Effects of movement protein mutations on the formation of tubules in plant protoplasts expressing a fusion between the green fluorescent protein and Cauliflower mosaic virus movement protein

Fusions between the green fluorescent protein (GFP) and the Cauliflower mosaic virus (CaMV) movement protein (MP) induce the formation of fluorescent foci and surface tubules in Arabidopsis thaliana leaf mesophyll protoplasts. Tubules elongate coordinately and progressively in an assembly process approximately 6 to 12 h following transfection of protoplasts with GFP-MP constructs. Tubules are not formed in protoplasts transfected by GFP-MP(ER2A), a MP mutation that renders CaMV noninfectious. A small number of short tubules are formed on protoplasts transfected by GFP-MP(N6) and GFP-MP(N13), two second-site revertants of ER2A that partially restore infectivity. Protoplasts cotransfected with cyan fluorescent protein (CFP)-MP(WT) and GFP-MP(ER2A) form tubules containing both MP fusions, indicating that although the GFP-MP(ER2A) cannot induce tubule formation, GFP-MP(ER2A) can coassemble or colocalize with CFP-MP(WT) in tubules. Thus, CaMV MP-induced tubule formation in protoplasts correlates closely with the infectivity of mutation ER2A and its revertants, suggesting that tubule-forming capacity in plant protoplasts reflects a process required for virus infection or movement.     

Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement

We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.     

Multiple domains within the Cauliflower mosaic virus gene VI product interact with the full-length protein

The Cauliflower mosaic virus (CaMV) gene VI product (P6) is a multifunctional protein essential for viral propagation. It is likely that at least some of these functions require P6 self-association. The work described here was performed to confirm that P6 self-associates and to identify domains involved in this interaction. Yeast two-hybrid analyses indicated that full-length P6 self-associates and that this interaction is specific. Additional analyses indicated that at least four independent domains bind to full-length P6. When a central domain (termed domain D3) was removed, these interactions were abolished. However, this deleted P6 was able to bind to the full-length wild-type protein and to isolated domain D3. Viruses lacking domain D3 were incapable of producing a systemic infection. Isolated domain D3 was capable of binding to at least two of the other domains but was unable to self-associate. This suggests that domain D3 facilitates P6 self-association by binding to the other domains but not itself. The presence of multiple domains involved in P6 self-association may help explain the ability of this protein to form the intracellular inclusions characteristic of caulimoviruses.     

Differential induction of symptoms in Arabidopsis by P6 of Cauliflower mosaic virus

The gene VI protein (P6) of Cauliflower mosaic virus (CaMV) functions as a virulence factor in crucifers by eliciting chlorotic symptoms in infected plants. The ability to induce chlorosis has been associated previously with P6 through gene-swapping experiments between strains and through the development of transgenic plants that express P6. The primary role that has been identified for P6 in the CaMV infection cycle is to modify the host translation machinery to facilitate the translation of the polycistronic CaMV 35S RNA. This function for P6 has been designated as the translational transactivator (TAV) function. In the present study, we have characterized an unusual variant of P6, derived from CaMV strain D4, that does not induce chlorosis upon transformation into Arabidopsis thaliana. The level of D4 P6 produced in transgenic Arabidopsis line D4-2 was comparable to the amount found in transgenic plants homozygous for W260 and CM1841 P6, two versions of P6 that induce strong chlorotic symptoms and stunting in Arabidopsis. A complementation assay proved that P6 expressed in the D4-2 line was functional, as it could support the systemic infection of a CM1841 mutant that contained a lethal frame-shift mutation within gene VI. This complementation assay allowed us to separately assess the contribution of CM1841 gene VI to symptom development versus the contribution of other CM1841 genes. Furthermore, a previous study had shown that the TAV activity of D4 P6 was comparable to that of W260 P6. That comparative analysis of TAV function, coupled with the characterization of the D4-2 transgenic line in the present paper, indicates that the TAV function of P6 may play only a minor role in the development of chlorotic symptoms.     

Cauliflower mosaic virus as a gene expression vector for plants

It is possible to replace the CaMV (cauliflower mosaic virus) ORF (open reading frame) II with foreign sequences without interfering with virus viability. Such recom-binants can induce the synthesis of substantial amounts of a foreign protein in infected plants and confer new properties to these plants. However, so far only three genes have been successfully cloned and expressed in this way. The expression mechanism of CaMV demands precise replacement of ORF II and probably certain structural features of the viral 35S RNA, which should not be disturbed by inserted sequences. Since these features are largely unknown, it cannot at present be pre-dicted whether an insert will be tolerated. It is more likely that larger inserts will disturb the viral gene expression mechanism than smaller ones.     

Triple gene block protein interactions involved in movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.     

Structure-based rationale for the rescue of systemic movement of brome mosaic virus by spontaneous second-site mutations in the coat protein gene.

