Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4.

Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-alpha-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function.

JAGGED LATERAL ORGAN (JLO) controls auxin dependent patterning during development of the Arabidopsis embryo and root.

The plant hormone auxin plays a role in virtually every aspect of plant growth and development. Temporal and spatial distribution of auxin largely depends on the dynamic expression and subcellular localization of the PIN auxin-efflux carrier proteins. We show here that the Arabidopsis thaliana JAGGED LATERAL ORGAN (JLO) gene, a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) gene family, is required for coordinated cell division during embryogenesis. JLO promotes expression of several PINFORMED (PIN) genes during embryonic and root development. Inducible JLO misexpression reveals that JLO activity is sufficient for rapid and high level PIN1 and PIN3 transcription. Genes of the PLETHORA (PLT) family respond to auxin and direct PIN expression, but PLT genes were severely underexpressed in jlo mutants. JLO controls embryonic patterning together with the auxin dependent MONOPTEROS/BODENLOS pathway, but is itself only mildly auxin inducible. We further show that all known auxin responses in the root require JLO activity. We thereby identify JLO as a central regulator of auxin distribution and signaling throughout plant development.

JLO regulates embryo patterning and organ initiation by controlling auxin transport.

In Arabidopsis, lateral organ initiation correlates with the formation of an auxin maximum in a group of cells at the periphery of the shoot apical meristem (SAM). This signal establishes founder cells that build the lateral organ. Primordia initiation is closely associated with the creation of a functional boundary that separates the newly formed primordium from the remainder of the meristem. In the June issue of Plant Cell, we have characterised the JLO (for Jagged Lateral Organ) gene of Arabidopsis, a member of the Lateral Organ boundary Domain gene family. JLO is expressed in boundaries and regulates both auxin transport, via a negative regulation of PIN auxin export carriers, and meristem fate by promoting the expression of the KNOX genes SHOOTMERISTEMLESS (STM) and BP/KNAT1. In this Addendum, we discuss the regulation of PIN genes by JLO, and propose a model for JLO function during embryonic and post-embryonic development.

Arabidopsis JAGGED LATERAL ORGANS is expressed in boundaries and coordinates KNOX and PIN activity.

Plant lateral organs are initiated as small protrusions on the flanks of shoot apical meristems. Organ primordia are separated from the remainder of the meristem by distinct cell types that create a morphological boundary. The Arabidopsis thaliana gain-of-function mutant jagged lateral organs-D (jlo-D) develops strongly lobed leaves, indicative of KNOX gene misexpression, and the shoot apical meristem arrests organ initiation prematurely, terminating in a pin-like structure. The JLO gene, a member of the LATERAL ORGAN BOUNDARY DOMAIN gene family, is expressed in boundaries between meristems and organ primordia and during embryogenesis. Inducible JLO misexpression activates expression of the KNOX genes SHOOT MERISTEMLESS and KNAT1 in leaves and downregulates the expression of PIN auxin export facilitators. Consequently, bulk auxin transport through the inflorescence stem is drastically reduced. During embryogenesis, JLO is required for the initiation of cotyledons and development beyond the globular stage. Converting JLO into a transcriptional repressor causes organ fusions, showing that during postembryonic development, JLO function is required to maintain the integrity of boundaries between cell groups with indeterminate or determinate fates.

The open reading frame VI product of Cauliflower mosaic virus is a nucleocytoplasmic protein: its N terminus mediates its nuclear export and formation of electron-dense viroplasms.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replication and assembly occur. In this study, the mechanism involved in viroplasm formation was investigated by in vitro and in vivo experiments. Far protein gel blot assays using a collection of P6 deletion mutants demonstrated that the N-terminal alpha-helix of P6 mediates interaction between P6 molecules. Transient expression in tobacco (Nicotiana tabacum) BY-2 cells of full-length P6 and P6 mutants fused to enhanced green fluorescent protein revealed that viroplasms are formed at the periphery of the nucleus and that the N-terminal domain of P6 is an important determinant in this process. Finally, this study led to the unexpected finding that P6 is a nucleocytoplasmic shuttle protein and that its nuclear export is mediated by a Leu-rich sequence that is part of the alpha-helix domain implicated in viroplasm formation. The discovery that P6 can localize to the nucleus opens new prospects for understanding yet unknown roles of this viral protein in the course of the CaMV infection cycle.

Cauliflower mosaic virus: still in the news.

SUMMARY Taxonomic relationship: Cauliflower mosaic virus (CaMV) is the type member of the Caulimovirus genus in the Caulimoviridae family, which comprises five other genera. CaMV replicates its DNA genome by reverse transcription of a pregenomic RNA and thus belongs to the pararetrovirus supergroup, which includes the Hepadnaviridae family infecting vertebrates. Physical properties: Virions are non-enveloped isometric particles, 53 nm in diameter (Fig. 1). They are constituted by 420 capsid protein subunits organized following T= 7 icosahedral symmetry (Cheng, R.H., Olson, N.H. and Baker, T.S. (1992) Cauliflower mosaic virus: a 420 subunit (T= 7), multilayer structure. Virology, 16, 655-668). The genome consists of a double-stranded circular DNA of approximately 8000 bp that is embedded in the inner surface of the capsid. Viral proteins: The CaMV genome encodes six proteins, a cell-to-cell movement protein (P1), two aphid transmission factors (P2 and P3), the precursor of the capsid proteins (P4), a polyprotein precursor of proteinase, reverse transcriptase and ribonuclease H (P5) and an inclusion body protein/translation transactivator (P6). Hosts: The host range of CaMV is limited to plants of the Cruciferae family, i.e. Brassicae species and Arabidopsis thaliana, but some viral strains can also infect solanaceous plants. In nature, CaMV is transmitted by aphids in a non-circulative manner.

P6 protein of Cauliflower mosaic virus, a translation reinitiator, interacts with ribosomal protein L13 from Arabidopsis thaliana.

The P6 protein of Cauliflower mosaic virus (CaMV) transactivates translation of the CaMV 35S polycistronic pregenomic RNA and its spliced versions, and thus allows synthesis of a complete set of viral proteins. Previous studies have shown that P6 interacts with plant L18 and L24 ribosomal proteins and initiation factor eIF3, and it has been proposed that these interactions are involved in the reinitiation of translation of polycistronic viral RNAs. This study characterizes a novel cellular partner of P6, the ribosomal protein L13 from Arabidopsis thaliana. Far-Western assays performed with several P6 deletion mutants have shown that L13 interacts with the miniTAV of P6, which represents the minimal domain for transactivation, suggesting that the P6-L13 interaction might also be involved in this process. L13 and L18 were found to bind to the same region within the miniTAV. Competition assays between L18 and L13 for binding to miniTAV suggest that interactions between P6 and these ribosomal proteins involve separate P6 molecules, and/or occur at different stages of translation or in the context of another function also mediated by P6.