The Plant Cell, Vol. 8, 1079-1090, June 1996 O 1996 American Society of Plant Physiologists

Changes in the Plasma Membrane Distribution of Rice Phospholipase D during Resistant lnteractions with Xanthomonas oryzae pv oryzae

Scott A. Young,a Xuemin Wang,b and Jan E. Leach a,’

a Department of Plant Pathology, 4024 Throckmorton Plant Sciences Center, Kansas State University, Manhattan, Kansas 66506-5502

Department of Biochemistry, 104 Willard, Kansas State University, Manhattan, Kansas 66506-5502

Phospholipase D (PLD; EC 3.1.4.4), which hydrolyzes phospholipids to generate phosphatidic acid, was examined in rice leaves undergoing susceptible or resistant interactions with Xanthomonas oryzae pv oryzae. RNA analysis of leaves un- dergoing resistant interactions revealed different expression patterns for PLD over 5 days relative to control plants or those undergoing susceptible interactions. By using an activity assay and immunoblot analysis, we identified three forms of PLD (1, 2, and 3). PLD 1 was observed only at 1 day after tissue infiltration. PLDs 2 and 3 were detected up to 3 days in all interactions. lmmunoelectron microscopy studies revealed PLD to be associated predominantly with the plasma membrane. In cells undergoing a susceptible response, PLD was uniformly distributed along the plasma membrane at 3, 6, 12, and 24 hr after inoculation. However, within 12 hr after bacterial challenge in resistant interactions, PLD was clustered preferentially in membranes adjacent to bacterial cells.

INTRODUCTION

In mammalian systems, phospholipase D (PLD) is a critical component in cellular signal transduction (Billah, 1993; Divecha and Irvine, 1995). PLD hydrolyzes phospholipids, generating phosphatidic acid (PA) and a free head group, such as cho- line, which serve as second messengers and bioactive molecules (Dennis et al., 1991). PA also can be further metabo- lized by PA phosphohydrolase to form diacylglycerol, which can function as an activator for protein kinase C in signal trans- duction pathways (Shukla and Halenda, 1991). Although clearly involved in signal transduction in mammals, PLD, which is of- ten the most abundant phospholipase in plants, shows evidence of playing a role in plant signaling or metabolism. Recently, evidence for PLD involvement in signaling in plants was provided by showing that G protein activators stimulate the enzyme in green alga and carnation petals (Munnik et al., 1995). PLD activity has been suggested to be associated with phospholipid metabolism in connection with storage lipid mobilization after seed germination (Herman and Chrispeels, 1980; Lee, 1989; Wang et al., 1993), mediation of cellular re- sponses to environmental stimuli (Di Nola and Mayer, 1986; Acharya et al., 1991), and membrane deterioration during senescence and stress injuries (McCormac et al., 1993; Samama and Pearce, 1993; Voisine et al., 1993; Ryu and Wang, 1995). Multiple forms of PLD were identified in castor bean

1 To whom correspondence should be addressed

tissues (Dyer et al., 1994; Ryu and Wang, 1995). Interestingly, these studies have demonstrated that different structural vari- ants of PLD are associated with developmental and growth conditions.

The role that PLD might play in plant defense responses, in particular the hypersensitive response (HR), is not known. The HR involves a cascade of dynamic events, many of which are comparable to those occurring during apoptosis or pro- grammed cell death in animals (Greenberg et al., 1994). The HR cascade is initiated by perception of the pathogen by the plant and leads to the production of rapid and localized en- dogenous signals that ultimately result in membrane damage, necrosis, and cell collapse (reviewed in Goodman and Novacky, 1994). Biochemical responses correlated with the HR are elec- trolyte loss from plant cells, activation of a K+/H+ exchange response, calcium influx, increased lipoxygenase activity, transient bursts of active oxygen, activation of specific defense- related genes, accumulation of antimicrobial compounds, and alterations of the plant cell wall (Atkinson, 1993; Goodman and Novacky, 1994). Because phospholipids, the substrates for PLD, are primary structural components in plant cell mem- branes, PLD could play a role in early stages of the HR, in particular, those involving membrane alterations or damage. Indeed, PLD activity increases in plants undergoing the HR (Huang and Goodman, 1970; Saini et al., 1990). Whether the increased activity in disease-stressed cells is due to de novo synthesis of the enzyme or, as observed for other stresses,

1080 The Plant Cell

due to decompartmentalization of PLD from its original intra-cellular stores (Yoshida, 1979; Willemot, 1983) is not known.

