CAUT - Cell autonomy
Donated by
- Ian Furner Department of Genetics, University of Cambridge
Click here to view all 77 of these lines.
Description
The cell-autonomy (CAUT) lines of Arabidopsis
Cell-autonomy is a property of particular genotypes and is useful to examine developmental and/or signalling interactions between cells and tissues. Cell-autonomy is studied by producing genetic mosaics and chimeras containing tissue of differing genotypes and analysing the resulting phenotypes. A trait is completely cell-autonomous if the genotype and phenotype of the tissue always correspond irrespective of the genotype of the adjacent tissue. Conversely a trait is non cell-autonomous if the phenotype of either tissue is affected by the genotype of the adjacent tissue.
Cell-autonomy has been studied in plants using a variety of methods to generate the chimeric or mosaic plants. Such methods include; grafting, site-specific recombination, transposon excision and radiation induced deletion. The latter method has been used for many elegant studies in maize but in only one study of Arabidopsis (Furner, I.J. et al. 1996. PMID 8631249). In these studies recessive cell-autonomous colour markers are included in the experimental design to allow the routine identification of the tissue containing the appropriate deletion. This is comparatively easy in maize as many markers at different locations are available. In Arabidopsis there is a shortage of colour markers and finding a good cell-autonomous colour marker near a gene of interest is not usually possible.
The CAUT lines are an attempt to overcome the shortage of useful markers by artificially generating markers using genetic complementation and Agrobacterium mediated transgenesis. The idea is simple; the recessive yellow ch-42 mutant can be complemented by transformation with the dominant wild-type CH-42 gene on a T-DNA. This has the net effect of translocating the CH-42 gene and the ability to synthesise chlorophyll to a novel and unique location in each transgenic plant. A recessive mutant trait of interest can be crossed into the yellow ch-42 mutant background. Subsequent crosses to an appropriate green T-DNA transformant can generate a situation with the recessive mutant trait on one chromosome and on the homologous chromosome the wild-type gene and the dominant green CH-42 marker.
Background and theory
The ch-42 allele on chromosome 4 (red) is inactive but the wild-type CH-42 function is provided by the T-DNA insert (yellow) on another chromosome (blue). The plant is initially heterozygous for the recessive gene b with the wild-type copy (B) linked in cis to the T-DNA. After irradiation (lower panel) phenotypically yellow sectors are selected and these have lost both the CH-42 copy on the T-DNA and the adjacent B gene. The recessive b mutant is the only copy and the tissue is both yellow and genotypically mutant. The analysis of phenotype of the sector and adjacent wild-type tissue can be used to look for autonomous and non-autonomous interactions.
Generation and mapping of the CAUT lines.
The yellow ch-42 mutant was transformed with a construct containing the wild type CH-42 gene. Green corrected plants were backcrossed to the yellow ch-42 parent until a 1:1 segregation of yellow to green plants was observed. Sequences flanking the T-DNA inserts were recovered by plasmid rescue and mapped on one of the five Arabidopsis chromosomes. The mapping technique changed over the course of the work; the earliest lines were mapped using the Lister and Dean recombinant inbred lines. Later lines were mapped using gridded BACs from the ABRC. Finally, sequencing using a left border primer and BLAST searches of the Arabidopsis genome sequence at TAIR was used to position the last inserts. The results of the project are shown in graphical form (below - please see the ordering page for a tabular representation). The lines containing these inserts were made homozygous and sent to NASC.
Working with the CAUT lines
The CAUT lines were designed for studies of cell-autonomy in the Arabidopsis shoot, leaves and flowers but in order to use them effectively you have to have some background in the development of the shoot apical meristem (SAM) and its derivatives. The Arabidopsis SAM consists of a group of about one hundred cells set up in three layers (L1, L2 and L3). It is set up late in embryogenesis and remains about the same size throughout vegetative and reproductive growth. Once set up the layers are clonally distinct and cells within them have characteristic fates. The L1 layer contributes to a one cell thick epidermis covering the plant. As it contains little chlorophyll sectors deficient in chlorophyll are not seen in this layer. The L2 layer contributes most of the green tissue in the leaves and flowers and a layer in the stem. The L3 layer contributes a core of tissue in the leaves and flowers and the centre of the stem. Chlorophyll deficient sectors can be visualised in green tissues derived from L2 and/or L3 but a colourless genetically wild-type epidermis derived from L1 overlies such sectors.
Cells at the periphery of the seed SAM typically make small contributions to the early leaves and cells nearer the centre of the structure make larger contributions to one or more leaves. Only cells at the centre of the seed SAM make large contributions to the late leaves and flowers (Furner and Pumfrey, 1992.Development 115; 755-764). Seed irradiation produces a large number of sectors affecting the early leaves and relatively few affecting the flowers. The axilliary meristems are clonally related to the L2 derived tissue at the centre of the leaf beneath them. By cutting back the bolting stem it is possible to encourage the development of the axilliary meristem above sectored leaves and get a bolting stem containing the chlorophyll deficient tissue. This process can be repeated several times until periclinal chimeras of the type; L1 wild-type, L2 and L3 yellow are obtained. Such plants can be used to study traits expressed only in flowers and to generate seeds to test sector genotype.
Large ch-42 sectors affecting L2 derived tissue of the inflorescence and silique
Setting up the experiment - step by step instructions.
