T4 AND TMV

1. FROM ,
SONA PETER P
MSC . BOTANY
VIMALA COLLEGE THRISSUR

3. BACTERIOPHAGE
• Also known as bacterial viruses
• bacteriophage that infects Escherichia coli bacteria
• British scientist- Twort -1915
• French scientist – De Herelle -1917
• Seen in sewages or fecal material
• In human & other animals –intestine
• 4 types – ssDNA ,dsDNA,ssRNA,dsRNA

4. T4 PHAGE
• Commonly known as T4 coliphage
• It is double stranded DNA phage
• It parasitizes Escherichia coli
• 5 parts – head , neck ,tail ,base plate &tail fibers
HEAD
• Icosahedron shape
• Elongated , bipyramidal prism
• 95 cm long ,65 nm wide
• Central DNA- contain hydroxymethy cytosine –instead of –cytosine
• DNA encloses capsid

5. HEAD TAIL CONNECTOR
• Also known as neck
• Region b/w head &neck
• Contain coller or disc & fiber like whiskers
TAIL
• Narrrow cylindrical
• 80 nm long & 18 nm wide
• Central helical & hollow core –viral DNA pass to host during infection
• Core surrounded by –contractile sheath-144 protein sub unit arranged
in24 ring of 6 in each ring
BASAL PLATE
• also known as end plate

6. • Hexagonal shape
• Tail pin or spike in each corner
TAIL FIBERS
• 6 in number in 6 corners
• Attachment of host cell wall during infection
• 130 nm long & 2 nm wide
• 2 portion – proximal half (attachment-base plate)
- terminal half ( attachment-host receptor)

9. LIFE CYCLE
1) LYTIC CYCLE
• Also known as virulent
• Infected cell release large no : of progeny viruses
• 5 phases-adsorption , penetration , synthesis , maturation ,assembly &
release
 ADSORPTION
• Phage-contact-bacterial cell –attach specific receptor site
• With help of tail fibers
• Certain cofactor (cation) necessary for adsorption
• Phage attack one bacterial sps do not attack other sps

10.  PENETRATION
• Viral nucleic acid-host cell
• Whole virus cannot penetrate – nucleic acid enter – leaving capsid
• Hole on bacterial cell –leakage of cell content
• Bacterial lysis occur without phage multiplication - LYSIS
 SYNTHESIS OF PHAGE COMPONENT
• After release of nucleic acid – bacterial cell –synthesis initiated
• Viral DNA directs – biosynthetic machinery in host cell – to shut down
normal cellular metabolisum – produce new virus particles.
• Early protein – contain viral DNA polymerase
• Late protein – contain sub unit of phage head & tail

11. • Nucleic acid replication carried by – viral DNA polymerase – copies of viral nucleic
acid
 MATURATION & ASSEMBLY
• Assembly of phage DNA
• Occur in nucleus or cytoplasam
• DNA wraped by protein coat
• Finally tail str added forming a virion (infected virus particle)
 RELEASE
• Release by lysis of infected bacterium
• Phage enzymes – weaken – cell wall – during replication of phage
• Enzyme conc: increase –last stage of growth

13. ONE STEP GROWTH OF PHAGE
• Infection period – maturation of progeny ( no symptom) – eclipsed phase
• Infection period – release of progeny –latent period
• Increased no : of progeny - rise period
• The average no: of phage per infected plant - burst size

17. 2) LYSOGENIC CYCLE
• Occur in alpha phage – attack – E.coli
• Phage – follow –lysogenic cycle – temperate phage
• Host bacterium in which cycle complete – lysogens or lysogenics strains
• Normal bacterium – transform - infected – lysogenization
• Close association b/w viral genome & bacterial genome – lysogeny
• Association b/w phage & bacterium – lysogenic state
• Viral DNA after –adsoption , penetration enters host cell
• viral genome integrated with bacterial genome. Viral genome –prophage
• Hybrid genome daughter genome

18. • Association break & viral genome-released –bacterial cytoplasam –
induction
• Viral genome enter lytic cycle form mature temperate phage – release –by
lysis of bacterial cell

23. MECHANISM OF REPLICATION
• Viral multiplication begin with adsorption & end with release of new
viruses
 Adsorption of virus on host cell
• Virus tail tip – attach –specific receptor site
• Interaction b/w tip & cytoplasmic membrane
• Adsorption can be reversible or ireversible
• Reversible – tip of tail fibers– attach –cell surface
• Ireversible – spikes or tail pin attach

24.  Penetration of nucleic acid
• 3 stages
1. Tail fiber of virus attach cell & hold tail against cell wall
2. Sheath contract – tail core in to the cell through cell wall & membrane
3. Virus inject DNA into host
• During penetration protein coat – outside the cell

