applications of bioinformatics in plant virus research r.usha department of plant biotechnology...
TRANSCRIPT
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Applications of Bioinformatics in Plant Virus Research
R.UshaDepartment of Plant
BiotechnologySchool of Biotechnology
Madurai Kamaraj University
Pondicherry University, 17/2/2006
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Lettuce Mosaic Grapevine fanleaf Tomato bushy stunt
Tobacco ringspot Abutilon mosaic Tulip breaking
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Virus classification into Families, Genera and Species based on :
Particle morphology
Genome properties
Biological properties
Serological properties
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Plant Viruses
Classification Based on the Nature of the Genome
DNA RNA
ssDNA dsDNAssRNA dsRNA
-ve sense +ve sense
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Particle morphology
Isometric
Rod shaped
Filamentous
Geminate
Bacilliform
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Genome properties
Nature of the genome: circular or linear.
Number of genome components: 1 component to 11
Number of genes: Common minimum : 3
Replication, Movement & Encapsidation.
Genome relatedness: at nucleic acid or protein level.
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Translation strategy
Genus Potyvirus:
Genus Furovirus
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The mode of transmission is a useful characteristic of some groups of plant viruses.
Aphid Whitefly Hopper Thrip
Nematode Mite
Plasmodiophorids
Biological properties:
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Genome sequences of viruses
There are now nearly 980 species of plant viruses belonging to 70 genera
According to a recent report a total of 8884 sequences of plant viruses, viroids and satellites have been deposited in the databases till August 2005.
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Structural studies on plant viruses
A number of icosahedral (spherical) plant viruses have been studied by X-ray crystallography
3-D structures have opened up avenues for engineering the plant viruses for the expression of epitopes from animal and human pathogens, towards the development of plant-based vaccines
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Virus Crystals
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Antiviral drug binding to HRV14
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CPMV (Wt) CPMV-HRV Chimera
Chimera
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Virus Family Symptom Particle
Cardamom
mosaic virus
Potyviridae
Geminiviridae
Soybean isolate of Mungbean yellow mosaic virus. Horsegram yellow mosaic virus
Bhendi yellow vein mosaic virus
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First Report : 1945
Widespread Disease.
Severe loss in yield.
Mosaic symptoms on diseased cardamom leaf.
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1 2
kDa
38
1.35
40723054
20361636
1018
506
bp
1 2
a b c d
9.497.46
4.40
1 2
kb
8.5 8.5
kb
9.40
7.46
4.40
2.37
1.35
2.37
97.4
68.0
45.0
29.0
14.3
1.8
kb
SDS PAGE RNA gel PCR Northern blot
Indian cardamom mosaic virus
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Virus Genes23 (1):81-88, August 2001.
© Kluwer Academic Publishers3-Terminal Sequence analysis of the RNA Genome of the Indian Isolate of
Cardamom Mosaic Virus: A New Member of Genus macluravirus of potyviridae
Thomas Jacob Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India
R. UshaDepartment of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India; Author for all correspondence: E-mail: [email protected]
AbstractCardamom mosaic virus, a possible member of the family Potyviridae has been associated with the mosaic disease (Katte disease) of small cardamom in India. A virus isolated from the symptomatic cardamom leaves was positive in ELISA only with antiserum to the Guatemalan isolate of cardamom mosaic virus and not with a number of other potyviruses. The size of the viral RNA (8.5 kb) and the molecular weight of the coat protein (CP) (38 kDa) were determined. A 1.8-kb product containing the partial nuclear inclusion body (NIb) gene, the entire coat protein gene and the 3 untranslated region (UTR) was amplified by reverse transcription (RT) and polymerase chain reaction (PCR), cloned and sequenced. The viral origin of the clone was confirmed by Northern hybridization with viral RNA. The experimentally determined N-terminal sequence of the CP matched with the deduced amino acid sequence of the CP gene. Sequence analysis of the clone suggests that the cardamom mosaic virus is a member of the Macluravirus genus of the family Potyviridae.
Keywordscardamom mosaic virus, macluravirus, potyvirusISSN 0920-8569
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1
2
5
Distribution of Cardamom mosaic virus in South India
3 4
1. Sirsi
2. Coorg
3. Palghat
4. Valparai
5. Idukki
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Symptoms produced by different strains of CdMV
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High genetic diversity in the coat protein and 3untranslated
regions among geographical isolates of Cardamom
mosaic virus from south India
T JACOB, T JEBASINGH, M N VENUGOPAL* and R USHA†
J. Biosci. | Vol. 28 | No. 5 | September 2003 | 589–595 | © Indian Academy of Sciences
Phylogenetic Tree of CdMV Strains
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Approaches for the development of transgenic virus-resistant cardamom by PDR:
Viral sequences used for the transgenesis:
Coat protein from Yeslur and Kursupara isolates (each with and without the 3’UTR).
Core coat protein
Hairpin RNA construct with the core CP coding region
NIb
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Bhendi Yellow Vein Mosaic Virus (BYVMV) Whitefly-transmitted Geminivirus
Family Geminiviridae genus Begomovirus
Reported first from India in 1924
Causes heavy yield loss in infected Abelmoschus esculentus.
Symptoms:Vein clearing, yellowing of leaves
Molecular characterization of Bhendi yellow vein mosaic virus.
Sequence analysis of BYVMV genomic components.
Demonstration of agroinfection to fulfill Koch's postulates.
Construction of binary vector clones for PDR
Tissue culture and transformation of bhendi
Analysis of transgenic lines for viral resistance.
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PHYLOGENETIC TREE BASED ON DNA A SEQUENCES
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GENOME ORGANIZATION AND COMPARISON OF
DNA A AND DNA
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Infectivity of BYVMV and DNA in bhendi plants.