- Genetics.
Choose one or two CAUT lines with inserts near your mutant of interest using the map and tables above and order them from NASC. You only need the map position of your mutation on either the Lister-Dean recombinant inbred map or a sequence position on the TAIR map. Grow up the mutant-ch-42 and the chosen CAUT line(s) and make several crosses between them to generate an F1. Grow this generation up and allow it to self-fertilise to generate a large F2 for irradiation. The genetics involved is illustrated below for the hypothetical recessive gene b.
All plants ch-42 homozygotes.
v
b/B+T-DNA(CH-42) F1
v
b/b : b/B+T-DNA(CH-42), : B+T-DNA(CH-42)/B+T-DNA(CH-42)
F2 1 yellow mutant : 2 green heterozygotes : 1green wild-type
v
Irradiate bulked seeds and find yellow sectors on seedlings. (Yellow plants are disregarded and green homozygotes do not show sectors).
Sector genotype; b/0 Plant genotype b/B+T-DNA(CH-42)
As the gametes are usually set in tissue derived from the L2 layer of the floral primordium seeds set in the sector and the adjacent wild type tissue can be used to asses the genotype of L2. Seeds set in the yellow sectors should give rise to 100% yellow and phenotypically mutant plants. Seeds set in the wild type tissue should segregate and give rise to 3 green wild type plants to one yellow mutant plants. If large samples are planted a few recombinant types are typically found. Sometimes seeds which give rise to green plants are found in samples from yellow sectors, these may be due to contributions to the gametes from cells in the L1 layer of the floral primordium.
- Seed irradiation and sector analysis.
In a typical experiment 10 batches each of 20 Mg of seeds are irradiated with unfiltered X-rays. Various doses can be used in these experiments but the highest level that can routinely be used is 16 kilorad. This dose delays leaf emergence by 3 days. Each batch of seed is broadcast to a filled seed tray in 10 gm of dry sand. It is important to keep the humidity high until the true leaves emerge, as the irradiated plants tend to dry out and die. Sectors are found on up to 1% of plants and sectored plants are picked off to new trays.
The ch-42 sectors are a characteristic yellow colour and sectors with other phenotypes are usually the result of unrelated events and should be discarded. The sectors tend to green up over time but this effect can be reduced by increasing the light intensity. Once branches with yellow L2 and L3 tissue have been generated by cutting back they can be marked with waterproof ink. This allows the seeds to be collected from the sector even though the dry silique has no chlorophyll and the sector cannot be seen. Sector phenotypes can be scored at whatever time and/or stage they normally appear. It is a good idea to retain a few yellow mutant homozygotes and a few green non-sectored plants as controls.
- Verification of the CAUT lines.
The CAUT lines are phenotypically identical and all are full green and kanamycin resistant. So it is fairly easy to mix them up and ruin your cell-autonomy experiment by using the wrong one. As such errors do happen in any lab a simple and robust method of line verification has been developed. The method is based on Southern blots, T-DNA probes and comparisons to blots run in the Furner lab (see thumbnails below).
The parental ch-42 mutant is tagged with a T-DNA containing a promoter less and silent neomycin phosphotransferase and an active hygromycin phosphotransferase conferring hygromycin resistance. The T-DNA also contains a pBR322 copy. Homozygous ch-42 plants are yellow in soil and grow to maturity. Southern blots of DNA from this line prepared with ECORI and probed with pUC18 have a single 9kb fragment corresponding to the tag.
The pCV002GC plasmid used to generate the CAUT lines also has a pBR322 region near the left border (Koncz et al, 1990 The EMBO Journal 9; 1337-1346.). There is one ECORI site in the T-DNA to the right of the pBR322 region. In the transformed plants a second ECORI site is found in the adjacent flanking plant DNA. The second site varies between integration events and therefore between lines. All of the lines are homozygous for a single correction but there can be multiple T-DNA copies at that site. The CAUT lines were made by introducing the correcting T-DNA into the ch-42mutant. After recurrent back crosses, the single correcting insertions were made homozygous. Southern blots of these lines made with genomic DNA digested with ECORI and probed with pUC18 show a characteristic pattern. All lines have a 9kb band corresponding to the tag at ch-42 on chromosome 4. In addition all lines have one or more bands of varying sizes corresponding to the correcting CH-42 insert at the new location. The fragment pattern is unique to each line and can be used as a distinctive fingerprint to verify the identity of a line.
Below are 7 blots corresponding to the inserts of the 5 chromosomes (chromosomes 1 and 4 are on two blots). The arrows are the positions of lambda HindIII fragments and the sizes (from the top) are: 23.1, 9.4, 6.6, 4.4, 2.3 and 2.0 KB. All lines have the 9kb fragment but there is slight variation in the migration of the fragment.
Chromosome 1 a
Chromosome 1 b
Chromosome 2
Chromosome 3
Chromosome 4 b
Chromosome 4 a
Chromosome 5
Related links
References
- Furner I.J., et al. 2008. The CAUT lines; a novel resource for studies of cell autonomy in Arabidopsis. The Plant Journal 53(4): 645-660. PMID. 18269574.
- Stirnberg P., Furner I.J. & Leyser H.M.O. 2007. MAX2 participates in an SCF complex which acts locally at the node to suppress shoot branching. The Plant Journal 50(1): 80-94. PMID. 17346265.