Post on 30-Mar-2018
Plant Pathology 602Plant-Microbe Interactions
Sophien Kamounkamoun.1@osu.eduThe Ohio State UniversityOhio Agricultural Research & Development Center
Lecture 2Molecular methods for studying host-pathogen interactions I
n Refresher on gene expression
n Strategies for molecular study of plantpathogens and their hosts
Plant Pathology 602Plant-Microbe Interactions
Outline - lecture 2
n Refresher on gene expression
n Strategies for molecular study of plantpathogens and their hosts
Plant Pathology 602Plant-Microbe Interactions
Outline - lecture 2
Refresher on gene expression - DNA: The stuff of life
Refresher on gene expression
Gene
Refresher on gene expression
Gene
Transcript
Refresher on gene expression
Gene
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Refresher on gene expression
Gene
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DNA
Refresher on gene expression
Gene
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DNA
RNA
Refresher on gene expression
Gene
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DNA
RNA
Protein
Gene
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DNA
RNA
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Transcription
Translation
Refresher on gene expression Central dogma of molecular biology
Gene
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DNA
RNA
Protein
Transcription
Translation
Replication
Refresher on gene expression
Gene
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DNA
RNA
Protein
DNA cloningAmplification of DNADNA sequence
Refresher on gene expression
Gene
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DNA
RNA
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DNA cloningAmplification of DNADNA sequence
cDNAsynthesized in vitro
Refresher on gene expression
Gene
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DNA
RNA
Protein
DNA cloningAmplification of DNADNA sequence
DNAcloning
cDNAsynthesized in vitro
Refresher on gene expression
Gene
Transcript
Product
DNA
RNA
Protein
DNA cloningAmplification of DNADNA sequence
DNAcloning
Protein PurificationBiochemical analyses
cDNAsynthesized in vitro
Refresher on gene expression
Gene
Transcript
Product
DNA
RNA
Protein
DNA cloningAmplification of DNADNA sequence
DNAcloning
Protein PurificationBiochemical analyses
cDNAsynthesized in vitro
Refresher on gene expression
n Structurally simpler
n Cells do not contain nucleus
n Genomic DNA generally
naked and circular
n Genes are generally present
in single sets (haploidy)
n Structurally more complex
n Cells contain a true nucleus
n DNA contained in
chromosomes in nuclei
n Cell organelles
(mitochondria, chloroplasts
etc…)
n Genes are present in two or
more sets (diploidy)
prokaryotes eukaryotes
Refresher on gene expressionTechnical challenges in the molecular geneticstudy of eukaryotes
n Structurally more complex
n DNA transformation can be limiting (cell walletc…)
n Large genomes (lots of noncoding sequences,gene families...)
n Diploid organisms (more than one gene to disrupt)
n Refresher on gene expression
n Strategies for molecular study of plantpathogens and their hosts
Plant Pathology 602Plant-Microbe Interactions
Outline - lecture 2
Strategies for molecular study of plantpathogens and their hostsGenetic approach
n Many methods are centered around a(molecular) genetic approach
n Basis of the genetic approach is to start withtwo individuals/populations that differ inphenotype and identify the genes thatdetermine the difference
n Variation could be natural or artificiallygenerated by mutagenesis
Genetic approach- Natural variation
Pathogen
Plant
ResistanceDisease
Strain A Strain B
Genetic approach- Mutagenesis
Pathogen
Plant
ResistanceDisease
Wild-type Mutant
X
Strategies for molecular study of plantpathogens and their hostsBiochemical approach
n A protein/secondary metabolite with aparticular property can be purified from thepathogen/plant
n The amino-acid sequence of the protein canthen be determined and the correspondinggene isolated
n Example: toxin or plant cell wall degradingenzymes isolated from pathogens
Biochemical approach
Pathogen
Plant
Toxic effectDisease
Strain A
Strategies to isolate pathogen genesinvolved in disease (virulence/avirulence)
n Gene disruption
n Gene silencing
n Map-based cloning
n Differential gene expression
n Biochemical approach
n Genomics (random sequencing of DNA)
Strategies to isolate plant genes involved indisease (resistance/susceptibility)
n Mutagenesis (transposon and others)
n Map-based cloning
n Differential gene expression
n Biochemical approach
n Candidate genes (similarity based)
n Model plants (Arabidopsis, rice)
Gene transfer = DNA transformation Fungi/oomycetes
n Proroplasts or zoospores
n Liposome mediated
n Electroporation
n Particle bombardment
n Agrobacterium-mediated
Reporter genes (ex: GFP or GUS)
n GFP: green fluorescent protein
n GUS: beta glucuronidase
Reporter genes (ex: GFP or GUS)
n GFP: green fluorescent protein
n GUS: beta glucuronidase
n Constitutive promoters
n Inducible promoters
promoter-reporter gene
mRNA
Gene transfer = DNA transformation Plants
n Stable or transient
n Agrobacterium-mediated
n Particle bombardment
n Transgene - exogenous recombinant DNAfragment(s) introduced into plants
n Transgenic plants - plants that containexogenous recombinant DNA, also commonlycalled Genetically Modified Organisms or GMOs
Gene transfer into plant cells
How are transgenic plants generated?
