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Plant Physiology 132:1610-1622 (2003) © 2003 American Society of Plant Biologists Altered Gene Expression in Three Plant Species in Response to Treatment with Nep1, a Fungal Protein That Causes NecrosisAlternate Crops and Systems Laboratory, U.S. Department of Agriculture/Agricultural Research Service, Beltsville Area Research Center-West, Beltsville, Maryland 20705 (S.E.K., B.A.B.); Department of Biology and Microbiology, University of Wisconsin, Oshkosh, Wisconsin 54901 (T.A.K.); and Plant Sciences Institute, Beltsville Area Research Center-West, Beltsville, Maryland 20705 (J.D.A.)
Nep1 is an extracellular fungal protein that causes necrosis when applied to many dicotyledonous plants, including invasive weed species. Using transmission electron microscopy, it was determined that application of Nep1 (1.0 µg mL–1, 0.1% [v/v] Silwet-L77) to Arabidopsis and two invasive weed species, spotted knapweed (Centaurea maculosa) and dandelion (Taraxacum officinale), caused a reduction in the thickness of the cuticle and a breakdown of chloroplasts 1 to 4 h after treatment. Membrane breakdown was most severe in cells closest to the surface of application. Differential display was used to isolate cDNA clones from the three species showing differential expression in response to Nep1 treatment. Differential gene expression was observed for a putative serpin (CmSER-1) and a calmodulin-like (CmCAL-1) protein from spotted knapweed, and a putative protein phosphatase 2C (ToPP2C-1) and cytochrome P-450 (ToCYP-1) protein from dandelion. In addition, differential expression was observed for genes coding for a putative protein kinase (AtPK-1), a homolog (AtWI-12) of wound-induced WI12, a homolog (AtLEA-1) of late embryogenesis abundant LEA-5, a WRKY-18 DNA-binding protein (AtWRKY-18), and a phospholipase D (AtPLD-1) from Arabidopsis. Genes showing elevated mRNA levels in Nep1-treated (5 µg mL–1, 0.1% [v/v] Silwet-L77) leaves 15 min after Nep1 treatment included CmSER-1 and CmCAL-1 for spotted knapweed, ToCYP-1 and CmCAL-1 for dandelion, and AtPK-1, AtWRKY-18, AtWI-12, and AtLEA-1 for Arabidopsis. Levels of mRNA for AtPLD-1 (Arabidopsis) and ToPP2C-1 (dandelion) decreased rapidly in Silwet-L77-treated plants between 15 min and 4 h of treatment, but were maintained or decreased more slowly over time in Nep1-treated (5 µg mL–1, 0.1% [v/v] Silwet-L77) leaves. In general, increases in mRNA band intensities were in the range of two to five times, with only ToCYP-1 in dandelion exceeding an increase of 10 times. The identified genes have been shown to be involved or are related to gene families that are involved in plant stress responses, including wounding, drought, senescence, and disease resistance.
The necrosis-inducing protein Nep1 is an extracellular protein produced by Fusarium oxysporum in liquid cultures (Bailey, 1995  ;
Bailey et al., 1997). The gene
for Nep1 has been cloned (GenBank accession no. AF036580) and partially
characterized (Nelson et al.,
1998; Bailey et al.,
2002). Antigenically related proteins of similar size and activity
are produced by Fusarium accuminatum and Fusarium avenaceum
(Bailey et al., 1997).
Subsequent to the original isolation and characterization of NEP1,
related gene and gene products have been identified (GenBank) as being
produced by species of Pythium, Streptomyces, Phytophthora, and other
genera, which demonstrates that Nep1-related proteins are widely produced by
taxonomically divergent microorganisms. Nep1 production was disrupted in
F. oxysporum f. sp. erythroxyli using gene replacement, thus
providing evidence that Nep1 production is not critical to development of
Fusarium wilt of Erythroxylum coca
(Bailey et al., 2002).
