Emergence of Indian Tomato Yellow Leaf Curl Viral (TYLCV) Disease: Insights from Evolutionary Divergence and

Molecular Prospects of Coat Protein

S. Prasanth Kumar1, Yogesh T. Jasrai*1, Himanshu A. Pandya1 and Rakesh M. Rawal2

1Bioinformatics Laboratory, Department of Botany, USSC, Gujarat University, Ahmedabad- 380 009.2Division of Medicinal Chemistry and Pharmacogenomics, Department of Cancer Biology, The Gujarat Cancer & Research Institute (GCRI), Ahmedabad- 380 016.

S. PRASANTH KUMAR Bioinformatics Laboratory, Department of Botany, Applied Botany Centre (ABC) Gujarat University, Ahmedabad, INDIA

Category: ORIGINAL RESEARCH PRESENTATION

Tomato Leaf Curl Disease (TLCD)Tomato (Solanum lycopersicum) Solanaceous plant

Symptoms: Yellowing of leaf lamina with upward leaf curl, Leaf distortion, Shrinking of the leaf surface and Stunted plant growth

Vector: Whitefly (Bemisia tabaci, Bemisia argentifolii)

Causal agent: Tomato yellow leaf curl virus (TYLCV)Family: GeminiviridaeGenera: Begomovirus Isolates: Known tropical and sub- tropical including Indian subcontinent

Other hosts: Eggplant, Potato, Tobacco and Pepper

Whitefly (Vector)†

Tomato plant showing symptoms†

Coat protein of Geminivirus*

Courtesy: † U.S. Dept. of Agriculture, * Protein Databank (PDB)

TYLCV Indian Isolates & Disease Management SUPPRESS WHITEFLY TRANSMISSION

Imidacloprid-based systemic insecticides (e.g., Admire® Pro, Provado®)

PROBLEMSSignificantly affects the fruiting

Contaminates soil nutrients

TYLCV-RESISTANT TOMATO VARIETIES(BHN Seed, Golden Valley Seed, Hazera Seed,

Sakata Seed, Seminis Seed, Zeraim Gedera)

PROBLEMUnder stress, resistant intolerable to symptoms

Disease can spread to nearby growing Solanaceous plants

AVOID GROWING SOLANACEOUS PLANTS NEARBY

PROBLEM Economical ?

New DelhiLucknow

Varanasi

MirzapurVadodara

Bangalore

TYLCV Indian Isolates

Objectives of the Study

Examine the evolutionary background of Coat Protein from Indian strains.

Study the molecular properties of coat protein facilitating its biochemical function, virulence.

Learn the electronic features of coat protein essential for interaction with a spectrum of bio-molecules (ssDNA, dsDNA and virulence protein).

Characterize the hotspot specific amino acid mutations in making Indian isolates, a newly emerging factor for TYLCD.

Methodology at a Glance

TYLCV Coat protein

Sequence Level Analysis

NLS Signal Phylogenetic Analysis

DNA binding properties

Structure Level Analysis

DNA binding properties

Coat protein modeling

Plant DNA Modeling Docking Simulation

Electrostatic Interaction & Molecular Assemblage

Sequence Level Analysis

GenBank

CAA88227.1 | AAD51286.1 | AAK19178.1 | AAL26553.1 |AAL78666.1 | AAO25668.1 | AAM21566.1 | AAB08929.1 | AAA92817.1 | CAA76209.1

Sequence positions

Dis

orde

r p

roba

bili

ty

MSKRPADMLIFTPASKVRRRLNFDSPSVSRAAAPIVRVTKAKAWANRPMN-terminal of Coat protein (Karyophilic)

DISORDERED PROFILE

Disopred

Nuclear Localization Signal Importin α-dependent NLS

Score: 10.5

Exclude NLS for further

analysis

Phylogenetic Analysis

(Truncated for Clarity)

Multiple Sequence Alignment (MSA) of Coat Protein from Indian strains

Coat proteins are also conserved in

their regions

Structure modeling of Coat Protein No close homolog having PDB data

No close structural neighbor

Structure Validation Only 1 Outlier

Ramachandran Plot (99.5 % favored model)

Coat Protein Active Site Prediction

Active Site Prediction Server

Active Site Residues: KDMHTVNSPLAIRYFCGEQ Cavity point x= 48.934 y= 58.666 z= -4.295

Volume of the Cavity = 14411 Å3

Coat protein DNA binding propertiesSequence-based DNA binding properties prediction

