S8 11

Slide1: 

Molecular Cloning of ICMV and Its Comparison with Other Whitefly Transmitted Geminiviruses T.Makeshkumar, V.G.Malathi1, G.Radhakrishnan1 and S.Edison Central Tuber Crops Research Institute Sreekariyam, Trivandrum, Kerala, India 1 IARI, New Delhi, India

Slide2: 

RF : 1750 mm Temp : Min Max 8 30 Soil: Sandy, Clay loam RF : 1450 mm Temp: Min Max 8 38 Soil :Sandy loam Silty clay, Alluvial RF : 1050 mm Temp: min max 10 40 Soil : Sandy loam RF : 947 mm Temp: min max 17 37 Soil : Red loam RF : 3000 mm Temp: min max 19 34 Soil : Laterite RF : 3560 mm Temp: min max 12 37 Soil : Laterite RF :1790 mm Temp: min max 15 38 Soil: Hilly, Red Soil Potato Belt (Temperate) Cassava Sweet potato Aroids Yams Tuber Crops Growing Areas in India

Slide3: 

Cassava Growing areas in India No. of states cassava grown – 13 Tamil Nadu Kerala Andhra Pradesh Area (ha) 85322 111922 25000 Production (t) 3266410 2531752 275000 Productivity (t/ha) 39 23 11 1999 - 2000

Types of CMV reported till today: 

Types of CMV reported till today

Importance of CMD in India: 

Importance of CMD in India Occur in more severe form in Kerala and Tamil Nadu Emerging as a problem in other states It causes yield loss ranging from 25 – 80% No cultivar is resistant to this disease Factors: Indiscriminate use of infected planting material Non adoption of rogueing / clean cultivation practices

Indian Cassava Mosaic Disease: 

Indian Cassava Mosaic Disease Field view

Slide7: 

Indian Cassava Mosaic Disease Mosaic Leaf Distortion

Cassava Mosaic Disease: 

Cassava Mosaic Disease Severe Symptom ICMV

Slide9: 

Crinkling / Distortion

Indian Cassava Mosaic Virus: 

Indian Cassava Mosaic Virus It is bipartite geminate particles having ss DNA as their genome It differ serologically and Nucleotide sequence level from ACMV & EACMV Nucleotide sequence of this virus reported by Hong et al (1993): DNA –A 2815 bp; DNA – B 2645 bp

Glimpse of Important work done in India: 

Glimpse of Important work done in India Survey and Surveillance of ICMD Symptomatology, Pathogen identification, Pathogenicity, Transmission studies, Disease indexing & Yield loss estimation Epidemiological studies, Degeneration of planting materials Various management strategies Production of virus free plants through meristem tip culture

The present study…..: 

The present study….. Cloning of ICMV genome Sequencing of AC1 gene and its comparison with other geminiviruses

Methodology: 

Methodology Isolation & Purification of Replicative forms of viral DNA Cloning of viral genome in pUC18 vector Identification of DNA A& B clones Sequencing of AC1 gene Comparison of AC1 sequences with other cassava mosaic viruses and other whitefly transmitted geminiviruses

Slide14: 

Fraction Numbers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Gel electrophoretic analysis of Replicative forms of viral DNA

Slide15: 

Fraction Numbers 1 2 3 4 5 6 7 8 9 10 Southern analysis of Replicative forms of viral DNA

Cloning Strategy…….: 

Cloning Strategy……. Linearising RF viral DNA with 3 different enzymes - Bam HI (DNA -B), Hind III & Kpn I ( DNA - A/B) Restricted Viral DNA ligated with pUC 18 vector DNA restricted with similar enzyme Transformation into competent cells of E. coli DH5 by CaCl2 method Rapid screening and identification of positive clones

Slide17: 

2815/1 Eco R I (105) Hind III (203) Eco RV (530) Kpn I (1613) Pst I (1649) Bgl II (1778) Kpn I (1788) Eco RI (1877) Eco RI (2052) Cla I (2459) DNA – A (2815 bp) Restriction map of ICMV DNA - A

Slide18: 

2645/1 Hind III (293) Bgl II (403) Kpn I (1901) DNA – B (2645 bp) Eco RI (2530) Bam HI (1510) Restriction map of ICMV DNA - B

