Download Viruses I - University of Florida

Characteristics of Viral and Viroid
Pathogens
Svetlana Folimonova
Assistant Professor
Dept. of Plant Pathology
University of Florida
Learning Objectives
• Introduction to viruses: prevalence, diversity,
virus structure, genome composition and organization (with
main focus on plant viruses)
• Plant virus life cycle
• Virus movement
• Plant-virus interactions (types of plant response, symptoms)
• Detection and identification
• Control of plant viruses
• Characteristics of viroids
Prevalence …
Virtually all known life forms have been
shown to be infected with viruses
Viruses are the major players in the genetic
universe
1 cm3 of seawater contains 106-109 virus particles
Suttle, C.A. (2005) Nature 437:356
There are millions of diverse bacteriophage species
in the water, soil, and gut
Edwards and Rohwer (2005) Nat. Rev. Microbiol. 3:504
Viruses dominate biosphere: there are 10-100
viruses per each living cell
Slide source: V. Dolja “Introduction to plant viruses”
VIRUSES
Non-cellular form of life
Obligate intracellular parasites
The fundamental characteristic is their
absolute dependence on a living host
organism for their reproduction
Exist as inert particles (virions)
outside the cell
Virions harbor viral genome protected
by protein shell
Diversity between viruses
• There is much biological diversity between viruses:
- Host range
- Type of disease
- Virus particle size (large or small?)
- Virus particle structure
- Virus genome type (DNA or RNA?) and size
- Virus genome replication strategy
Virus structure
Virus particles (virions) are built of a nucleic
acid and a protein shell
Icosahedral (spherical) virions
(protein subunits are arranged
with cubic symmetry)
Elongated (helical) virions
Slide source: V. Dolja “Introduction to plant viruses”
Structure of TMV virion
95% protein, 5% RNA
have been reported to retain
infectivity at room temp.
for at least 50 years
15 x 300 mµ
- 2130 protein subunits;
- each sununit is associated
with 3 nts;
-49 nts and 16 1/3 protein
subunits per turn
- ~ 130 helix turns of protein
8
subunits
Virus structure
“complex” structure
enveloped viruses
“simple structure”
10
A. Smallpox virus B. Orf virus C. Rhabdovirus D. Paramyxovirus E. Bacteriophage T2 F.
Flexuous-tailed bacteriophage G. Herpes virus H. Adenovirus I. Influenza virus J.
Filamentous flexuous virus K. Tobacco mosaic virus L. Polyoma/papilloma virus M. 11
Alflafa mosaic virus N. poliovirus O. Bacteriophage phiX174.
Virions under EM
Tobacco mosaic virus rigid rods
Small pox
virus –
enveloped
Tobacco vein mottling
virus – flexuous rods
a geminivirus –
twinned icosahedra
Vesicular
stomatitis virus
- enveloped
Genome composition and
organization
13
Viruses infecting
plants
Genome
No.
