INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

In most plant-pathogen interactions, the occurrence of disease is the exception, not the rule. A complex network of signaling between the host and the pathogen usually results in plant disease resistance. A general type of resistance against a broad range of pathogens is known as non-host resistance. This inherent plant resistance response is a complicated interaction that involves the recognition of multiple elicitors. Such elicitors may be components of the pathogen such as chitosan (Chit), a de-acetylated derivative of fungal cell wall chitin, or pathogen exudates. These elicitors may also be derived from the host and include various plant polysaccharides from the digested cell wall matrix (for review, see Dixon and Lamb, 1990). In pea (Pisum sativum L. cv Alcan), such recognition signals the transcriptional induction of at least 20 defense genes (Wagoner et al., 1982; Hadwiger and Wagoner, 1983; Loschke et al., 1983; Riggleman et al., 1985).

Disease resistance, in general, is initiated by a recognition event(s) that triggers a rapid hypersensitive response (HR). This differs from the susceptible response, which is associated with a weaker or delayed HR. Whereas little is currently known with regard to the induction mechanism of pathogenesis-related (PR) gene expression in response to numerous pathogens and chemically diverse elicitors, the HR in each of these interactions shares some of the following features: activation of PR genes coding for proteins such as -glucanases, chitinases, defensins, and enzymes involved in phytoalexin, lignin, and lignan synthesis; membrane depolarization; oxidative burst; activation of endonucleases and DNA cleavage (Greenberg, 1997; Mittler and Lam, 1997); and activation of transcription factors (Zhou et al., 1997).

Our laboratory previously reported that certain DNA-damaging agents such as actinomycin D (ActD) and mitomycin C (MMC) induce phytoalexin accumulation at a level comparable to that of biotic elicitors in pea tissue (Schwochau and Hadwiger, 1969; Hadwiger and Schwochau, 1971). Likewise, psoralen treatment in the presence of UV₃₆₅, which crosslinks the pea DNA, increases pisatin levels, and induces the expression of some pea PR genes (Parsons and Hadwiger, 1998). The biotic elicitors in the pea system include intact spores of the bean pathogen, Fusarium solani f. sp. phaseoli (Fsph), as well as Fsph fungal components such as Chit (Kendra et al., 1989; Hadwiger et al., 1994; Hadwiger, 1999) and Fsph DNase, an endonuclease exuded from fungal mycelium (Gerhold et al., 1993; Hadwiger et al., 1995). Each of these biotic agents elicit disease resistance responses, including the activation of PR genes (Chang et al., 1992; Hadwiger et al., 1995).

Because many DNA-damaging agents are well characterized in their specific mode of action (Chabner et al., 1996), we can examine the effects of these different abiotic agents to gain insight into a potential mechanism that would explain how certain defense gene promoters are activated during a host-pathogen interaction. The abiotic agents are characterized by their ability to cause DNA strand breaks. It has been proposed that these agents eventually disrupt the normal conformation of chromatin and possibly facilitate global plant defense gene activation (Hadwiger, 1988).

In this study, we evaluated reporter gene expression of PR gene promoter:GUS chimeric genes transferred into the heterologous host tobacco. The promoter from the pea disease resistance response gene DRR206 (Fristensky et al., 1988; Culley et al., 1995) was used as the focus of the study. The DRR206 structural gene shares homology with a dirigent protein involved in lignan biosynthesis (Davin et al., 1997; Gang et al., 1999). Promoters of pea genes Phe ammonia lyase (PAL) and chalcone synthase (CHS) were also assayed since they encode important enzymes in the production of phytoalexins, including pisatin. Promoters from the pea gene DRR206, Arabidopsis PAL gene (Huang and McBeath, 1994), and a bean CHS gene (Schmid et al., 1990) linked to the -glucuronidase (GUS) reporter gene were used to evaluate gene expression in response to the various agents in transgenic tobacco plants. The mRNA accumulation patterns of these genes and the kinetics of GUS catalytic activity indicate that DNA damage or chromatin alteration in response to both biotic and abiotic inducers may influence the promoter activity of these genes, even in a heterologous system. Most importantly, the inducible nature of DRR206 presented herein suggests that the DRR206 promoter may be a valuable tool in engineering disease resistance in combination with the appropriate defense gene.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Activation of Defense Gene Expression in Pea

