In-silico identification and characterization of R-genes ...

3 downloads 0 Views 5MB Size Report
Vinay Kumar Singh,. Enrollment No. : 342113. Supervisor : Dr. Brahma Deo Singh,. Professor Emeritus. School of Biotechnology, Faculty of Science. Banaras ...
Supervisor : Dr. Brahma Deo Singh, Professor Emeritus School of Biotechnology, Faculty of Science Banaras Hindu University, Varanasi

In-silico identification and characterization of R-genes in Cajanus cajan

Ph.D. Student: Vinay Kumar Singh, Enrollment No. : 342113

1.

Introduction

 Plants are attacked by a wide spectrum of pathogens, being the targets of viruses, bacteria, fungi, protozoa, nematodes and insects.  Over the course of their evolution, plants have developed numerous defense mechanisms including chemical and physical barriers that are constitutive elements of plant cell responses locally and/or systemically.  Resistance genes (R-genes) allowed the examination of their localization in plant cells and the role they play in signal transduction during the plant resistance response to biotic and abiotic stress factors.  R-genes are genes in plant genomes that convey plant disease resistance against pathogens by producing R-proteins.

1.

Introduction

 Once the R-protein has detected the presence of a pathogen, the plant can mount a defense against the pathogen. Because R-genes confer resistance against specific pathogens, it is possible to transfer an R-gene from one plant to another and make a plant resistant to a particular pathogen.  R-protein activation often trigger a hypersensitive response to prevent damage due to spontaneous inappropriate activation.

•Van Loon LC. Systemic induced resistance. In: Slusarenko AJ, Fraser RSS, Van Loon LC, editors.Mechanisms of Resistance to Plant Diseases. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2000. pp. 521–574. •Lukasik, E, and Takken, FL, STANDing strong, resistance proteins instigators of plant defence, Curr Opin Plant Biol., 12, 427-436, 2009.

Functional domains arrangement of the identified plant resistance (R) genes

Major 8 classes of plant resistance (R) genes based on the arrangement of the functional domains. LRR Leucine rich repeats; NBS - Nucleotide-binding site; TIR Toll/Interleukin-1- receptors; C-C - Coiled coil; TrD - Transmembrane domain; PEST - Protein degradation domain (proline-glycine-serine-threonine); ECS Endocytosis cell signaling domain; NLS - Nuclear localization signal; WRKY - Amino acid domain; HM1 Helminthosporium carbonum toxin reductase enzyme.

Reported genes for each class of plant resistance (R)

Bacterial pathogens Fungal pathogens Viral pathogens Oomycetes pathogens Nematodes Insects

Pigeon pea (Cajanus cajan) The pigeon pea (Cajanus cajan; 2n = 22), also known as tropical green pea, a perennial member of the family Fabaceae. Pigeon peas contain high levels of protein and the important amino acids methionine, lysine, and tryptophan. Pigeon peas are an important legume crop of rainfed agriculture in the semiarid tropics. The Indian subcontinent, eastern Africa and Central America, in that order, are the world's three main pigeon pea producing regions.

PIGEON PEA DRAFT GENOME Taxonomy ID: 3821

Accession: PRJNA48381 ID: 48381 Accession: PRJNA48383 ID: 48383

3.

Materials and methods

In this work many tools and techniques were used sequence comparisons (sequence alignment) in order to detect similarities and differences among them to understand its biology. In silico biology tools and techniques includes a wide range of applications, which are listed below. 1. Sequence comparison to infer their relatedness (homology) between model and target system. 2. Identification of gene structures, distributions of introns and exons and the regulatory elements, intrinsic features of the different sequences, such as active sites, post-translational modification sites and disordered regions. 3. Unraveling the evolutionary process and assessment of genetic diversity of the sequences and the organisms. 4. Designing of Primers and confirmation of in silico deduced candidate gene using molecular biology technique. 5. Homology modeling and Protein-Protein interaction to understand the functional role of identified candidate genes. 6. Pathogen protein and fungicide interaction to investigate the binding affinity for selection of better fungicides.

