REDUCING THE EFFECTS OF VIRAL

DISEASE

VIRUS PROPERTIESInfectious – must be transmissible horizontally

Intracellular – require living cells

RNA or DNA genome, not both*

Most all have protein coat*

May of may not have lipid envelope

May have broad or narrow host range

Replication involves eclipse (breaking apart of virus particles) and reassembly

Use host factors for to complete replication cycle

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

GENOME STRUCTURE FAMILIES AND GENERA NOTESRNA, 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:

RhabdovirusUnassigned genus: Tenuivirus

Bunyaviridae possess a lipidic envelope in addition to their nucleocapsid

RNA, double stranded Family: Reoviridae, Genera: Fijivirus, Phytoreovirus,

OryzavirusFamily: 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

Basically plant virus genome comprises of; Coding regions that

–expresses the proteins required for viral infection cycle

–Movement through the plant

–Interaction with host –Movement between

hosts Non-coding regions

that control the expression and

replication of the genome

Control sequences that can also be found in

coding regions

Same nucleotide sequence in a viral

genome could code for upto 12 or more polypeptides.

There could be an ORF in each of the three

reading frames of both +ve & -ve sense strands,

that give rise to six polypeptides

–ORF is a sequence that commences with AUG initiation codon and is

capable of expressing a protein of 10KDa or

more. Read through and

frame shift are quite common

Most of the ss +ve sense RNA genome code for ~4-7

proteins. In addition to coding regions for proteins, genomic n/a contains

nucleotide sequences with recognition and control

function that are important for virus replication. These

control and recognition functions are found mainly in the 5’ & 3’ non-coding sequences of the ssRNA

viruses, however, they may also occur internally even in

coding sequences. Viruses make very efficient use of the limited amount of genomic n/a they possess.

28

General properties of plant viral genome

MAJOR VIRAL DISEASES OF ECONOMIC CROPS IN PAKISTAN. 

Tobacco mosaic virus TMV•

Genus

Tob

amov

irus

15 m

embe

rs

nake

d, ri

gid

rod,

+ u

nseg

men

ted

ss R

NA

•No

fam

ily a

ssig

natio

n by

ICTV

30 nm

Helical symmetry

RNA genome18 nm 2 nm

NC protein

STRUCTURAL FEATURES OF TMV

CAPSID

TMV genome organization

5’ cap

6,395 nt

30 K

MT-Hel

UAG (leaky)

183K

Replicase RdRp

tRNAhis17.6 K

Movement MP

Capsid CP

126 K

TMV Life cycle 5’ cap Host

RbVirus 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 complement

ary 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’

RdRp + Strand (genome)

Neg. strand

promoters

Transcription by RdRp

MP mRNACP mRNA

TMV Life cycle (contt)

Tobacco mosaic virus

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

functionRNA2 -Coding sequences ---two CP subunits along with

MPGenome directly translated on entry into the cellPolyproteins made representing the entire coding

sequenceCleaved into active proteins by specific proteases

Other members of Comoviridae--nepoviruses

COMOVIRUSES

AAAAA(A)n

RNA 1

VP-g

RNA

Polyprotein

Traslation

200 kDa

Proteolytic cleavage

Proteases cofactor 32 kDa VP-g58 kDa Protease

24 kDaCore 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

RNA 2

Translation of the cowpea mosaic virus (CPMV) genome. VPg, genome-linked viral protein.

Rhizomania disease of sugar beet first reported in Italy in 1959since 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

GENOME ORGANIZATION OF BNYVV

Samples for soil-bait testingSoil 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.

SamplingSamples 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 preparationFor 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.

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

Natural defense in plants against viruses. Illustration of the Hypersensitive response (HR) and the Extreme Resistance (ER)

mechanisms where production of secondary metabolites confers resistance against infecting virus

1. Passive defense (barriers such as rigid cell wall) 2. Active defense (triggered upon the recognition of the encountered pathogen )

Natural response of plants against viral attack, where production of secondary metabolites

cause extreme or moderate resistance

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.

ways of controlling viral diseases

Schematic representation of the strategies opted to engineer virus resistant transgenic plants.

Major milestones in virus resistance strategies drawn to scale, starting form cross protection to

RNA-mediated gene suppression

Pathogen Derived Resistance

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

Coat Protein-Mediated Protection

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.

Economically important nepovirus+Ve sense RNA with genome separated on two molecules

RNA1 and RNA2CP coded on RNA2 and synthesized as polyprotein

Which later undergoes processing to release CP

Arabis mosaic virus (ArMV)

5.Comparison with the sequence obtained by computer translation of mRNA sequence and 5’ end identified

CP VECTOR GENERATION

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

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

RNA2 genome

polyprotien processedSequenced Nterminus

cDNA synthesis

Coat Protein

cDNA

PCR amplification

Restriction endonucleasedigestion of primers

cDNA (CP)promoter terminatorSelection cassetteLeft border Right borderIntroduced into tobacco via

Agrobacterium-based transformation system

strategies for the construction of ARMV transformation vectors and transgenic plants

Possible mechanism behind this resistance include

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

cucumber mosaic virus cucumovirus (CMV) symptoms – reduced – carrying satellite

Transgenic tobacco and tomato plant with CMV satellites – china – (1990 - 1992)

not good- sever disease symptoms

Satellites mutates very fast

Recombination bt satellites – serious consequences

Satellite Sequences

• 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

Antisense RNAs

Mechanism of action of antisense

oligonucleotide in suppressing the

expression of target gene at transcriptional stage. RNase H is the

endonuclease responsible for digestion

of duplex RNA, thus blocking translation of

target mRNA.

Ribozymes

• Ribozymes are small antisense RNA molecules – catalytic cleavage of target RNA

• Similar to those used for antisense approach except that a short catalytic sequence is embedded within a target region

• Block the replication of RNA by forming dsRNA hybrid and cut a key region of the virus genome.

• But not effective unless the antisense sequence is designed against a very conserved region .

Gene silencing in transgenic plants

• It was firstly reported when introduction of additional chalcone synthase gene was transformed to petunia flower to intensify the voilet color of flower

• Obtained flowers (transgenic lines) were both

1. intensed violet colored type- transgenesis worked

2. white colored as well-Both native and transgenic chalcone synthase turned off

• Stable integration and expression of transgene is required for commercial exploitation.

• Expression of introduced gene can be abberant - GENE SILENCING!!!

Mechanism of post-transcriptional gene silencing (PTGS)

RNAIII double stranded- specific ribonuclease•Drosophila---Dicer•Plants---3 Dicer like protiens (DCLs) DCL 2 cleaves dsRNA fro replcating virusesDCL 3 cleaves dsRNAs derived from endogenous transcripts through the activity of RdRps 2 & 6DCL 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.

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