Viral diseases can be reduced through several methods. Coat protein mediated resistance involves transforming plants with the viral coat protein gene, which allows the plant to resist infection from that virus or related viruses. Antisense RNAs can also be used, where small untranslatable RNAs pair with and degrade target viral RNA sequences. RNA interference is a natural antiviral response in plants where dicer enzymes produce small interfering RNAs that guide the RNA induced silencing complex to cleave homologous viral RNA transcripts.

1. REDUCING THE
EFFECTS OF VIRAL
DISEASE

2. VIRUS PROPERTIES

4. Typically, a combination of characters are used and some of the most important are

5. GENOME STRUCTURE FAMILIES AND GENERA NOTES
RNA, single stranded, positive sense
(acts as mRNA directly)
Families: Bromoviridae, Cornoviridae,
Potyviridae,
Sequiviridae, Tombusviridae,
Luteoviridae
Example of unassigned genera: Tobamo- &
Tobavirus
70% of the known plaant viruses, both segmented and
non segmented (two or more RNAs in different virus
particles)
RNA, single stranded, negative sense
(RNA needs to be copied before it can
act as mRNA)
Families: Bunyaviridae, Genus: Tospovirus:
Family: Rhanbdoviridae, Genus: Rhabdovirus
Unassigned genus: Tenuivirus
Bunyaviridae possess a lipidic envelope in addition to
their nucleocapsid
RNA, double stranded Family: Reoviridae,
Genera: Fijivirus, Phytoreovirus, Oryzavirus
Family: Partiviridae,
Genera: Alpha- & Betacryptovirus
The plant members of the Reoviridae family have a
genome consisting of 10-12 segments of RNA: each
has one ORF that produces a protein
DNA, double stranded Family: Caulimoviridae,
Genera: Caulimovirus and Badnavirus
The only plant viruses of this group are the
Caulimoviruses; their genome consists of one double
stranded circular DNA molecule with specific single
stranded discontinuities in both strands; it codes for
six or eight ORFs located on one strand only; DNA
replication occurs by a process of reverse
transcription ( i.e. via RNA intermediate) similar to
that of the animal retroviruses
DNA, single stranded Family: Geminiviridae The only plant viruses possessing either or two
molecules of single stranded genomic DNA; DNA
replications via double- stranded DNA intermediates;
ORFs located on both viral strand and its complement

9. MAJOR VIRAL DISEASES OF ECONOMIC CROPS IN PAKISTAN.

10. 30 nmHelical symmetry
RNA genome18 nm 2 nm
NC protein
STRUCTURAL FEATURES OF TMV
CAPSID

13. TMV genome organization
5’ cap
6,395 nt
30 K
MT-Hel
UAG (leaky)
183K
Replicase RdRp
tRNAhis
17.6 K
Movement MP
Capsid CP
126 K

14. TMV Life cycle
5’ cap Host Rb
Virus entry trough
abrasions on plant tissue.
Inside cell associates
with ER
spontaneous release of
few capsid (CP) subunits
5' end of genome is
uncovered
Host ribosome attaches
to viral RNA, moves down
displacing more CP units
Ribosome meets start
codon, translates first
two proteins (126K ,183 K)
while uncoating continues
“co-traslational
disassembly”
126 K ( MET-Hel) & 183 K
( RdRp) use viral RNA as
template to make full
length complementary
neg. strand RNA
Neg. RNA strand used by
viral replicase
(RdPp/MET-Hel ) as
template for +RNA
Also, neg. RNA strand
has internal promoters
used by replicase to make
mRNA for 30K protein
(MP) and 17.5 K (CP)
MP combines with viral
+RNA to move it into new
plant cells through
plasmodesmata
Accumulation of +RNA &
CP proteins stimulates
assembly of progeny
virions. massive TMR
replication occur in the X-
bodies (viroplasmas)
5’

15. RdRp + Strand (genome)
Neg. strand
promoters
Transcription by
RdRp
MP mRNA
CP mRNA
TMV Life cycle (contt)

17. Segmented genome—split into two parts RNA1 and RNA2
RNA1 –coding sequences for core polymerase, a protease,
and a VPg (genome linked viral protein)
VPg attached to 5’ end of the molecule and fulfills a cap
function
RNA2 -Coding sequences ---two CP subunits along with MP
Genome directly translated on entry into the cell
Polyproteins made representing the entire coding sequence
Cleaved into active proteins by specific proteases
Other members of Comoviridae--nepoviruses
COMOVIRUSES

