J. Wellink

RNA-Mediated Virus Resistance: Role of Repeated Transgenes and Delineation of Targeted Regions.

Resistance to cowpea mosaic virus (CPMV) in transgenic Nicotiana benthamiana plants is RNA mediated. In resistant CPMV movement protein (MP) gene-transformed lines, transgene steady state mRNA levels were low, whereas nuclear transcription rates were high, implying that a post-transcriptional gene-silencing mechanism is at the base of the resistance. The silencing mechanism can also affect potato virus X (PVX) RNAs when they contain CPMV MP gene sequences. In particular, sequences situated in the 3[prime] part of the transcribed region of the MP transgene direct elimination of recombinant PVX genomes. Remarkably, successive portions of this 3[prime] part, which can be as small as 60 nucleotides, all tag PVX genomes for degradation. These observations suggest that the entire 3[prime] part of the MP transgene mRNA is the initial target of the silencing mechanism. The arrangement of transgenes in the plant genome plays an important role in establishing resistance because the frequency of resistant lines increased from 20 to 60% when transformed with a transgene containing a direct repeat of MP sequences rather than a single MP transgene. Interestingly, we detected strong methylation in all of the plants containing directly repeated MP sequences. In sensitive lines, only the promoter region was found to be heavily methylated, whereas in resistant lines, only the transcribed region was strongly methylated.

Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus

The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed “Secoviridae” to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae.

Modification of Phytohormone Response by a Peptide Encoded by ENOD40 of Legumes and a Nonlegume

The gene ENOD40 is expressed during early stages of legume nodule development. A homolog was isolated from tobacco, which, as does ENOD40 from legumes, encodes an oligopeptide of about 10 amino acids. In tobacco protoplasts, these peptides change the response to auxin at concentrations as low as 10−12 to 10−16 M. The peptides encoded by ENOD40 appear to act as plant growth regulators. Sequence alignment of full ENDO40 gene sequences from soybean, pea, alfalfa, and tocacco plants.

Evidence for the involvement of the 58K and 48K proteins in the intercellular movement of cowpea mosaic virus.

Infection of cowpea cells with cowpea mosaic virus (CPMV) is accompanied by the appearance of tubular structures containing virus-like particles which protrude from or penetrate the cell wall. Immunogold labelling of sections of infected cells using antisera against a CPMV M RNA translation products, and Protein A-gold, showed that the 58K and/or 48K tentative transport proteins of CPMV were located in or on these tubular structures. Furthermore, these proteins were detected in small electron-dense areas near the tail-end of the tubules. The possible function of these structures in virus movement from cell to cell is discussed.

Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid proteins.

Summary Insertions and deletions have been introduced into an infectious cDNA clone of M RNA of cowpea mosaic virus (CPMV), in the coding regions of the 58K/48K and capsid proteins. Transcripts derived from these mutant clones appeared to be replicated in cowpea protoplasts as detected by immunofluorescent staining and Northern blotting. However in cowpea plants, mutations in either region restricted the replication of the viral RNAs to the inoculated cells and thus prevented a successful systemic infection of the plant. These results indicate that the M RNA-encoded 58K/48K proteins are involved in cell-to-cell transport of CPMV, and that the virus can spread only if the RNA is encapsidated in particles.

Cowpea Mosaic Virus Infection Induces a Massive Proliferation of Endoplasmic Reticulum but Not Golgi Membranes and Is Dependent on De Novo Membrane Synthesis

ABSTRACT Replication of cowpea mosaic virus (CPMV) is associated with small membranous vesicles that are induced upon infection. The effect of CPMV replication on the morphology and distribution of the endomembrane system in living plant cells was studied by expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi membranes. CPMV infection was found to induce an extensive proliferation of the ER, whereas the distribution and morphology of the Golgi stacks remained unaffected. Immunolocalization experiments using fluorescence confocal microscopy showed that the proliferated ER membranes were closely associated with the electron-dense structures that contain the replicative proteins encoded by RNA1. Replication of CPMV was strongly inhibited by cerulenin, an inhibitor of de novo lipid synthesis, at concentrations where the replication of the two unrelated viruses alfalfa mosaic virus and tobacco mosaic virus was largely unaffected. These results suggest that proliferating ER membranes produce the membranous vesicles formed during CPMV infection and that this process requires continuous lipid biosynthesis.

The movement proteins of cowpea mosaic virus and cauliflower mosaic virus induce tubular structures in plant and insect cells.

The movement proteins (MP) of cowpea mosaic virus and cauliflower mosaic virus (CaMV) are associated with tubular structures in vivo which participate in the transmission of virus particles from cell to cell. Both proteins have been expressed in plant protoplasts and insect cells. In all cases, immunofluorescent histochemistry showed that the MPs accumulate intracellularly as tubular extensions projecting from the cell surface. Additionally, electron microscopy revealed intracellular MP aggregates in CaMV MP-expressing cells. The data presented establish common features for the tubule-forming MPs: no other virus gene products are required for tubule formation and unique plant components (e.g. plasmodesmata) are not essential for tubule synthesis.

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, 110K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMV-specific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active. Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, 110K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A).oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.

Cowpea Mosaic Virus 32- and 60-Kilodalton Replication Proteins Target and Change the Morphology of Endoplasmic Reticulum Membranes

ABSTRACT Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have expressed individual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in Nicotiana benthamiana plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and 60K, when expressed individually, accumulate in only low amounts but are found associated with membranes mainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to high levels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides, expression of 32K and 60K results in necrosis of the inoculated N. benthamiana leaves, suggesting that 32K and 60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levels without causing necrosis.

The involvement of cowpea mosaic virus M RNA-encoded proteins in tubule formation.

On the surface of cowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M cDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wild-type virus.