ICTV Virus Taxonomy Profile: Caulimoviridae.

Caulimoviridae is a family of non-enveloped reverse-transcribing plant viruses with non-covalently closed circular dsDNA genomes of 7.1-9.8 kbp in the order Ortervirales. They infect a wide range of monocots and dicots. Some viruses cause economically important diseases of tropical and subtropical crops. Transmission occurs through insect vectors (aphids, mealybugs, leafhoppers, lace bugs) and grafting. Activation of infectious endogenous viral elements occurs in Musa balbisiana, Petunia hybrida and Nicotiana edwardsonii. However, most endogenous caulimovirids are not infectious. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caulimoviridae, which is available at ictv.global/report/caulimoviridae.

A model for intracellular movement of Cauliflower mosaic virus: the concept of the mobile virion factory.

The genomes of many plant viruses have a coding capacity limited to <10 proteins, yet it is becoming increasingly clear that individual plant virus proteins may interact with several targets in the host for establishment of infection. As new functions are uncovered for individual viral proteins, virologists have realized that the apparent simplicity of the virus genome is an illusion that belies the true impact that plant viruses have on host physiology. In this review, we discuss our evolving understanding of the function of the P6 protein of Cauliflower mosaic virus (CaMV), a process that was initiated nearly 35 years ago when the CaMV P6 protein was first described as the 'major inclusion body protein' (IB) present in infected plants. P6 is now referred to in most articles as the transactivator (TAV)/viroplasmin protein, because the first viral function to be characterized for the Caulimovirus P6 protein beyond its role as an inclusion body protein (the viroplasmin) was its role in translational transactivation (the TAV function). This review will discuss the currently accepted functions for P6 and then present the evidence for an entirely new function for P6 in intracellular movement.

Association of the P6 protein of Cauliflower mosaic virus with plasmodesmata and plasmodesmal proteins.

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.

Genetic diversity and tissue and host specificity of Grapevine vein clearing virus.

Grapevine vein clearing virus (GVCV) is a new badnavirus in the family Caulimoviridae that is closely associated with an emerging vein-clearing and vine decline disease in the Midwest region of the United States. It has a circular, double-stranded DNA genome of 7,753 bp that is predicted to encode three open reading frames (ORFs) on the plus-strand DNA. The largest ORF encodes a polyprotein that contains domains for a reverse transcriptase (RT), an RNase H, and a DNA-binding zinc-finger protein (ZF). In this study, two genomic regions, a 570-bp region of the RT domain and a 540-bp region of the ZF domain were used for an analysis of the genetic diversity of GVCV populations. In total, 39 recombinant plasmids were sequenced. These plasmids consisted of three individual clones from each of 13 isolates sampled from five grape varieties in three states. The sequence variants of GVCV could not be phylogenetically grouped into clades according to geographical location and grape variety. Codons of RT or ZF regions are subject to purifying selection pressure. Quantitative polymerase chain reaction assays indicated that GVCV accumulates abundantly in the petioles and least in the root tip tissue. Upon grafting of GVCV-infected buds onto four major grape cultivars, GVCV was not detected in the grafted 'Chambourcin' vine but was present in the grafted 'Vidal Blanc', 'Cayuga White', and 'Traminette' vines, suggesting that Chambourcin is resistant to GVCV. Furthermore, seven nucleotides were changed in the sequenced RT and ZF regions of GVCV from a grafted Traminette vine and one in the sequenced regions of GVCV from grafted Cayuga White but no changes were found in the sequenced regions of GVCV in the grafted Vidal Blanc. The results provide a genetic snapshot of GVCV populations, which will yield knowledge important for monitoring GVCV epidemics and for preventing the loss of grape production that is associated with GVCV.

A survey of resistance to Tomato bushy stunt virus in the genus Nicotiana reveals that the hypersensitive response is triggered by one of three different viral proteins.

