SEARCHING FOR NEW GENES IMPLIED IN SYSTEMIC ACQUIRED RESISTANCE OF CUCUMBER BY DIFFERENTIAL DISPLAY
R KEMPFand H KAUSS
Universität Kaiserslautern, FB Biologie, Erwin-Schrödinger-Strasse, D-67663 Kaiserslautern, Germany
Background and objectives
In plants exhibiting systemic acquired resistance (SAR), one of the defence strategies is the direct induction of certain genes encoding proteins (e.g. chitinase, 1,3-beta-glucanase). In addition, complex mechanisms (e.g. HR, papillae formation), which are induced only on a secondary challenge infection, appear also to play an important role. In hypocotyls of etiolated cucumber seedlings exhibiting SAR due to root pre-treatment with INA or BTH, the class-III chitinase is not present prior to infection, but is expressed on adhesion and germination of Colletotrichum lagenarium spores, without a requirement of penetration of the epidermal cells . In the same tissue, competence for elicitation of hydrogen peroxide is not constitutive but requires gentle cuticle abrasion followed by a conditioning period of several hours, and it is this process which is enhanced by the resistance inducers . Both these physiological responses appear to imply transcriptional activation of genes hitherto not considered to be important in SAR. We have used differential display to identify such genes, aiming to find new molecular markers for SAR.
Materials and methods
5-day-old control (grown on water) or resistant (grown on 40 µM BTH or 100 µM INA) cucumber (Cucumis sativus L.) seedlings were incubated in a suspension of spores of C. lagenarium (melanin-deficiant mutant). Alternatively, the hypocotyls were gently abraded with SiC. Total RNA was isolated 5 h later and cDNA synthesized by reverse transcription using anchored oligo-dT primers. The generated cDNAs were used as a template for subsequent PCR amplification with different combinations of the same anchored oligo-dT primers and 20-decamer arbitrary primers. Differentially displayed PCR products were used to re-amplify the cDNAs of interest, and cloned. After sequence analysis the data have been compared to the GenBank databases using the BLAST tools.
Results and conclusions
To identify new genes that are implied in SAR of cucumber, nine different sets of probes have been analysed by differential display: seedlings (grown on water, BTH or INA) that were gently abraded and conditioned for 6 h; seedlings (grown on water, BTH or INA) that were incubated 6 h in a spore suspension of C. lagenarium; and seedlings (grown on water, BTH or INA) without further treatment. Several cDNA fragments could be identified which are directly and specifically induced only in SAR cucumber seedlings without any further treatment. In addition, we also isolated differentially displayed cDNAs which represent genes that show a potentiation of their transcription, but are activated only on subsequent spore application and/or conditioning. The differential expression of some of the isolated cDNA fragments has been verified by Northern blot analysis. Sequence analysis of the isolated cDNA fragments showed homologies to genes that code for proteins with a potential role in SAR.
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