The accumulation of mRNA for pea genes DRR206, CHS, and PAL was assessed following the application of DNA-damaging agents. The mechanisms of action of these various agents are outlined in Figure 1 (Kopka et al., 1985; Chabner et al., 1996). DRR206 mRNA was detected at low levels by wounding since its RNA product was detectable in the water control. However, its expression was undetectable in the untreated control where the intact pods were frozen immediately without splitting (Fig. 2). The abiotic agents that cross-link or intercalate DNA, MMC, and ActD, respectively, were able to induce all three genes within 6 h. The topoisomerase inhibitor etoposide (Etop) was only marginally effective in the activation of these defense genes (Fig. 2). UV irradiation, which induces the formation of pyrimidine dimers, generated levels of defense gene activation response lower than the maximal level obtained with some other DNA-specific agents (Fig. 2). Hydrogen peroxide (H₂O₂), capable of generating free radicals, resulted in relatively low PAL and DRR206 mRNA accumulation (Fig. 2). Low accumulation of gene-specific mRNA is evident in endocarp tissue treated with the pyrimidine analog, 5-fluorouracil (FU) (Fig. 2). Cisplatinum (CisP), another DNA cross-linking/alkylating agent, caused a low accumulation of pea gene DRR206 RNA and high levels of PAL and CHS RNA at 3 h, and all genes were active within 6 h (Fig. 2). However, the induction by CisP was lower compared with that of MMC. Treatment with netropsin (Netro), a DNA minor groove-binding compound, resulted in only low to moderate levels of induction (Fig. 2).

View larger version (44K): [in this window] [in a new window]   Figure 1.   A schematic presentation of abiotic agents used in this study.

View larger version (32K): [in this window] [in a new window]   Figure 2.   Northern analysis of pea pod RNA following treatments with biotic and abiotic agents at 3 and 6 h. The blot was hybridized with pea DRR206, PAL, and CHS probes (see "Materials and Methods"). The agents are abbreviated and were applied at varying concentrations: No trt, no treatment; MMC (50 µg mL1); NaCl (1.2 mg mL1); ActD (12.5 µg mL1); Etop (12.5 µg mL1); Netro (125 µg mL1); CisP (50 µg mL1); UV (589 J m2); H2O2 (10 mM); FU (200 µg mL1); Chit (1 mg mL1); DNase, purified fungal exudate from Fusarium solani spp (20 units µL1); Fspi (>107 spores mL1); and Fsph (>107 spores mL1).

As expected, biotic elicitors were potent inducers of all three defense genes (Fig. 2). High inoculum levels of both the true pea pathogen, F. solani f. sp. pisi (Fspi), and the bean pathogen, Fsph, were used to evaluate the speed at which the representative pea genes were activated. At these levels, both fungi generated significant increases in CHS-homologous RNA 3 h after treatment. Chit and DNase released by Fsph, both efficiently increased PAL, CHS, and DRR206 RNA at both time points.

Cytology of Elicitor Activity Inside the Pea Cell

To determine if various DNA-specific agents mimic the nuclear-altering effects of the biotic elicitors, cytological analyses were conducted. Nuclear changes were evaluated with a fluorescent nuclear dye, 4',6'-diamidino-2-phenylindole (DAPI), while cell viability was evaluated by trypan blue staining. Cell viability assays were also conducted with fluorescein diacetate (data not shown), a compound that is cleaved to yield fluorescein in the presence of lipase activity in the cell (Guilbault and Kramer, 1964). Overall, the percent cell death observed was low in the whole plant tissues (intact pea pod endocarp). Unlike protoplast preparations or cell suspensions, whole tissues are not subjected to stresses inherent to those preparations.

Cytological analyses in the presence of DAPI revealed that this dye may have penetrated more efficiently into cells that did not accumulate callose. UV-irradiated cells did not accumulate detectable callose in the plant cell walls even after 24 h, which may explain how DAPI intercalated more efficiently in those cells exposed to UV prior to staining. In contrast, callose deposition was readily observed in pea pods treated with biotic elicitors and H₂O₂ within 9 h. Over a period of 24 h following treatment, UV, CisP, Chit, and the fungal pathogens caused the most nuclear distortions/collapse (Fig. 3). Note that for the Fspi treatment, the germinated spores could not be removed from the tissue after 9 h, possibly quenching the fluorescence of many plant nuclei. Therefore, the number recorded in this particular instance is likely to be underestimated. H₂O₂-treated cells undergoing nuclear collapse had an increased degree of yellow background fluorescence around the entire perimeter of the cell after 18 h (data not shown), suggesting total nuclear collapse.

