1. Arabidopsis Molecular Genetics HORT 301 – Plant Physiology October 31, 2007 Reading 1 (Alonso and Ecker (2006) Nature Reviews Genetics Reading 2 (Page and Grossniklaus (2002) Nature Reviews Genetics Function of all plant genes Assumption – majority of genes in plants have conserved function, including those in crops

2. Lecture outline: Genetic terminology Arabidopsis thaliana, the current plant genetic model system Resources for gene function determination Phenotypic selection or screening of a T-DNA mutant population – forward genetics, identification of the mutated gene Candidate gene based on predictions of function (phenotype) – reverse genetics

3. Terminology Genetics – study of heredity/inheritance and function of genetic material Gene – individual functional unit of the DNA that includes the promoter and encodes the messenger mRNA that is translated into a protein, i.e. locus Locus – usually two alleles at each, contributed by two homologous chromosomes in a diploid (2n) organism Molecular genetics – application of molecular biology to genetics, i.e. gene identification by DNA structure, genetic engineering Genotype (genes) to phenotype (appearance and function)

4. Inheritance – single gene dominant and recessive phenotypes Griffiths AF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM. 1996. An Introduction to Genetic Analysis. 6th ed. W.H. Freeman & Company Yellow – 416 (3) Green – 130 (1) Y (dominant) and y (recessive) are alleles

5. Allele – alternative state of a gene that may be different in each of the two chromosomes of the pair, dominant (Y) or recessive (y) allele Mutation – change in DNA structure of a gene, alteration in gene function Plant breeders improve crops by transferring “mutations” that confer better phenotypes/traits!!!! See the maize example

6. Arabidopsis thaliana (L.) Heynh, plant genetic model – (Brassicaceae, mustard or crucifer family) common name Arabidopsis, rosette-type plant

7. Morphological, developmental and molecular genetic attributes of Arabidopsis that make this plant a genetic model: Small size – numerous plants can be grown in a small area Short life cycle – six to eight weeks, 6 to 8 generations per year Produces numerous seed – several thousand seeds per plant

8. Diploid (2n) – n = 5 (chromosomes) Self-fertile – amenable to classic genetic manipulation by self or cross pollination Natural variation – better alleles for certain traits in other accessions or ecotypes Transformable using Agrobacterium tumefaciens – foreign DNA is easily transferred into the genome (complete set of genes and accompanying DNA)

9. Genome size is relatively small - ~120 x 10 6 bp, 26,819 genes encode proteins, 31,762 total including genes that encode miRNA, pseudogenes, and transposable elements, etc. http://www.arabidopsis.org/portals/genAnnotation/genome_snapsho t.jsp http://www.arabidopsis.org/portals/genAnnotation/genome_snapsho t.jsp Genome sequence information is available, also cDNA sequence, http://signal.salk.edu/cgi-bin/tdnaexpress http://signal.salk.edu/cgi-bin/tdnaexpress Bioinformatic information, http://signal.salk.edu/, http://mips.gsf.de/proj/plant/jsf/index.jsp http://signal.salk.edu/ http://mips.gsf.de/proj/plant/jsf/index.jsp mRNA expression data, https://www.genevestigator.ethz.ch/at/ https://www.genevestigator.ethz.ch/at/ Mutations for nearly every gene – mutants are available, http://www.biosci.ohio-state.edu/pcmb/Facilities/abrc/abrchome.htm http://www.biosci.ohio-state.edu/pcmb/Facilities/abrc/abrchome.htm

10. Genetic resources for gene function identification - T-DNA insertional tagging mutagenesis - Agrobacterium-mediated transformation Agrobacterium tumefaciens – crown gall disease, bacterial pathogen of plants that transfers DNA (T-DNA) into the plant genome during the infection process Tumor cells produce carbon and nitrogen sources for use by the bacteria Agrobacterium infection and tumor development

11. “Disarmed” Agrobacterium strains are used for genetic manipulation (engineering) of plants – bacteria are no longer pathogenic but are still capable of T-DNA transfer T-DNA is inherited as a single dominant gene (locus)

12. T-DNA binary vector (plasmid) composition, e.g., pSKI015 - right border (RB) and left border (LB) DNA between the borders is inserted into the plant genome (DNA), selectable marker gene Selectable marker gene – e.g. herbicide resistance gene to “select” transformed plants Insertions are random, 1.5 insertions per event

13. 4X 35S T-DNA Vector – * pSKI015 Transformation Mutant Plants Plant Genomic DNA T - DNA or Disruption Activation LB 3’-ocs-bar-mas-5’ OriC RB Agrobacterium T-DNA insertional (tagging) mutagenesis Activation sequence – e.g., 4X 35S that can activate expression of a native gene depending on the insertion position

14. Primary interest is to alter the function (cause a mutation) of every gene by T-DNA insertional mutatgenesis It is estimated that ~300,000 random insertions will “saturate the genome”, a mutation in each gene, a population of 300,000 plants Process is referred to as “tagging” “Tag” – insertion of the T-DNA “tags” the region in the genome because the T-DNA sequence can be located in the genome because of sequence Identification of the flanking sequence

15. Floral transformation of Arabidopsis Each seed that is transformed has a unique mutation Transformation procedure - inflorescences are dipped into a solution containing Agrobacterium Plants are grown in the greenhouse and seeds are collected

16. Selection of transformed plants – typically based on resistance to a toxic agent (transgene expression results in detoxification), in this instance the herbicide bialaphos Population is maintained through seed, i.e. inherited Generation of T-DNA tagged population Herbicide selection of transformants Propagation and collection of seed

17. Phenotypic screening of the T-DNA mutant population – forward genetics Identify Salt Responsive Mutants in a T-DNA Insertion Population T 1 Generation Select Transformed Plants (Heterozygous, Bialaphos R, ~0.2%)  T 2 Generation Primary Screen for Mutants (NaCl sensitivity or insensitivity)  T 3 Generation Confirm Genetic Stability of Phenotype (Homozygous, Recessive/Dominant )  Locate the “Tag” and Identify the Flanking Genomic Sequence

18. Root Development (C24) Luciferase Imaging (C24RD29A::LUC) Shoot Development (Col-0 sos3-1) Stress Isolation of NaCl mutants using different approaches

19. Identification of the mutation in Arabidopsis genome – location of the T- DNA insertion (tag) and determination of flanking sequence Most mutations are recessive, both alleles of the locus are homozygous for the mutation

20.  -importin gene – At5g49310 Arabidopsis chromosomes

21. Reverse genetics – candidate gene identification based on prediction of function and phenotypic effects Ca 2+ /CaM regulated transcription factor that regulates drought stress responses of plants, GTL family Phylogenetic dendogram

22. Mutant lines are available to the public (2) Generate a collection of mutations in selected genes : Arabidopsis Biological Resource Center (ABRC) providesT-DNA insertion lines in Arabidopsis genome to public (www.arabidopsis.org) AtGTL1 (At1g33240) gtl1-2 LR L LB L+LB L+LB L+LBL+LB no T- DNA homozygousheterozygou s No T-DNA homozygous heterozygous Select homozygous T-DNA insertion line

23. Evaluate phenotypes