Ethylene biosynthesis-inducing xylanase (EIX) from the fungus Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattem, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen.

1251-Labeled ethylene biosynthesis-inducing xylanase (EIX) was used to study the movement of this protein in tobacco (Nicotiana tabacum) tissues. A biologically active 1251-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. 1251-Labeled EIX was extracted from treated leaves, and EIX translocation in the leaf was blocked by preincu-bation 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 trans-located to the leaf mesophyll, where it directly elicits plant defense responses. EIX2 is a 22-kD protein isolated from the fungus Tricho-derma viride (7, 8). When applied to tobacco (Nicotiana taba-cum cv Xanthi) leaf tissue, EIX induces ethylene formation and hypersensitive necrosis, as well as other plant defense responses (1-3). There is evidence suggesting that the elicitor activity of EIX is independent of the endo-fl-1,4-xylanase activity, i.e. that the protein is an elicitor by itself (. Dean, personal communication). A number of enzymes and peptide elicitors have been isolated from fungi (5, 13, 19,20, 22,23). Proteinaceous elicitors with hydrolytic enzymic activity would generally be assumed to produce elicitors that are released from the plant or pathogen cell wall by action of the enzymic activity rather than the protein itself being the elicitor compound.

We report the use of Ni2" and Co2l on free-hand sections of soybean (Glycine max L.) and Bidens sp to localize polygalacturonates. In soybean only the hourglass cells of the seedcoat stain intensely. In the pod the epidermis of the outer pod wall and a few layers of subepidermal cells stain lightly, while that part of the funiculus adjacent to the seedcoat palisade epidermal cells stains heavily and the neck of the funiculus close to the pod also stains. In Bidens stem sections, the walls of the collenchyma stain most intensely.We are interested in the architecture of the cell walls of dicotyledonous plants. The general plan of a typical wall involves cellulose microfibrils embedded in a more or less amorphous matrix; the microfibrils resist tension, the matrix resists compression. The matrix includes hemicelluloses and pectins. Growing cells produce walls appropriate to their place in the cell body by controlling the relative rates of synthesis and secretion of the various wall components. In the case of pectin, because it is secreted with its uronic acid residues largely in the methyl ester form, dramatic changes in physical properties can be achieved in muro. Secretion ofpectin methyl esterase (PME) or perhaps activation of PME already in place generates uronic acids with pKs in the range of 3.5 to 5. Thus protons are generated in muro. These lower the pH and raise the osmotic potential of the wall. PME preferentially hydrolyzes uronic acid esters in blocks, thereby generating blocks of polygalacturonate residues that can bind Ca2" to form egg box structures with a high linear charge density (2). These structures consist of dimers and larger aggregates (6). The gelling ability of partially deesterified pectins in dilute solutions is remarkably sensitive to degree of esterification and concentration of Ca2" (and Ba2 , Sr", Cu2", Cd2", and Co2" but not K+ or Mg2+; Ni2" was not tried) (8

Tissue printing onto membranes such as nitrocellulose is a technique employed to study the localization of proteins, nucleic acids, and soluble metabolites from freshly cut tissue slices. We probed tissue prints of young and mature celery (Apium graveolens) petioles with antibodies raised against two proteins, spinach ribulose-1,5-bisphosphate carboxylase and tomato fruit catalase. The purposes of this study were to determine if these proteins are developmentally regulated and to determine if the patterns and intensities of cross-reactivity of antibodies on tissue blots corresponded only to the presence of specific epitopes or was related to the amount of protein present in any given area on the tissue prints. Different and distinct cross-reactivity patterns were observed with each of the two antibodies used. Tissue prints from young and mature tissues also showed differences in antibody cross-reactivity. Comparison of Coomassie blue staining patterns with antibody reactivity patterns showed that there is little relationship between tissue protein concentration and antibody reactivity.Most studies conceming regulation of protein levels involve the use of complex tissues or organs. Data obtained from such studies are extremely valuable but do not necessarily indicate in which cells or tissues the changes are occumng. Determining the amount and distribution of proteins in specific tissues or cells could yield valuable insights into cell development. Tissue printing is a relatively new technique that is being used to study cell-specific localization of macromolecules (Cassab et al., 1985; Cassab and Vamer, 1987;Spruce et al., 1987; Cassab et al., 1988; Cassab and Vamer, 1989; McClure and Guilfoyle, 1989; Vamer and Taylor, 1989; Lagrimini, 1991; Ye and Vamer, 1991; Bailey et al., 1992; Taylor, 1992b). The technique was described by Cassab and Vamer (1987) and consists of transfemng cellular materials from the freshly cut surface of tissues to nitrocellulose membranes.We sought to develop this method in a more quantitative way, i.e. measuring the amount of material transferred from one cell type or tissue. Quantitative interpretation in antibody reactivity on tissue prints is difficult if one tries to explain differences in the degree of cross-reactivity without knowing the amount of protein transferred from each tissue; specific signal might simply reflect the bulk transfer of protein from regions of high cell density and yield no information on the MATERIALS AND METHODS Plant TissueCelery petioles (Apium graveolens L.) were purchased from local markets. The petioles (Fig. 1A) selected represent two developmental stages: Y (nongreen petioles from the center of the stalk) and more M (green extemal petioles). The sections printed of both Y and M tissues were taken from the central portion of each petiole used. The arrows show this area only in the most M petiole. Tissue PrintingFreehand cross-sections of approximately 1-to 3-mm thickness were made with a razor blade from celery petioles (Vamer and Taylor, 1989;T...