%
dsDNA
30
3
ssDNA
166
17
dsRNA
45
4.6
(-) ssRNA
100
10.2
(+) ssRNA
635
65
Total plant viruses: 976
Source: Matthew’s
Plant Virology
by R. Hull
Functions of viral gene products
- structural proteins
- proteins involved in nucleic acid synthesis
(RdRp, RdDp or reverse transcriptase,
methyl transferase, helicase)
- proteases
- proteins involved in virus movement and
transmission by vectors
Most of the ss (+)-sense RNA genomes code
for about 4 to 7 proteins (CTV – 12)
15
Genome organization of Tobacco mosaic virus
Genomic RNA 6.4 kb
“leaky’ UAG
5’cap
69 ntsNTR
MT
HEL
MT
HEL
126 kDa
183 kDa
3’ NTR
tRNA-like
structure
RdRp
CP
MP
17.6kDa, CP
30 kDa, MP
CTV genome (19.3 kb)
Replication
Movement
1a
L1 L2
MT
p33
IDR
HEL
HSP70h
CPm
p18
p20
1b
RdRp
p6
p61
CP
p13
p23
Virion assembly
RNA silencing
suppressors
Virus Life Cycle
1
Invasion
Invasion through
leaves:
Cytoplasm
Cell
Wall
vectoring insects;
mechanical damage
Invasion through
roots:
vectoring nematodes or
fungi;
mechanical damage
Exception: seed and pollen transmission
Slide source: V. Dolja “Introduction to plant viruses”
Virus Life Cycle
2
Genome uncoating, expression
and replication
uncoating
translation
replication
Slide source: V. Dolja “Introduction to plant viruses”
Virus Life Cycle
3
Particle (virion) assembly
Slide source: V. Dolja “Introduction to plant viruses”
Virus Life Cycle
4
Cell-to-cell movement
Cell-to-cell movement – from the initially infected cell to the vascular
bundle. Exception: phloem-limited viruses are usually injected directly
into the phloem by their vector.
Slide source: V. Dolja “Introduction to plant viruses”
Virus Life Cycle
5
Systemic transport through phloem
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
Long-distance movement – through the phloem. Further cell-to-cell
movement establishes systemic infection in the young leaves.
Slide source: V. Dolja “Introduction to plant viruses”
Virus movement pathway
From Carrington et al. (1996) Plant Cell
Vol. 8 (10):1669-1681
Cell walls between different types of cells represent significant barriers for the virus during its
cell-to-cell movement, loading into phloem and further unloading out of vascular tissue into
parenchyma cells in systemic leaf.
Structure of plasmodesmata and comparison to
viral particles
Viral encoded “movement proteins (MPs)” facilitate
these steps. Most MPs are multifunctional.
1) MPs are required for movement
2) MPs bind to virus genomes
3) MPs interact with plant cytoskeleton
4) MPs localize to plasmodesmata
5) MPs alter and gate plasmodesmata, increase their size
exclusion limit
Virus genome may be associated with MP or
coat protein during cell-to-cell movement
26
Genome organization of Tobacco mosaic virus
Genomic RNA 6.4 kb
“leaky’ UAG
5’cap
69 ntsNTR
MT
HEL
MT
HEL
126 kDa
183 kDa
3’ NTR
tRNA-like
structure
RdRp
CP
MP
17.6kDa, CP
30 kDa, MP
Functional characteristics from the pioneer
research on TMV 30-kDa MP:
•It binds single stranded RNA in a sequence nonspecific manner to form
a stable RNP complex
•It interact with the host PD machinery to induce an increase in the
SEL(size exclusion limit) from 1kDa ~ 30kDa
•It gates PD at the leading edge of TMV infection.
•It is present within the PD structure
•It mediates the cell-to-cell trafficking of itself and viral progeny
RNA, leading to the infection of neighboring cells
•It associates with microtubules and microfilaments as well as with ER
28
Movement proteins are a general feature of many
plant viruses
Lucas. 2006. Virology. 344:169-184
Viral proteins involved in systemic transport
Genus
Virus
Viral
factors
Genus
Virus
Viral
factors
30
Systemic movement of TMV expressing GFP
in Nicotiana benthamiana
31
Virus Life Cycle
6
Plant-to-plant transmission
.
Aphids
Whiteflies
Mealybugs
Leafhoppers
Treehoppers
Beetles
Mites
Thrips
Nematodes
Slide source: V. Dolja “Introduction to plant viruses”
Plant-virus interactions
33
Types of plant response to inoculation with a virus
Plant is immune: virus does not replicate in protoplasts nor
in cells of the intact plant
Infection is limited to initially infected cells: replication, but
no cell-to-cell movement (could be due to an ineffectual virus
movement protein)
Infection is limited to initial leaf: replication, cell-to-cell movement,
but no long distance movement (inability to enter phloem or due to
a host resistance gene-mediated response, i.e. hypersensitive
response when the plant resistance gene product recognizes
some viral product, local lesions formation)
34
Hypersensitive response
Examples:
Tobamoviruses
tobacco N gene recognizes the replicase protein
tobacco N’ gene recognizes the coat protein
tomato Tm2 gene recognizes the movement protein
pepper L gene recognizes the coat protein
eggplant recognizes the coat protein
Potexviruses
potato Nx recognizes the coat protein
potato Nb recognizes the replicase protein
35
Types of plant response to inoculation with a virus
(cont.)