In this study, we screened 22 Nicotiana spp. for resistance to the tombusviruses Tomato bushy stunt virus (TBSV), Cucumber necrosis virus, and Cymbidium ringspot virus. Eighteen species were resistant, and resistance was manifested in at least two different categories. In all, 13 species responded with a hypersensitive response (HR)-type resistance, whereas another five were resistant but either had no visible response or responded with chlorotic lesions rather than necrotic lesions. Three different TBSV proteins were found to trigger HR in Nicotiana spp. in an agroinfiltration assay. The most common avirulence (avr) determinant was the TBSV coat protein P41, a protein that had not been previously recognized as an avr determinant. A mutational analysis confirmed that the coat protein rather than the viral RNA sequence was responsible for triggering HR, and it triggered HR in six species in the Alatae section. The TBSV P22 movement protein triggered HR in two species in section Undulatae (Nicotiana glutinosa and N. edwardsonii) and one species in section Alatae (N. forgetiana). The TBSV P19 RNA silencing suppressor protein triggered HR in sections Sylvestres (N. sylvestris), Nicotiana (N. tabacum), and Alatae (N. bonariensis). In general, Nicotiana spp. were capable of recognizing only one tombusvirus avirulence determinant, with the exceptions of N. bonariensis and N. forgetiana, which were each able to recognize P41, as well as P19 and P22, respectively. Agroinfiltration failed to detect the TBSV avr determinants responsible for triggering HR in N. arentsii, N. undulata, and N. rustica. This study illustrates the breadth and variety of resistance responses to tombusviruses that exists in the Nicotiana genus.

Transient expression of cauliflower mosaic virus (CaMV) P6-GFP complements a defective CaMV replicon to facilitate viral gene expression, replication and virion formation.

Over the past decades, several studies have examined the subcellular localization of the cauliflower mosaic virus (CaMV) P6 protein by tagging it with GFP (P6-GFP). These investigations have been essential in the development of models for inclusion body formation, nuclear transport, and microfilament-associated intracellular movement of P6 inclusion bodies for delivery of virions to plasmodesmata. Although it was shown early on that the translational transactivation function of P6-GFP was comparable to wild type P6, it has not been possible to incorporate a P6-GFP gene into an infectious clone of CaMV. Consequently, it has not been possible to formally prove that a P6-GFP fusion is comparable in function to the unmodified P6 protein. Here we show that transient expression of P6-GFP can complement a defective CaMV replicon through gene expression, replication and encapsidation, which validates the relevance of P6-GFP subcellular localization studies for understanding the development of CaMV infections.

Excision and episomal replication of cauliflower mosaic virus integrated into a plant genome.

Transgenic Arabidopsis (Arabidopsis thaliana) plants containing a monomeric copy of the cauliflower mosaic virus (CaMV) genome exhibited the generation of infectious, episomally replicating virus. The circular viral genome had been split within the nonessential gene II for integration into the Arabidopsis genome by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were assessed for episomal infections at flowering, seed set, and/or senescence. The infections were confirmed by western blot for the CaMV P6 and P4 proteins, electron microscopy for the presence of icosahedral virions, and through polymerase chain reaction across the recombination junction. By the end of the test period, a majority of the transgenic Arabidopsis plants had developed episomal infections. The episomal form of the virus was infectious to nontransgenic plants, indicating that no essential functions were lost after release from the Arabidopsis chromosome. An analysis of the viral genomes recovered from either transgenic Arabidopsis or nontransgenic turnip (Brassica rapa var rapa) revealed that the viruses contained deletions within gene II, and in some cases, the deletions extended to the beginning of gene III. In addition, many of the progeny viruses contained small regions of nonviral sequence derived from the flanking transformation vector. The nature of the nucleotide sequences at the recombination junctions in the circular progeny virus indicated that most were generated by nonhomologous recombination during the excision event. The release of the CaMV viral genomes from an integrated copy was not dependent upon the application of environmental stresses but occurred with greater frequency with either age or the late stages of plant maturation.

Comparative analysis of the capacity of tombusvirus P22 and P19 proteins to function as avirulence determinants in Nicotiana species.

