Plant Viruses: An Overview

Viruses are nucleic acid protein complexes which multiply in living cells by hijacking the hosts’ replicative machinery.

One quarter of all known viruses (>2000) attack plants.

Nomenclature –

from original plant they were isolated from – and a description of the symptoms

e.g. Tobacco mosaic virus, Prunus necrotic ringspot virus.

Tomato spotted wilt virus.

Poor system since symptoms vary so greatly.

Symptoms:

Plant viruses do not cause disease through toxin production but disruption of the cellular processes – hence many viral infections resemble nutrient deficiencies.

- Infection with all viruses cause “dwarfing and stunting”

- Localised chlorotic / necrotic lesions

- Patternings

Mosaic symptoms – arise from 100,000 – 10,000,000 viruses per cell

Ring-spots –chlorotic / necrotic rings.

- Other viruses have no obvious additional symptoms – the latent viruses.

Classification: Functional Morphology

1. Shape – coat protein dependent.

- Rod - (15 x 300nm) “spherical” polyhedral diameter – 17nm in diameter

- “Bacillus-like” 52-75nm x 300-350nm.

2. Genome- type and form of nucleic acid

Tobacco Mosaic Virus: A viral paradigm

Genome A single RNA stranded (+) virus

There is a leaky STOP codon at the end of the 130KDa gene allowing read through to form the 180KDa protein.

Genome – function

Note: Coat protein serves not only a protective role but helps in systemic dispersal.

INFECTION PROCESS

Very easily by plant rubbing. Wounding is important -

Virus is very stable. Purified TMV is infectious after 50years storage in a fridge.

Uncoating

Uncoating begins 2-3min after infection.

The + RNA is effectively mRNA so can bind ribosomes.

Ribosomes bind to 5’ end and displace coat protein only until the entire 180kDa region is exposed.

The expressed replicase binds to the 3’ end to uncoat the rest of the virus.

The process is complete within 30min.

Replication

+ strand is encapsidated, so need negative strand to for further viral genomes.

Also this will serve as a template for the synthesis of subgenomic RNAs for MP + CP and CP alone.

This is necessary since both MP and CP are encoded on the “opposite” – strand.

130kDa / 180KDa show homology to RNA dependent RNA polymerase - 180KDa protein is essential for replication not so the 130KDa.

Replicase complex –

Replicase also makes the “incomplete” RNA copies known as “subgenomics”

The use of subgenomics allows simultaneous RNA replication and MP / CP translation to occur.

Otherwise this would be attempted on the same molecule!!

Where is this occurring?

After penetration of the tissue –

(1) the nascent viroids accumulated into “viral factories” –

…in ER where replication predominates

(2) Then disassociate as MP – RNA complexes which are associated within the cytoskeleton.

(3) MP-vRNA accumulates at plasmodesmata.

Accumulation and distribution of fluorescent MP:GFP in the population of infected tobacco BY-2 protoplasts and N. benthamiana at given times after inoculation. Note in (B, C, J, K), MP:GFP becomes detectably associated with small irregular fluorescent structures – viral factories in the endoplasmic reticulum. In (D, E, L, M), the irregular fluorescent structures decrease in size and appear to be interlinked with filaments (microtubules). Finally, (G, N) fluorescence becomes associated with the plasma membrane (plasmodesmata). Bars = 10 µm.

Movement

Movement is governed by source - sink relationships. Hence symptom tend to appear in the youngest leaves

Cell-to-cell (influx + efflux) using plasmodesmata.

These are “symplastic” connections between plant cells.

The center of the plasmodesmata is termed the desmotubule and is made up of

compressed endoplasmic reticulum. The region between the desmotubule and the plasma membrane is the

cytoplasmic sleeve, the major conduit through which molecules pass from cell to cell. Electron dense

substructures line the cytoplasmic sleeve between the DT and the plasma membrane.

Both globular particles and

elongated spokes appear to interconnect these two membranes and may act to expand or contract the cytoplasmic

space to increase or restrict transport. Cross-sectional views reveal that PD are subdivided into 2.5-nm diameter

microchannels

Plasmodesmata allow the movement of 1mm 8 to 10 cells per day in leaf parenchyma.

Cell-to-cell movement (influx + efflux) aided by the 30KDa (P30) “movement protein” which enlarges plasmodesmatal channels.

Four main functions

(i) Binds TMV RNA

(ii) Interacts with the cytoskeleton to facilitate transport

(iii) > the “pore size” of the plasmodesmata

(iv) Interacts with a cell wall receptor

Movement of MP-Nucleic acid complexes

Stage 1 – Nucleic acid become coated with MP

The particle size is now 2-2.5nm

P30 binding to DNA is not sequence specific.

· In vitro P30 can bind to any RNA molecule –

· MPs from other viruses can substitute for the TMV MP and move TMV RNA to the plasmodesmata.

Stage 2: The RNA-MP interact with the cytoskeleton

· On microtubules – composed of tubulin

· On microfilaments – composed of F-actin.

These are the “superhighway” by which the MP-RNA is targeted to the plasmodesmata.

Stage 3: Interaction with the plasmodesmata.

Plasmodesmata have a usual pore size of 1.5cm

Exclusion limit is 0.75- 1.00 KDa without MP

>10KDa with MP

Stage 4: Interact with cell wall receptor

Once reaches the cell wall/plasmodesmata interacts with a p38 protein.

P38 appears to mediate RNA / and/or protein trafficking.

One working model predicts the following.

Note, phosphorylation of MP stops their interaction with P38 and causes their partial release from the viral RNA - but also the movement of virus to the next cell.

Have virus hitched onto a mechanism that plants use to control gene expression?

The “Knotted” story.

The knotted1 mutation was first isolated as a dominant mutation in maize and produced a “knotted” leaf phenotype (see above).

Comparison of the patterns of KN1 mRNA expression and protein accumulation shows that they do not coincide. While expression of the mRNA is excluded from the outer cells in the embryo (see A above) and shoot meristem, but that is where the KN1 protein is localised (see B above) !!

It has been shown that KN1 movement from the site of transcription to protein accumulation is regulated by plasmodesmata i.e. that have important developmental role.

Could the virus MP-RNA interaction with plasmadesmata be exploiting this function?

Systemic Movement Entry into phloem –Entry into the phloem

(i) In the phloem companion cell – virus disassembles

(ii) A CP-MP-viral complex is this formed to enter sieve element.

(iii) In the phloem CP and vRNA form viral assembly complex.