In silico biology tools and databases

Comparative analysis for Gene identification & characterization

Functional Structural Sequential

Identification & Characterization of Gene Family Members from Cajanus cajan

Construction of 3D Structure of domain from Cajanus cajan

Correlation of Sequential and Structural Classification for Functional Elucidation of Candidate Gene/Protein

Selection of Candidate Gene

Gene/CDS

Candidate Gene

Cis-acting Elements

Protein

Functional Motifs

Gene Behavior

Structure – Sequence Relationships

2.

Objectives

The objectives of the present study are summarized as follows: 1. In-silico identification of candidate genes from R-gene family in C. cajan using comparative genomics approach. 2. Comparative phylogenetic analysis of R-gene family members of C. cajan with A. thaliana (thale cress) and G. max (soybean) homologous. 3. In-silico characterization, expression analysis during developmental stages and mining of cis-regulatory elements located in the upstream regions. 4. Confirmation of in-silico detected candidate gene in C. cajan genome employing molecular biological techniques, design of primers and PCR amplification. 5. Homology modeling of candidate R-proteins (linking sequence-structurefunction relationship) to deduce functional role in plant defense mechanism and metabolism. 6. Protein-protein functional association network, protein-fungicide interaction study using computational investigation.

4.

Results

Based on available genome sequence of C. cajan (AFSP01000000), near about 400 genes were identify with TIR/NBS/ LRR domain (http://www.insilicogenomics.in/r/cc.html), of which 103 TIR domain associated genes were obtained. After complete gene organization only 21 genes were observed that had all three domains (TIR, NBS and LRR domain) in their genic region. Further, these 21 TNL genes were subjected to in silico characterization and classification on the basis of maximum similarity algorithm. Based on phylogenetic classification of the 21 TNL genes were divided into two subgroups of 14 and 7 genes and the larger group was further divided into two sub-sub groups of 8 and 6 genes. Motif identification of these genes also confirms that all genes contain 3 domains with multilevel consensus sequence and have TIR (IPR000157), NBS (IPR002182) and LRR (IPR001611, IPR011713) domains, respectively. Complete gene organization revealed that the maximum gene size was 5,016bp (CDS), which encoded a protein of 1671aa.

Phylogenetic classification of the identified103 TIR proteins from C. cajan

In-silico analysis of TIR proteins from Cajanus cajan 103 TIR domain containing genes Interproscan revealed that 60 of these genes had TIR and NBS domains, 21 genes had TIR, NBS and LRR domains, while 22 genes had only TIR domain 100 84

TNL1 AFSP01009574.1 2925 6890 TNL2 AFSP01009575.1 478 4737 1

99

TNL3 AFSP01037926.1 1442 5088 TNL4 AFSP01003638.1 19 4806 1

67

TNL5 AFSP01018800.1 406 8068 1 97

100

I

TNL6 AFSP01018801.1 3298 8916 TNL7 AFSP01012046.1 2885 7387

53 100

Group-I

TNL8 AFSP01032097.1 6 5817 1 TNL9 AFSP01031006.1 3371 9966 TNL10 AFSP01015817.1 688 3804

65 100

TNL11 AFSP01033098.1 1428 5758 TNL12 AFSP01027600.1 402 4059

II

TNL13 AFSP01008515.1 2685 8991

38 99

TNL14 AFSP01021591.1 5396 1029

94

TNL15 AFSP01052619.1 93 3773 1

100

TNL16 AFSP01045452.1 143 2232

III

TNL17 AFSP01027210.1 7240 1254

Group-II

TNL18 AFSP01011182.1 1410 6922

98

TNL19 AFSP01003411.1 5339 9781

100

TNL20 AFSP01018306.1 11554 180

77 100

IV

TNL21 AFSP01006468.1 1716 8658

Phylogenetic tree of proteins encoded by the 21 TNL genes (genes having TIR-NBS-LRR domains) from C. cajan.