We have used an agroinfiltration assay for a comparative study of the roles of tombusvirus P22 and P19 proteins in elicitation of hypersensitive response (HR)-like necrosis and the role of P19 in silencing suppression in Nicotiana species. The advantage of agroinfiltration rather than expression in plant virus vectors is that putative viral avirulence proteins can be evaluated in isolation, eliminating the possibility of synergistic effects with other viral proteins. We found that tombusvirus P22 and P19 proteins elicited HR-like necrosis in certain Nicotiana species but, also, that Nicotiana species could recognize subtle differences in sequence between these proteins. Furthermore, Nicotiana species that responded with systemic necrosis to virion inoculations responded to agroinfiltration of tombusvirus P19 with a very weak and delayed necrosis, indicating that the rapid HR-like necrosis was associated with putative resistance genes and a plant defense response that limited the spread of the virus. Tombusvirus P19 proteins also appeared to differ in their effectiveness as silencing suppressors; in our assay, the P19 proteins of Cymbidium ringspot virus and Tomato bushy stunt virus were stronger silencing suppressors than Cucumber necrosis virus P20. Finally, we show that agroinfiltration can be used to track the presence of putative plant resistance genes in Nicotiana species that target either tombusvirus P19 or P22.

Cauliflower mosaic virus P6 inclusion body formation: A dynamic and intricate process.

During an infection, Cauliflower mosaic virus (CaMV) forms inclusion bodies (IBs) mainly composed of viral protein P6, where viral activities occur. Because viral processes occur in IBs, understanding the mechanisms by which they are formed is crucial. FL-P6 expressed in N. benthamiana leaves formed IBs of a variety of shapes and sizes. Small IBs were dynamic, undergoing fusion/dissociation events. Co-expression of actin-binding polypeptides with FL-P6 altered IB size distribution and inhibited movement. This suggests that IB movement is required for fusion and growth. A P6 deletion mutant was discovered that formed a single large IB per cell, which suggests it exhibited altered fusion/dissociation dynamics. Myosin-inhibiting drugs did not affect small IB movement, while those inhibiting actin polymerization did. Large IBs colocalized with components of the aggresome pathway, while small ones generally did not. This suggests a possible involvement of the aggresome pathway in large IB formation.

Intracellular transport of viruses and their components: utilizing the cytoskeleton and membrane highways.

Plant viruses are obligate organisms that require host components for movement within and between cells. A mechanistic understanding of virus movement will allow the identification of new methods to control virus systemic spread and serve as a model system for understanding host macromolecule intra- and intercellular transport. Recent studies have moved beyond the identification of virus proteins involved in virus movement and their effect on plasmodesmal size exclusion limits to the analysis of their interactions with host components to allow movement within and between cells. It is clear that individual virus proteins and replication complexes associate with and, in some cases, traffic along the host cytoskeleton and membranes. Here, we review these recent findings, highlighting the diverse associations observed between these components and their trafficking capacity. Plant viruses operate individually, sometimes within virus species, to utilize unique interactions between their proteins or complexes and individual host cytoskeletal or membrane elements over time or space for their movement. However, there is not sufficient information for any plant virus to create a complete model of its intracellular movement; thus, more research is needed to achieve that goal.

Combinatorially selected peptides for protection of soybean against Phakopsora pachyrhizi.

Phakopsora pachyrhizi, the fungal pathogen that causes Asian soybean rust, has the potential to cause significant losses in soybean yield in many production regions of the United States. Germplasm with durable, single-gene resistance is lacking, and control of rust depends on timely application of fungicides. To assist the development of new modes of soybean resistance, we identified peptides from combinatorial phage-display peptide libraries that inhibit germ tube growth from urediniospores of P. pachyrhizi. Two peptides, Sp2 and Sp39, were identified that inhibit germ tube development when displayed as fusions with the coat protein of M13 phage or as fusions with maize cytokinin oxidase/dehydrogenase (ZmCKX1). In either display format, the inhibitory effect of the peptides on germ tube growth was concentration dependent. In addition, when peptides Sp2 or Sp39 in either format were mixed with urediniospores and inoculated to soybean leaves with an 8-h wetness period, rust lesion development was reduced. Peptides Sp2 and Sp39, displayed on ZmCKX1, were found to interact with a 20-kDa protein derived from germinated urediniospores. Incorporating peptides that inhibit pathogen development and pathogenesis into breeding programs may contribute to the development of soybean cultivars with improved, durable rust tolerance.