Plant is immune: virus does not replicate in protoplasts nor
in cells of the intact plant
Infection is limited to initially infected cells: replication, but
no cell-to-cell movement (could be due to an ineffectual virus
movement protein)
Infection is limited to initial leaf: replication, cell-to-cell movement,
but no long distance movement (inability to enter phloem or due to
a host resistance gene-mediated response, i.e. hypersensitive
response when the plant resistance gene product recognizes
some viral product, local lesions formation)
Systemic infection
36
Types of plant response to inoculation with a virus
(cont.)
Does systemic infection equal disease?
37
Papaya ringspot virus
Types of plant response to inoculation with a virus
(cont.)
Does systemic infection
equal disease?
Papaya ringspot virus
Tolerance (latent infection: little or no obvious symptoms)
Disease (severe symptoms, death)
Recovery (due to RNA silencing; virus replicates and causes
symptoms in new leaves but with time and further plant growth
symptoms disappear)
Resistance (SAR: active host response to infection by diverse
pathogens that cause necrotic cell death, resulting in diminished
susceptibility to later pathogen attack; VIGS: active response by
the host induced by infection with a virus that leads to specific38
degradation of viral RNAs)
Symptoms
Systemic symptoms
General:
Abnormal growth and developmental malformations (reduced
growth, dwarfing, stunting of plants, etc.)
Reduced life span of plants, death
Foliage symptoms:
Mosaics
Yellows
Ring spots
Other:
Symptoms on stems,
fruit, roots
Local symptoms
Necrotic lesions
None
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Some
Examples:
A potyvirus in
tobacco
Peanut stunt virus in white clover
Cowpea chlorotic mottle
virus in cowpea
Barley stripe mosaic virus
in barley
Tobacco mosaic virus in tobacco
Necrotic local lesions on N. tabacum
Glurk leaf, demonstrating Holmes’ Ngene resistance following inoculation
with TMV. Photo: K.-B. G. Scholthof.
45
Tomato spotted wilt virus in peanut
Leaf roll virus on grape leaves. (Courtesy of A. Baudoin)
49
Citrus tristeza virus
in citrus
Citrus tristeza virus in citrus
Symptoms
Systemic symptoms
General:
Abnormal growth and developmental malformations (reduced
growth, dwarfing, stunting of plants, etc.)
Reduced life span of plants, death
Foliage symptoms:
Mosaics
Depend on virus strain, host,
Yellows
time, environmental conditions,
Ring spots
presence of other viruses!
Other:
Symptoms on stems,
fruit, roots
Local symptoms
Necrotic lesions
None
52
Symptoms of the
mosaic strain of PVY in
potato
Symptoms of the
necrotic strain of PVY in
potato
Pictures courtesy of Nan Jing Ko
53
Sweet orange cv. Valencia grafted on sour orange
rootstock affected by Citrus tristeza virus (CTV)
Stem pitting induced
by CTV on a
grapefruit trunk.
Pictures by L. Navarro IVIA, Valencia
54
Vein-clearing on TMV-infected
tobacco. Picture credit W.O.
Dawson
55
Effect of temperature on symptom development and Cymbidium ring spot virus spreading
of virus-infected N. benthamiana plants. Szittia et al. (2003) EMBO 22:633-640.