View larger version (42K): [in this window] [in a new window]   Figure 3.   Percentage of pea pod endocarp cells undergoing nuclear distortions/collapse with the various biotic and abiotic treatments at 3, 9, 18, and 24 h. Pod halves were stained with DAPI, observed, and counted under fluorescence microscopy in triplicate experiments. Treatments same as in Figure 2. An asterisk indicates P < 0.05 compared with the water control at the same time point.

Cytological analyses in the presence of trypan blue indicated that a portion of the cells that had previously undergone nuclear distortion, subsequently underwent cell death. Thus, these cells apparently sustained DNA damage at irreparable levels. But for the majority of treatments, the percent cell death was less than 10% (Fig. 4). Treatments with Etop, CisP, fluorouracil, and biotic agents were associated with a higher percentage of cells with nuclear distortion than the actual percentage of cells that underwent cell death, suggesting that an efficient DNA-repair process is present in pea. In endocarp tissue treated with ActD, Netro, or UV, there was little difference between the percent nuclear distortion and the percent cells undergoing cell death. It is interesting that the DNA crosslinking agent MMC, a potent inducer of all three defense genes, did not cause an excessive level of nuclear distortion or cell death.

View larger version (30K): [in this window] [in a new window]   Figure 4.   Percentage of pea pod endocarp cells experiencing cell death at 3, 9, 18, and 24 h. Stained with trypan blue, observed, and counted under light microscopy in quadruple experiments. Treatments same as in Figure 2. An asterisk indicates P < 0.05 compared with the water control at the same time point.

Promoter Activation in Transgenic Tobacco

To determine if any of the defense gene promoters respond to these biotic and DNA-damaging agents following transfer to a different plant system, promoters from bean CHS, Arabidopsis PAL, and pea DRR206 genes were evaluated in tobacco. Tobacco plants used had been transformed with DNA constructs containing promoters from a representative of each of these defense genes and the 35S promoter linked to the GUS reporter gene. Each transgenic tobacco plant was treated with the same abiotic and biotic elicitors used in the pea analysis. However, the treatment concentrations were higher because the sensitivity level of mature tobacco leaf in preliminary experiments appeared lower than that of immature pea pod endocarp tissue. Fluctuations of GUS activity occur typically among replicated samples taken from the same leaf (for review, see Gatz, 1997). Thus, the GUS expression system was used primarily to determine qualitatively whether the elicitors did or did not activate the specific promoters in a heterologous tobacco system.

Evaluation of the pea DRR206 promoter-linked GUS fusion gene (Fig. 5) revealed activation by a majority of the treatments: the DNA cross-linking agent MMC, the intercalator ActD, the topoisomerase inhibitor Etop, as well as by H₂O₂, which generates free radicals. Netro the DNA minor groove binding compound, the base analog FU, and the alkylating agent CisP were ineffective in inducing the pea DRR206 promoter in tobacco. The natural tobacco pathogen, Pseudomonas syringae pv tabaci (Ps tabaci), also activated this pea promoter along with the other biotic elicitors. Live Ps tabaci induction exceeded that of its nutrient broth yeast (NBY) culture medium. The latter induction exceeded the water control, possibly due to eliciting components contributed by the yeast extract. The overall pattern of GUS activity expressed in tobacco in response to these elicitors did not closely correlate with that observed in the pea northern analysis (Fig. 2). Unlike the activation of the DRR206 promoter in transgenic tobacco, most of the abiotic agents did not efficiently activate the Arabidopsis PAL promoter-linked GUS gene above the level of the water control (Fig. 5). Only MMC, H₂O₂ and the biotic elicitors activated the Arabidopsis promoter, as did the natural tobacco pathogen, Ps tabaci. The tobacco plant with the bean CHS promoter-linked with the GUS gene generally expressed lower total GUS activity (Schmid et al., 1990) with all of the elicitors. The DNA cross-linking agent MMC and all the biotic elicitors were able to induce the CHS promoter as did all of the biotic elicitors. Some of the elicitors that were able to induce the specific CHS promoter in pea did not consistently activate the corresponding promoter in the transgenic tobacco (Fig. 5).

