Publications - Stress and Develop Biology

The Arabidopsis genome contains 28 genes with sequence homology to the Arabidopsis NDR1 gene and the tobacco HIN1 gene. Expression analysis of eight of these genes identified two (NHL25 and NHL3 for NDR1/HIN1-like) that show pathogen-dependent mRNA accumulation. Transcripts did not accumulate during infection with virulent Pseudomonas syringae pv. tomato DC3000 but did accumulate specifically when the bacteria carried any of the four avirulence genes avrRpm1, avrRpt2, avrB, or avrRps4. Furthermore, expression of avrRpt2 in plants containing the corresponding resistance gene, RPS2, was sufficient to induce transcript accumulation. However, during infection with an avirulent oomycete, Peronospora parasitica isolate Cala-2, only NHL25 expression was reproducibly induced. Salicylic acid (SA) treatment can induce expression of NHL25 and NHL3. Studies performed on nahG plants showed that, during interaction with avirulent bacteria, only the expression of NHL25 but not that of NHL3 was affected. This suggests involvement of separate SA-dependent and SA-independent pathways, respectively, in the transcriptional activation of these genes. Bacteria-induced gene expression was not abolished in ethylene- (etr1-3 and ein2-1) and jasmonate- (coi1-1) insensitive mutants or in mutants impaired in disease resistance (ndr1-1 and pad4-1). Interestingly, NHL3 transcripts accumulated after infiltration with the avirulent hrcC mutant of Pseudomonas syringae pv. tomato DC3000 and nonhost bacteria but not with the virulent Pseudomonas syringae pv. tomato DC3000, suggesting that virulent bacteria may suppress NHL3 expression during pathogenesis. Hence, the expression patterns and sequence homology to NDR1 and HIN1 suggest one or more potential roles for these genes in plant resistance.

In-vivo imaging of transgenic tobacco plants (Nicotiana tobacum L.) expressing firefly luciferase under the control of the Arabidopsis phenylalanine ammonia-lyase 1 (PAL1)-promoter showed that luciferase-catalyzed light emission began immediately after the substrate luciferin was sprayed onto the leaves and reached a plateau phase after approximately 60 min. This luminescence could easily be detected for up to 24 h after luciferin application although the light intensity declined continuously during this period. A strong and rapid increase in light emission was observed within the first minutes after wounding of luciferin-sprayed leaves. However, these data did not correlate with luciferase activity analysed by an in-vitro enzyme assay. In addition, Arabidopsis plants expressing luciferase under the control of the constitutive 35S-promoter showed similar wound-induced light emission. In experiments in which only parts of the leaves were sprayed with luciferin solutions, it was shown that increased uptake of luciferin at the wound site and its transport through vascular tissue were the main reasons for the rapid burst of light produced by preformed luciferase activity. These data demonstrate that there are barriers that restrict luciferin entry into adult plants, and that luciferin availability can be a limiting factor in non-invasive luciferase assays.

Transient influx of Ca2+ constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae–derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca2+]cyt), which peaked at ∼1 μM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca2+]cyt signature was achieved by elicitor concentrations sufficient to stimulate Ca2+ influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca2+]cyt but not the rapidly induced [Ca2+]cyt transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca2+ influx across the plasma membrane or of Ca2+ release from internal stores suggests that the elicitor-induced sustained increase of [Ca2+]cyt predominantly results from the influx of extracellular Ca2+. Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca2+]cyt, and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca2+]cyt is causally involved in signaling the activation of pathogen defense in parsley.