Sequence Logo representation Domain1 -TIR

Domain2 - NBS

Domain3 - LRR

Domain details with their Sequence Logo representation

TNL gene members organization CcTNL19 CcTNL04 CcTNL21 CcTNL13

CcTNL01 CcTNL02 CcTNL18 CcTNL07 CcTNL10 CcTNL20 CcTNL05 CcTNL06 CcTNL14 CcTNL17 CcTNL12 CcTNL09

CcTNL08 CcTNL11 CcTNL03 CcTNL16

CcTNL15

Complete gene organization for the 21 TNL genes.

Computational mapping of the 14 of the 21 predicted C. cajan TNL genes

Conclusion  In silico study revealed ~400 genes availability in C. cajan draft genome with TIR/NBS/ LRR domain, of which 103 TIR domain associated genes were obtained (http://www.insilicogenomics.in/r/cc.html).  Total 21 genes were observed that had all three domains (TIR, NBS and LRR domain) in their genic region.  Sequence analysis revealed the presence of six highly conserved motifs Motif1 (WPVFYDVDPSHVRHQ), Motif 2 (VVFSPNYASSTWCLDELVKI), Motif 3 (WKYDVFLSFRGEDTR), Motif 4 (FTGHLYAALCRKGINTFMDD), Motif 5 (GDEIWPALMQAIEGS) and Motif 6 (KKDMEMVQKWRMALTEAANL),

which can play important role in host response to pathogen, plant immunity, self-association and signaling .

In-silico characterization, computational expression analysis during developmental stages and mining of cisregulatory elements located in the upstream regions.

Computational expression analysis during developmental stages

Clustering of analyzed expression data of identified CcTNL genes based on Glycine max expression atlas.

Mining of cis-regulatory elements

Clustering of Cis-acting elements present in 21 genes Based on available sequence, no upstream sequences were retrieved for CcTNL15, CcTNL12, CcTNL02 and CcTNL06

Selection of candidate gene, promoter region analysis and primer designing

Candidate gene selection • In Arabidopsis, the immune receptors RPS4 and RRS1 are required to activate defense to three different pathogens, namely, Colletotrichum higginsianum, Ralstonia solanacearum and Pseudomonas syringae (Narusaka et al. 2009). • It has been found that both the above selected Rproteins provide a dual resistance-gene system against fungal and bacterial pathogens. • Based on both AT5G45250 and AT5G45260 proteins similarity search it has been found that one gene CcTN1 (AFSP01036179.1: 5650-7568) showed the maximum similarity (query coverage: 84% and identity: 41%) Narusaka, M, Shirasu, K, Noutoshi, Y, Kubo, Y, Shiraishi, T, Iwabuchi, M, Narusaka, Y, RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens, Plant J., 60, 218-226, 2009.

 Based on identified gene full length gene prediction was done and retrieval of 1000bp upstream region was also performed for investigation of cis-acting elements analysis.  The total available genic region 4676bp (Introns + Exons) was retrieved for amplified gene sequence, and the predicted mRNA was found 2712bp with 903aa.  From start codon total 323bp sequences upstream sequences were retrieved for cis-acting elements analysis.  After signal scan two major elements WBOXATNPR1 and WRKY71OS were identified, which are mainly associated with disease resistance and pathogenesis, this investigation also confirm that identified gene play very important role during pathogens attack.  Further full length protein was taken for similarity search against C. cajan EST database, similarity search revealed that one candidate EST (GR464368.1) was 99% identical to selected gene.  The accession id GR464368 belongs to ICPL20102_CW45_A12 Fusarium wilt challenged (10 DAI :days after inoculation) pigeonpea genotype ICPL20102 root cDNA library C. cajan cDNA clone.

Primer sequence for TIR domain amplification of the candidate gene (AGCT01041475.1_9515_9937). Primer

Sequence

Length

Tm (°C)

GC (%)

Product size (bp)