View larger version (41K): [in this window] [in a new window]   Figure 5.   Promoter activation using the GUS-reporter gene in transgenic tobacco leaves 9 h following treatments with abiotic and biotic agents. Triplicate leaf discs were assayed for GUS activity via fluorescence min1 emitted. Constitutive GUS expression was driven by the CaMV 35S promoter, 35S::GUS; Xanthi, N. tabacum cv Xanthi is the non-transformed wild-type; DNase, purified fungal exudate from Fusarium solani spp.; A600 = 4.0.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Only a few recent reports exist on the effects of chemical DNA-damaging agents on plants (Iona et al., 1998; Vonarx et al., 1998; Albinsky et al., 1999). By comparing the patterns of defense genes expression with biotic and abiotic agents, we have demonstrated that DNA damage, or the likely alteration of chromatin that accompanies this damage, can be a signal for the inducible expression of the plant defense genes assessed in this study. Yet we have also demonstrated that significant differences in this expression pattern occur in different plant species. Some of these differences in gene expression may be related to the slight differences in transcriptional machinery within the species and positional effects related to the site of insertion within the tobacco genome. But certain elicitors consistently induce the expression of defense genes, regardless of the genetic background.

The molecular mechanism(s) triggering non-host resistance responses remain unclear. However, some biotic elicitors have the potential to directly cause chromatin alterations. For example, marked alterations of chromatin are detectable by sedimentation velocity analysis and electron microscopy in pea cells 30 min following the inoculation with Fsph (Hadwiger and Adams, 1978). Specifically, the pea cell chromatin in the inoculated tissue was dispersed relative to the water controls. The polycationic compound Chit released from Fsph has a high affinity for DNA and has been shown to enter the plant nucleus (Hadwiger et al., 1981) and induce pisatin synthesis (Hadwiger and Beckman, 1980). Moreover, Chit is also capable of causing DNA strand breakage in vitro (Kashige et al., 1994). Therefore, Chit may have a dual function in signaling either by competing with histones or non-histone proteins for binding sites within the chromatin and a distortional function in that Chit may contribute to DNA strand breakage. Chit, alternately, may signal by binding to receptors on the plant cell membrane (Kauss et al., 1989), triggering a kinase cascade that eventually modulates chromatin architecture. Another biotic elicitor, a DNase released by the Fsph pathogen, is capable of causing single strand breaks in plant DNA (Gerhold et al., 1993). Cleavage of DNA within the accessible regions of chromatin by DNase and/or the initiation of repair processes could potentially alter chromatin structure and signal transcriptional changes. Topoisomerases, which also cleave DNA, are known to play a critical role in relaxing supercoils generated during transcription. The specific distribution of topoisomerase II within cellular chromatin is indicative of the involvement of topoisomerase II in active transcription. That is, DNA conformational changes occurring in transcription can be modulated by topoisomerases as suggested by the twin-supercoiled-domain model of transcription. In this model, positive and negative supercoils are generated ahead of and behind the transcription complex, respectively (Liu, 1989). Thus, supercoiling may also be alleviated by all elicitors capable of causing DNA strand breakage.

Among the abiotic compounds used in this study, ActD has been the most thoroughly characterized in regard to its mode of action. DNA-intercalating topoisomerase poisons, such as ActD, are readily incorporated within the DNA and are known to cause DNA strand breakage that can restructure local regions of the chromatin (Workman and Kingston, 1998). ActD has been predominantly used as a transcription inhibitor (Chabner et al., 1996) that inhibits RNA polymerase at concentrations of one molecule of ActD per 270 bp (Hyman and Davidson, 1970). However, the inducing concentration of ActD applied to pea tissue is approximately 1 molecule per 10,000 bp (Hadwiger et al., 1974), indicating that the induction is probably unrelated to the inhibition of RNA polymerase. Prior to DNA cleavage, ActD intercalation can have a major effect on DNA conformation (Sobell et al., 1977). Alteration of DNA by its intercalation has been proposed to effect DNA/chromatin on a global scale (Ross et al., 1979), which precedes transcription inhibition. Hence, incorporation of a DNA-specific compound in a promoter/enhancer region may induce specific genes prior to overall transcription inhibition.

The antitumor drug MMC is known to crosslink DNA (Iyer and Szybalski, 1963) and induce pea defense gene expression and the synthesis of the phytoalexin, pisatin (Hadwiger and Schwochau, 1971). It is interesting that while levels of defense gene induction were markedly elevated in response to the MMC concentration in these experiments, levels of cell death were comparable with those of the water control. Thus, our results suggest that cell death is not essential for defense gene activation. Instead, in consideration of the DNA crosslinking activity of MMC (Iyer and Szybalski, 1963), the alterations of DNA/chromatin following MMC treatment are more likely than cell death to influence defense gene expression.