The CaMV transactivator/viroplasmin interferes with RDR6-dependent trans-acting and secondary siRNA pathways in Arabidopsis.

Several RNA silencing pathways in plants restrict viral infections and are suppressed by distinct viral proteins. Here we show that the endogenous trans-acting (ta)siRNA pathway, which depends on Dicer-like (DCL) 4 and RNA-dependent RNA polymerase (RDR) 6, is suppressed by infection of Arabidopsis with Cauliflower mosaic virus (CaMV). This effect was associated with overaccumulation of unprocessed, RDR6-dependent precursors of tasiRNAs and is due solely to expression of the CaMV transactivator/viroplasmin (TAV) protein. TAV expression also impaired secondary, but not primary, siRNA production from a silenced transgene and increased accumulation of mRNAs normally silenced by the four known tasiRNA families and RDR6-dependent secondary siRNAs. Moreover, TAV expression upregulated DCL4, DRB4 and AGO7 that mediate tasiRNA biogenesis. Our findings suggest that TAV is a general inhibitor of silencing amplification that impairs DCL4-mediated processing of RDR6-dependent double-stranded RNA to siRNAs. The resulting deficiency in tasiRNAs and other RDR6-/DCL4-dependent siRNAs appears to trigger a feedback mechanism that compensates for the inhibitory effects.

Plant SNAREs SYP22 and SYP23 interact with Tobacco mosaic virus 126 kDa protein and SYP2s are required for normal local virus accumulation and spread.

Viral proteins often interact with multiple host proteins during virus accumulation and spread. Identities and functions of all interacting host proteins are not known. Through a yeast two-hybrid screen an Arabidopsis thaliana Qa-SNARE protein [syntaxin of plants 23 (AtSYP23)], associated with pre-vacuolar compartment and vacuolar membrane fusion activities, interacted with Tobacco mosaic virus (TMV) 126 kDa protein, associated with virus accumulation and spread. In planta, AtSYP23 and AtSYP22 each fused with mCherry, co-localized with 126 kDa protein-GFP. Additionally, A. thaliana and Nicotiana benthamiana SYP2 proteins and 126 kDa protein interacted during bimolecular fluorescence complementation analysis. Decreased TMV accumulation in Arabidopsis plants lacking SYP23 and in N. benthamiana plants subjected to virus-induced gene silencing (VIGS) of SYP2 orthologs was observed. Diminished TMV accumulation during VIGS correlated with less intercellular virus spread. The inability to eliminate virus accumulation suggests that SYP2 proteins function redundantly for TMV accumulation, as for plant development.

Cauliflower Mosaic Virus TAV, a Plant Virus Protein That Functions like Ribonuclease H1 and is Cytotoxic to Glioma Cells.

Recent comparisons between plant and animal viruses reveal many common principles that underlie how all viruses express their genetic material, amplify their genomes, and link virion assembly with replication. Cauliflower mosaic virus (CaMV) is not infectious for human beings. Here, we show that CaMV transactivator/viroplasmin protein (TAV) shares sequence similarity with and behaves like the human ribonuclease H1 (RNase H1) in reducing DNA/RNA hybrids detected with S9.6 antibody in HEK293T cells. We showed that TAV is clearly expressed in the cytosol and in the nuclei of transiently transfected human cells, similar to its distribution in plants. TAV also showed remarkable cytotoxic effects in U251 human glioma cells in vitro. These characteristics pave the way for future analysis on the use of the plant virus protein TAV, as an alternative to human RNAse H1 during gene therapy in human cells.

A Novel Assay Based on Confocal Microscopy to Test for Pathogen Silencing Suppressor Functions.

In plants, RNA silencing is an important mechanism for gene regulation and defense that is targeted by proteins of viral pathogens effecting silencing suppression. In this chapter we describe a new assay to probe silencing suppressor activity using Agrobacterium infiltration of Nicotiana benthamiana and confocal microscopy. The key element in this assay involves the use of a reporter construct that is transiently expressed at a much lower level than free GFP, and this increases the sensitivity of detection of weak silencing suppressors such as the P6 protein of Cauliflower mosaic virus. Although initially developed for virus silencing suppressors, this technique could also prove valuable to characterize the potential for weak silencing suppressors in the effector repertoires of fungi, bacteria, nematodes, and oomycetes.