18. AAAAA(A)n
VP-g
RNA
Polyprotein
Traslation
200 kDa
Proteolytic cleavage
Proteases
cofactor
32 kDa VP-g58 kDa Protease
24 kDa
Core polymerase 87 kDa
AAAAA(A)n
Traslation
RNA
Polyprotein 105 kDa
95 kDa
58 kDa
48 kDa
transport
CP L 37 kDa
CP S 23 kDa

25. Rhizomania disease of sugar beet first reported in Italy in 1959
since been reported in more than 25 countries
disease causes economic loss to sugar beet (Beta vulgaris var. saccharifera) by
reducing yield. caused by Beet necrotic yellow vein virus (BNYVV), which is
transmitted by the soil fungus, Polymyxa betae
virus can survive in P. betae cystosori for more than 15 years.
symptoms also known as ‘root madness’, include root bearding, stunting, chlorosis
of leaves, yellow veining and necrosis of leaf veins.
virus spread by movement of soil, primarily on machinery, sugar beet roots,
stecklings, other root crops, such as potato, and in composts and soil.
Water is important in the spread of the fungal vector; drainage water, ditches
and irrigation with water from infected crops can favour the disease.
THE DEVELOPMENT IN SUGAR BEET

26. GENOME ORGANIZATION OF BNYVV

27. Samplesfor soil-bait testing
Soil samples from the field can be tested for rhizomania by growing susceptible beet in the soil (bait testing) in
a glasshouse or in growing chambers. A total of 2.5 kg of field soil should be taken by walking in a W shape
across each of the sampling areas. Each sample should be separately identified and placed in a labelled plastic
bag.
Sampling
Samples should be taken from identified yellow patches in beet crops (identified by aerial photography, etc.). A
fork or spade should be used to dig up the roots (especially in dry hard baked soils). Care should be taken to lift
the beet whole as the root tip and laterals with ‘rat tails’ can easily break off and be left behind in the ground.
Each sample should consist of the lower third of the taproot of 5 or 6 plants showing symptoms. Each sample
should be separately identified and placed in a labelled plastic bag.
Sample preparation
For laboratory-based tests, the sugar beet samples should be thoroughly washed in cold water to remove loose
soil from the roots and dried on absorbent paper. Samples should then be placed in labelled plastic bags for
processing.

28. Infected plants?
Storage organs of sugar beet plants
(leaves removed)
Healthy plants
Seedlings grown
In sterile soil
Bait seedlings
grown In test soil ELISA
4 weeks
Test soil sample
The soil bait scheme
•ELISA test
•RT-PCR test
•Immunocapture PCR
•TaqMan® RT-PCR
•Electron microscopy tests
CONFORMATION TESTS

31. The use of disease-free planting material.
Virus-free stocks are obtained by virus
elimination through heat therapy and/or
meristem tissue culture. This approach is
effective for seed-borne viruses, but is
ineffective for viral diseases transmitted by
vectors
Adopting cultural practices that minimize
epidemics, for example by crop rotation,
quarantine, rouging diseased plants and using
clean implements. Pesticides may also be used
to control viral vectors, but the virus may be
transmitted to the plant before the vector is
killed
Classical cross protection, in which a mild
strain of the virus is used to infect the crop,
and protects the crop from super-infection by
a more severe strain of the virus. Successful
against closterovirus citrus tristeza virus
(diseases of citrus trees) potyviruses papaya
ringspot virus, yellow zucchini yellow mosaic
virus, cucumber mosaic viru (associated
satellite RNAs)
Use of disease resistant planting material.
Natural resistance against viruses may be bred
into susceptible lines through classical
breeding methods or transferred by genetic
engineering.
Engineered cross protection. This involves
integration of pathogen-derived or virus
targeted sequences into DNA of potential host
plants, and conveys resistance to the virus
from which the sequences are derived.