TIR1F

TGCATCAGAAACCAAAGCAG

20

59.99

45.0

TIR1R

TTCTTCATGTTGTGGCCTGA

20

60.24

45.0

606

M

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15

16 17 18

19

500 bp 250 bp

M

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

500 bp

250 bp

M = Thermo Scientific Gene Ruler 50 bp DNA Ladder; 1-38 are pigeonpea genotypes as: Bahar (1), IPA-204 (2), KPL-43 (3), BDN-2010 (4), BDN-2029 (5), IPA-8F (6), BDN-2001-9 (7), IPA-234 (8), ICP-9174 (9), BWR-23 (10), BSMR-846 (11), IPA-9F (12), IPA-16F (13), BDN-2004-1 (14), BWR-133 (15), MAL-31 (16), NDA-1 (17), ICP-9150 (18), Amar (19), MAL-13 (20), MA-6 (21), MAL-18 (22), ICP-2376 (23), LRG-41 (24), BSMR-301 (25), MAL-23 (26), MA-3 (27), MAL-34 (28), MA-23 (29), MA Deo-89 (30), MA PTH-2 (31), JKM-7 (32), ICP-11887 (33), ICP-7200 (34), ICP-8862 (35), KPBR-80-2-1 (36), C. Cajanifolius (37) and C. scarabaeoides (38).

M

S1

S2

S3

1000bp

500bp

Primer TIR 1 amplified a PCR product of approx. 606 bp in the pigeonpea genotypes BSMR-846 (S1; FW resistant) and MAL-13 (S3; FW moderately resistant); however it amplified a more intense band in the pigeonpea genotype, Bahar (S2; FW susceptible).

AFV93476.1 Cajanus cajan 100

AFV93475.1 Cajanus cajan

84

AFV93474.1 Cajanus cajan

99

XP 006593121.1 Glycine max

99

XP 007132920.1 Phaseolus vulgaris XP 003607698.1 Medicago truncatula PIR E71437 Arabidopsis thaliana NP 001060018.1 Oryza sativa

40

30

20

10

0

Phylogenetic classification of Cajanus cajan TIR domain with different organisms

Sequence alignment of C. cajan TIR domain

Alignment of Cajanus cajan TIR domain with different organisms Based on this in silico investigation it was found that the identified TIR motifs were involved in resistance to disease, plant innate immunity and apoptosis.

Functional domain analysis

Conclusion • Total 606bp band was successfully isolated in different C. cajan genotypes. • The selected candidate gene translational product concluded the presence of R-protein domain and TIR domain signature region. • The cis-acting elements prediction (WBOXATNPR1 and WRKY71OS) and EST detection (GR464368.1) through comparative analysis also concluded that the amplified candidate gene can be involved in disease resistance mechanism during fungal infection.

Homology modeling and structural classification of candidate R-proteins

Study of five candidate R-proteins C. cajan R gene

A. thaliana homolog

Gene R1 (CcTN1)

AT5G17680.1

Gene R2

AT5G36930.1

Gene R3

AT5G43470.1 (RPP8)

Gene R4

AT5G06860.1 (PGIP1)

Gene R5

AT5G46330.1

Description Disease resistance protein (TIR-NBS-LRR class), putative; FUNCTIONS IN: transmembrane receptor activity, ATP binding; INVOLVED IN: signal transduction, defense response, apoptosis, innate immune response; LOCATED IN: intrinsic to membrane; EXPRESSED IN: 14 plant structures; EXPRESSED DURING: 6 growth stages; CONTAINS InterPro DOMAIN/s: Leucine-rich repeat, typical subtype (InterPro:IPR003591), NB-ARC (InterPro:IPR002182), Leucine-rich repeat (InterPro:IPR001611), Disease resistance protein (InterPro:IPR000767), TollInterleukin receptor (InterPro:IPR000157) Disease resistance protein (TIR-NBS-LRR class) family; FUNCTIONS IN: transmembrane receptor activity, ATP binding; INVOLVED IN: signal transduction, defense response, apoptosis, innate immune response; LOCATED IN: intrinsic to membrane; EXPRESSED IN: 19 plant structures; EXPRESSED DURING: 13 growth stages; CONTAINS InterPro DOMAIN/s: NB-ARC (InterPro:IPR002182), TollInterleukin receptor (InterPro:IPR000157), Disease resistance protein (InterPro:IPR000767) Confers resistance to Peronospora parasitica. In arabidopsis ecotype Dijon-17, HRTmediated signaling is dependent on light for the induction of hypersensitive response and resistance to turnip crinkle virus Encodes a polygalacturonase inhibiting protein involved in defense response. PGIPs inhibit the function of cell wall pectin degrading enzymes such as those produced by fungal pathogens. PGIP1 is induced by fungal infection. Suppressed in the proton sensitive stop1-mutant, but the transcription level was recovered by transformation of STOP2. Knockout mutant showed severe damage in the root tip in low Ca and low pH medium. Encodes a leucine-rich repeat serine/threonine protein kinase that is expressed ubiquitously. FLS2 is involved in MAP kinase signalling relay involved in innate immunity. Essential in the perception of flagellin, a potent elicitor of the defense response. FLS2 is directed for degradation by the bacterial ubiquitin ligase AvrPtoB.