We describe spontaneous second-site reversions within the coat protein open reading frame that rescue the systemic-spread phenotype and increase virion stability of a mutant of brome mosaic virus. Based on the crystal structure of the related cowpea chlorotic mottle virus, we show that the modified residues are spatially clustered to affect the formation of hexamers and pentamers and therefore virion stability.     

Cauliflower mosaic virus: Pathways of infection

This short review summarizes what is known, and points out some of the unknown features, about the molecular biology of the natural spread of cauliflower mosaic virus into a susceptible host and its subsequent replication in that host.     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

Pathogenic interactions between variants of cauliflower mosaic virus and Arabidopsis thaliana

Pathogenic interactions between genetic variants of cauliflower mosaic virus (CaMV) and Arabidopsis thaliana were characterized to identify combinations potentially useful in molecular genetic analysis. Infections of a glabrous mutant (gl1) of Arabidopsis ecotype Columbia (Col-0 gl1) by 30 CaMV isolates were assessed by recording symptom character. Thirteen isolates failed to cause symptoms; the remainder induced symptoms that varied between mild and very severe. Some CaMV isolates produced symptoms in Arabidopsis that differed significantly in severity or character from those produced in a standard host Brassica rapa (turnip). A greater variety of symptom types was observed in a single Arabidopsis ecotype infected with a range of CaMV isolates than was found in a range of Arabidopsis ecotypes infected with a single, typical CaMV isolate (Cabb B-JI). One isolate, Bari-1, that was asymptomatic but accumulated virus in Arabidopsis ecotype Col-0 gl1, caused mild symptoms in ecotype Ler gl1. A hybrid virus constructed from CaMV isolates Cabb B-JI and Bari-1 produced symptoms in Arabidopsis variants that were more severe than in either parental isolate. From a screen of EMS-mutagenized Arabidopsis, one mutant (Col-0 dv1) with a pale-green, dark-vein phenotype which had an altered symptom response to CaMV, was isolated. From this, a phenotypically near-normal revertant (Col-0 dv1R) spontaneously arose, but which showed altered responses to CaMV. Infection of Col-0 dv1R by CaMV isolate Bari-1 elicited symptoms unlike the parent Arabidopsis ecotype (Col-0 gl1). Also, Col-0 dv1 and Col-0 dv1R expressed an uncharacteristic necrotic reaction to CaMV.     

Identification of Arabidopsis proteins that interact with the cauliflower mosaic virus (CaMV) movement protein

Gene I of cauliflower mosaic virus (CaMV) encodes a protein that is required for virus movement. The CaMV movement protein (MP) was used in a yeast 2-hybrid system to screen an Arabidopsis cDNA library for cDNAs encoding MP-interacting (MPI) proteins. Three different clones were found encoding proteins (MPI1, -2 and -7) that interact with the N-terminal third of the CaMV MP. The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants. A non-infectious MP mutant, ER2A, with two amino acid changes in the N-terminal third of the MP failed to interact with MPI7, while an infectious second-site mutant, that differed from ER2A by only a single amino acid change, interacted in the 2-hybrid system. MPI7 is encoded by a member of a large, but diverse gene family in Arabidopsis. MPI7 is related in sequence, size and hydropathy profile to mammalian proteins (such as rat PRA1) described as a rab acceptor. The gene encoding MPI7 is expressed widely is Arabidopsis plants, and in transgenic plants the MPI7:GFP fusion protein is localized in the cytoplasm, concentrated in punctate spots. In protoplasts transfected with CFP:MP and MPI7:YFP, CFP:MP colocalized to some of the sites where MPI7:YFP is expressed. At these sites, fluorescence resonance energy transfer (FRET) between fluorophores was observed indicating an interaction in planta between the CaMV MP and MPI7.     

Dimerization of recombinant tobacco mosaic virus movement protein

The p30 movement protein (MP) is essential for cell-to-cell spread of tobacco mosaic virus in planta. We used anion-exchange chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain highly purified 30-kDa MP, which migrated as a single band in native PAGE. Analytical ultracentrifugation suggested that the protein was monodisperse and dimeric in the nonionic detergent n-octyl-beta-D-glucopyranoside. Circular dichroism (CD) spectroscopy showed that the detergent-solubilized protein contained significant alpha-helical secondary structure. Proteolysis of the C-tail generated a trypsin-resistant core that was a mixture of primarily monomers and some dimers. We propose that MP dimers are stabilized by electrostatic interactions in the C terminus as well as hydrophobic interactions between putative transmembrane alpha-helical coiled coils.     