Some abiotic agents/elicitors used in this study require that cells be actively dividing to cause DNA strand breakage or to incorporate into the DNA. But the endocarp cells of pea pods 1 to 2 cm in length used in these experiments have entered a cell elongation stage and have essentially ceased cell division. Etop is known to form a ternary complex with topoisomerase II and DNA, which in dividing cells results in double-stranded DNA breaks and further inhibits the function of the topoisomerase in re-annealing DNA (Chabner et al., 1996). Also, unlike ActD, Etop is a nonintercalative topoisomerase inhibitor, suggesting that intercalation or the DNA damage incurred by crosslinking may be required for the induction of the PR genes assessed in this study. The base analog FU was not an efficient elicitor, which may relate to an inefficient incorporation of this pyrimidine analog into DNA in tissue undergoing limited DNA replication.

Considerable interest has been directed to the accumulation of H₂O₂, superoxides, and other reactive oxygen intermediates that are detected very early in most plant-pathogen interactions, and all of these have been shown to be capable of damaging DNA (Levine et al., 1996). These reactive oxygen intermediates reportedly play a role in programmed cell death of both plants and animals (for review, see Jabs, 1999). In our study, DRR206 and PAL genes surprisingly were not induced in immature pea endocarp cells even at the substantial levels of H₂O₂ tested. This may be due to efficient DNA-repair, free-radical scavenger systems, or that H₂O₂ is unrelated to the signaling of defense genes in the pea endocarp system. In this study, exogenously applied H₂O₂ (10 mM) did not induce the expression of pea defense genes DRR206 or PAL over water control levels within 3 h, whereas CHS was induced at relatively low levels. This was unexpected since H₂O₂ has been reported to be an integral part of plant defense in vivo, having a dual role as a local trigger of programmed cell death and as a diffusible signal for neighboring cells (Levine et al., 1994). The low accumulation levels of PR gene mRNAs in H₂O₂-treated pea endocarp tissue occurred in association with a slight increase in the level of non-viable cells. In general, with notable exception of MMC, the number of non-viable cells in this study is positively correlated with the levels of defense gene expression. Such results have been interpreted as "cross-talk" between the pathways leading to cell death and those leading to defense gene expression in other systems. In consideration of this correlation, our results indicate that the defense gene induction process is initiated before the onset of cell death. Furthermore, Ryerson and Heath (1996) concluded that H₂O₂ does not cause the same type of cell death as the fungal pathogen or fungal exudates, a type of programmed cell death characterized by DNA laddering.

UV-induced pyrimidine dimerization leads to a rapid nucleotide excision repair and thus DNA strand breaks may exist only transiently (Xu et al., 1998). The maximum potential induction of defense genes following UV irradiation may also be partially reduced by an increase in cell death (Fig. 4), which Danon and Gallois (1998) associated with the characteristic apoptotic DNA laddering. Despite the fact that plants are capable of repairing such DNA damage, the dosage of UV irradiation in these experiments clearly led to the highest relative level of cell death. However, the relative induction of DRR206 in response to UV irradiation, although enhanced, did not exceed the levels of induction of DRR206 by biotic elicitors.

In general, pea gene DRR206 was induced later than PAL or CHS, perhaps because pea gene DRR206 is used in secondary metabolism subsequent to PAL; pea gene DRR206 is known to possesses sequence homology with an enzyme in the metabolic pathway for lignan production (Davin et al., 1997; Gang et al., 1999). These data with biotic elicitors are consistent with PAL and CHS functioning earlier in the metabolic pathway. Although the PAL and CHS genes are highly conserved within the plant kingdom, when the promoters are transferred into another distantly related plant species, there are very subtle differences in inducibility. This may be explained by the promoter/enhancer regions being quite different among plant species and additionally with differences in the roles of cis- and trans-acting regulatory DNA elements (Martini et al., 1993; Kato et al., 1995).