Cloning result….: 

Cloning result…. - Out of 82 colonies screened, 17 of them positive

Slide20: 

M 1 2 3 4 5 1 2 3 4 5 M Bam H I Hind III Gel electrophoretic analysis of positive clones restricted with Bam HI + Bgl I or Hind III + Bgl I 2.8 kb

Slide21: 

M 1 2 3 4 5 6 7 M Kpn I Gel electrophoretic analysis of positive clones restricted with Kpn I + Bgl I 2.8 kb

Slide22: 

Southern analysis of positive clones restricted with Bam HI + Bgl I or Hind III + Bgl I M 1 2 3 4 5 1 2 3 4 5 M Bam H I Hind III

Slide23: 

M 1 2 3 4 5 6 7 M Kpn I Gel electrophoretic analysis of positive clones restricted with Kpn I + Bgl I

Slide24: 

Clone B3 Clone B4 Bam H1 Eco R I Hind III Kpn I Pst I Sal I Xba I Bam H1 Eco R I Hind III Kpn I Restriction analysis of selected clones with seven restriction enzymes 5.6kb 2.8 kb

Slide25: 

Clone B4 Clone H2 Pst I Sal I Xba I Pst I Sal I Xba I Bam H1 Eco R I Hind III Kpn I Restriction analysis of selected clones with seven restriction enzymes 5.6kb 2.8 kb 1.0 kb 4.3 kb

Slide26: 

Clone K2 Bam H1 Sal I Hind III Kpn I Pst I Eco R I Xba I 5.6kb 2.8kb Restriction analysis of selected clones with seven restriction enzymes

Primers used for sequencing….: 

Primers used for sequencing…. Primer 1 (N– terminal of AC1) 5’AAGCTTATGTCACCACCTAAGCGCTTTCAAATAAC 3’ Primer 2 (C-terminal of AC1) 5’GGATCCTTAGCAGCTCTGTGTTGGACCTTGATTGG 3’ M13 Forward and Reverse Primer

Slide28: 

ttagcagctctgtgttggaccttgattggtacctgagtatagtggtccttcgagggagatgaaggtcgcatttttcagagcccaggcttttaatgcgctattcttttcctcgtccaggaattctttatagctggaattggggcctggattgcagaggaagatagtgggaatacctcctttaatttgaactggcttaccgtacttggtgtttgattgccagtcctctgggcccccatgaattcttttgaagtgctttaggtagtggggatcgacgtcatcaatgacgttgtaccatgcatcattgttgtagaccttaggacttaaatccaggtgtccacataggtaattatgtggacccaatgcacgggaccacatggtcttgcccgtccgactatcgccttcgattacgattgattttggtctcaaaggccgcgcacgacccatcacgttttcgtggaaccactcgtcgagttcttctggaactcggtcaaacgaagagagagggaaagggttctcatatggaggtggtggttttgtaaaaatccgatccaggtttgaactgatgtgatggaagtccctaagataatccctaggtgctaattctctaaggatcttaagagcctctgatttactgccgctgttaagtgccgcggcgtaagcgtcgtttgcagattgttgaccccctctggctgatcgtccatcgatctgaaaagttccccatctccaagtatcaccgtccttgtcgatgtaggacttgacgtcggagctggatttagctccctgaatttttggatggaaatgtgctgaccttgttggtgataccaggtcgaagaatcgctgattctggcatttgtatttgccttcgaactggatgagcacgtgcagatggggctccccattctcatgtagttccctgcatattttgataaatttagggtttgtaggtgtttggaagttcctaatctgagagagagtctcttctttcgttaaggagcatcgtgggtaagtgaggaaatagtttttagcgtttatttgaaagcgcttaggtggtgacat Nucleotide sequence of AC1 gene

Slide29: 

Amino acid sequence of AC1 gene MSPPKRFQINAKNYFLTYPRCSLTKEEALSQIRNFQTPTNPKFIKICRELHENGEPHLHVLIQFEGKYKCQNQRFFDLVSPTRSAHFHPNIQGAKSSSDVKSYIDKDGDTWRWGTFQIDGRSARGGQQSANDAYAAALNSGSKSEALKILRELAPRDYLRDFHHISSNLDRIFTKPPPPYENPFPLSSFDRVPEELDEWFHENVMGRARPLRPKSIVIEGDSRTGKTMWSRALGPHNYLCGHLDLSPKVYNNDAWYNVIDDVDPHYLKHFKRIHGGPEDWQSNTKYGKPVQIKGGIPTIFLCNPGPNSSYKEFLDEEKNSALKAWALKNATFISLEGPLYSGTNQGPTQSC