BACTERIA PLANT CELLWITH TRANSGENE
TRANGENIC PLANT
n Agrobacterium - The
natural genetic engineer!!!!
n A bacterium that can
naturally transfer genes into
plants
Gene transfer into plant cells
n Particle bombardment- A method for introducingDNA into plant cells usinga gene gun
Gene transfer into plant cells
http://www2.oardc.ohio-state.edu/plantranslab/
n Totipotency - unique ability of isolated plantcells to regenerate into a whole plant
Plant regeneration
http://www2.oardc.ohio-state.edu/plantranslab/
Transient transformation assays in plants
n Agroinfiltration
n Virus based (example Potato Virus X)
n Transformation of protoplasts
(electroporation)
Agroinfiltration AssayExample: HR elicitors
T-DNA
Vir
Chromosome
35S promoter-elicitor
LB RB
A. tumefaciens
Expression of HR elicitor genes fromthe Potato Virus X genome
166K 25K 8K CP12K
PVX
166K 25K 8K CP12K
PVX-INFelicitor
Gene disruption or gene knockout
n Gene disruption can be obtained by insertinga piece of DNA inside the target genecreating a “disruption” or mutation
n Inserted DNA is generally an artificiallyengineered fragment and usually contains agene for antibiotic resistance for selection
n Insertion can be random or can be targetedthrough the process of homologousrecombination
Gene disruption or gene knockout
Antibiotic resistance
Target gene Disrupted geneMUTANT!!!
Gene disruption or gene knockoutChallenges for eukaryotes
n Most fungal species are haploid but in diploidspecies disruption of the two copies isnecessary to obtain a null mutant
n DNA transformation can be limiting
n Rates of homologous recombination varybetween organisms (example: very low frequency
in Phytophthora)
Gene silencing or RNAi (RNA intereference)
n Introduction of a gene in an antisense (wrong)orientation or sometimes even in a senseorientation triggers a gene silencing response inthe eukaryotic cell
n “Foreign” DNA is silenced that is the gene is notexpressed anymore or the RNA gets degraded
n If the “foreign” DNA is similar to a gene of thetransformed organism, silencing will affect boththe transgene and the endogenous genes thusresulting in a mutant phenotype
Gene silencing - Example: Virus inducedGene Silencing (VIGS)phytoene desaturase in tobacco results in bleaching
Source: D. Baulcombe lab
TRV TRV::sgt1
Virus Induced Gene Silencing tocharacterize plant response to elicitors Map-based cloning
n If individuals with different phenotypes are availablethen genetic crosses can be set up and segregation ofthe differing genes can be noted
n Molecular markers (mutation scattered throughout thegenome and identified using molecular tools such asrestriction enzyme digests or PCR) can be identifiedin this segregating population
n Markers that are genetically linked to the segregatinggene can be used to identify the target gene
Map-based cloning - genetic map
1 2 3 4
Differential gene expression
n Pathogen and plant genes that are highlyexpressed during infection are likely to beinvolved in the infection process
n Patterns of gene expression are revealed by thepopulations of mRNA present in the cell
n Methods to compare and subtract different RNA(cDNA) populations exist and can be applied tothe identification of infection induced genes
The interaction transcriptome
Genomics (random sequencing of DNA)
n Genomics or random sequencing of genomicDNA and/or cDNA can be applied to the study ofplant-microbe interactions
n Various computer analyses of the sequencedgenes can be used to identify candidate genes(bioinformatics)
n Functional genetic assays need to be performedto test the function of the candidate genes(functional genomics)
Example: cDNA sequencing (ExpressedSequence Tags)
ORFATG TAG5’UTR 3’UTR
AAA
The functional genomics paradigm
Sequence Candidategenes
Phenotype
bioinformatics Functional assay
Paradigm shifts in biological researchAdapted from Peltonen and McKusick, Science 291:1224
Structural genomics --> Functional genomicsGenomics --> ProteomicsMap-based --> Sequence-based gene discoveryMonogenic traits --> Multifactorial traitsAnalysis of one gene --> Analysis of multiple genes
in gene families, pathways, or systems
Gene action --> Gene regulationEtiology (mutation) --> Pathogenesis (mechanism)One species --> Several species
n Short generation time
n Small genomes
n Genome sequences available
n Powerful mutagenesis tools
n DNA transformation and functionalassays available
n Resistant to diverse pathogens
Model plants (Arabidopsis, rice) Glossary of genetic terms
n http://www.weihenstephan.de/~schlind/genglos.html
n http://www.genome.gov/glossary.cfm
The interaction transcriptome