Nep1 kills cells of many different dicotyledonous plants but does not
damage monocots (Bailey, 1995
In addition to necrosis, tobacco (Nicotiana tabacum) cellular
responses to Nep1 treatment include ethylene production, active oxygen
production, altered cell respiration, K+ and H+ channel
fluxes, and altered gene expression
(Jennings et al., 2001
The Necrotic Response to Nep1 Spotted knapweed and dandelion were much more sensitive to Nep1 than Arabidopsis was in these studies. Spotted knapweed and dandelion had 25% and 75% leaf necrosis 24 h after treatment with 1 and 5 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77), respectively. Necrotic spots on spotted knapweed and dandelion were uniformly spread and coalesced, sometimes covering whole leaves by 48 h after treatment. Arabidopsis had only 3% necrosis after treatment with 5 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77), and necrosis caused by the 1 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77) was not measurable macroscopically on Arabidopsis. Necrosis on Arabidopsis was observed as a tip burn that did not develop further after 24 h. The Silwet-L77 treatment (0.1%, v/v) did not cause measurable necrosis on any of the three plant species.
Application of Nep1 (1 µg mL–1, 0.1% [v/v] Silwet-L77) caused a noticeable decrease in the thickness of the leaf cuticle between 1 and 4 h after treatment (Figs. 1 and 2). Changes in dandelion (data not shown) and Arabidopsis (Fig. 2) were apparent at the 1- and 4-h time points, and changes in spotted knapweed were observed at the 4-h time point (Fig. 1). The cuticle appears in micrographs as a darker layer on the upper (abaxial) surface of epidermal cell walls. The dark staining is due to the presence of waxes and esters in the cuticle that react with the osmium tetroxide used as a postfixative on these samples. The decrease in cuticle thickness took on two forms. In the treated spotted knapweed leaves, the cuticle appeared to be degrading and "sloughing off" (as indicated by arrows), compared with the cuticle in the Silwet-L77-treated (0.1%, v/v) leaf, which remained a solid dark gray layer (Fig. 1B, arrows). In dandelion (data not shown) and Arabidopsis (Fig. 2B), the cuticle (as indicated by arrows) of Nep1-treated (1 µg mL–1, 0.1% [v/v] Silwet-L77) leaf tissues appeared to be compressed to a thinner, more darkly stained layer. This is in comparison with the cuticles in Silwet-L77-treated (0.1%, v/v) tissues, where the cuticle was thicker and more lightly stained (Fig. 2A).
Chloroplasts withstood the fixation process better than most other organelles, allowing their detailed description. Data concerning the responses of other organelles and membranes were inconclusive. Nep1 (1 µg mL–1, 0.1% [v/v] Silwet-L77) had a marked effect on the appearance of chloroplasts and chloroplast membranes in all three species studied (Figs. 1 and 2). In general, application of Nep1 caused breakdown of the thylakoid and granal membrane structures that was noticeable in dandelion (data not shown) and Arabidopsis (Fig. 2D) at the 1- and 4-h time points; changes in spotted knapweed (Fig. 1D) were observed at the 4-h time point. Also noticeable was the disappearance of starch granules from chloroplasts of treated leaves (Fig. 2D). This is of note because all of the leaves were harvested at the same time of day and under the same light conditions. The chloroplasts themselves also appeared to swell and become misshapen after treatment with Nep1 (1 µg mL–1, 0.1% [v/v] Silwet-L77), and the outer chloroplast membrane appeared to deteriorate, which was especially evident in Arabidopsis (Fig. 2D). Last, chloroplasts from Nep1-treated (plus 0.1% [v/v] Silwet-L77) leaves often appeared to have more lipophilic bodies compared with chloroplasts from Silwet-L77-treated (0.1%, v/v) leaves (Figs. 1D and 2D). Note that all of the observations above pertain to chloroplasts nearer the point of Nep1 application. Chloroplasts on the opposite side of the leaf from the application site showed no change in appearance (data not shown).
