Refresher on gene expression - DNA: The stuff of life

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1 Plant Pathology 602 Plant-Microbe Interactions Lecture 2 Molecular methods for studying hostpathogen interactions I Sophien Kamoun kamoun.1@osu.edu The Ohio State University Ohio Agricultural Research & Development Center Plant Pathology 602 Plant-Microbe Interactions Outline - lecture 2 n n Strategies for molecular study of plant pathogens and their hosts Plant Pathology 602 Plant-Microbe Interactions - : The stuff of life Outline - lecture 2 n n Strategies for molecular study of plant pathogens and their hosts

2 Central dogma of molecular biology Replication ion ion Translation Translation

3 cloning Amplification of sequence cloning Amplification of sequence c synthesized in vitro cloning Amplification of sequence cloning Amplification of sequence c synthesized in vitro cloning c synthesized in vitro cloning Purification Biochemical analyses cloning Amplification of sequence prokaryotes n Structurally simpler n Cells do not contain nucleus eukaryotes n Structurally more complex n Cells contain a true nucleus c synthesized in vitro cloning n Genomic generally naked and circular n s are generally present in single sets (haploidy) n contained in chromosomes in nuclei n Cell organelles (mitochondria, chloroplasts Purification Biochemical analyses etc ) n s are present in two or more sets (diploidy)

4 Technical challenges in the molecular genetic study of eukaryotes Plant Pathology 602 Plant-Microbe Interactions n Structurally more complex n transformation can be limiting (cell wall etc ) n Large genomes (lots of noncoding sequences, gene families...) n Diploid organisms (more than one gene to disrupt) Outline - lecture 2 n n Strategies for molecular study of plant pathogens and their hosts Strategies for molecular study of plant pathogens and their hosts tic approach n Many methods are centered around a (molecular) genetic approach n Basis of the genetic approach is to start with two individuals/populations that differ in phenotype and identify the genes that determine the difference n Variation could be natural or artificially generated by mutagenesis tic approach- Natural variation Strain A Strain B Pathogen Plant Disease Resistance tic approach- Mutagenesis Wild-type Pathogen Plant Disease Mutant X Resistance Strategies for molecular study of plant pathogens and their hosts Biochemical approach n A protein/secondary metabolite with a particular property can be purified from the pathogen/plant n The amino-acid sequence of the protein can then be determined and the corresponding gene isolated n Example: toxin or plant cell wall degrading enzymes isolated from pathogens

5 Biochemical approach Strain A Pathogen Plant Strategies to isolate pathogen genes involved in disease (virulence/avirulence) n disruption n silencing n Map-based cloning n Differential gene expression n Biochemical approach n Genomics (random sequencing of ) Disease Toxic effect Strategies to isolate plant genes involved in disease (resistance/susceptibility) transfer = transformation Fungi/oomycetes 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) 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 m

6 transfer = transformation Plants transfer into plant cells n Stable or transient n Agrobacterium-mediated n Particle bombardment n Transgene - exogenous recombinant fragment(s) introduced into plants n Transgenic plants - plants that contain exogenous recombinant, also commonly called tically Modified Organisms or GMOs How are transgenic plants generated? transfer into plant cells BACTERIA PLANT CELL WITH TRANSGENE TRANGENIC PLANT n Agrobacterium - The natural genetic engineer!!!! n A bacterium that can naturally transfer genes into plants transfer into plant cells Plant regeneration n Particle bombardment - A method for introducing into plant cells using a gene gun n Totipotency - unique ability of isolated plant cells to regenerate into a whole plant

7 Transient transformation assays in plants Agroinfiltration Assay Example: HR elicitors n Agroinfiltration n Virus based (example Potato Virus X) n Transformation of protoplasts (electroporation) 35S promoter-elicitor LB T- Chromosome Vir A. tumefaciens RB Expression of HR elicitor genes from the Potato Virus X genome PVX 166K 25K 8K CP 12K PVX-INF 166K 25K 12K 8K elicitor CP disruption or gene knockout n disruption can be obtained by inserting a piece of inside the target gene creating a disruption or mutation n Inserted is generally an artificially engineered fragment and usually contains a gene for antibiotic resistance for selection n Insertion can be random or can be targeted through the process of homologous recombination

8 disruption or gene knockout disruption or gene knockout Challenges for eukaryotes Antibiotic resistance Target gene Disrupted gene MUTANT!!! n Most fungal species are haploid but in diploid species disruption of the two copies is necessary to obtain a null mutant n transformation can be limiting n Rates of homologous recombination vary between organisms (example: very low frequency in Phytophthora) silencing or i ( intereference) n Introduction of a gene in an antisense (wrong) orientation or sometimes even in a sense orientation triggers a gene silencing response in the eukaryotic cell n Foreign is silenced that is the gene is not expressed anymore or the gets degraded n If the foreign is similar to a gene of the transformed organism, silencing will affect both the transgene and the endogenous genes thus resulting in a mutant phenotype silencing - Example: Virus induced Silencing (VIGS) phytoene desaturase in tobacco results in bleaching Source: D. Baulcombe lab Virus Induced Silencing to characterize plant response to elicitors Map-based cloning TRV TRV::sgt1 n If individuals with different phenotypes are available then genetic crosses can be set up and segregation of the differing genes can be noted n Molecular markers (mutation scattered throughout the genome and identified using molecular tools such as restriction enzyme digests or PCR) can be identified in this segregating population n Markers that are genetically linked to the segregating gene can be used to identify the target gene

9 Map-based cloning - genetic map Differential gene expression n Pathogen and plant genes that are highly expressed during infection are likely to be involved in the infection process n Patterns of gene expression are revealed by the populations of m present in the cell n Methods to compare and subtract different (c) populations exist and can be applied to the identification of infection induced genes The interaction transcriptome Genomics (random sequencing of ) Example: c sequencing (Expressed Sequence Tags) n Genomics or random sequencing of genomic and/or c can be applied to the study of plant-microbe interactions n Various computer analyses of the sequenced genes can be used to identify candidate genes (bioinformatics) n Functional genetic assays need to be performed to test the function of the candidate genes (functional genomics) 5 UTR ATG TAG ORF 3 UTR AAA

10 The functional genomics paradigm Sequence bioinformatics Candidate genes Phenotype Functional assay Paradigm shifts in biological research Adapted from Peltonen and McKusick, Science 291:1224 Structural genomics --> Functional genomics Genomics --> Proteomics Map-based --> Sequence-based gene discovery Monogenic traits --> Multifactorial traits Analysis of one gene --> Analysis of multiple genes in gene families, pathways, or systems action --> regulation Etiology (mutation) --> Pathogenesis (mechanism) One species --> Several species Model plants (Arabidopsis, rice) n Short generation time n Small genomes n Genome sequences available n Powerful mutagenesis tools n transformation and functional assays available n Resistant to diverse pathogens Glossary of genetic terms n glos.html n

11 The interaction transcriptome

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