Selected domains from 5 candidate genes with templates details Domain

Primary Accession No.

Domain Start

Domain End

Template PDB ID

Organism

Identity

Positivity

TIR_1

AFSP01036179.1

5692

6123

3JRN.pdb

Arabidopsis Thaliana

46%

65%

TIR_2

AGCT01009452.1

1675

2480

3OZI.pdb

Linum usitatissimum

48%

66%

NBS

AFSP01050545.1

1430

2281

3J2T.pdb

Homo sapiens

30%

48%

LRR_1

AFSP01013557.1

2379

3317

1OGQ.pdb

76%

81%

LRR_2

AFSP01032181.1

1751

3322

4MNA.pdb

Phaseolus vulgaris Arabidopsis thaliana

55%

70%

Domain details with template PDB id, identity and positivity

Predicted models for selected domains

TIR2

LRR1

Ramachandran plot and secondary arrangement for TIR2 domain

Ramachandran plot and secondary arrangement for LRR 1 domain

RAMPAGE Statistics Domain

Percent of residues in the allowed region 3.5% 1.8% 6.7%

Percent of residues in the outlier region

TIR_1 TIR_2 NBS

Percent of residues in the favoured region 96.5% 98.2% 91.5%

LRR_1 LRR_2

96.5% 90.0%

3.5% 9.2%

0.0% 0.8%

0.0 0.0% 1.8%

Ramachandran plot statistics for modeled domains

Overall quality factor details of TIR2 and LRR 1 domains

Model and template comparative quality details with observed resolutions, overall quality factor and PMDB ID for selected domains

Messaoudi A, Belguith H, Ben Hamida J. Three-Dimensional Structure of Arabidopsis thaliana Lipase Predicted by Homology Modeling Method. Evol Bioinform Online. 2011;7:99-105. doi: 10.4137/EBO.S7122. Epub 2011 Jun 28. PubMed PMID: 21792274; PubMed Central PMCID: PMC3140413.

Conclusion • Structural classification revealed that the TIR domain having classification lineage (3.40.50.10140) had α-β class with AlphaBeta barrel architecture and TIM Barrel, and Rossmann fold type topology. • In case of the NBS, the classification lineage was (3.40.50.300), and it had P-loop containing nucleotide triphosphate hydrolases with Alpha-Beta 3-Layer (aba) sandwich architecture and Rossmann fold type topology. • In case of the LRR domain, the classification linage was (3.80.10.10), with Alpha-Beta Horseshoe architecture and Leucine-rich repeat, LRR (right-handed beta-alpha superhelix) topology. • Structural classification of predicted domains implicated that C. cajan identified R-domains have major role in self-association, signaling, and auto-regulation with respect to plant immunity and defense.

In-silico protein-protein interaction

Identified R-genes and their interacting partners with highest confidence S.N.

Gene Name

Interacted Partners

1.

Gene R1

2.

Gene R2

AT5G46530, CYTC-2, CYTC-1, AT1G53350, AT1G18340 and AT1G12290 AT5G45490, DAR5, CYTC-2, CYTC-1, RPP4, RPP13, AT1G65190, AT5G42825, HR4 and AT1G29790

3.

Gene R3

EDS1, AT1G31540, RIN4, PBS2, AT3G48080, CRT1, AT4G36290, COP1, CPC and SGT1B

4.