25.  protein synthesis
• Phage utilize host protein machinery to synthesis protein
• Contain early gene & late gene
• 2 cls of phage messenger RNA - early messenger & late messenger
1. Early messenger – 2 – cls – immediate & delayed
• Immediate – break down host DNA
• Delayed – code for phage enzymes – produce – unique phage DNA
2. Late messenger – produce structural component of new phage
• Include lysosome – lysis bacterial cell – release mature virus

26.  Break down of bacterial chromosomes
• 3 step – unfolding , distruption , degradation
1. Unfolding – chromosome undergo unfolding & loss compactness – result
–relaxation of helical twist
2. Distruption –central nucleoid distrupted – DNA become associated with
the membrane at numerous point
3. Degradation – DNA cleaved by endonuclease to small fragments that are
further break down by another nuclease into acid soluble fragment

27.  Arrest of the host cell development
• Arrest of replication , transcription & translation
• Degradation of host DNA coded nucleases -primary cause of host DNA
arrest
 Replication of DNA
• T4 DNA polymerase cannot synthesis DNA de novo
• Begin with synthesis of RNA primer
• T4 DNA polymer extends DNA strand on primer
• On one end they have 3’ exonuclease – similar to E.coli DNA polymerase
• Lack 5’ exonuclease enzymes
• 3’ exonuclease – read the function & correct the errors
• T4 DNA binding protein – gene 32 – requried for replication &

28. • Gene 32 have more affinity towards ssDNA than dsDNA - maintain
ssDNA in that state its self
• DNA polymerase come into contact ssDNA & procced with replication
• DNA synthesis by short segment , okasaki fragments – they are
bidirectional , discontinous & 5’ 3’ direction - jointed by enzyme ligase
• T4 DNA replication – a rolling circle mechanism – through that a continues
DNA strand is obtained
• Last okasaki fragment are not joined because they have single stranded
terminations – covalently joined by ligase.This give rise to multiple
genome length molecule called concatemers

31.  MATURATION
• Genome wraped by protein coat to for mature phage
• Bacterial cell lysis & 200 mature phage per cell are liberated
• Maturation occur in cytoplasam
 RELEASE
• Take place by lysis of cell
• Lysis brought by phage lysozyme – distrupted inner membrane & attack cell
wall
• Lysozyme remain attach on tip of the tail later it help to drill hole on the cell
wall of the bacterium for injection

33. HELICAL ( CYLINDRICAL) VIRUSES
 The helical viruses are elongated, rod shaped, rigid or flexible.
 There capsid is a hollow cylinder with a helical structure.
 Capsid consists of monomers arranged helically in rotational axis
.
 capsid may be naked e.g. TMV or envelope
 TMV is the most serious pathogen causing mosaic on tobacco leaves
 The virus remains active in extracted host plant juice even up to 25 years
 It is a very resistant virus

34.  The tobacco mosaic virus affects all dicotyledonous plants of which most important
are tobacco and tomato
 does not affect any monocotyledonous plants.
 Adolph Mayer in 1886 first pointed out the mosaic pattern on leaves of affected
tobacco plants – he also say that it can transmitted to healthy plant by mechanical
means - mosaikkrankhet
 Earlier than this, in 1892 Iwanowski demonstrated that tobacco mosaic virus. He was
able to demonstrate that a diseased tobacco plant juice was able to induce mosaic
disease in healthy tobacco plants.
 1898 the first scientific proof of the existence of a virus was given by Beijerinck.
 In 1935 Stanley first isolated crystals of tobacco mosaic virus and indicated their Para
crystalline nature.
 The tobacco mosaic virus affects photosynthetic tissue of the host leading to
distortion, blistering and necrosis. It also causes dwarfing of affected plants.
 It reduce yield & quality

35.  STRUCTURE
• Franklin et al (1957) have described the structure of TMV
• belong to cls helical viruses
• Rod shaped
• 95% protein & 5% RNA
• 300nm long & 15-18 nm diameter
• Protein sub unit around central hole - The protein coat is technically called
‘capsid ‘
• Tmv made of capsid – capsid made of protein –each protein made up of 158
aminoacid
• Each protein contain furrow in which RNA embedded
• Tmv protein lack disulphid bond - contain only one SH group

36. • Tmv on degradation in cold dil. Alkaline at PH 10 gives smaller protein
fractions called – A protein
• Natural protein present in infected plant other than A protein & tmv
particle – X protein

37. • Different kind of inclusion :
 Crystalline inclusion
1. Hexagonal inclusion
 Microscopic
 Plate like bodies with hexagonal shape
 Unstable
 Seen in epidermal living cell
2. Spindle like or needle like inclusion
 Spindle or needle in shape
 Seen in cytoplasam or nuclei of tmv infected cell
 Tmv rodes arranged in a para crystalline manner .becoz of that they are
spindle or needle in shape