The goal of our study was to determine whether the expres-sion, activity, and location of PLD are consistent with apostulated role for the enzyme in the HR, specifically in resis-tant interactions between rice and the bacterial blight pathogenXanthomonas oryzae pv oryzae. Resistance in rice to X. o. ory-zae is correlated with increases in extracellular peroxidases,the accumulation of lignin, a reduction in bacterial multiplica-tion in the leaves, and the onset of the HR (Reimers and Leach,1991; Reimers et al., 1992; Quo et al., 1993; Young et al., 1995).Induction of resistance in rice-X. o. oryzae interactions is de-pendent on interactions between single resistance genes inthe plants and corresponding avirulence genes in the patho-gens (Hopkins et al., 1992). In this study, we used bacterialstrains isogenic for one avirulence gene (avrXaW) and ricecultivars near-isogenic for the corresponding resistance gene(Xa10) to investigate changes in PLD transcript accumulation,PLD isoform content, and PLD distribution in rice tissues un-dergoing resistant and susceptible responses. We describethe differential changes in PLD mRNA and three PLD vari-ants in rice during resistant and susceptible interactions withX. o. oryzae. Furthermore, the data presented here show achange in the plasma membrane distribution of PLD in ricecells undergoing resistant but not susceptible interactions.

RESULTS

Differential Expression of PLD and Phospholipase Cin Resistant and Susceptible Interactions betweenX. o. oryzae and Rice

In resistant interactions between X. o. oryzae and rice, macro-scopic symptoms characteristic of the HR, that is, browningand desiccation of the tissue, are observed within 24 to 36 hrafter infiltration. The HR is induced only in specific combina-tions of bacterial strains and rice cultivars. The combinationsof X. o. oryzae strains PXO86 or PXO99A(pBUavrXa10.F1),which carry the avrXalO gene, and ricecultivar IRBB10, whichcontains the Xa10 resistance gene, result in the HR. Combi-nations involving strain PXO99A, which lacks the avrXaWgene, produce a susceptible response, that is, a spreading le-sion with water-soaked tissue, within 1 day. Within 4 days, themajority of the tissue is blighted, tan, and desiccated.

A PLD cDNA clone isolated from castor bean (var Hale)(Wang et al., 1994) was used to investigate the patterns of tran-script accumulation in rice leaves over 5 days after varioustreatments (Figure 1A). Because a single band (~3 kb) of ricemRNA hybridized with the castor bean PLD during RNA gelblot analysis (data not shown), the quantification of the PLDtranscript in subsequent experiments was performed by slotblot analysis followed by densitometeric scanning (Figure 1B).Transcripts of an actin gene, which is constitutively expressed,

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Figure 1. Relative Accumulation of PLD and PLC mRNA Transcriptsin Rice Infected with X. o. oryzae.

(A) Total RNA was isolated from IRBB10 rice leaf tissue that was notinfiltrated with water (untreated, U) or was infiltrated with water (W),a virulent strain (susceptible, S), or an avirulent strain of X. o. oryzae(resistant, R) over a time course of 0 to 5 days after infiltration. Ap-proximately six times more desiccated tissue was needed to obtainRNA compared with undesiccated tissue. RNA (15 ng per slot) wastransferred onto nitrocellulose filters. Filters were successively hybrid-ized with nonradioactive-labeled PLD, PLC, and actin (act) gene probesto confirm equal RNA loading.(B) Signals were quantified by using a scanning densitometer, andrelative transcript levels were calculated.

were monitored as a control. The PLD transcript increasedwithin 1 day after infiltration in water-treated leaves or in leavesundergoing susceptible and resistant interactions with X. o.oryzae (Figures 1A and 1B). However, in untreated tissue, PLDmRNA did not change during the 5-day interval. Thus, the ac-cumulation of PLD mRNA in the treated tissues at 1 day wasmost likely caused by the infiltration process. In the suscepti-ble interactions, PLD mRNA gradually increased over 4 daysand then was barely detected at day 5; at this time, the lesionshad spread up and down the leaves and were tan and desic-cated. The expression pattern of PLD mRNA was different forthe resistant interactions in that the PLD mRNA peaked ear-lier (2 days) in resistant interactions than in susceptibleinteractions (4 days). By 5 days after inoculation, PLD mRNAhad decreased to levels comparable to the levels at day 0 inall treatments.