Gene R4

AT5G48140, AT5G44840, PGA1, AT3G14040, AT3G07820, PAD3, PGA4, AT2G41380, AT4G22530 and PGIP2

5.

Gene R5

BAK1, BIK1, KAPP, RIN4, AT5G57110, BTI1, BTI2, RPM1, AT5G41260 and Rps2

Conclusion • Based on protein-protein functional association network investigation it has been concluded that the identified 5 candidate genes can mainly involved in signal transduction, defense response, apoptosis, innate immune response, DNA repairing, regulation of transcription, N-terminal protein myristoylation (proteinprotein and protein-lipid interactions), protein amino acid phosphorylation, lipid metabolic process and carbohydrate metabolic process. • The identified genes can confer resistance to fungus, bacteria and virus and may involved in hypersensitive response to cadmium ion and plant immunity.

Pathogen protein-fungicide interaction study using computational investigation

• The pathogenic fungus Cercospora sp. is mainly involved in cercospora leaf spot (CLS) disease of plants.

The leaf spot disease, are small, brown, circular spots on leaves, which coalesce as they increase in size and lead to severe defoliation

• The 17β-Hydroxysteroid dehydrogenase (17βHSD) enzyme is a target for several commercially important fungicides that are used to control fungal diseases in plants.

• The availability of C. canescens BHU genome sequence with GenBank Accession: ANSM00000000.1 (~34Mb) gives us opportunities for bioinformatics analysis leading to identification and characterization of candidate genes for fungicide development and disease management.

The crystal structure pdb files of T4NR (PDB ID: 1JA9) complex with NADPH and pyroquilon from M. grisea and 17β-HSD (PDB ID: 3IS3) from fungus C. lunatus were used as templates for homology modeling.

Ho ology

odeli g of 7β-HSD protein in C. canescens

Three dimensional model of 17β-HSD protein (a) and RAMPAGE derived Ramachandran plot (b).

7β-HSD protein model verification using RAMPAGE & PDBSum

Ramachandran plot by PDBSum server (a) and Model quality factor by ERRAT server (b).

Quality assessment

ProSA results for Target (a) and Template model (b)

Active site residues identification

Sequence alignment for conserved region and active site residue identification using CLC sequence viewer

Cleft ide tifi atio of 7β-HSD protein model

Selected Fungicides

Compound: CID 91665 (Pyroquilon)

Compound: CID 39040 (Tricyclazole)

Docking of Pyroquilon with 17β-HSD protein

Do ki g of Tri y lazole ith 7β-HSD protein

Active site identification Compounds

Interacted Residues in 17β-HSD Protein

Pyroquilon (Control)

Arg26, Ile28, Val105, Thr198, Arg26, Ile28, Asn101, Ser102, Thr149, Thr200, Met202, Phe203, Val206 Gly103, Gly23, Gly29, Lys169, Gly27, Val104, Met202, Ser150, Tyr165, Thr200, Pro195, Val105, Leu124, Pro238, Asp201, Arg26, Ile28, Val105, Thr198, Thr198, Val199, Ser151, Val193, Phe203, Thr200, Met202, Phe203, Val206, Gly196, Ile241, Val206, Asp205, Asn152, Ala194, Gly197, Tyr210, His162, Phe107, Tyr210 Thr153, Arg234, Asp240, Arg243, Val263, Gly265, Gly236, Asp264, His209, Leu232, Asn235, Ser207, Gly266, Ser230, Ala226, Gln223, Ala267, Leu157, Val159, Arg222, Pro231, Ala229, Ala227, Ala268, Asp156, His228, Met225, Ile211, Pro160, Lys161, Gly108, Ala259, Ser158, Gln208, Glu221, Gly214, Glu215, Pro212, Asn213

Tricyclazole

Predicted Active Residues (Based on MetaPocket2.0 Server)

Reported Active Residues (Based on PDB IDs: 3IS3,1JA9)

Gly23, Gly25, Arg26, Gly27, Ile28, Ala48, Asn49, Ser50, Ala73, Asp74, Asn101, Ser102, Gly103, Val105, Leu124, Thr149, Ser150, Ser151, Asn152, Lys169, Tyr165, Pro195, Gly196, Gly197, Thr198, Thr200, Asp201, Met202, Phe203, Ser207, Tyr210, Ala267 and Ala268