38. 3. Amorphous inclusion
 X bodies - The X-bodies are amorphous, protoplasmic more or less
vacuolate inclusions. They are induced by common tmv strain injected to
cell
 Angled layer inclusion – they are arranged in an angle like str
 REPLICATION
1. UNCOATING
• In uncoating protein coat should be removed
• After entry (30min) into cell – protein coat is removed – becoz they can
exist as an organisum in living cell
• This naked viral RNA known as – elipse stage

39. 2. DEGRADATION
• It is removal of protein sub units
• Removal by invitro & invivo
• In invitro – protein sub unit – treat with alkaline
• In invivo – removal of protein sub unit from 3’ end
3. MULTIPLICATION
• Start when uncoated viral RNA reach nucleous
• RNA content increase lead to increase in size of nucleous
• New RNA produced is viral RNA & released to cytoplasam
• Assembely of TMV particle occur in cytoplasam or nucleus
• Elipse stage last fro 6 hrs & 1st progeny appear in cytoplasam

40.  Replication of viral RNA
• Viral RNA cannot categorized becoz ,they contain all genetic information about virus
• They serves as messenger
• They eliminates transcription step in protein synthesis
• Viral RNA multiplies its own & they independent on host cell DNA
• They are self duplicating polycistronic messenger
• polycistronic messenger -a messenger which contain codon for 2 or more proteins
• Viral RNA have 2 functions
1) Serves as messenger , immediately bind to ribosomes
2) Parental viral RNA is then replaced from ribosomes & start replication of viral RNA
during that time they behave as an template
3) Synthesis of viral proteins – they are influenced by transcription & translation

41. 4) Self assembly of virus – once protein sub units & viral RNA is synthesized, the 2
must aggregate to form virus – protein sub unit form capsid when this takes place
around a nucleic acid molecule a complete virus particle is self assembled
• Self assembly has 2 step – formation of initial complex &growth of helical rods

42. Viral entry & uncoating
Virus initial entry (wound)
Uncoating
Transcription
Translation of Mrna
Replication of genome
in cytoplasm in nucleus
chloroplast
Assembly
Release & (transfer
through plasmodesmata)

44. LIFE CYCLE

45. PROPERTIES OF VIRUS (TMV)
• Highly resistance – can survive 25 years
• Resistance to acids , alkaline and salt
• Resistance to temperature
• On direct sunlight no effect on viruse
• 4 % formaldehyde sol destroy tmv completely
• Dried tobacco leaf can survive 31 years

46.  TRANSMISSION
• From one plant to another by contact
• Soil : vires lie in the soil with plant debris after the harvest of the crop &
infect the new crop , when sown in the same field
• Smokers : spread by the fingers of smokers , unbrust piece of cigarette,
chewers sputum
• Store house : new stock place in same house where infected leave were
stored
• Seed : not common
mosaic virus in legume & cucurbits transmitted by seed
• Air & water : virus can transmitted but bacteria & fungus cannot
• Insects : by aphids , jassids & white flies
• Grafting : when infected plant tissue graft with healthy plant – through cell
sol it can transfer

47. • Contact :
• Tools :
 SYMPTOM
• Young leaf –vein clearing – molting –leaves enlarge – black green spot
• After infection, it developes symptoms of lightning of leaf colour along the
veins in early stages.
• There after it turns into light and dark green mosaic symptoms
• Lack chlorophyll – green clr disappeared & become yellowish spot –
CHLOROSIS
• Yellow spot spread on green background look like mosaic patter hence
know as – MOSAIC DISEASE

48. • RINGSPOT - formation of concentric rings or
broken rings of infected dead cells. The ringspot may be chlorotic rings rather than
necrotic rings.
• LEAF ABNORMALITIES - Due to virus infection leaves show abnormal growth
like leaf curling, leaf rolling, crinkling, etc. the other abnormalities may also develop in
leaves such as smaller blistered and thickened
Leaves
• FLOWER SYMPTOMS - The colour breaking means streaks or sector of tissue
with such colour that are different from the normal one. This happens due to loss or
increase of anthocyanin pigment in petals
• HISTOLOGICAL ABNORMALITIES -In leaves showing mosaic symptoms.
Mesophylls cells are smaller and less differentiated
• CYTOLOGICALABNOMALITIES - Chloroplasts may be rounded swollen and
clumped together in the cells . Chloroplast may be fragmented and colour may turn
to colour less.

49. Systemic
ringspotting caused
by TMV in
Nicotiana clevelandii
ringspots- spreading
and fusing
with one another

51.  CONTROL MEASURES
• Avoid infected soil
• Seed bed should sterlized
• Avoid contamination in hands – use soap
• Previous year’s plant debris should be destroyed by burning.
• Care should be taken to avoid contamination through hands and cultivation
implements
• Since pipe tobacco, cigarettes and chewing tobacco are all sources of
primary inoculum, smoking or chewing of any kind of tobacco should be
avoided
• Diseased plants should be removed and burnt to stop further spread of the
disease
• Growing resistant varieties produces good results

52. • Care should be taken to avoid contamination through hands and cultivation
implements