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Transgenic Strategies for
Developing Crops Resistant to
Geminiviruses
Student
D. Raghu (II Ph.D., Biotechnology)
08-807-002
Chairman
Dr. D. Sudhakar
Professor, DPMB&B,CPMB
Virus
“virus is an obligate intracellular parasites that
cannot reproduce independently”
• Latin – “toxin or poison”
• Nucleocapsid
• Enveloped viruses – possess an envelop around the
protein coat
• Virus core – additional protein layer between capsid and
the nucleoid
• Replicate inside the cells of another organism
• Electron microscope
Plant Pathogenic Virus
RNA virus
DNA virus
dsRNA
ssRNA (-)
dsDNA
ssRNA (+)
ssDNA
ssRNA (RT)
Plant pathogenic viruses - 450 species
Plant virus and shape
crops
Why to study Geminivirus?
(Vanderschuren et al., 2007)
Geminivirus disease complex
Geminivirus
Whitefly
Plant
Geminivirus Taxonomy
• Group II (ssDNA)
Group
• Geminiviridae
Family
Genera
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Mastrevirus
Curtovirus
Begomovirus
topocuvirus
Host and Vector
Genus
Type member
Host range
Vector
Genome
Mastrevirus
Maize streak virus (MSV)
Monocot and
a few dicots
Leafhoppers
Monopartite
Curtovirus
Beet curly top virus (BCTV)
Dicots
Leafhoppers
Monopartite
Begomovirus
Bean golden mosaic virus
(BGMV)
Dicots
Whiteflies
Mono and
bipartite
Topocuvirus
Tomato pseudo-curly top
virus (TPCTV)
Dicots
Treehopper
Monopartite
General characteristics
• Genome comprised of one or two
circular ss-DNA molecules
• each of which is ∼2.5–3.0 kb: Total
genome size 2.5–5.0 kb
• The smallest known genome for an
independently replicating virus
• Bidirectional transcription and
overlapping genes for efficient
coding of proteins
• Distinguishing feature is their
twinned icosahedral virions
The Latin “geminus” meaning twin
Genome organization of Geminiviridae
The Geminivirus DNA replication cycle
RCR-Rolling Circle Replication
mechanism of virus
Interference of geminivirus in the host
Genus I. Mastrevirus
Maize streak virus
• Monopartite genome
• Transmitted by leafhopper vectors to monocotyledonous plants
• H-Maize, Sugarcane, wheat, Bajra, Chickpea, Millets, Bean
leafhopper
Maize streak virus
• One of the oldest known plant viral
diseases
• Economically it is the most damaging
disease in maize in sub-Saharan Africa
resulting in up to 100% yield loss
• Endemic in Africa where wild grasses
are its natural hosts
Cicadulina mbila, the leafhopper vector of Maize streak virus
Genus II. Curtovirus
Beet curly top virus
• Monopartite genome
• Transmitted by leafhoppers to dicotyledonous plants
• Ambisense nature
• Host: pepper, melons, beans, tomato, spinach and ornamentals
Beet curly top virus
• Symptoms - vein clearing, curling,
general malformations and become
leathery and brittle
• Stunted, turn yellow, and the phloem
shows necrosis, early infection usually
results in early death (Brunt et
al.,1996)
Beet
• In the late 1990s BCTV emerged as a
serious problem of chilli cultivation in
southern New Mexico and destroyed
nearly 80% of the crop
chilli
Genus III.Topocuvirus
Tomato pseudo-curly top virus
• Monopartite genome
• Transmitted by tree hoppers to dicotyledonous plants
Tomato pseudo-curly top virus
• Virus is transmitted in a semipersistent manner,retained when the
vector moults
• Symptoms - vein-clearing, leaf
curling and cupping and shoot
proliferation
• Stunted and set few fruit
• Host - Ambrosia sp., Solanum
nigrum
Genus IV. Begomovirus
Bean golden yellow mosaic virus
Bipartite
• Transmitted by whiteflies
• Dicotyledonous plants
• Bipartite genomes (A and B
components)
• With some exceptions (e.g.,
Tomato yellow leaf curl virus,
Cotton leaf curl virus, Tomato
leaf curl virus…) for which no B
components have been found
A
whiteflies
B
Transgenic strategies
1. Pathogen-derived resistance through the expression of
viral proteins
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•
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Replication associated protein