Slide30: 

TomLCV - Bangalore CLCuV - India CLCuV - Pak OEV ArEV TLCV BYVMV - Madurai ICMV-H ICMV - K SqLCV PLCV AYVV AbMV BGMV SGMV BDMV ACMV - Nigeria ACMV - Uganda TYLCV WACMV - Cameroon EACMV - Malawi Phylogenetic analysis of ICMV –K with other cassava viruses and whitefly transmitted geminiviruses

Slide31: 

Sequence similarities of the Common regions of ICMV – K with other Cassava mosaic viruses Viruses % similarity ICMV – Hong 99 ACMV – Nigeria 52 ACMV – Uganda 53 ACMV – Cameeroon 63 ACMV – Kenya 61 ACMV – Malawii 57 EACMV – Malawii 64

Slide32: 

TAS ELISA of CMD infected plants with panel of Monoclonal antibodies of ACMV, EACMV & ICMV Varieties EACMV ACMV ICMV 17 23 33 58 60 H 97 0 0 0 2 2 H165 0 0 0 3 3 H226 0 0 0 4 4 Sree Vishakham 0 0 0 3 3 Sree Prakash 0 0 0 4 4 Sree Jaya 0 0 0 3 3 Malayan – 4 0 0 0 2 2 MD plants of H226 0 0 0 0 0 A405 nm after O/N incubation at 4oC with substrate was scored as follows: 4 (>1.5), 3 (1.1-1.5), 2(0.6-1.0), 1(0.2-0.5) and 0 (<0.2)

Slide33: 

TAS ELISA of different types of CMD symptoms in H226 tested against MAb of ICMV A405 nm after O/N incubation at 4oC with substrate was scored as follows: 4 (>1.5), 3 (1.1-1.5), 2(0.6-1.0), 1(0.2-0.5) and 0 (<0.2) Symptom Score SCR 58 SCR 60 Grade 0 (App. Healthy) 1 1 Grade 1 (mild mosaic) 2 2 Grade 2 (25% mosaic) 3 3 Grade 3 (50% mosaic) 4 4 Grade 4 (75%mosaic & 4 4 distortion ) Grade 5 (Total deformation) 4 4 MD plants 0 0

Slide34: 

Nucleic acid based detection of ICMD 1 2 3 4 5 1 6 11 5 10 15 Detection of ICMV in ICMD infected plants of different cultivars by NASH using ICMV DNA – A probe Cultivars used : H-226 (1), H 165 (2), Sree Jaya (3), Sree Vishakham (4), H-97 (6), M-4 (7), Sree Prakash (9), Sree Sahya (12), Symptomless Plts of H226 (8), H165 (11) & H97 (13), MD plants of H226 (5 & 10) and MNJ (15)

Summary: 

Summary Replicative forms of ICMV DNA was isolated & Purified ICMV genome was cloned into pUC18 vector DNA - A clones was identified from Hind III site clones & DNA - B clones from Bam H I clones DNA - A&B clones were further confirmed by Southern analysis and by restriction with several enzymes AC1 gene was sequenced which had 1055bp which encoded for 351 a.a Contd...

Slide36: 

Comparison of AC1 gene(ICMV –K) with published sequence of ICMV-H revealed changes in 11 bases and no change in a. a sequence Phylogenetic analysis of ACI of ICMV – K with other cassava viruses showed marked differences Reaction of panels of MAb with CMD infected plants confirmed the presence of ICMV only Increase in symptom score gave the increase in the reaction value of MAb reflected the virus concentration Detection of CMD infected plants by NASH showed varying intensity of signals depending upon the susceptibility of cultivar to ICMV

Future thrusts….: 

Future thrusts…. Development of transgenic cassava resistant to ICMD Screening of ICMV populations across the country for its variability Development of fast, quick reliable diagnostic methods