Lecture Notes - Set No. 1


Minimum number of genes in a plant RNA virus could be two: a coat protein and an RNA replicase gene (as is the case with RNA phages). Evidence indicates there are usually 3-5 gene products.

Plant positive-stranded RNA viruses frequently possess divided genomes (refer to Figure 14-4 in handout).  In addition, viral genomes are separately encapsulated. Viral genomes consisting of two or three different nucleic acid components, all required for infection are called bipartite, tripartite, or multipartite viruses. More than a single species of genomic RNA.  Refer to pages 271-273 in textbook.

Multipartite viruses are potentially at an evolutionary disadvantage. Infectivity dilution curve for Alfalfa mosaic virus (requiring B, M, Tb particles for infectivity) is steeper than for tobacco necrosis virus (single particle). Partition of genome could potentially hinder transmission or infection by a virus.


Kasinis in 1962, described the first satellite viruses. These viruses are serologically unrelated to their helpers and the two genomes exhibit little if any sequence similarity. Satellite viruses are dependent for its replication on the presence of a second, independently replicating virus.

Satellite RNAs have no coat protein of their own and are encapsulated with the help of other viral RNAs.



Due to the inability to observe plant viruses visually by observing them directly through the light microscope, virologists must resort to the following methods of detecting their presence and in diagnoses.

1. Ability to transmit disease via plant sap by rubbing plant, grafting, dodder or insect transmission.

2. Indexing - indicator plants - sensitive to specific virus and will react a certain way if exposed..

3. Visual inspection with EM.

4. By eliminating possibility that symptoms are not due to other sources (e.g., herbicide, nutritional deficiencies.

5. Serological Tests (ELISA - enzyme-linked immuno sorbent assay). Refer to Figure 14-24; page 499 in the handout.

ELISA tests are extremely sensitive (small amounts of antisera are needed) results are quantitative, large samples can be run at same time (96 well plates), results can be gathered in a few hours instead of days. ELISAs along with serial dilutions of plant sap and applications of this to the leaves of susceptible hosts (by counting the number of lesions) can be used to quantify the amount of virus present.


TOBACCO MOSAIC (Refer to Fig 14-31 on page 509 in handout or the following two internet links Picture No. 1 or Picture No. 2)
Management of Tobacco Mosaic Disease
Tobacco Mosaic Virus: The Prototype Plant Virus

The stability of the TMV virus particle accounts for its having been the first virus to be identified, purified to homogeneity, and then biochemically and biologically characterized.

Small coat protein subunits (capsomeres) aggregate to form a helical protein coat or capsid (see Fig 2.10 and 2.11 on pages 46 and 47).   The virus particle contains an axial channel that is 4 nm wide and the viral RNA lies within a groove in the surrounding protein helix.  The nucleic acid core is not in the axial channel, but passes about halfway between the interior channel and the exterior surface of the rod. The overall particle is rod-shaped, narrow, and rigid.  The pitch of the helix is 2.3 nm, and each turn contains 16 1/3 coat protein molecules.  A full-length virion contains 130 helical turns.

TMV particle is resistant to nucleases and proteolytic enzymes.  TMV particles will fall apart in both alkaline and acid solutions.  Denaturation is often reversible, as long as temperature and pH are not too extreme.  Removal of the denaturant allows the native structure of the viral protein to re-form and near its isoelectric point (pH 4 to 6), the TMV coat protein aggregates to form rod-shaped particles that look exactly like TMV virions.

When virus is subjected to neutral pH with either detergents (e.g., SDS) or 6 M urea or by extraction with phenol then RNA can be extracted in an intact form.  When isolated TMV RNA are added to native TMV protein, these form stable "reconstituted" virus, which is more stable (stable from pH 3 to 9) then protein alone (unstable below pH of 4 and above pH 6).

Protein and RNA are more infectious than naked RNA alone (nearly 1000 times the amount of naked RNA is required to cause infection).