In addition to the potential role of the pathogen and DNA damage-induced chromatin alteration in the mechanism of defense gene induction, there are numerous other potential mechanisms that may explain this type of defense gene regulation. For example, compounds such as Netro and UV₃₆₀-activated psoralen are known to bind AT-rich DNA (Kopka et al., 1985; Parsons and Hadwiger, 1998) sequences found within promoters of many PR genes as well as in other plant gene promoters. The DNA crosslinking activity of UV-activated psoralens has been detected within the vicinity of the induced PR genes DRR206 and DRR49 (Parsons and Hadwiger, 1998), suggesting that the alteration caused by at least one elicitor may be localized rather than having a general effect on the chromatin. Likewise, enzymes involved in DNA repair may be regulated by specific transcription factors (Sakamoto et al., 1998) that are induced following the application of these agents. Overall, the percentage of cells undergoing nuclear distortion in this study exceeded the percentage subsequently undergoing cell death, suggesting the involvement of DNA repair. Little is known about the actual mechanisms of plant DNA repair, however, a number of plant DNA repair homologs have been reported. The discovery in plants of an MSH2-homolog to a human excision repair gene indicates that some mechanisms for DNA repair may be conserved across the two kingdoms (Xu et al., 1998). In other words, the induction of defense responses may be closely linked with plant DNA repair or stress response mechanisms. Such responses in animal cells, including the initiation of programmed cell death, are associated with a DNA damage-inducible p53 protein. However, Klosterman et al. (2000) demonstrated that a p53-like transcription factor homolog of animal p53 is not present in plants. Yet it remains plausible that certain other inducible DNA repair mechanisms in plants may coordinately regulate defense gene expression.

In summary, this study indicates that PR genes are inducible by agents with the potential to intercalate, crosslink, and otherwise cause breaks in DNA. Chemically different elicitors such as ActD and Chit both have the potential to interact with host DNA, and both can induce similar patterns of total transcript levels (Loschke et al., 1983). Many DNA-damaging agents cause nuclear distortion and eventually nuclear collapse, but we have observed that plant cells apparently have mechanisms to recover partially from such stress, preventing a portion of the cells from undergoing cell death. In this study, only a relatively low percentage of the treated cells undergo irreversible cell death in response to certain agents such as MMC while the levels of defense gene activation are high. Therefore, it is not likely that the inductive signal is initiated by cell death. Instead, the signal is more likely to be associated with the rapidly occurring changes in the chromatin. Finally, the action of DNA-specific elicitors is variable, both in activating the gene DRR206, CHS, and PAL promoters inherently in peas and when they are transferred to tobacco, suggesting that DNA alteration-induced promoter activation can be modulated by additional factors. But given the complexity of DNA and all its associated proteins, chromatin may serve as a receptor for both direct signaling of DNA-specific agents and indirect signaling pathways that are transduced through cellular routes prior to their influence on transcription.

Understanding the mode of activation of promoters such as that of pea gene DRR206 could improve strategies for genetically engineering disease resistance in crop plants. We have shown that the promoter of the pea DRR206 gene is activated at low levels in response to wounding, but is expressed at a high level when treated with natural pathogens and DNA-damaging agents. Such a promoter is ideal for the regulatable expression of defense genes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Materials and Treatment

Pea (Pisum sativum L. cv Alcan) seeds were obtained from Roger's Seed Company (Boise, ID). Immature pea pods ranging from 1 to 2 cm in length were split, 25 µL of each treatment was applied to the endocarp surface, and the split pods were incubated at room temperature in a humid environment. For RNA extraction after 3 and 6 h, four pod halves were frozen in liquid nitrogen. For cytological analyses, the pods were soaked in fluorescein diacetate stain at 3 or 9 h after treatment and were viewed using fluorescence microscopy. Cell death counts and distorted nuclei were observed after staining with trypan blue or DAPI, respectively, at 3, 9, 18, and 24 h.

Four transgenic Nicotiana tobacum lines were used in this study: 206-17(3-2), containing a 2.7-kb promoter/enhancer from P. sativum DRR206 (accession no. U11716)::GUS gene fusion (D.E. Culley, unpublished data); SO1, containing a 1.8-kb Arabidopsis PAL promoter::GUS gene fusion (Huang and McBeath, 1994); CG-8, containing a 1.4-kb bean CHS promoter::GUS gene fusion (Schmid et al., 1990); and pBI101, containing a 800-bp 35S::GUS gene fusion (Jefferson et al., 1987). All tobacco lines were maintained in the greenhouse and treated at approximately the six-leaf stage by infiltrating 25 µL of each suspension or solution into tobacco leaf panels with a needleless syringe. Triplicate leaf disc samples were removed with a cork borer (1.6-cm diameter) from the infiltrated area after 9 h and immediately frozen in liquid nitrogen before assaying for GUS activity.