35. The concept of pathogen-derived resistance (PDR) strategy is based on the insertion of resistant genes that
are derived from the pathogen (virus) into the host plant
Strategies of Pathogen Derived Resistance
PROTEIN ACCUMULATION
Coat Protein Mediated Resistance,
Movement Protein Mediated Resistance
Replicase Protein Mediated Resistance
NUCLEIC ACID SEQUENCES
Replicase Mediated Resistance

37. Coat protein (CP) gene of tobacco mosaic virus (TMV) was used in the first demonstration of virus-derived
resistance in transgenic plants
Coat protein-mediated resistance (CP-MR) is the phenomenon by which transgenic plants expressing a plant virus
coat protein (CP) gene can resist infection by the same or a homologous virus
The major function of coat proteins (CPs) is disassembly of challenging virus accompanied by a later function in
assembly of progeny virus. In addition CPs has a role in
viral RNA translation
targeting the viral genome to its site of replication
 severity of the infection
Coat protein gene is transformed in plants which ultimately form coat protein using host cell machinery. As the
plant encounters the pathogen (virus), protein mediated response become visible
CP-MR has been reported for more than 35 viruses representing more than 15 different taxonomic groups
including the tobamo-, potex-, cucumo-, tobra-, carla-, poty-, luteo-, and alfamo- virus groups. The resistance
requires that the CP transgene be transcribed and translated.

38. 5.Comparison with the sequence obtained by computer translation of
mRNA sequence and 5’ end identified
1.Precise location of the CP gene sequence through In Silico
analysis
2.Single CP subunit (64 kDa) present at 3’ end of RNA2 (3’
end defined by stop codon)
3.Identification of 5’ end more difficult as it is located in
the region coding for polyprotein
4.N terminal amino acid obtained by sequencing CP purified
by variuos phase separtions and centrifugation in linear
sucrose gradients

39. 6.PCR primers design
•Complementary to about 30 nucleotides of virus
•Several sites for restriction endonuclease site at 5’
end.
•Primer for N terminal end of sequence contains AUG
start condon
7.PCR carried out using cDNA as template.
Amplified DNA digested with restriction sites to
allow the amplified CP construct to be ligated into
E.coli vector cloning vector
8.Cassette vector (pMON316)
•35 CaMV promoter with transcription enhancers
at N terminal
• TMV signal to optimize the level of translation
at N terminal
• NOS terminator signal at C terminal
9.Complete sequence digested out of intermediary
plasmid and ligated into binary vector pBIN19 used
for Agrobacterium based transformation.
10.Transgenic plant lines obtained after Agrobacterium
mediated transformation and screened for protein
expression levels using ELISA

40. RNA2 genome
polyprotien processed
Sequenced Nterminus
cDNA synthesis
Coat Protein
cDNA
PCR
amplification
Restriction endonuclease
digestion of primers
cDNA (CP)promoter terminatorSelection cassetteLeft border Right border
Introduced into tobacco via
Agrobacterium-based
transformation system

41. Coat protein produced from transgene is capable of subunit-subunit interaction in
which direct association of a small number (1 to 6) of transgene-derived CP
molecules with the challenge virus during disassembly takes place. This
interaction will ultimately prevent binding of ribosomes to the RNA of the
invading virus, and hence infection
Binding of coat protein to the host factors responsible for disassembly of the
virion. This underlying mechanism will only be true for a plant containing a
mutated transgene of coat protein. The mutated coat protein will offer a
competitive inhibition to the coat protein of invading virus for binding to host
factor involved in viral disassembly Thus blocking the viral infection
Coat protein may confer resistance against a specific virus by interacting with
nuclear inclusion protein b (a replication protein), this possibility is specific
for Potyviruses only

44. Satellite Sequences

46. • Antisense RNAs refer to small untranslatable RNA molecules that pair with a
target RNA sequence on homology basis and thereby exert a negative control on
interaction of target RNA with other nucleic acids or protein factors
• Further, RNase H cause an increase in rate of degradation of double stranded
RNA
• This phenomenon completely operates on homology basis with target sequence.
• Block the specific gene expression.
• E.g. Beta 1,3-glucanase was down regulated by antisense RNA in Tobacco +
tolerance mosaic virus+ delayed spread+ reduced virus yield

53. RNAIII double stranded- specific ribonuclease
•Drosophila---Dicer
•Plants---3 Dicer like protiens (DCLs)
DCL 2 cleaves dsRNA fro replcating viruses
DCL 3 cleaves dsRNAs derived from endogenous transcripts through
the activity of RdRps 2 & 6
DCL 1 --- production of microRNAs
siRNA duplexes bind to the complex that contains another nuclease to
form RNA induced silencing complex (RISC)
Associated ATP dependant helicase then unwinds the duplexes
RISC then target the homologous single stranded RNA transcripts
and cleaves the RNA molecues.