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Sonia Phillips PhD Supervisors: H van Heerden IA Dubery Molecular Characterization of Elicitor-responsive Genes in Cotton.

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Presentation on theme: "Sonia Phillips PhD Supervisors: H van Heerden IA Dubery Molecular Characterization of Elicitor-responsive Genes in Cotton."— Presentation transcript:

1 Sonia Phillips PhD Supervisors: H van Heerden IA Dubery Molecular Characterization of Elicitor-responsive Genes in Cotton

2 Background Cotton is a multi-billion rand industry (> $ 500 billion) and Gossypium hirsutum accounts for > 95% cotton fibre Verticillium wilt = vascular plant disease caused by Verticillium dahliae, a destructive soil-borne, multi- host fungus (> 400 plant species afflicted) Cotton production severely hampered by V. dahliae: in SA yield losses are around 30 % and can be as high as 70 % Symptoms: Wilting Discolouration Stunted growth Defoliation Smaller fruits Poor fiber quality Ultimately - death Soil fumigation and fungicides are costly and have a negative ecological and health impact – alternative eco-friendly control measures are required: need to investigate the genetic components of plant immunity to V. dahliae DDRT-PCR with V. dahliae elicitor in cotton (Michelle Zwiegellar, 2003, MSc dissertation) ID several ESTs differentially induced upon treatment of cell suspensions with V. dahliae elicitor

3 Objectives Obtain, analyze and characterize the full genomic sequences (+ promoter) of 3 genes represented by ESTs ID in DDRT-PCR study by genome walking, RACE, PCR, RT-PCR, bioinformatics analysis and Southern blots:  C4B5 (kinase?)  C4B4 (arm repeat?)  C1B10 (?) Elicitation studies: Isolate elicitor from V. dahliae cell wall Investigate if CWD elicitor induces PR protein production Viability studies to determine appropriate elicitor concentrations for expression analysis RT-qPCR to determine the time frame and kinetics of induction with CWD elicitor Molecular characterization and genomic assignment of GhLIPN homoeologs (GhLIPN N and I) GhLRK = Lectin receptor-like kinase (RLK) gene GhARM = Armadillo-repeat gene GhLIPN = Lipin gene

4 Polyploidy in cotton AADD tetraploid 2n = 52 1 – 2 mya G. darwinii G. mustelinum G. tomentosum AA diploid 2n = 26 represented by G. herbaceum and G. arboreum DD diploid 2n = 26 represented by G. raimondii Common ancestor 5 – 10 mya Hybridization and Endoreduplication New allotetraploid species B Endoreduplication New autotetraploid species A AutoployploidyAlloployploidy

5 Genome Walking Genomic DNA digested with four different restriction enzymes to obtain a genomic library from each Genomic DNA isolated from the young leaves of Gossipium hirsutum cv. ‘OR-19’ Adapters ligated to digested fragments. Primary PCR with first gene-specific primers and adaptor primer 1 SP 1 AP 1 Secondary or ‘nested’ PCR with the second gene- specific primers and adaptor primer 2 SP 2 AP 2 Major bands cut from the gel, cloned, sequenced and characterized

6 5` RACE PCR product ready for agarose gel analysis and cloning SP 1 (A) n 3`5` Synthesis of first strand cDNA with gene-specific primer SP 1 and reverse transcriptase mRNA (A) n 3` 3` cDNA Degradation of the mRNA template by the RNase H activity of reverse transcriptase 3` (A) n AAA Tailing of the purified cDNA with dATP by terminal transferase Purify cDNA Amplification of the tailed cDNA by primary PCR using the oligo (dT)- anchor primer and a nested gene-specific primer SP2 (A) n AAAA (T) n TTTTV SP 2 Oligo dT-anchor primer *V = A, C or G SP 3 Inner PCR anchor primer Secondary nested PCR with the PCR anchor primer and a SP 3 primer

7 3` RACE Amplification of the cDNA by PCR using gene-specific primers SP 4 and the outer PCR anchor primer SP 4 Outer PCR anchor primer *V = A, C or G cDNA synthesis using the oligo (dT)- anchor primer by reverse transcriptase 5`mRNA AAAAAAA VTTTT 3` Degradation of the mRNA by the RNase H activity of reverse transcriptase cDNA TTTT PCR product ready for agarose gel analysis and cloning SP 5 Inner PCR anchor primer Nested secondary PCR, with the inner PCR anchor primer and gene-specific primer SP5

