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 Grapevine fanleaf Tomato bushy stunt Tobacco ringspot Abutilon mosaic Tulip breaking

Virus classification into Families, Genera and Species based on :  Particle morphology  Genome properties  Biological properties  Serological properties

Plant Viruses Classification Based on the Nature of the Genome DNA RNA ssDNAdsDNA ssRNAdsRNA -ve sense +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

 The mode of transmission is a useful characteristic of some groups of plant viruses. Aphid Whitefly Hopper Thrip Nematode Mite Plasmodiophorids  Biological properties:

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

CPMV (Wt) CPMV-HRV Chimera Chimera

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

First Report : 1945 Widespread Disease. Severe loss in yield. Mosaic symptoms on diseased cardamom leaf.

1 2 kDa bp 1 2 a b c d kb 8.5 kb kb SDS PAGE RNA gel PCR Northern blot Indian cardamom mosaic virus

Virus Genes 23 (1):81-88, August © 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 , India R. Usha Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai , India; Author for all correspondence: 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

1 2 5 Distribution of Cardamom mosaic virus in South India Sirsi 2.Coorg 3.Palghat 4.Valparai 5.Idukki

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 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

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 

Infectivity of BYVMV and DNA  in bhendi plants.

DNA A DNA  Structural Relationship between DNA A and DNA  Half the size of DNA A Functional relationship and evolutionary origin?

GC-Rich TAATATTAC SATELLITE DNA Stem-Loop

C1 ORF FEATURES OF DNA  A rich region 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 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

BYVMV  CLCuRVCLCuRV Comparison of DNA 

Multiple Sequence Alignment of DNA  TAATATT

Multiple Sequence Alignment of DNA  A-Rich Region

ORF Prediction

Multiple Sequence Alignment of c1 protein

Phylogenetic tree of DNA  (c1 protein)

DNA  DNA A

The earliest recorded plant virus disease Eupatorium yellow vein disease Poem by Empress Koken 752 A.D. 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

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

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.

(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 A B (A) The positions of surface exposed loops in the monomer. Conserved residue positions are shown in blue and variable residues are in pink colour.

Yellow mosaic disease of soybean

Healthy Soybean Naturally infected A DNA alone 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 legume- infecting 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.