Coat protein-mediated protection
Movement protein
2. Pathogen-derived resistance without protein expression
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•
Gene silencing
Antisense RNA
3. Resistance due to the expression of non-pathogen
derived antiviral agents
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•
•
•
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Virus-induced cell death
DNA binding protein
GroEL-mediated protection
Peptide aptamers
InPAct
Pathogen-derived resistance through the
expression of viral proteins
Replication associated protein (Reps)
• Viral gene transcrioption regulation
• Initiation & termination of viral replication
• Regulation of host gene expression
Eg. Interaction of geminiviral Rep with host pRBR induce viral
DNA synthesis
• Driving cells into “S” phase
• Activating the expression of “S” phase specific factors
Pathogen-derived resistance through the
expression of viral proteins
Coat protein-mediated resistance
• Systemic infection by monopartite geminiviruses (Rojas et al., 2001)
• Tomato plants expressing CP of the monopartite begomovirus (TYLCV)
exhibited delayed symptom Development
• CP of bipartite geminiviruses is not absolutely necessary , as NSP can
substitute (Pooma et al., 1996)
• CP-mediated strategy against bipartite geminiviruses will not produce
a high level of resistance
Pathogen-derived resistance through the
expression of viral proteins
Movement protein (MP) - mediated resistance
• Cell-to-cell and long distance systemic spread
• Used to engineer resistance to various begomoviruses
• Transgenic plants expressing the defective movement protein were
resistant to both ToMoV & CaLCuV
(Shepherd et al., 2009)
Pathogen-derived resistance without protein
expression
Gene silencing - mediated resistance
Pathogen-derived resistance without protein
expression
Antisense RNA - mediated resistance
Resistance due to the expression of nonpathogen - derived antiviral agents
Virus - induced cell death
• Death of infected cells and their neighbours induced by host innate
defensive hypersensitive (Shepherd et al., 2009)
• Transgenic plant shows resistance to geminivirus by combined action of
the barnase & barstar proteins of B. amyloliquefaciens
• Barnase – viral “V” sense promoter (expressed during virus infection)
• Barstar – viral “C” sense promoter (repressed during virus infection)
• Absence of geminivirus infection, barnase & barstar equally expressed
• Presence of infection
• Barnase is over expressed
• Cell die before infecting virus can replicate & move
Resistance due to the expression of nonpathogen - derived antiviral agents
DNA binding proteins
• Zinc finger proteins are high affinity for the “Rep–specific direct repeats “
in the “virion-ori “ of different geminiviruses
• Block the binding of “Rep” to “virion- ori” of geminivirus
• Transgenically expressed artificially designed Zinc finger protein provide
resistant against geminiviruses
Resistance due to the expression of nonpathogen - derived antiviral agents
GroEL – mediated resistance
• Chaparon
• Homologue of GroEL produced by endosymbiotic bacteria from B. tabaci
• Higher affinity to TYLCV coat protein
• Vector – virus interaction protect the virus from distruction during its
passage through insect haemolymph
Eg. B. tabaci GroEL gene expressed in transgenic tomatoes under phloem
specific promoter, protected the plants from the TYLCV infection (Rudolph et al., 2003
Resistance due to the expression of nonpathogen - derived antiviral agents
Peptide aptamers
• Short recombinant protein, ~ 20 amino acid length
• Strongly binds with target protein and destructs the function
• Transgenic N. benthamiana - nucleoprotein of the Tomato spotted wilt
Virus
(Lopez et al., 2006)
Tansgenic virus resistance strategies (Table)
Resistance due to the expression of nonpathogen - derived antiviral agents
InPAct system
Conclusion