Proof that the viral RNA was the sole determinant of tobacco mosaic disease was obtained by a mixed reconstitution of RNA from Holmes ribgrass mosaic virus (RMV) with the protein subunits from TMV.  Reconstituted virus caused localized lesions on plants instead of a systemic infection and formed new RMV virus (RMV RNA + protein coat containing histidine and methionine - not found in TMV). Refer to Figure 2.13 on page 50 in handout #2

Assembly of Helical Viruses

Aggregates of 33 protein molecules form the double disk.  This combines with viral RNA.  Attachment of the nucleic acid to the protein aggregate begins at the origin of assembly site (OAS) about 800 nucleotides from the 3' terminus of TMV common strain RNA.  Rod growth toward the 5' terminus of the viral RNA is rapid, involving addition of double disks; encapsidation of the 3' terminus proceeds more slowly, through the addition of A protein monomers or small aggregates. Refer to Figure 6.6 in the textbook or to Figure 2.14 on page 50 in handout #2).  Cotranslation disassembly - the protein coat is displaced at the 5' end by ribosomes in host cell.


3' end of TMV RNA ends with the sequence -C-C-C-A and can be charged with an amino acid (histidine).  This region is non-coding and be folded into a tRNA-like structure preceded by a series of four pseudoknots.  Why?  Four possibilities exist.

  1. Donating an amino acid during some stage of protein synthesis.
  2. Facilitating translation by disrupting base pairing between the 3' and 5' - terminal regions of the viral RNA
  3. Acting as a recognition site for the viral replicase to initiate negative-strand synthesis
  4. A molecular fossil from the original RNA world where tRNA-like structures tagged RNAs for replication and prevented the uncontrolled loss of nucleotides from third 3' terminus.
Subgenomic mRNAs and translational read-through in TMV replication.

Five open reading frames or ORFs are found in the genome of TMV.  Subgenomic mRNAs and translational read-through are two strategies employed by TMV to regulate gene expression.
Plant positive-sense RNA viruses have developed several other mechanisms to facilitate and/or regulate the expression of individual genes.  5 strategies of regulating gene expression.


CUCUMBER MOSAIC (Refer to Fig 14-44 on page 531 in handout)
Control and Management
BARLEY YELLOW DWARF (Refer to Fig 14-42 on page 526 in the handout)
Management of Barley Yellow Dwarf Disease

Subviral Pathogens and Other Virus-like Infectious Agents Satellite Viruses

Satellite viruses were first observed in plants.  Their replication is dependent on the presence of a helper virus that provides the replicase, but the satellite virus is not required for helper virus replication.  There is little or no sequence similarity between the genomes of satellite viruses and those of their helper viruses.   Satellite viruses generally produce their own capsid protein.

Example: The helper virus for satellite tobacco necrosis virus (STNV - a 18 nm particle), is tobacco necrosis virus (TNV -  a small 30 nm icosahedral virus). STNV contains a monocistronic mRNA for the synthesis of its 22kDa coat protein.  Note that satellite viruses are generally smaller than helper viruses.

STNV is an obligatory parasite of its helper, dependent for its replication on the presence of TNV in the cells it enters.

Specificity of this dependence is illustrated by (1) the inability of plant viruses other than TNV to act as helper and (2) variation in the ability of certain TNV strains to support the replication of different strains of STNV.  The presence of STNV greatly suppresses the replication of TNV.

Satellite RNAs

Cucumber mosaic virus (an epiphytotic of lethal necrotic disease among tomato plants in France in 1972).  This was unusual because of the severity and atypical nature of the symptoms associated with infections of tomato plants (See description of cucumber mosaic in prior text).  Enhanced response of tomatoes to this disease were associated with host used for virus propagation; virus grown in tobacco or tomato yielded an enhanced necrotic response, those propagated in cucurbit hosts yielded a reduce necrotic response.  RNA extractions of necrotic tomato plants revealed the presence of variable amounts of a small RNA species in addition to the expected three genomic and one subgenomic RNAs.  This 5th RNA species was designated CARNA 5 (i.e., CMV-associated RNA 5 or CMV satRNA), a satellite RNA.

When CARNA 5 is present with CMV, symptoms of the disease are enhanced in tomato plants, while in tabasco pepper the presence of CARNA 5 tends to attenuate  the disease symptoms caused by CMV.  Other factors including sequence changes in the satellite RNA, use of a different helper virus strain, and changes in environmental conditions appear to greatly enhance or suppress symptoms caused by the helper virus.

Genome Structure of Satellite RNAs

Satellite RNAs have been found associated with members of five different plant virus groups.  With the exception of TCV (turnip crinkle virus) RNA C, satellite RNAs exhibit only limited sequence homology within the genomes of their respective helper viruses.  Satellite RNAs appear to from two size classes - large that are similar in size to the genomes of satellite viruses and much smaller.


