., 4 www.plantphysiol.org on September 4, 2017 Published by Downloaded from Copyright © 2006 American Society of Plant Biologists. All rights reserved. 2000), bodenlos (iaa12) (Hamann et al., 1999), vascular network defective 1 to 6 (van16) (Koizumi et al., 2000; Koizumi et al., 2005), scarface (Deyholos et al., 2000; Sieburth and Deyholos, 2006), lop1/tornado1 (Carland and McHale, 1996), cotyledon vein pattern 1 and 2 (cvp1, cvp2) (Carland et al., 2002; Carland and Nelson, 2004), orc (Willemsen et al., 2003), fackel (Jang et al., 2000), and ifl1 (Zhong and Ye, 1999), show either reduced vascular formation or discontinuous vascular patterning. Furthermore, all these mutants also display defects in other aspects of plant growth and development. Very few mutants exhibit increased vascular formation. Therefore, the in vitro Zinnia TE induction system has long been used for vascular differentiation studies (Ye, 2002). Virus-induced gene silencing (VIGS) circumvents lethality by suppressing the expression of essential genes in developed plants, and thus serves as an optimal technique for such research. Here, we describe the identification of RPN9, which affects broad-spectrum viral systemic transport and regulates vascular development, from a viral transport suppressor screening using VIGS. 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” (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). In plants, the 26S proteasome degrades various regulators of diverse cellular processes, including cell division, stress responses, and hormone-signaling pathways (Chinnusamy et al., 2003; Moon et al., 2004; Smalle and Vierstra, 2004). 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). However, our knowledge of these functions is still limited. The 19S RP is divided further into 2 subcomplexes known as the “base” and the “lid”. The “base” contains 6 ATPase subunits, RPT1 to 6, and 3 non-ATPase subunits, RPN1, 2 and 10. The RPT subunits use ATP hydrolysis to facilitate channel opening, polyubiquitin 5 www.plantphysiol.org on September 4, 2017 Published by Downloaded from Copyright © 2006 American Society of Plant Biologists. All rights reserved. chain binding and protein unfolding (Lam et al., 2002; Hartmann-Petersen et al., 2003). RPN10 binds polyubiquitin chains and contributes to the turnover of multiple proteasome targets (Mayor T, 2005). In plants, RPN10 regulates ABA signaling by targeting ABA signaling proteins for degradation (Smalle et al., 2003). RPT2a is involved in the maintenance of Arabidopsis meristems (Ueda et al., 2004). RPN1 is essential for Arabidopsis embryogenesis (Brukhin et al., 2005). The “lid” contains 8 subunits, RPN3, RPN5-9 and RPN11-12. The functions of “lid” subunits are poorly understood. Some of them may function in a target-specific manner presumably by recruiting specific E3 ligases or carrier proteins to deliver ubiquitinated targets to the 26S proteasome (Smalle and Vierstra, 2004). Arabidopsis RPN12 is required for normal cytokinin responses, likely by identifying specific proteins controlling these processes for degradation (Smalle et al., 2002; Smalle and Vierstra, 2004). However, the roles of RPN9 and other subunits of the 19S RP in higher plants, particularly during morphogenesis, are still unknown. Here, we demonstrate that down regulation of RPN9, but not 2 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 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. Results 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 TMVchallenged and untreated N. benthamiana plants. The cDNA library was cloned into the 6 www.plantphysiol.org on September 4, 2017 Published by Downloaded from Copyright © 2006 American Society of Plant Biologists. All rights reserved. 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 3 or 4 leaf above the VIGS agro-infiltration sites) 2 weeks after Agrobacteria inoculation for visualization of the viral infection and systemic spread in the silenced plants (Jin et al., 2002). One of the genes that attenuated viral systemic spread after silencing was RPN9 (DQ226994), which encodes a subunit of the 26S proteasome regulatory complex. The ons