Genetic studies have previously implicated the prp1 gene family in the defence of potato against infection with the late blight fungus Phytophthora infestans. Here, we show that the concentrations of PRP1 mRNA as well as protein rapidly increase in potato leaves after fungal infection and stay at high levels during an extended period of the infection cycle. After separation of subcellular components by differential centrifugation, PRP1 protein was identified in the cytosolic fraction. Expression studies with chimeric promoter/β‐glucuronidase gene constructs in transgenic potato plants provided evidence that transcription of the prp1‐1 gene, representing one member of the prp1 gene family, is at least partly responsible for the accumulation of PRP1 mRNA and protein upon fungal infection. After expression of the prp1‐1 coding sequence in Escherichia coli, the corresponding 26–kDa protein exhibited glutathione S‐transferase activity with Km values of 9.8 mM and 0.11 mM for the artificial standard substrate 1‐chloro‐2,4–dinitrobenzene and glutathione, respectively. Photoaffinity labeling of the protein with tritiated 5–azido‐indole‐3–acetic acid suggested that the phytohormone indole‐3–acetic acid or a structurally related compound serve as a regulator or substrate of the prp1‐1 encoded glutathione S–transferase. This assumption was further supported by the inhibitory effect of the phytohormone on the enzyme activity in vitro. The implications of these findings for a potential involvement of indole‐3–acetic acid in the control of defence reactions are discussed.
The transcriptional start site of the rRNA operon from Zea mays chloroplasts has been identified by a combination of S1 mapping and Southern hybridization with in vitro capped chloroplast RNA as radioactive probe. This is the first example in which the transcriptional start site of a choloroplast gene has been established by identification of the triphosphate‐bearing RNA terminus. The start site is located at position −117 proximal to the 16S rRNA gene and is preceded by −10 and −35 sequences homologous to prokaryotic promoters. The primary transcript directed by this promoter does not include tRNAValGAC sequences which are coded further upstream between positions −302 and −231. A major processing site of the primary rRNA transcript was identified at position −30 which is embedded in a secondary structure typical for prokaryotic RNase III processing sites. Several putative processing and start sites of the tRNAValGAC transcipts have been mapped by primer extension with reverse transcriptase.
Transcription of at least one member of the potato prp1 gene family, prp1-1, is activated at early stages of potato infection with the late blight fungus Phytophthora infestans. In this paper we present evidence that mRNA encoded by prp1-1 does not accumulate in response to abiotic environmental cues which stimulate transcription of other defence-related genes. Regulatory elements were identified in the 5' terminal region of prp1-1 by assaying the expression pattern of chimeric promoter/beta-glucuronidase gene constructs in transgenic potato. A 273 bp fragment comprising the promoter sequence between positions -402 and -130 was sufficient for rapid and strictly localized transcriptional activation at infection sites during the development of late blight disease. Like the native promoter, this truncated promoter did not mediate transcriptional activation in response to other abiotic stimuli. The use of the identified regulatory region to generate conditional mutations selectively at infection sites is discussed.
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