Taking into account the three plant species being studied, a total of 29 cDNA fragments were cloned and sequenced based on their consistent differential expression on two replicate differential display gels. Of the 29 clones, 15 showed no significant homology to known sequences using blastx and tblastx. Nine of the 15 unidentified clones were isolated from spotted knapweed and five were isolated from dandelion. Two clones, one from spotted knapweed and one from dandelion, showed homology to unknown Arabidopsis proteins. The remaining 11 clones (Table I), which showed significant homology to known genes or gene families, were selected for further study.
Eleven cDNA clones (Table I) were used as probes on northern blots of total RNA from Silwet-L77 (1%, v/v) and Nep1-treated (1 µg mL–1, 0.1% [v/v] Silwet-L77) leaves for the three plant species being studied without consideration of the origin of the clone. Only CmTIF-1 and CmCAL-1 hybridized to total RNA from plant species other than the species from which the clone was obtained (data not shown). CmTIF-1 hybridized to total RNA from all three species but did not show differential expression, and CmCAL-1 hybridized with total RNA from spotted knapweed and dandelion showing differential expression in both species. Only the cDNA clone/plant species combinations that showed significant hybridization to northern blots in the studies of the 1 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77) treatments were used when probing northern blots of total RNA from leaves treated with 5 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77). Unless otherwise indicated, the results for studies using the 1 µg mL–1 Nep1 rate (plus 0.1% [v/v] Silwet-L77) were similar to those presented for the 5 µg mL–1 Nep1 rate (plus 0.1% [v/v] Silwet-L77), and the data are not shown. CmB-Glu-1 showed low level induction in Silwet-L77-treated (0.1%, v/v) spotted knapweed 4 h after treatment (data not shown) and was not studied further.
Spotted Knapweed
Total RNA (20 µg lane–1) extracted from spotted knapweed leaves after treatment with Nep1 (5 µg mL–1 Nep1, 1% [v/v] Silwet-L77) had approximately two times more CmSER-1 mRNA than samples extracted from Silwet-L77-treated (1%, v/v) leaves at all time points sampled (Fig. 4). The quantity of CmSER-1 mRNA in Silwet-L77-treated spotted knapweed leaves remained steady at 15 min, 1 h, and 4 h.
Dandelion
ToCYP-1 mRNA accumulated in response to Nep1 treatment. The Nep1 rate strongly influenced the time course of ToCYP-1 mRNA accumulation (Fig. 6). Fifteen minutes after treatment, total RNA from the 5 µg mL–1 Nep1-treated (plus 0.1% [v/v] Silwet-L77) dandelion leaves had 11 times more ToCYP-1 mRNA than Silwet-L77-treated (1%, v/v) dandelion leaves. The high level of ToCYP-1 mRNA was maintained for 4 h. Treatment of dandelion with 1 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77) resulted in ToCYP-1 mRNA accumulation at 1 and 4 h but not at 15 min.
The quantity of ToPP2C-1 mRNA detected on northern blots of total RNA isolated from Nep1-treated (5 µg mL–1, 1% [v/v] Silwet-L77) dandelion leaves remained steady between 15 min and 4 h after treatment (Fig. 7). The quantity of ToPP2C-1 mRNA detected decreased on northern blots of total RNA isolated from Silwet-L77-treated (1%, v/v) dandelion leaves between 15 min and 4 h. As a result, six times more ToPP2C-1 mRNA was detected on northern blots of total RNA from Nep1-treated dandelion leaves compared with northern blots of total RNA from Silwet-L77-treated dandelion leaves 4 h after treatment.
Arabidopsis
Fifteen minutes after treatment, 4.3 times more AtWRKY-18 mRNA was detected on northern blots of total RNA (20 µg lane–1) extracted from Nep1-treated (5 µg mL–1, 1% [v/v] Silwet-L77) Arabidopsis leaves compared with northern blots of total RNA from Silwet-L77-treated (0.1%, v/v) Arabidopsis leaves (Fig. 9). AtWRKY-18 mRNA levels decreased between 15 min and 4 h after Nep1 treatment. AtWRKY-18 mRNA levels were most variable 1 h after treatment for Nep1- and Silwet-L77-treated Arabidopsis leaves.