Viroids are the smallest known agents of infectious disease - small (246-375) nucleotides), highly structured, single-stranded RNA molecules lacking both a protein capsid and detectable messenger RNA activity.

The first viroid disease to be studied was potato spindle tuber disease. Disease was first recognized and described by Schulz and Fosom in 1923, but it wasn't until Diener demonstrated in 1971, that the fundamental differences between the structure and properties of its causative agent, potato spindle tuber viroid (PSTVd).  Vd for viroid.

  1. Viroids exist in vivo as nonencapsidated, low-molecular-weight RNAs;
  2. Infected tissues do not contain virus-like particles;
  3. Only a single species of low-molecular-weight RNA is required for infectivity;
  4. Viroids do not code for any proteins;
  5. Despite their small size, viroids are replicated autonomously in susceptible cells and no helper virus is required.
  6. Single-stranded viroid RNAs are resistance to digestion by ribonucleases and possess a high degree of thermal stability (not easily denatured).
The genome of viroids, such as PSTVd are organized into a series of short double helices and small internal loops which form the basis for five domains.  Conserved central domain (highly conserved and site where cleavage and ligation to form circular progeny occur), pathogenicity domain (modulate symptom expression), variable domain (greatest sequence variability among otherwise closely related viroids), and two terminal domains (replication and evolution).

  1. Addition of new genes (to express a new gene product).
  2. Suppression of a gene or a gene product (in the plant - e.g., against ethylene gas production to prevent senescence of fruits or against a potential plant virus - e.g., TMV).
Potential benefits of transgenic plants

Genetic engineering can produce plants that are:

In general, transformed plants that express a viral coat protein are resistant to infection by both that virus and related viruses.   Viral coat protein may interfere with viral uncoating of the invading pathogen.  Coat protein-mediated resistance now available shows a lot of promise in inferring genetic resistance to various viral pathogens, including TMV.  Other promising strategies being investigated include the expression of antisense viral RNAs (which interfere with viral replication) and viral satellite RNAs, and noncoat viral genes (producing high resistance to specific strains).

Potential problems

Strategies for introducing novel genes into plants

1.  Agrobacterium-mediated gene transfer

Agrobacterium tumefaciens is the causal agent for crown gall in wide range of host plants.  It enters through wounds and injuries and causes a localized region of uncontrolled cell division (a tumor or gall) on the plant.  The bacterial cell contains a Ti or Tumor-inducing plasmid.  The Ti plasmid contains genes that call for the production of opines (C and N source for the bacterium) and regulate cytokinin and auxin production in plants (causing hyperplasia - excessive cell division = tumor).

  1. Ti genes removed by using restriction enzymes
  2. Target gene/gene for antibiotic resistance are introduced
  3. Modified Ti plasmid introduced back into A. tumefaciens
  4. Bacteria allowed to infect desired host (usually a tissue culture of the host)
  5. Bacteria incorporates its DNA into host
  6. Antibiotics added to growing callus to (a) eliminate the bacterium and (b) to select against untransformed cells
  7. Callus in tissue culture used to regenerate whole plant
2.  Bombardment  3.  Electroporation 4.  Plant virus-mediated transformation
Mycoviruses and the Biological Control of Chestnut Blight

Mycoviruses are probably widespread in fungi in nature, despite the fact that relatively few have been isolated and characterized, and still fewer have been experimentally transmitted to test fungi and demonstrated to be infectious.  A survey of 50 isolates of Rhizoctonia solani from the field, 49 were found to have RNA resembling that of mycoviruses.

Typical mycovirus particles are isometric in shape and have a diameter between 25-48 nanometers.  Some mycoviruses are rod-shaped or have some other form.  This nucleic acid core usually consists of dsRNA and infrequently contains dsDNA.  Some mycoviruses appear to be multipartite.  Strains of Penicillium chrysogenum, from which the antibiotic penicillin is commercially produced appears to be infected by a mycovirus.  Mycoviruses appear to be transmitted by means of cytoplasm and through spores.  Mycoviruses appear to be retained in the cytoplasm of a fungal strain indefinitely.

Many mycoviruses do not cause symptoms in their hosts, despite the fact that cells may harbor large numbers of virus particles.  Viral infection in the edible mushroom Agaricus brunnescens causes a degeneration of the mycelium and development of malformed basidiocarps, resulting in a reduction of yield.

Chestnut Blight and Hypovirulence

This page was assembled by Martin J. Huss, who can be reached at mhuss@astate.edu.
Last revised on: October 31, 2002.