Because phospholipase C (PLC) also has been suggestedas playing a role in the HR (Atkinson, 1993), the expressionpattern of PLD was compared with that of a PIP2-specific PLC,

Phospholipase D in Rice 1081

which hydrolyzes phospholipids to generate diacylglycerol anda phosphorylated head group (Shi et al., 1995). The same blotwas hybridized with the PLC cDNA isolated from soybean (Shiet al., 1995). In both the untreated and water-treated leaves,PLC mRNA levels did not change, indicating that the PLCmRNA was not induced by the infiltration process. However,in the susceptible and resistant interactions, both PLC and PLDmRNA increased within 1 day after inoculation. Relative to theactin control, the increase in PLC transcript levels was higherthan that observed for PLD. Similar to PLD, PLC transcript ac-cumulation peaked at 2 days in resistant interactions anddeclined thereafter. In susceptible interactions, PLC transcriptlevels never reached those observed for the peak levels in resis-tant interactions. However, at 3 and 4 days, the PLC transcriptlevels were higher in the susceptible than the resistantinteractions.

Analysis of gel blots containing BamHI- and EcoRI-digestedrice genomic DNAs was used to estimate copy numbers ofPLD and PLC in rice. The castor bean PLD probe hybridizedstrongly with a single band in both BamHI- (~5 kb) and EcoRI-(~8 kb) digested DNAs (data not shown). Two bands withweaker signals were observed in both digests. The PLC proberevealed two strong bands in both enzyme digests, that is, ~4and 10 kb in BamHI and ~7 and 12 kb in EcoRI digests (datanot shown). These results, which were obtained after high-stringency washes, suggested that the rice genome containsat least one PLD that is highly related to the castor bean PLDand possibly two PLCs that are highly related to soybean PLC.

Fractionation and Multiple Forms of PLDin Rice Leaves

Microsomal and cytosolic PLD from untreated and inoculatedrice leaves was fractionated. If protein fractions were sepa-rated under denaturing conditions, a single protein with anapparent M, of 92 kD was detected by castor bean anti-PLDspecific antibodies in the microsomal fraction from untreatedrice leaves (Figure 2A, lane 3). The apparent molecular massof PLD is similar to that of PLD in castor bean (Figure 2A, lane6). The anti-PLD antibodies reacted faintly with a single bandin the cytosolic protein fraction and did not react with proteinsin the extracellular fluids (Figure 2A, lanes 4 and 5, respec-tively). Similar results were obtained for rice leaves 24 hr afterinoculation with X. o. oryzae in either susceptible or resistantinteractions (data not presented). These data suggest that amajority of PLD is associated with microsomal membranes inrice leaves.

Three different forms of PLD were detected previously incastor bean after separation in nondenaturing gels (Dyer etal., 1994). To determine whether different forms were presentin rice, proteins concentrated from the microsomal fraction wereseparated on nondenaturing polyacrylamide gels without de-tergents, transferred onto a polyvinylidene difluoride membrane,and probed with antibodies raised against the 92-kD PLD from

castor bean. Two PLD forms were detected in untreated(uninoculated) rice leaves (Figure 2B, lane 1), whereas threeforms were detected in rice leaves 24 hr after infiltration withwater (data not shown) or inoculation with either a virulent (datanot shown) or avirulent strain of X. o. oryzae (Figure 2B, lane 2).

To verify that the immunopositive bands were PLD, PLD ac-tivity was assayed in gel slices after resolution of microsomalproteins by nondenaturing PAGE. The positions at which ac-tivity was detected in the gel were consistent with positionsat which PLD was immunologically detected (Figures 2B and2C). PLD activities in untreated leaf tissue were resolved intotwo peaks: one migrated at 18 mm and another at 20 mm onthe gel (Figure 2C). By contrast, the PLD activity profile from

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Figure 2. Analysis of Fractionated PLD Protein and Activity from RiceLeaf Tissue.(A) Shown is a protein gel blot analysis of the total protein extract (lane2), microsomal fraction (lane 3), cytosolic fraction (lane 4), and extracel-lular fraction (lane 5) from healthy rice leaves and purified PLD fromcastor bean (lane 6). Equivalent volumes of proteins were separatedby SDS-PAGE (on a 10% polyacrylamide gel) and transferred to a poly-vinylidene difluoride membrane. The blot was incubated with antiserumraised against castor bean PLD. Molecular mass markers (Bio-Rad)are in lane 1.(B) and (C) The microsomal proteins (70 i*g per lane) containing PLDsextracted from untreated (lane 1) rice leaf tissues and tissues under-going a resistant response 1 day after inoculation (lane 2) wereseparated by nondenaturing PAGE in 10% polyacrylamide gels. (B)shows an immunoblot of nondenaturing gel incubated with PLD anti-bodies. PLDs 1,2, and 3 (shown at right) correspond to the PLD variants.(C) shows a PLD activity profile in which gel slices (2 mm per slice)were assayed. PLD protein, as detected immunologically, comigratedwith PLD activity.