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DNA A DNA
Structural Relationship between DNA A and DNA
Half the size of DNA A
Functional relationship and evolutionary origin?
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GC-Rich
TAATATTAC
SATELLITE DNA
Stem-Loop
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0500 1000
1350
C1 ORF FEATURES OF DNA
A rich region
GC rich conserved region
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DNA A SEQUENCE OF BYVMV
2551 ATTTTTGGAA TTGATGACAA AACGCCTTGG AGGCATGTTG ACTATTTTTG
2601 AGACCCGATT GACCGCTCTT ACAACTCTCC CCAGTATATC GGGTCCCTAT
2651 ATATAGTGAG ACCCAAATGG CATAATTGTA ATAAAACAAC TTTAATTTGA
2701 AATTCAAACG AAAAGGCTAA AGCGGCCATC CGTATAATAT T
1 ACCGGATGGC CGCGCGATTT TTTAAGTGGT GGGTCCAGAA CGCACGACG
51 TGCAGACTCA AAGCTTAGAT AACGCTCCTT CGGCTATAAG TACGTGCGCA
101 CTAAGTTTCA ATTCAAAAAA TGTGGGATCC ACTATTAAAC GAATTTCCGG
151 ATACGGTTCA CGGGTTTCGT TGTATGCTAT CTCTAAAATA TTTGCAACTT
DNA BETA SEQUENCE OF BYVMV
1151 ACTTTTAAGT TATATCGCGC GTCGTAGTGC GCTTAAAAAG TTATCTTCTC
1201 TCTCTTCAGT TCCGATAAAA ACCTAATTTC CCGATGATCG GAGTCGAATT
1251 TTCCGACACG CGCGGCGGTG TGTACCCCTG GGAGGGTAGA AACCTCTACG
1301 CTACGCAGCA GCCTTAGCTA CGCCGGAGCT TAGCTCGTCC ACGTTCTAAT
1351 ATT
1 ACCGTGGGCG AGCGGAGTCT GAGTCGTTGT GGAACCCTCT TATGAATGAA
51 GTTTATGGGT GATTTCTAGT ATATGGAGGA AATTGTGGAT GAGAAAAGGA
101 ATCAAGTTTT GGTTTTGCAA ATTATTTTAG ATAACAGTCT CCTAATAATA
151 ATTAATATGC AAACATATTA CTAACAAAAT TAAATTATTA TCTTATTATC
DNA A STEM LOOP
GCCATCCG TATAA
|||||||| T
CGGTAGGC CATTA
DNA BETA STEM LOOP
GCTCGTCCACG TTCTA
||||||||||| A
CGAGCGGGTGC CATTAT
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BYVMV
CLCuRV
Comparison of DNA
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Multiple Sequence Alignment of DNA TAATATT
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Multiple Sequence Alignment of DNA
A-Rich Region
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ORF Prediction
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Multiple Sequence Alignment of c1 protein
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Phylogenetic tree of DNA (c1 protein)
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DNA DNA A
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The earliest recorded plant virus disease
Eupatorium yellow vein disease
Poem by Empress Koken
752 A.D.
Saunders et al., (2003). Nature 422, 831.
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Distribution of diseases associated with monopartite begomoviruses and DNA Beta.
TRENDS in Plant Science Vol.8 No.3 March 2003
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Pentamer viewed from
(1)Top
(2) Bottom
(3) Side
Space-filling model colored based on
(A) chain
(B) secondary structure yellow is β sheet, red is α helix and blue is turn.
(C) Model showing strands
Fivefold related
subunits of BYVMV
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A:Salt bridges
B: Hydrophobic interactions
C: Amino acids involved in the whitefly transmission
D: Positions of the Conserved and variable amino acids of BYVMV
(Blue highly conserved, white less conserved, pink highly variable and the remaining residues are yellow in colour)
Fivefold of BYVMV showing the different interactions in the fivefold-related symmetry related sub units.
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(B) The positions of surface exposed loops in the pentamer. Subunits are colored according to the secondary structure; yellow is β sheet, red is α helix, white is random coil and blue is turn.
The positions of surface exposed loops both in the monomer and pentamer
AB
(A) The positions of surface exposed loops in the monomer. Conserved residue positions are shown in blue and variable residues are in pink colour.
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Yellow mosaic disease of soybean
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Healthy Soybean Naturally infected
A DNA alone B DNA alone A DNA +B DNA
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The genetic variability in plant virus populations is an important aspect of plant virology.
For example, two different individuals of the same plant virus isolate are often more divergent at the nucleotide level than are humans and chimpanzees.
Three major mechanisms, which drive the genetic variation in virus populations, are:
mutation
recombination
reassortment
Evolution and adaptation leads to emergence of highly pathogenic virus genotypes.
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The detection of recombination from DNA sequences is relevant to the understanding of evolutionary and molecular genetics.
RDP: (Martin & Rybicki, 2000; Martin, et al., 2005) utilizes a pair-wise scanning approach for the detection of recombination.
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Schematic representation of the recombinant regions in legume-infecting begomoviruses from South and South-East Asia.
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Lab members who have contributed to the studies on
CdMV:
Thomas Jacob, Archana Somanath, T.Jebasingh,C.Manohari, Dr.S.Backiyarani, Kasin Yadunandam.
BYVMV:
Joyce Jose, P.Pravin Kumar, P. Gopal, Dr. B. Sinilal, Phaneeswara Rao.
SYMV & HgYMV:
K. R. Girish, R.M.Packialakshmi, A.D.Barnabas.
Other viruses:
L.N.Kaza, Somdeb Mitra, Anuja Guria, Diwakar Kumar,
Neetu Srivastava, Kasin Yadunandam.
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