Fifteen minutes after treatment, 1.8 times more AtPLD-1 mRNA was detected on northern blots of total RNA (20 µg lane–1) extracted from Silwet-L77-treated (1%, v/v) Arabidopsis leaves compared with northern blots of total RNA from Nep1-treated (5 µg mL–1, 1% [v/v] Silwet-L77) Arabidopsis leaves (Fig. 10). AtPLD-1 transcript decreased in Silwet-L77-treated Arabidopsis leaves to almost undetectable levels 1 h after treatment. AtPLD-1 mRNA decreased more slowly in Nep1-treated Arabidopsis leaves between 15 min and 4 h. As a result, AtPLD-1 mRNA levels for samples from Nep1-treated Arabidopsis leaves were at least 1.7 times that detected for samples from Silwet-L77-treated leaves 1 and 4 h after treatment.
When AtWI-12 and AtLEA-1 were used as probes on northern blots, they produced similar expression profiles (Figs. 11 and 12). Fifteen minutes and 1 h after treatment, AtWI-12 and AtLEA-1 mRNA levels for samples from Nep1-treated (5 µg mL–1, 1% [v/v] Silwet-L77) Arabidopsis leaves were at least three times that observed for samples from Silwet-L77-treated (0.1%, v/v) Arabidopsis leaves. The mRNA levels for AtWI-12 and AtLEA-1 decreased between 1 and 4 h after Nep1 treatment.
Structural Changes Caused by Nep1 Plant cells respond to Nep1 by rapid structural changes, including the thinning of the cuticle and disruption of chloroplasts. Similar changes could be seen by transmission electron microscopy within 1 h of Nep1 treatment in all three species studied, long before macroscopic necrosis was observed. Arabidopsis was less sensitive to Nep1 at the macroscopic level, but the types of structural changes caused by Nep1 to Arabidopsis closely resembled changes observed in Nep1-treated dandelion.
The chloroplast changes we observed included, most strikingly, a complete
breakdown of the internal chloroplast membranes, breakdown of the outer
chloroplast membrane, loss of starch grains, and a "swelling" of
the chloroplasts. In studies of the hypersensitive response in plant cells,
similar changes to chloroplasts and other cell membrane structures have been
observed (Goodman, 1972
The rapid (1–4 h) changes in cuticle thickness in response to Nep1
treatment were unexpected and raise the possibility that Nep1 directly acts on
the cuticle. The change in cuticle thickness in response to Nep1 was
consistent across the three species and at multiple time points. Many
compounds within the cuticle, such as the epicuticular waxes
(Eglinton and Hamilton, 1967
AtPK-1 in Arabidopsis and ToPP2C-1 in dandelion putatively code for a
protein kinase and a protein phosphatase 2C, respectively
(Table I). Reversible protein
phosphorylation is mediated by protein kinases and protein phosphatases and
serves as a primary regulator of many signal transduction pathways
(Ganguly and Singh, 1999
AtWRKY-18 is the gene for a salicylic acid/pathogen-induced WRKY
DNA-binding protein in Arabidopsis (Table
I). The WRKY DNA-binding proteins make up a superfamily of
transcription factors that are involved in the regulation of gene expression,
including genes involved in plant defense and senescence
(Du and Chen, 2000
CmCAL-1 was induced by Nep1 treatment in spotted knapweed and dandelion.
CmCAL-1 is closely related to genes for calmodulin-like proteins in
Arabidopsis and many other plant species, but did not hybridize to northern
blots of total RNA from Nep1-treated Arabidopsis under the conditions used.