1082 The Plant Cell

rice leaves 24 hr after inoculation with an avirulent strain ofX. o. oryzae showed three PLD activity peaks at 10, 18, and20 mm (Figure 2C). The relative activities of each form of PLDvaried between different treatments. Activity associated withthe 10-mm peak (PLD 1) was less than both the 18- and 20-mmpeaks (PLD 2 and PLD 3, respectively). In both the untreatedand treated rice tissue, PLD activity was higher in the 20-mmpeak (PLD 3) than in the others.

Change of PLD Variants in Rice during Resistant andSusceptible Interactions

To examine whether different PLD isoforms were present dur-ing pathogen attack, the PLD profiles were monitored inuntreated or water-treated rice leaves or leaves undergoingsusceptible or resistant interactions with X. o. oryzae over a5-day period (Figure 3A). PLD 1 was detected in proteins ex-tracted from rice leaves within 1 day of infiltration with waterand in susceptible and resistant interactions. However, un-treated leaf tissue did not contain the PLD 1 band at the timesexamined. This result suggests that PLD 1 is associated withthe perturbation during the infiltration process. PLDs 2 and3 were present in both untreated and water-treated rice leavesfrom 0 to 5 days (Figure 3A). However, after 3 days, variantsof PLDs 2 and 3 disappeared in susceptible interactions. In-terestingly, in the resistant interactions, the PLD 3 variant wasnot detected after 3 days, but PLD 2 was still present at 5 daysafter inoculation. These results indicate a dynamic regulationof the different PLD variants during interactions with pathogens.

PLD protein content of the microsomal fractions was ana-lyzed by SDS-PAGE and detected by immunoblotting withanti-PLD antibodies. RNA gel blot analysis showed an increasein the expression of PLD during susceptible and resistant in-teractions. However, a visual comparison of relative amountsof immunologically reacting protein in the blots indicated noincrease in accumulation of PLD but instead showed somedecrease in the protein (Figure 3B).

Localization and Change in Distribution of PLD in thePlasma Membrane of Mesophyll Cells

The antibodies raised against PLD were used for subcellularlocalization of PLD at 3, 6, 12, and 24 hr in rice undergoingresistant or susceptible interactions with X. o. oryzae. Macro-scopic symptoms of the HR (browning of the infiltration site)were first visible 24 to 36 hr after infiltration in resistant inter-actions. No obvious cellular reorganization was observed bytransmission electron microscopy at 3 hr after infection witheither the susceptible or resistant interactions (Figure 4A). At24 hr, both bacterial accumulation and collapse of themesophyll cells were observed in most cells at the site of in-filtration (Figure 4B). In resistant interactions, membrane

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Figure 3. Immunoblot Analysis of Microsomal PLD Variants over Timein Rice Leaf Tissues Undergoing Resistant and Susceptible Responses.

(A) The microsomal proteins (80 ug per lane) containing PLDs wereextracted from rice leaf tissue that were not infiltrated (untreated, U)or were infiltrated with water (W), a virulent X. o. oryzae strain (sus-ceptible, S), or an avirulent X. o. oryzae strain (resistant, R) over a timecourse of 0 to 5 days after infiltration (lanes 0 to 5, respectively). Theproteins were resolved on nondenaturing polyacrylamide gels andtransferred to nitrocellulose. Blots were probed with castor bean anti-PLD antibodies. PLDs 1, 2, and 3 (shown at right) correspond to thePLD variants.(B) Microsomal proteins from 0 to 5 days (lanes 2 to 7) of leaves fromIRBB10 rice infected with X. o. oryzae PXO86 (a resistant interaction)were analyzed using protein gel blots probed with anti-PLD antibod-ies. The proteins (50 ng per lane) were subjected to SDS-PAGE in 10%polyacrylamide gels. Molecular mass markers are in lane 1.

degeneration was distinct only at sites at which bacteria wereadjacent to the cell wall; it was rarely observed at 12 hr butcommonly observed at 24 hr. In susceptible interactions, col-lapse of the mesophyll cells was not observed at 24 hr, andmembranes were generally intact even when bacteria were

Phospholipase D in Rice 1083

adjacent to the wall (data not shown). PLD was detected pri- marily in the plasma membrane at all four time points after infection in both susceptible and resistant interactions (Figures 4C to 41). Examination of the immunogold-labeled tissues by scanning electron microscopy confirmed that PLD was located on the membrane (Figures 5A and 58).

