Characteristics of ethylene biosynthesis-inducing xylanase movement in tobacco leaves. Plant Physiol 100: 2059-2065

Loyola University Maryland, Baltimore, Maryland, United States
Plant physiology (Impact Factor: 6.84). 01/1993; 100(4):2059-65. DOI: 10.1104/pp.100.4.2059
Source: PubMed


(125)I-Labeled ethylene biosynthesis-inducing xylanase (EIX) was used to study the movement of this protein in tobacco (Nicotiana tabacum) tissues. A biologically active (125)I-labeled EIX was obtained using chloramine-T as the oxidizing agent. Labeled EIX was detected in the far most edges of the leaf 5 min after it was applied to the petiole of a detached leaf. EIX was distributed uniformly throughout the leaf, including the mesophyll area within 5 to 15 min, after which there was only little change in the distribution of radioactivity in the leaf. (125)I-Labeled EIX was extracted from treated leaves, and EIX translocation in the leaf was blocked by preincubation of labeled EIX with anti-EIX antibodies, indicating that the intact peptide moves in the leaf. Injection of anti-EIX antibodies into the intercellular spaces of the leaf mesophyll prevented induction of necrosis by EIX, suggesting the mesophyll as the site of EIX action. EIX was translocated both to upper and lower parts of the plant when applied to a whole plant through the petiole of a cut leaf. Radioactivity was found in all leaves and in the stem, although some leaves accumulated much more EIX than others; EIX was not found in the roots. There was no difference between the accumulation pattern of EIX in fresh and ethylene-treated leaves or between sensitive (Xanthi) and insensitive (Hicks) tobacco cultivars. These data support the hypothesis that intact EIX protein is translocated to the leaf mesophyll, where it directly elicits plant defense responses.

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Available from: Bryan A Bailey, Feb 14, 2014
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    • "In previous studies, we showed that the GCC box functions as a cis-acting element that is responsive to ethylene (Ohme-Takagi and Shinshi, 1995; Shinshi et al., 1995). Furthermore, it has also been demonstrated that TvX induces the biosynthesis of ethylene (Bailey et al., 1990; Dean and Anderson, 1991; Sharon et al., 1992). Lotan and Fluhr (1990) suggested, moreover, that TvX elicits the accumulation of a subset of PR proteins via an ethyleneindependent pathway in tobacco. "
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    ABSTRACT: In cultured XD6S tobacco cells, xylanase from Trichoderma viride (TvX) induced the expression of a luciferase reporter gene that was under the control of a GCC box, which is an 11 bp sequence (TAAGAGCCGCC) that is found in the 5'-upstream region of pathogen-responsive defence genes that include genes for class I basic chitinase. TvX-induced biosynthesis of ethylene was not required for the TvX-activated transcription. The TvX-induced, GCC box-mediated transcription of the reporter gene was completely blocked not only by staurosporine, an inhibitor of serine/threonine protein kinases, at 1 microM, but also by calyculin A, an inhibitor of protein phosphatases 1 and 2A, at 0.2 microM. It appeared also that protein synthesis de novo was required for the GCC box-mediated transcription of the reporter gene. Accumulation of mRNAs for various ERFs (ethylene-responsive transcription factors), which have been shown to bind specifically to the GCC box, was also induced by TvX prior to increases in the level of mRNA for a class I basic chitinase. In particular, the level of mRNA for EFR2 reached a maximum from 3 to 6 h, whereas levels of mRNAs for ERF3 and ERF4 were highest 0.5 h after the start of treatment of TvX and decreased thereafter. Moreover, induction of accumulation of the mRNA for ERF2 was inhibited by staurosporine and calyculin A. These results suggest that ERF2 might play a major role in TvX-induced, GCC box-mediated transcription of genes and that both protein kinase(s) and protein phosphatase(s) might be involved, as positive regulators, in the signal transduction pathway that leads to expression of ERF2 and subsequent GCC box-mediated transcription of genes.
    Preview · Article · Jan 2000 · The Plant Journal
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    • "Similar xylanases have been identified in xylan-induced filtrates of phytopathogenic fungi (Dean et al. 1989; Wu et al. 1997) . When applied to cut roots or petioles EIX was translocated in the xylem tissues and induced symptoms in leaves both above and below the point of application (Bailey et al. 1991; Sharon et al. 1992).. Injection of EIX into the leaf-mesophyll intercellular spaces induced ethylene production, localized cell death as well as other plant defense responses in Nicotiana tabacum cv Xanthi (Bailey et al. 1990; Lotan and Fluhr 1990; Avni et al. 1994) and cell suspensions (Bailey et al. 1992; Felix et al. 1993; Yano et al. 1998). These are characteristic responses of plants to exogenously applied elicitors (Keen et al. 1990; Blein et al. 1991; Felix et al. 1993). "
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    ABSTRACT: An ethylene-inducing xylanase (EIX) from Tricohoderma viride is a potent elicitor of ethylene biosynthesis, localized cell death and other defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum). Wild species of tomato, such as Lycopersicon cheesmanii and Lycopersicon pennellii, do not respond to EIX treatment. The F1 progeny of a L. esculentum√óL. cheesmanii and a L. esculentum√óL. pennellii cross responded to EIX treatment with an increase in ethylene biosynthesis and the induction of localized cell death. The F2 progeny of the above mentioned crosses segregated 3:1 (responding:non-responding). We mapped the EIX-responding locus (Eix) to the short arm of chromosome 7 using a population of introgression lines (ILs), containing small RFLP-defined chromosome segments of L. pennellii introgressed into L. esculentum. RFLP analysis of 990 F2 plants that segregated for the introgressed segment mapped the Eix locus 0.1 cM and 0.9 cM from the flanking markers TG61 and TG131, respectively. Using the marker TG61 we isolated a yeast artificial chromosome (YAC) clone that carries 300-kb DNA segments derived from the Eix region. By mapping the ends of this YAC clone we show that it spans the Eix locus. Thus, positional cloning of the Eix locus appears feasible.
    Full-text · Article · Dec 1999 · Theoretical and Applied Genetics
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    • "This fragment was used as a probe to screen a T. viride cDNA library constructed in Lambda ZAP II (Stratagene, La Jolla, CA). Screening was performed by standard procedures (Sambrook et al., 1989). "
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    ABSTRACT: Ethylene-inducing xylanase (EIX) elicits plant defense responses in certain tobacco (Nicotiana tabacum) and tomato cultivars in addition to its xylan degradation activity. It is not clear, however, whether elicitation occurs by cell wall fragments released by the enzymatic activity or by the xylanase protein interacting directly with the plant cells. We cloned the gene encoding EIX protein and overexpressed it in insect cells. To determine the relationship between the two activities, substitution of amino acids in the xylanase active site was performed. Substitution at glutamic acid-86 or -177 with glutamine (Gln), aspartic acid (Asp), or glycine (Gly) inhibited the beta-1-4-endoxylanase activity. Mutants having Asp-86 or Gln-177 also lost the ability to induce the hypersensitive response and ethylene biosynthesis. However, mutants having Gln-86, Gly-86, Asp-177, or Gly-177 retained ability to induce ethylene biosynthesis and the hypersensitive response. Our data show that the xylanase activity of EIX elicitor can be separated from the elicitation process, as some of the mutants lack the former but retain the latter.
    Full-text · Article · Nov 1999 · Plant physiology
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