Calmodulin and calmodulin-related genes in Arabidopsis and other plant species
are regulated by physical inducers, including rain, wind, touch, and wounding,
resulting in rapid transcript accumulation and altered development
(Braam and Davis, 1990
Signaling cascades can be triggered by the activation of
phospholipid-cleaving enzymes such as phospholipases C, D (PLD), and A(2)
(Qin et al.,
1997
Nep1 (5 µg mL–1, 0.1% [v/v] Silwet-L77)
induces accumulation of ToCYP-1 transcript in dandelion within 15 min of
treatment and is the most highly up-regulated of the genes studied. ToCYP-1
putatively codes for a cytochrome P450 protein
(Table I). Cytochrome P450
enzymes form a large superfamily of genes
(Nelson et al., 1993
AtWI-12 is related to an elicitor inducible protein in tobacco (59%
identity) and the wound-induced protein WI12 in common ice plant
(Mesembryanthemum crystallinum; 51% identity). Wounding, methyl
jasmonate, and pathogen infection induced local WI12 expression in common ice
plant (Yen et al., 2001
CmSER-1 is most closely related to serpin genes found in barley
(Table I), wheat (Triticum
aestivum), Avena fatua, and Cucurbita maxima in
addition to putative serpins found in Arabidopsis
(Table I). Serpins, Ser
proteinase inhibitors, are well described in higher animals where they
function in divergent biological processes
(Silverman et al., 2001
It remains unclear if the identified responses to Nep1 treatment should be
characterized as a general stress response to a toxin or some form of induced
resistance (Jennings et al.,
2001
Using differential display, we were able to identify cDNA clones from
multiple plant species potentially associated with signal transduction
pathways that are responsive to Nep1, including calcium/calmodulin,
DNA-binding/gene activation, lipid metabolism, reversible phosphorylation, and
phytoalexins biosynthesis. Based on sequence homologies, the cDNA clones
identified are in most cases closely related to genes involved in plant
defense and/or stress responses, including wounding, salicylic acid, drought,
disease resistance, and senescence. Nep1 induced ethylene biosynthesis in many
different plant species and Nep1 has been shown to induce
1-aminocyclopropane-1-carboxylic acid synthase and
1-aminocyclopropane-1-carboxylic acid oxidase transcript accumulation in
tobacco (Jennings et al.,
2001
Nep1 Purification
Nep1 was purified from culture filtrates of Fusarium oxysporum f.
sp. erythroxyli grown for 6 d in Czapek-Dox broth plus 1% (w/v)
casamino acids (Bailey, 1995
Seeds of spotted knapweed (Centaurea maculosa) were collected from local populations in Polo, IL, and seeds of dandelion (Taraxacum officionale) were collected from local populations in Beltsville, MD. Seeds of Arabidopsis (wild type) and the two weed species were planted in 10.2-cm pots filled with Scott's Redi-earth, and plants were grown in ambient light and temperature conditions in a greenhouse. Plants were used in experiments 28 to 32 d after planting.
Nep1 was combined with 1,1,1,3,5,5,5-heptamethyltrisiloxanyl propylmethoxy-poly[ethylene oxide] (Silwet-L77; Witco Corporation, Friendly, WV) for foliar spray applications to plants. Plants were treated with Nep1 (1 or 5 µg mL–1) in 0.1% (v/v) Silwet-L77. Plants treated with Silwet-L77 (0.1%, v/v) were included as a control. Foliar sprays were applied with a sprayer (model 15; Binks, Glendale Heights, IL) at 15 psi and at a rate of 86 mL m–2 (860.9 L ha–1). All sprays were applied between 10 AM and 2 PM when stomata were fully open. Plants were maintained in greenhouse conditions until sampled. Leaves from Silwet-L77(0.1%, v/v) and Nep1-treated (plus 0.1% [v/v] Silwet-L77) plants were collected 15 min, 1 h, and 4 h after spray application, frozen in liquid nitrogen, and stored at –80°C.
