Transcript Document

Applications of Bioinformatics
in Plant Virus Research
R.Usha
Department of Plant Biotechnology
School of Biotechnology
Madurai Kamaraj University
Pondicherry University, 17/2/2006
Lettuce Mosaic
Tobacco ringspot
Grapevine fanleaf
Abutilon mosaic
Tomato bushy stunt
Tulip breaking
Virus classification into Families, Genera and
Species based on :
 Particle morphology
 Genome properties
 Biological properties
 Serological properties
Classification Based on the Nature of the Genome
Plant Viruses
RNA
DNA
ssDNA
dsDNA
ssRNA
-ve sense
dsRNA
+ve sense
Particle morphology
Isometric
Rod shaped
Filamentous
Geminate
Bacilliform
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.
 Translation strategy
Genus Potyvirus:
Genus Furovirus
 Biological properties:
 The mode of transmission is a useful characteristic of some groups of
plant viruses.
Aphid Whitefly
Hopper
Thrip
Nematode
Mite
Plasmodiophorids
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.

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
Virus Crystals
Antiviral drug binding to HRV14
Chimera
CPMV (Wt)
CPMV-HRV Chimera
Virus
Family
Cardamom
mosaic virus
Potyviridae
Geminiviridae
Soybean isolate of
Mungbean yellow
mosaic virus.
Horsegram yellow
mosaic virus
Bhendi yellow vein
mosaic virus
Symptom
Particle
Mosaic symptoms on
diseased cardamom leaf.
First Report : 1945
Widespread Disease.
Severe loss in yield.
Indian cardamom mosaic virus
1
2
1
kDa
2
kb
68.0
2
kb
9.49
7.46
97.4
1
bp
9.40
8.5
8.5
4.40
2036
1636
1.8
38
2.37
29.0
7.46
4072
3054
4.40
45.0
kb
2.37
1018
506
1.35
1.35
14.3
a
b
SDS PAGE
RNA gel
c
PCR
d
Northern blot
Virus Genes
23 (1):81-88, August 2001.
© Kluwer Academic Publishers
3-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. Usha
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India;
Author for all correspondence: E-mail: [email protected]
Abstract
Cardamom 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.
Keywords
cardamom mosaic virus, macluravirus, potyvirus
ISSN 0920-8569
Distribution of Cardamom mosaic virus in South India
1
2
1.
Sirsi
2.
Coorg
3.
Palghat
4.
Valparai
5.
Idukki
3
4
5
Symptoms produced by different strains of CdMV
High genetic diversity in the coat protein and 3untranslated
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
Approaches for the development of transgenic virusresistant 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
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.
PHYLOGENETIC TREE BASED ON DNA A
SEQUENCES
GENOME ORGANIZATION AND COMPARISON OF
DNA A AND DNA b
Infectivity of BYVMV and DNA b in bhendi plants.
Structural Relationship between DNA A and DNAb
Half the size
of DNA A
DNA A
DNAb
Functional
relationship
and
evolutionary
origin?
SATELLITE DNA
TAATATTAC
GC-Rich
Stem-Loop
FEATURES OF DNA b
C1 ORF
0
500
1000
A rich region
1350
GC rich
conserved
region
DNA A SEQUENCE OF BYVMV
2551
ATTTTTGGAA TTGATGACAA AACGCCTTGG AGGCATGTTG ACTATTTTTG
2601
AGACCCGATT GACCGCTCTT ACAACTCTCC CCAGTATATC GGGTCCCTAT
2651
ATATAGTGAG ACCCAAATGG CATAATTGTA ATAAAACAAC TTTAATTTGA
GCCATCCG
2701
AATTCAAACG AAAAGGCTAA AGCGGCCATC CGTATAATAT T
||||||||
ACCGGATGGC CGCGCGATTT TTTAAGTGGT GGGTCCAGAA CGCACGACG
CGGTAGGC
1
51
TGCAGACTCA AAGCTTAGAT AACGCTCCTT CGGCTATAAG TACGTGCGCA
101
CTAAGTTTCA ATTCAAAAAA TGTGGGATCC ACTATTAAAC GAATTTCCGG
151
ATACGGTTCA CGGGTTTCGT TGTATGCTAT CTCTAAAATA TTTGCAACTT
DNA A STEM LOOP
TATAA
T
CATTA
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
DNA BETA STEM LOOP
1
ACCGTGGGCG AGCGGAGTCT GAGTCGTTGT GGAACCCTCT TATGAATGAA
GCTCGTCCACG TTCTA
51
GTTTATGGGT GATTTCTAGT ATATGGAGGA AATTGTGGAT GAGAAAAGGA
|||||||||||
101
ATCAAGTTTT GGTTTTGCAA ATTATTTTAG ATAACAGTCT CCTAATAATA
CGAGCGGGTGC CATTAT
151
ATTAATATGC AAACATATTA CTAACAAAAT TAAATTATTA TCTTATTATC
A
Comparison of DNA b
C
L
C
u
R
V
b
BYVMVb
Multiple Sequence Alignment of DNA b
TAATATT
Multiple Sequence Alignment of DNA b
A-Rich Region
ORF Prediction
Multiple Sequence Alignment of c1 protein
Phylogenetic tree of DNA b (c1 protein)
DNA b
DNA A
The earliest recorded plant virus disease
Poem by
Empress Koken
752 A.D.
Eupatorium yellow vein disease
Saunders et al., (2003). Nature 422, 831.
Distribution of diseases associated with
monopartite begomoviruses and DNA Beta.
TRENDS in Plant Science Vol.8 No.3 March
2003
Fivefold related
subunits of
BYVMV
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 of BYVMV showing the different interactions in the fivefoldrelated symmetry related sub units.
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)
The positions of surface exposed loops both in the
monomer and pentamer
A
(A) The positions of surface exposed loops in
the monomer. Conserved residue positions are
shown in blue and variable residues are in pink
colour.
B
(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.
Yellow mosaic disease of soybean
Healthy Soybean
A DNA alone
Naturally infected
B DNA alone
A DNA +B DNA
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.
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.
Schematic representation of the recombinant regions in legumeinfecting begomoviruses from South and South-East Asia.
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.