Details of interacted residues and comparison with predicted and reported active residues

Docking calculation along with score, area and ACE Compound Name

Score

Area

ACE

Pyroquilon

2900

333.70

-126.91

Tricyclazole

3024

323.10

-194.88

In-vitro effect of Tricyclazole on production of cercosporin by C. canescens The Effect of Tricyclazole on production of Cercosporin 3 2.65 2.5

Mean

2

1.5

Fresh weight of fungal colonies (g)

1.39

Cercosporin quantity (µg/mm2 fungal colony) 1.08

1

1.00 0.87

0.61 0.5

0.44 0.32

0 1

2

3

4

The effect of Tricyclazole on production of cercosporin Cercosporin: A photoactivated toxin involved in plant disease Daub ME, Ehrenshaft M. THE PHOTOACTIVATED CERCOSPORA TOXIN CERCOSPORIN: Contributions to Plant Disease and Fundamental Biology. Annu Rev Phytopathol. 2000;38:461-490. PubMed PMID: 11701851.

Conclusion  17β-HSD protein can play a significant role in leaf spot disease caused by Cercospora sp. This study sought to identify suitable inhibitors from Pubchem compound database and homology modeling for the prediction of three dimensional structure and active site identification for 17β-HSD protein.  From a selection of potential fungicides Pyroquilon and Tricyclazole, we identified Tricyclazole as a suitable 17β-HSD inhibitor, with strong binding affinity towards 17β-HSD.  In-vitro experiment showed that Tricyclazole at 100 µg/ml reduced 10 times cercosporin production.  Based on in-silico and in-vitro experiments it has been found that Tricyclazole can be a better fungicide target for leaf spot disease in pigeonpea (C. cajan) caused by the fungal pathogen Cercospora species.

Abstract Published • Singh VK, Singh S, Kayastha AM, Singh NK and Singh BD (2012) In-silico identification, sequential and structural classification of TIR-NBS-LRR resistance gene family from Pigeonpea (Cajanus cajan) presented in IV International conference on Legume Genetics and Genomics (ICLGG), Hyderabad during Oct 2- 7, 2012. Page No. 95, P-EAD-28. • Singh VK, Kayastha AM, Singh NK and Singh BD(2013) Identification of TIR- and non-TIR-NBS-LRR disease resistance gene (R) analogues in Cajanus cajan: in silico characterization, genetic variation and functional divergence. International Conference on Biotechnology, New Delhi. Page No. 151.

Paper published 1. Singh V.K., Singh A.K., Singh N.K., Singh B.D. Toll and interleukin-1 receptor (TIR) domains in Cajanus cajan: an in-silico perspective. World Res J of Bioinform. 2014, 1(1):1-8. 2. Singh V.K., Chand R., Singh B.D., In silico 17 βHydroxysteroid dehydrogenase fungicide for leaf spot disease (Cercospora sp)., Onl J Bioinform. 2014, 15 (2): 198-209.

3. 2 Manuscripts in pipeline

Database Developed

Cajanus cajan R-genes details

Acknowledgement     

Supervisor Em. Prof. B.D. Singh Prof. S. Singh (Department of Botany) Coordinator, School of Biotechnology Prof. A.M. Kayastha Coordinator of DBT funded SUB-DIC, Centre for Bioinformatics Prof. S.M. Singh The eminent scientists of School of Biotechnology Prof. Ashok Kumar, Prof. A.K. Tripathi, Dr. Arvind Kumar  Prof. R. Chand (Department of Mycology and Plant pathology, IAS, BHU)  Dr. A.K. Singh (Department of Genetics and Plant Breeding, CARS, Korea, Chhattisgarh)  All non-teaching staff of School of Biotechnology and Centre for Bioinformatics I would like to extend thanks to the supreme power. The strength that supre e po er is really enormous which underlies that self-belief that I have through the vast ocean, called LIFE .

“Most of the iologi al i estigatio s i st e tury will be in silico” Walter Gilbert (Nobel Laureate)