In Situ Hybridization to RNA in Whole Arabidopsis Plants
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Abstract The importance of determining spatial distribution and expression of biomolecules in living organisms has been increasingly recognized because of the relevance to biological function. High resolution spatial information at the level of the cell or organism can be obtained through in sztu detection techniques, including in situ hybridization, mnnunohistochemistry ( 1 ), and expression of reporter genes such as the green fluorescent protein ( 2 , 3 ). For plant materials, tissue printing is also widely used ( 4 ). This chapter focuses on in situ detection of RNA in mhabidopszs at the level of the whole plant. Other m situ detection protocols for Arabzdopsis plants are described in Chapters 35 and 36 . Whole plant zn sztu hybridization can reveal the spatial distribution of a DNA or RNA species in whole plants. Melcher et al. ( 5 ) first developed an in situ hybridization protocol to detect cauliflower mosaic virus (CaMV) DNA m individual turnip leaves, and Leisner et al. ( 6 ) modified the technique to detect CaMV DNA m whole Arabidopsis plants. The followrng protocol was developed for in situ detection of viral RNA in Arabidopsis, and has been used to study long-distance movement of RNA viruses (7−9). An example is shown in Fig. 1 . The protocol should also be amenable for zn situ detection of abundant mRNAs in Arabldopsis plants after minor modtfications. In brief, the plant 1s placed in a Petri dish, fixed with 95% ethanol, digested with pronase to remove barriers for probe access, treated with 0.2N HCl to denature the RNA, neutralized with 2X SSC, dried m an, baked for 2 h at 80�C under vacuum, prehybridized, and hybridized with a specific radioactive DNA probe. The plant is then washed, transferred to a piece of acetate sheet, dried m air, wrapped in plastic wrap, and subjected to autoradiography. The steps before autoradiography require 3–4 d. The exposure time for detection of turnip crinkle virus (TCV) RNA with a 32 P-end-labeled oligonucleotide probe in infected Arabidopsis is as short as 30 min without an intensifying screen. This is a delicate technique, and some practice is needed for best results.
Fig. 1. Time course of turnip crinkle virus (TCV) movement in Arubidopsis. One of the two oldest leaves of 2-wk-old Arabidopsis ecotype Col-0 (TCV-susceptible) or Di-0 ( TCVresistant ) seedlings were inoculated by TCV or by inoculation buffer alone(Mock). Numbers on top of the panel denote number of days postinoculation when the plants were collected and frozen at -80” C. The plants were subjected to the whole plant in situ hybridization analysis using an oligonucleotide probe specific for TCV genomic RNA, as described in the text. Arrowheads point to the inoculated leaf. Outlines of leaves were drawn on a clear plastic sheet and photographed with the autoradiogram.
