Cloning and expression analysis of cDNAs corresponding to genes activated in cucumber showing systemic acquired resistance after BTH treatment

Abstract

Background: Infection of plants by necrotizing pathogens can lead to the rapid and localized induction of a complex set of defense responses resulting in a restriction of pathogen growth and spread. Subsequently, an increase of plant resistance against a broad spectrum of pathogens is observed systemically. This plant immunity is known as Systemic Acquired Resistance. To identify components of the transduction pathway, we cloned and analysed the expression pattern of several mRNAs accumulating in cucumber plants after induction of Systemic Acquired Resistance.

Results: We tested on cucumber different compounds known to induce systemic acquired resistance. Among these, BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester) proved to be very effective. mRNA RT-PCR differential display was used to identify mRNA sequences induced 24 hours after the application of 10 microM BTH to cucumber plants. A cDNA library constructed from cucumber plants sprayed with 10 microM BTH was screened to get corresponding full length cDNAs. Among the identified cDNAs were those coding for a putative ras-related GTP-binding protein, a putative beta-1,4-N-Acetylglucosaminyltranferase III and a putative pathogenesis related protein. The time course of accumulation of the three corresponding mRNAs was analysed by northern blotting in plants treated by BTH or in plants infected by Colletotrichum lagenarium.

Conclusions: The mRNA RT-PCR differential display technique allowed the identification of three genes possibly involved in Systemic Acquired Resistance in cucumber. Pathogenesis-related proteins are known to be involved in plant defence against pathogens. GTP-binding protein and N-acetylglucosaminyltranferase III have been reported to be components of signal transduction pathways in mammals and plants.

Figure 1

Time course of PR-8 mRNA accumulation in cucumber leaves in response to chemical treatments. Total RNA was extracted from cucumber leaves at various time after spraying with water, 5 mM salicylic acid, 1 mM BTH, 50 mM L-α-amino butyric acid (ABA), 0.78 mM 2-thiouracil (2T-U) or from untreated leaves (UT). RNA (10 μg) was fractionated by electrophoresis on agarose gel. Northern blot was probed with α-³²P labeled PR-8 cDNA (loading of equal amounts of RNA was confirmed by ethidium bromide staining).

Figure 2

Local acquired resistance induced by 10 μM or 50 μM BTH. Four-week-old cucumber plants (with two true fully expanded leaves) were sprayed with H₂O, 10 μM BTH, 50 μM BTH or left untreated. The second true leaf was infected with C. lagenarium 5 days later. Plants were photographed 2 weeks postinoculation.

Figure 3

Zoom in on an auto-radiogram of differentially displayed RT-PCR products. The RT-PCR were performed with primers 5'T₁₁GA and 5'GATCTAACCG (lane 1 to 4), primers 5'T₁₁GA and 5'GATCAATCGC (lane 5 to 8), primers 5'T₁₁GA and 5'TACAACGAGG (lanes 9 to 12). Uneven lanes: H₂O treated plants. Even lanes: 10 μM BTH treated plants. The arrows show bands corresponding to transcripts accumulating only or presenting a higher expression in BTH treated plants.

Figure 4

Slot blotting of PCR amplified fragments identified in differential display experiments. PCR amplified DNA from four dd11 clones, nine dd6 clones and four dd5 clones respectively were hybridized with the ³²P labeled RT-PCR corresponding products from either H₂O or BTH treated plants. The arrows show DNA from clones containing differentially displayed fragments.

Figure 5

Time course of mRNA accumulation in cucumber leaves after BTH treatment (A) or C. lagenarium inoculation (B). A: Total RNA was extracted from cucumber leaves at various time after application of 10 μM BTH or water (mock treatment). RNA (15 μg) from leaves 1 and 2 (treated) and leaves 3 and 4 (untreated) was fractionated by electrophoresis on agarose gels. Identical Northern blots were probed with ³²P-labeled PR-8, CPR and CGT cDNAs. B: Total RNA was extracted from cucumber leaves at various time after C. lagenarium inoculation. RNA (15 μg) from untreated leaves (UT), leaves 1 (infected) and leaves 2, 3 and 4 (non infected) was fractionated by electrophoresis on agarose gels. Identical Northern blots were probed with ³²P-labeled PR-8, CPR and CGT cDNAs. Equal loading of RNA was confirmed by ethidium bromide (Et Br) staining. A representative gel is shown.