Coat protein DNA binding propertiesStructure-based DNA binding properties prediction

+ 63 representative DNA binding proteins Pscore = 0.31 Parea = 2102.26

A protein with Pscore > 0.12 & Parea > 250 is considered as dsDNA binding protein

DNA Structure ModelingSequence: Plant DNA Conformation: Eukaryotic (B-DNA) DNA Modeling rule: Watson-Crick accounted Base-pair: 99 Base-pair parameters: Default Bending: Normal Temperature: physiological pH (7.4) Ion concentration: Mg2+, Na+ present

Cross view

Lateral view

DNA Structure Modeling

Plant DNA

TYLCV Coat protein

Binding energy: -264139 Kcal/mol HADDOCK score: 12.0 Kcal/molRMSD from the overall lowest energy structure: 4.3 ÅVan der Waals energy: 66.9 Kcal/molElectrostatic energy: -827.4 Kcal/molDesolvation energy: 125.7 Kcal/mol Buried Surface Area: 2099.6 Å3

Molecular Interface

Plant DNA

TYLCV Coat protein

Major grove of DNA

Loop conformation of protein

Electrostatic potential at Interface

Units are in

Plant DNA

TYLCV Coat protein

Neutral patches as interface

Electrostatic potential of Coat protein

Protein family –Worldwide Isolates

pfam00844: Gemini_coat Geminivirus coat protein/ nuclear export factor BR1 family

Evolutionary Divergence

P/Q Q/H

E/D

Mutation

Conserved residue

Amino acids crucial for Systemic Infection,Particle Formation, and Insect Transmission*

*Noris et al., 1998. J. Virol. 72(12): 10050–10057

Molecular Assemblage

Molecular Assemblage built using coordinates of coat protein

Coat proteinCoat protein

Ribbon Model Surface Model

Major Outcomes

TYLCV Indian isolates are conserved among themselves and diverged with respect to geographical locations.

Amino acid mutations in the hotspot of protein (essential for systemic infection, particle formation and insect transmission) provides signs of evolutionary divergence.

Coat protein’s N-terminal is karyophilic in nature as demonstrated by NLS Signal and the member of a nuclear export family BR1 (result of Pfam annotation). This karyophilic nature is further confirmed by Disordeness peak.

Coat protein beside virulence in nature, also known to interact NON-SPECIFICALLY with other viral proteins and double stranded DNA (both viral and plasmid DNA in vitro*).

*Liu et al., 1997. J. Gen. Virol 78, 1265–1270.

Major Outcomes

Phylogenetic analysis of coat protein revealed that Vadodara isolate is a close neighbor of Varanasi and Kello strains.

We further emphasize the role of coat protein virulence in the emergence of evolutionary compatibility, distinguished new Indian species in disease prevention and the need of studies to safeguard plant biodiversity and breeding for resistant varieties.

Coat protein’s neutral patches contributes in making interaction with DNA. We explored the positive patches to interact with negatively charged DNA, but it was ruled out primarily due to the geometrical constraints rather a charge-charge interaction. Sequence- and Structure- based approaches validated the DNA binding properties and are scattered throughout the protein.

Loop geometrical components of coat protein plays a vital role in interacting with DNA and helical content was found to contribute for molecular assemblage

Major Citations

Noris et al., 1998. Amino Acids in the Capsid Protein of Tomato Yellow Leaf Curl Virus That Are Crucial for Systemic Infection, Particle Formation, and Insect Transmission. J. Virol. 72(12): 10050–10057. Liu et al., 1997. Maize streak virus coat protein binds single- and double stranded DNA in vitro. J. Gen. Virol. 78, 1265–1270. Henryk Czosnek. Tomato Yellow Leaf Curl Virus Disease Management, Molecular Biology, Breeding for Resistance. Springer Verslag, ISBN 978-1-4020-4768-8 (Chap. 3 to 5). Zhang et al., 2001. Structure of the Maize Streak Virus Geminate Particle. Virol. 279, 471-477. Chakraborty et al., 2003. Tomato leaf curl Gujarat virus, a New Begomovirus Species Causing a Severe Leaf Curl Disease of Tomato in Varanasi, India. Virol. 93(12): 1485-1495.

Image Courtesy: U.S. Dept. of Agriculture- Tomato disease photographs Protein Databank (PDB)-Structure coordinates

Thank you for your kind attention