56
Recovery phenotype was
first described in 1928
(a) Typical symptoms of TRSV in N. benthamiana plants at 10 days p.i. (b) Recovery
of a plant from initial infection at 40 days. Siddiqui et al. (2008) J. Gen Virol 89:150208.
Virus synergism in mixed infections
Tobacco plants infected singly with either potatovirus X (PVX) or
potatovirus Y (PVY) show mild symptoms, whereas doubly
infected plants develop a severe synergistic disease in
systemically infected leaves.
59
Detection and identification…
your ideas here?!
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Detection and identification
Virus biology observations:
- Host range
- Symptoms (possible causes by nutrient deficiencies,
toxicities, insects, etc. need to be eliminated and confirmed
by transmission to a healthy plant)
- Modes of transmission (vegetatively, mechanically,
by plant sap passed through bacterial filters, insect vectors)
Virus properties:
- LM (inclusion bodies) and EM (virus particles)
- Virus purification
- Serology (only for known viruses for which antisera are
available)
- dsRNA isolation
- PCR, RT-PCR (for known viruses)
- Virus genome sequencing
- small RNA preparation, sequencing using Next generation
sequencing technologies, virus genome seq. assembly
- Microarray
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Control of plant viruses
Prevention of infection!!!
Control of plant viruses
- Keep out of an area through quarantine, inspection, and certification programs
that would prevent or limit local (within state, between states) or international
(between countries) virus movement and warranty the use of virus-free seed,
tubers, budwood
-
Eradication of diseased plants
-
Controlling the insect vectors (oil sprays, repellents, insecticides, predators…)
-
Breeding plants for resistance
- Transgenic resistance (natural resistance genes, viral sequences, genes
from other sources) Today, 80% of Hawaiian papaya is genetically engineered, and
there is still no conventional or organic method to control ringspot virus. 13% of the
zucchini grown in the US was genetically modified to resist three viruses.
-
Systemic acquired resistance
-
Mild isolate protection (cross-protection)
Large-scale utilization of cross-protection
Virus system
Host plant
Country
Tobacco mosaic virus
tomato
Tomato mosaic virus
tomato, pepper
China
Tien and Chang (1983)
Cucumber mosaic
virus with satellite
pepper
China
Tien and Chang (1983)
Cocoa swollen shoot
virus
cocoa
West Africa
Zucchini yellow
mosaic virus
squash, melon,
watermelon
Israel
Apple mosaic virus
apple
New Zealand
Passion fruit
woodiness virus
passion fruit
Australia
Papaya ringspot virus
papaya
Taiwan, Thailand,
Mexico, Florida, Hawaii
Netherlands, U.K.,
Japan, France, USA
Reference
Rast (1972), Fletcher
(1975), Oshima (1975),
Marrou (1978)
Posnette and Todd (1951)
Yarden et al. (2000)
Chamberlin et al. (1964)
Simmonds (1959)
Yeh and Gonsalves (1984)
Cross-protection by Citrus tristeza virus
is one of the best examples of cross-protection of major economic importance
A total of 50 million orange trees have been protected with a mild strain in Brazil
(Costa and Muller, 1980). Cross-protection was also used to control the disease
in South Africa, Australia, Peru and other countries.
Viroids
- Pathogenic agents in plants
- Unencapsidated naked small circular
RNA molecules, a few hundred nucleotides
long, with high degree of secondary structure
- Lack the protein coat of viruses
- Do not code for any protein
- Replicate in the nuclei of infected cells
- Rapidly move from cell to cell and through
the phloem
- Readily transmitted mechanically in the field,
vegetative propagation, some - by pollen and
66
seed
Potato spindle tuber viroid was the first to be
identified and is the most studied.
Viroids in citrus:
Citrus exocortis viroid – 375 nts
Citrus viroid III – 297 nts
Citrus viroid IV – 284 nts
Citrus bent leaf viroid – 318 nts
68
Suggested reading material
‘Matthew’s
Plant Virology’ by Roger Hull (2002)