Tracing the Lineage of Two Traits Associated with the Coat Protein of the Tombusviridae: Silencing Suppression and HR Elicitation in Nicotiana Species.

In this paper we have characterized the lineage of two traits associated with the coat proteins (CPs) of the tombusvirids: Silencing suppression and HR elicitation in Nicotiana species. We considered that the tombusvirid CPs might collectively be considered an effector, with the CP of each CP-encoding species comprising a structural variant within the family. Thus, a phylogenetic analysis of the CP could provide insight into the evolution of a pathogen effector. The phylogeny of the CP of tombusvirids indicated that CP representatives of the family could be divided into four clades. In two separate clades the CP triggered a hypersensitive response (HR) in Nicotiana species of section Alatae but did not have silencing suppressor activity. In a third clade the CP had a silencing suppressor activity but did not have the capacity to trigger HR in Nicotiana species. In the fourth clade, the CP did not carry either function. Our analysis illustrates how structural changes that likely occurred in the CP effector of progenitors of the current genera led to either silencing suppressor activity, HR elicitation in select Nicotiana species, or neither trait.

Cauliflower mosaic virus gene VI product N-terminus contains regions involved in resistance-breakage, self-association and interactions with movement protein.

Cauliflower mosaic virus (CaMV) gene VI encodes a multifunctional protein (P6) involved in the translation of viral RNA, the formation of inclusion bodies, and the determination of host range. Arabidopsis thaliana ecotype Tsu-0 prevents the systemic spread of most CaMV isolates, including CM1841. However, CaMV isolate W260 overcomes this resistance. In this paper, the N-terminal 110 amino acids of P6 (termed D1) were identified as the resistance-breaking region. D1 also bound full-length P6. Furthermore, binding of W260 D1 to P6 induced higher beta-galactosidase activity and better leucine-independent growth in the yeast two-hybrid system than its CM1841 counterpart. Thus, W260 may evade Tsu-0 resistance by mediating P6 self-association in a manner different from that of CM1841. Because Tsu-0 resistance prevents virus movement, interaction of P6 with P1 (CaMV movement protein) was investigated. Both yeast two-hybrid analyses and maltose-binding protein pull-down experiments show that P6 interacts with P1. Although neither half of P1 interacts with P6, the N-terminus of P6 binds P1. Interestingly, D1 by itself does not interact with P1, indicating that different portions of the P6 N-terminus are involved in different activities. The P1-P6 interactions suggest a role for P6 in virus transport, possibly by regulating P1 tubule formation or the assembly of movement complexes.

Plant-virus interactions.

A variety of techniques have been used to examine plant viral genomes, the functions of virus-encoded proteins, plant responses induced by virus infection and plant-virus interactions. This overview considers these technologies and how they have been used to identify novel viral and plant proteins or genes involved in disease and resistance responses, as well as defense signaling. These approaches include analysis of spatial and temporal responses by plants to infection, and techniques that allow the expression of viral genes transiently or transgenically in planta, the expression of plant and foreign genes from virus vectors, the silencing of plants genes, imaging of live, infected cells, and the detection of interactions between viral proteins and plant gene products, both in planta and in various in vitro or in vivo systems. These methods and some of the discoveries made using these approaches are discussed.

The Role of Viruses in the Phytobiome.

Viruses are an important but sequence-diverse and often understudied component of the phytobiome. We succinctly review current information on how plant viruses directly affect plant health and physiology and consequently have the capacity to modulate plant interactions with their biotic and abiotic environments. Virus interactions with other biota in the phytobiome, including arthropods, fungi, and nematodes, may also impact plant health. For example, viruses interact with and modulate the interface between plants and insects. This has been extensively studied for insect-vectored plant viruses, some of which also infect their vectors. Other viruses have been shown to alter the impacts of plant-interacting phytopathogenic and nonpathogenic fungi and bacteria. Viruses that infect nematodes have also recently been discovered, but the impact of these and phage infecting soil bacteria on plant health remain largely unexplored.