Biotic Elicitors

Fusarium solani f. sp. phaseoli strain W-8 (ATCC no. 38135) and Fusarium solani f. sp. pisi strain P-A (ATCC no. 38136) were maintained on potato dextrose agar (DIFCO Laboratories, Detroit) for 3 to 4 weeks after which macroconidia were scraped off the plates and suspended in sterile water (1 × 10⁷ spores mL¹). Nitrous acid-cleaved Chit (prepared from native crab-shell Chit according to Ride and Drysdale, 1972) was suspended in water to a concentration of 1 mg mL¹. DNase activity from F. solani f. sp. phaseoli exudate (Fsph DNase) was purified and the DNase activity quantified as previously described (Hadwiger et al., 1995). One unit of DNase activity converts one-half of 0.25 µg supercoiled plasmid DNA to linear DNA in 1 h at 37°C. Fsph DNase was diluted to 20 units µL¹ for pea pod treatments. For tobacco leaf treatments, solutions of Fsph DNase were infiltrated at 30 units µL¹. Pseudomonas syringae pv tabaci (Ps tabaci) was grown overnight in 1 mL of nutrient broth yeast extract medium to attain an A₆₀₀ reading of 4.0 and infiltrated into tobacco leaves. As a control, the tobacco leaves also were infiltrated with the NBY extract medium.

Abiotic Elicitors

DNA-damaging agents obtained from Sigma (St. Louis) and were prepared as stock solutions: actinomycin D in water (0.5 mg mL¹), MMC in water (1 mg mL¹), Etop in 50% (v/v) ethanol (1.25 mg mL¹), FU in water (2 mg mL¹), and CisP in dimethyl sulfoxide (1 mg mL¹). Netro (Fluka, Buchs, Switzerland) was prepared in water (2.667 mg mL¹). These solutions were stored at 4°C and diluted with water prior to use. Pea endocarp tissues were treated with the following concentrations: MMC (50 µg mL¹), actinomycin D (12.5 µg mL¹), CisP (50 µg mL¹), Etop (12.5 µg mL¹), FU (200 µg mL¹), H₂O₂ (10 mM), and Netro (125 µg mL¹). Tobacco leaves were infiltrated with MMC (250 µg mL¹), actinomycin D (50 µg mL¹), CisP (250 µg mL¹), Etop (250 µg mL¹), FU (1 mg mL¹), H₂O₂ (7%, v/v), or Netro (500 µg mL¹). These concentrations were determined as optimal in dose-response experiments (data not shown).

The pea endocarp tissue was also treated with UV irradiation at 254 nm, performed with a mineral light lamp (model R-51, UV Products, San Gabriel, CA) at a dosage of 589 J m².

GUS Assays

GUS activity was assayed as described by Gallagher (1992) with several modifications. Tobacco leaf discs were ground using a Dremel 7360 drill with pestle bit, and suspended in 300 µL of GUS extraction buffer (50 mM NaHPO₄, pH 7.0, 10 mM -mercapthoethanol, 10 mM EDTA, 0.1% [v/v] sarcosyl; 0.1% [v/v] Triton X-100). Duplicate aliquots (5 µL) of each extract were added to wells of a 96-well microtiter plate containing 45 µL of 2 mM 4-methyl umbelliferyl--glucuronide (Sigma) dissolved in GUS extraction buffer and 20% (v/v) methanol and incubated at 37°C. After 10- and 45-min incubation periods, 20 µL of the reaction was added to 180 µL of stop buffer (0.2 M NaCO₃). Fluorescence was read with a Fluoralite 1,000 microtiter plate fluorometer (excitation wavelength 365 nm; emission wavelength 450 nm; Dynatech Laboratories, Chantilly, VA). GUS activity was expressed as fluorescence per minute.

To determine the authenticity of the GUS assay as a measure of gene activity, RNA from tissue treated with Ps tabaci, or Fsph, along with RNA from non-transformed wild-type plants and the transgenic plants expressing the constitutive 35S::GUS fusion tobacco RNA were probed (data not shown) with a 2-kb EcoRI/HindIII fragment containing the GUS gene from pBI101 (CLONTECH Laboratories, Palo Alto, CA).

RNA Extraction and Electrophoresis

RNA was extracted according to Chomczynski and Sacchi (1987) with modifications. About 150 mg of tissue was frozen in liquid nitrogen and ground with 1 mL of extraction buffer (0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate, pH 5, 5% [v/v] glycerol, 38% [v/v] water-saturated acid phenol, pH 4.0) and incubated at room temperature for 5 to 10 min. Chloroform (200 µL) was added and the mixture incubated at room temperature for 2 to 15 min. Following a 15-min centrifugation from the supernatant, total RNA was precipitated after the addition of a 0.5 volume of isopropanol. Following centrifugation, the pellet was suspended in 0.1% (v/v) diethyl pyrocarbonate (DEPC)-treated water. Ten micrograms of RNA was electrophoresed on a 1.2% (v/v) agarose gel with 0.67 M formaldehyde, and RNA transferred in 10× SSC (1.5 M NaCl, 150 mM trisodium citrate) onto a Zeta-probe nylon membrane (Bio-Rad Laboratories, Richmond, CA).