Multiple resistance phenotypes to Lettuce mosaic virus among Arabidopsis thaliana accessions

With the aim to characterize plant and viral factors involved in the molecular interactions between plants and potyviruses, a Lettuce mosaic virus (LMV)-Arabidopsis thaliana pathosystem was developed. Screening of Arabidopsis accessions with LMV isolates indicated the existence of a large variability in the outcome of the interaction, allowing the classification of Arabidopsis accessions into seven susceptibility groups. Using a reverse genetic approach, the genome-linked protein of LMV, a multifunctional protein shown to be involved in viral genome amplification and movement of potyviruses, was established as the viral determinant responsible for the ability to overcome the resistance of the Niederzenz accession to LMV-0. Preliminary genetic analyses from F2 and recombinant inbred lines available between susceptible and resistant Arabidopsis accessions revealed the existence of at least three resistance phenotypes to LMV with different genetic bases. One dominant resistance gene, designated LLM1, involved in blocking the replication or cell-to-cell movement of the LMV-0 isolate in the Columbia accession, was mapped to chromosome I and shown to be linked to the marker nga280. At the same time, genetic analyses of segregating F2 populations were consistent with the restriction of the systemic movement of the LMV-AF199 isolate in Columbia being controlled by two dominant genes and with the complete resistance to all tested LMV isolates of the Cape Verde islands (Cvi) accession being conferred by a single recessive resistance gene. Sequencing of the eukaryotic translation initiation factor 4E genes from the different LMV-resistant Arabidopsis accessions showed that these factors are not directly involved in the characterized resistance phenotypes.     

Agroinfiltration of Cauliflower mosaic virus gene VI elicits hypersensitive response in Nicotiana species

Cauliflower mosaic virus strain W260 induces hypersensitive response (HR) in Nicotiana edwardsonii and systemic cell death in N. clevelandii. In contrast, the D4 strain of Cauliflower mosaic virus evades the host defenses in Nicotiana species; it induces chlorotic primary lesions and a systemic mosaic in both hosts. Previous studies with chimeric viruses had indicated that gene VI of W260 was responsible for elicitation of HR or cell death. To prove conclusively that W260 gene VI is responsible, we inserted gene VI of W260 and D4 into the Agrobacterium tumefaciens binary vector pKYLX7. Agroinfiltration of these constructs into the leaves of N. edwardsonii and N. clevelandii revealed that gene VI of W260 elicited HR in N. edwardsonii 4 to 5 days after infiltration and cell death in N. clevelandii approximately 9 to 12 days after infiltration. In contrast, gene VI of D4 did not elicit HR or cell death in either Nicotiana species. A frameshift mutation introduced into gene VI of W260 abolished its ability to elicit HR or cell death in both Nicotiana species, demonstrating that the elicitor is the gene VI protein.     

Specific enrichment of miRNAs in Arabidopsis thaliana infected with Tobacco mosaic virus.

RNA silencing is a broadly conserved machinery and is involved in many biological events. Small RNAs are key molecules in RNA silencing pathway that guide sequence-specific gene regulations and chromatin modifications. The silencing machinery works as an anti-viral defense in virus-infected plants. It is generally accepted that virus-specific small interfering (si) RNAs bind to the viral genome and trigger its cleavage. Previously, we have cloned and obtained sequences of small RNAs from Arabidopsis thaliana infected or uninfected with crucifer Tobacco mosaic virus. MicroRNAs (miRNAs) accumulated to a higher percentage of total small RNAs in the virus-infected plants. This was partly because the viral replication protein binds to the miRNA/miRNA* duplexes. In the present study, we mapped the sequences of small RNAs other than virus-derived siRNAs to the Arabidopsis genome and assigned each small RNA. It was demonstrated that only miRNAs increased as a result of viral infection. Furthermore, some newly identified miRNAs and miRNA candidates were found from the virus-infected plants despite a limited number of examined sequences. We propose that it is advantageous to use virus-infected plants as a source for cloning and identifying new miRNAs.     

Host responses in life-history traits and tolerance to virus infection in Arabidopsis thaliana

Knowing how hosts respond to parasite infection is paramount in understanding the effects of parasites on host populations and hence host-parasite co-evolution. Modification of life-history traits in response to parasitism has received less attention than other defence strategies. Life-history theory predicts that parasitised hosts will increase reproductive effort and accelerate reproduction. However, empirical analyses of these predictions are few and mostly limited to animal-parasite systems. We have analysed life-history trait responses in 18 accessions of Arabidopsis thaliana infected at two different developmental stages with three strains of Cucumber mosaic virus (CMV). Accessions were divided into two groups according to allometric relationships; these groups differed also in their tolerance to CMV infection. Life-history trait modification upon virus infection depended on the host genotype and the stage at infection. While all accessions delayed flowering, only the more tolerant allometric group modified resource allocation to increase the production of reproductive structures and progeny, and reduced the length of reproductive period. Our results are in agreement with modifications of life-history traits reported for parasitised animals and with predictions from life-history theory. Thus, we provide empirical support for the general validity of theoretical predictions. In addition, this experimental approach allowed us to quantitatively estimate the genetic determinism of life-history trait plasticity and to evaluate the role of life-history trait modification in defence against parasites, two largely unexplored issues.