Leaf tissues for each plant species were collected 1 and 4 h after
treatment and were prepared for transmission electron microscopy. One leaf was
sampled from three plants of each species for each treatment at each time
point. Tissue squares (1 mm x 1 mm) treated with Silwet-L77 (0.1%, v/v)
or 1 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77)
were dissected under 2.5% (w/v) glutaraldehyde/2% (w/v) paraformaldehyde in 50
mM PIPES buffer (pH 7.6) and were fixed for 24 h at 4°C
(Karnovsky, 1965 Thin sections of embedded material were cut using a ultramicrotome (MT-5000; Sorvall, Kendro Laboratory Products, Newtown, CT) and were picked up onto 200 mesh, formvar-coated nickel grids. Sections were stained with 2% (w/v) uranyl acetate for 10 min, rinsed, then stained with calcined lead stain for 10 min. Sections were viewed and negatives were taken using a transmission electron microscope (EM-10CA; Zeiss, Jena, Germany). Negatives were then scanned and saved as tiff files using a scanner (UMAX Powerlook 1100, UMAX Technologies, Inc., Dallas) connected to a computer (PowerMac G4; Apple Computers, Cupertino, CA).
Total RNA was extracted from leaf tissues using a modified phenol
extraction procedure (Goldsbrough et al.,
1986
DNA was removed from total RNA by DNaseI treatment (MessageClean kit; Genhunter, Nashville, TN). Total RNA was isolated from each of the three plant species 15, 60, or 240 min after treatment with 1 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77). Total RNA isolated from plant tissues 240 min after treatment with Silwet-L77 (0.1%, v/v) was included as the control. RNA was reverse transcribed, and the resulting cDNA was amplified by PCR using arbitrary 13 mers and anchored primers labeled with 5'-rhodamine (RNA Spectra Red kit 1; GenHunter). Eight arbitrary primers (H-AP1 through H-AP8) were paired with three anchored primers for a total of 24 primer combinations. Fluorescently labeled PCR product was separated on a 30 x 40 cm, 6% (w/v) denaturing polyacrylamide gel in Tris borate-EDTA buffer (60 W constant power for 3.5 h), and was imaged at 200 µm resolution using a Cy3 setting (532 nm excitation laser, 555 nm emission filter with a 20-nm band pass filter) on a Typhoon 8600 Variable Mode Imager (Molecular Dynamics/Amersham Biosciences, Piscataway, NJ). Differential display was carried out on replicate total RNA samples for each plant species primer combination. Apparent differences in band intensities in control versus Nep1-treated tissues on the imaged gel were used to locate bands of interest. The opened gel was placed on top of a full-scale printout of the fluorescent image of the gel, and the region containing the band of interest was excised and frozen at –20°C. The number of bands isolated was 186 for spotted knapweed, 266 for dandelion, and 133 for Arabidopsis. Because replicate gels were run, ideally each band should have been isolated twice. Twenty-nine bands of interest were identified by careful comparisons of display patterns and intensities between replicate gels (approximately 10% if all the bands were duplicated) for use in this study. Of the 29 bands, 15 were isolated from spotted knapweed, eight from dandelion, and six from Arabidopsis.
Only DNA bands showing consistent differential expression on two replicate differential display gels were further processed. cDNA was eluted from excised gel slices and was reamplified by PCR using the primer set previously used for differential display PCR, but without fluorescently labeled anchored primer (RNA SpectraKit; GenHunter). cDNA probes were subcloned using the PCR-Trap Cloning System (GenHunter). A subset of the probes were reamplified, filter purified (Geneclean Turbo for PCR; Bio 101, Carlsbad, CA), and sequenced before subcloning as a means of tentatively identifying genes of potential interest based on sequence homology. All subcloned cDNA probes were sequenced to verify identifications made before subcloning (BigDye v. 3.0 dye terminator kit on an ABI 3100 Prism; Applied Biosystems, Piscataway, NJ).