8 Viability Study V. dahliae elicitor (  g/ml) % Viability

9 2171 – 2501 bp C4B5 EST A StuI DraI EcoRI PvuII M B StuI DraI EcoRI PvuII M Upstream from EST Primary PCRSecondary/nested PCR 2240 bp StuI DraI EcoRI PvuII M A B StuI DraI EcoRI PvuII M Downstream from EST Primary PCRSecondary/nested PCR 657 bp

10 1080 bp 11 22 2 11 255 bp

11 279 bp 132 bp 2306 – 2583 bp 2306 – 2436 bp 2171 – 2501 bp C4B5 EST 11 22

12 1: SacI 2: XbaI 3: HindIII 4: EcoRI 2277 bp 2007 bp 30 bp 240 bp 309 bp 2171 – 2501 bp Full genomic sequence obtained from genome walking: 3011 bp

13 Elicitor/pathogen-responsive Myb/Myc TF-binding Dof TF-binding Hormone-responsive

14 Lectin domain Ser/Thr Protein Kinase domain TM SP

15 Predicted carbohydrate binding site Additional loop N-ter N28 N52 C-ter Activation segment C-ter N-ter N577 Catalytic loop Phosphate anchor Lectin domainProtein kinase domain Homology to legume lectin from bark of Robinia pseudoacacia: 30% ID, E-score = 9.8e -25, EP = 100% Homology to PK domain from AvrPto: 33% ID; E- score = 1e -35, EP = 100%

16 * * * * * * p-value > 0.05

17 C4B4 EST StuI DraI EcoRI PvuII M B StuI DraI EcoRI PvuII M A Secondary/nested PCR Primary PCR Downstream from EST 1078 bp B StuI DraI EcoRI PvuII M A StuI DraI EcoRI PvuII M Secondary/nested PCR Primary PCR Upstream from EST 2883 bp

18 22 22 11 11 832 bp bp 11

19 11 22 22 11 22 11 bp 402 bp 231 bp 1762 bp C4B4 EST

20 Retrotransposon (777 bp) 1: SacI 2: XbaI 3: EcoRI 4: HindIII 2759 – 3218 bp 1780 bp 1713 bp 67 bp 986 bp Full genomic sequence obtained from genome walking: 4131 bp

21 Elicitor/pathogen-responsive

22 Hormone-responsive

23 Dof TF-binding

24 Myb/Myc TF-binding

25 SP ARM Armadillo-type fold ABC

26 Homology to human β-catenin protein from ARM repeat family 14% ID; E-score = 3.6e -25, estimated precision = 100%

27 ** * * * * p-value > 0.05

28 C1B10 EST B a StuI DraI EcoRI PvuII M SnaBI SspI PdiI Eco47III M SacI XbaI HindIII EcoRV M b StuI SacI XbaI HindIII EcoRV M SspI PdiI M DraI EcoRI PvuII M Eco47III SnaB Secondary/nested PCRPrimary PCR 1462 bp B StuI DraI EcoRI PvuII M Secondary/nested PCR A StuI DraI EcoRI PvuII M Primary PCR Downstream from EST 1649 bp Upstream from EST StuI DraI EcoRI PvuII M StuI DraI EcoRI PvuII M a A b Primary PCRSecondary/nested PCR 676 bp

29 22 11 – 3685 bp 11 22 – 4073 bp 22 11 – 5243 bp 11 22 – 6542 bp

30 11 22 C1B10 EST bp 379 bp

31 : XbaI 2: EcoRI 3: HindIII Genomic sequence GhLIPN N: bp GhLIPN I: bp Transcribed sequence GhLIPN N: bp GhLIPN I: bp ORF Both: bp Promoter GhLIPN N: bp GhLIPN I: bp Several indels: mostly SNPs; 13 and 17bp

32

33 Sequence divergence: 2.5% (ungapped); between introns: 2.97%, exons: 2.27%

34 * Identical aa : Conserved aa (16). Semi-conserved aa (5) 40 mismatches (4.5%)

35 Therefore GhLIPN I likely derives from the ancestral D-genome and GhLIPN N from the ancestral A- genome

36 Elicitor/pathogen-responsive Dof TFBS Myb/Myc TFBS Hormone-responsive CAAT-box

37 N-terminal lipin C-LIP HAD-like domain BNLS

38 Homology to phosphatase domain of a polynucleotide kinase (HAD superfamily) 18% ID; E-score = 3.7e -07, estimated precision = 100% Haloacid dehalogenase (HAD) domain

39 * p-value > 0.05 * * * *

40 Hypothetical Gene Interaction Model


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