Probes

Pea DRR206 RNA was hybridized with an 800-bp EcoRI/HindIII fragment from plasmid pDC206-13I-800 (Culley et al., 1995). PCR primers were constructed from P. sativum cDNA sequences for both PAL and CHS in the GenBank database (accession nos. D10001 and X63335, respectively) with the Wisconsin Sequence Analysis programs of the Genetics Computer Group (GCG; release beta 9.0) (Devereux et al., 1984). Two primer sets were prepared: PAL forward primer (5'-CAGTAGCAGCAGCCATAAC-3') and reverse primer (5'-AACCAGAACCAACAGCAG-3'), CHS forward primer (5'-CTCAAGGAGAAATTCCAAC-3') and reverse primer (5'-AGCCACGCTATGAAGAAC-3'). The PCR reaction consisted of 3 µL of genomic pea DNA (50 ng µL¹), 5 µL of water, 4 µL each (5 pmol) of forward and reverse primers, 2 µL 10× reaction buffer containing 25 mM Mg²⁺, 1.6 µL dNTPs (2.5 mM), and 0.4 µL Taq polymerase (5 units µL¹). PCR amplification was performed in a Hybaid PCRSprint thermocycler (Midwest Scientific, St. Louis) with an initial 3-min step at 94°C, followed by 30 cycles of 20 s at 94°C, 20 s at 54°C, and 1.5 min at 72°C, and a final extension time of 7 min at 72°C. The amplified products were ethanol-precipitated and dissolved in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

RNA Hybridization

Transblot membranes were incubated at 47°C to 65°C for 2 h to overnight in prehybridization solution (10× Denhardt's solution [for 50× Denhardt's, 1% {w/v} polyvinylpyrrolione, 1% {w/v} bovine serum albumin fraction V, 1% {w/v} ficoll 400], 3× SSC, 10% [w/v] dextran sulfate, 7% [w/v] SDS, 167 µg mL¹ of single-stranded salmon sperm DNA). Probes were labeled with a DECAprime II random primer labeling kit (Ambion, Austin, TX) according to the manufacturer using 50 µCi of [³²P]dATP with specific activity of 3,000 Ci mmol¹ (NEN, Boston) and 50 ng of template DNA. The labeled probe was added to the prehybridization solution and incubated for 18 to 36 h at 60°C. The membranes were washed twice in 2× SSC (for 20×, 3 M NaCl, 0.3 M trisodium citrate)/0.1% (w/v) SDS for 30 min at room temperature, and twice more for 30 min at the hybridization temperature. Membranes were exposed to Kodak BioMax film with a BioMax intensifier screen (Kodak, Rochester, NY) at 70°C.

The uniformity of mRNA applied to each lane was verified in the pea northern blot by hybridization with a 3-kb HindIII fragment of a conserved, constitutive soybean actin gene (cloned in plasmid pSAC3, accession no. V00450).

Cytology

Fluorescent DAPI (Sigma) was used as a probe for DNA to determine the number of cells with nuclear distortions and/or collapse. The stock solution (1 mg mL¹) was prepared in water and stored at 4°C in the dark. The cell viability stain, fluorescein diacetate (Sigma), was dissolved in acetone as a 1 mg mL¹ stock solution and stored at room temperature in the dark. Each one-half of an immature pea pod (1 to 2 cm in length) was soaked in either 1 mL of DAPI (5 µg mL¹) for 10 min or in fluorescein diacetate (77 µg mL¹ diluted in water) for 15 min and rinsed in water. Pod halves were observed and photographed under a fluorescence microscope (model BX60 System, Olympus, Bellevue, WA).

Cell death counts were made after staining the treated pea pods with trypan blue (Sigma) dissolved in water at 1 mg mL¹. The endocarp was flooded with trypan blue for 5 min, rinsed in water, and observed at 200× magnification by light microscopy.

All of the cell counts were done in duplicate with each experiment repeated three or four times. The percentage of distorted or dead cells in a 200× field (approximately 850 cells) was determined by dividing the number of cells of interest by the total number of cells. The data were analyzed by analysis of variance using the general linear model procedure of SAS (SAS Institute, Cary, NC).