Twenty micrograms of total RNA was denatured in glyoxal/dimethyl sulfoxide
load dye at 50°C for 40 min (NorthernMax-Gly load dye; Ambion, Austin, TX)
and was electrophoresed at 50 V for 4 h on a 1.2% (w/v) agarose gel containing
300 mM Bis-Tris, 100 mM PIPES, and 10 mM
EDTA, at pH 8.0 (1x BisTns-PIPES-EDTA buffer). RNA was attached to
GeneScreen Plus membrane (Perkin-Elmer Biosystems, Boston) by upward transfer
in 10x SSC buffer. After crosslinking RNA to the membrane using the
autocrosslink setting (UV Stratalinker 8600; Stratagene, La Jolla, CA),
membranes were air-dried and stored between sheets of Whatman 3MM (Whatman
International, Ltd., Maidstone, Kent, UK) paper at –20°C. cDNA
probes were gel purified on a 1.5% (w/v) agarose gel (Geneclean II; Bio 101).
For each probe, 35 ng of cDNA was labeled with [ Regardless of plant species, probes were initially hybridized to three replicate blots, each containing total RNA from Nep1- (Nep1 1 µg mL–1, 0.1% [v/v] Silwet-L77) and Silwet-L77-treated (0.1%, v/v) tissues for the three species, spotted knapweed, dandelion, and Arabidopsis. Only probe/species combinations that showed significant hybridization in the studies using the 1 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77) rate were included in studies using the 5 µg mL–1 Nep1 (plus 0.1% [v/v] Silwet-L77) rate. Radioactively labeled cDNA probes were denatured at 95°C to 100°C for 5 min. The probe was added to 5 mL of fresh ExpressHyb solution, and blots were incubated with continuous shaking at 68°C for 2 h. Blots were washed according to the ExpressHyb protocol, covered in plastic wrap, exposed to x-ray film at –70°C with two intensifying screens, and/or exposed to a storage phosphor screen (Molecular Dynamics/Amersham Biosciences), and imaged at 200 µm resolution on a Typhoon 8600 Variable Mode Imager (Molecular Dynamics/Amersham Biosciences). After imaging, the replicate blots were washed, scanned to verify that no probe remained, and probed a second time with radioactively labeled 28S ribosomal cDNA probe.
Blots probed with 28S ribosomal cDNA were imaged and band volumes were calculated using ImageQuant Version 5.2c software (Molecular Dynamics/Amersham Biosciences). To correct for variations in loading volume on each replicate blot, the ratio of 28S ribosomal cDNA band volume for each treatment combination (plant species/time/Nep1 rate) was determined relative to the 15 min Silwet-L77 (thus arbitrarily set to one) control sample. The band volumes for blots probed with cDNAs of interest were corrected by multiplication by the appropriate 28S ribosomal cDNA ratio. For ease of presentation, the corrected band volumes were converted to ratios relative to the 15-min Silwet-L77 (thus arbitrarily set to one) control sample for each treatment combination on each replicate blot. The band volume ratios for each treatment combination were averaged using data from three replicate blots. The figures include the average band volume ratios (plus or minus 1 SE) for each treatment combination in addition to representative autoradiograms. The three replicates for each treatment combination consisted of two samples from an initial experiment and a third sample from a subsequent experiment carried out approximately 6 months after the initial experiment. Received December 27, 2002; returned for revision January 30, 2003; accepted February 16, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.019836. * Corresponding author; e-mail baileyb{at}ba.ars.usda.gov; fax 301–504–5823.
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TOP
ABSTRACT
RESULTS
(
-32P] dCTP
using a Random Primed DNA Labeling kit (Applied Biosystems, Foster City, CA)
and gel filtered (Edge Gel Filtration Cartridges; Edge BioSystems,
Gaithersburg, MD). Blots were prehybridized in ExpressHyb solution (BD
Biosciences/CLONTECH, Palo Alto, CA) at 68°C for 1 h. 
and regulation
of plant PLD