The immunogold labeling of PLD in samples obtained at 3, 6, 12, and 24 hr after infection was quantified after trans- mission electron microscopy (Figures 6 and 7). Labeling in both the susceptible and resistant interactions at all times was relatively low in the cell wall, cytoplasm, chloroplast, vacuole, and extracellular spaces of mesophyll cells. More labeling was observed in the plasma membranes of mesophyll cells than in organelle membranes during both the susceptible and resis- tant interactions (approximately five gold particles per centimeter). The total number of particles per mesophyll cell did not vary significantly over the time course of infection (Fig- ure 6). Little to no labeling was detected in the membranes of organelles.

Interestingly, in the resistant interactions at 12 and 24 hr af- ter infection, the gold particles detecting PLD were clustered in the plasma membrane predominantly at sites at which bac- teria were adjacent to the mesophyll cell (Figures 4E, 4F, and 7). Although more gold particles were associated with these plasma membrane sites at 6 hr, the values were not signifi- cantly different than at 3 hr after infection. In addition to the increase in gold particles associated with the plasma mem- brane shown in Figure 7, the number of particles also increased in the cytoplasm immediately adjacent to these sites during resistant interactions (from 0.2 f 0.1 gold particles per cm2 at 3 hr to 1.2 f 0.6 at 24 hr). The number of gold particles in the plasma membrane of the same mesophyll cell decreased over time at sites distant from those at which bacteria were adjacent to the cell wall (Figures 4H and 7). In mesophyll cells that did not have bacteria adjacent to the cell wall in resistant interactions, no specific clustering of gold particles was ob- served in the plasma membrane (5.5 f 0.2 particles per cm at 3 hr versus 5.2 k 0.3 particles per cm at 24 hr) or in the cytoplasm (0.2 f 0.1 particles per cm2 at 3 hr versus 0.2 f 0.2 particles per cm2 at 24 hr). The change in distribution of PLD was observed only in resistant interactions. In the sus- ceptible interactions over the same time course, no change in the specific pattern of labeling was observed, and the label was randomly distributed in the plasma membrane even when bacteria were found adjacent to the cell wall (Figures 4G, 41, and 7).

Specificity of anti-PLD antibodies to the antigen was deter- mined by first treating tissues with F(ab’), fragments from anti-PLD antibodies. Binding of anti-PLD antibodies to PLD was blocked, and not much labeling was observed in tissues during both incompatible and compatible interactions 24 hr after infection (Table 1). Anti-POCla F(ab’);! fragments did not block the antigenic sites of PLD, and binding of the anti-PLD antibody to PLD was detected (Table 1). These experiments confirmed that the binding of anti-PLD antibodies observed in the tissue sections is specific to PLD.

Table 1. Specificity of Anti-PLD Antibody Binding to PLD at 24 hr in Rice Undergoing Resistant lnteractions

Number of Gold Particles (mean/cm2 or cm f SEM)^ after First Treatmentb

Mesophyll Anti-PLD Anti-PLD Anti-POCla Tissue Antibody F(ab% F(ab%

Wall 0.5 f 0.22 0.1 f 0.1 0.65 f 0.17 Membrane 5.0 f 0.63 0.05 f 0.05 5.25 f 0.33 Cytoplasm 0.3 & 0.09 0.05 f 0.05 0.4 & 0.14 Chloroplast 0.33 f 0.12 0.05 f 0.05 0.3 f 0.13 Vacuole 1.6 f 0.33 O 1.7 f 0.27 Extracellular 0.08 & 0.04 0.05 f 0.05 0.1 f 0.06

a The mean number ( f SEM) of gold particles on the wall and mem- brane of mesophyll cells was determined per centimeter of tissue, whereas the number of gold particles in the cytoplasm, chloroplasts, vacuoles, or extracellular spaces was determined per square centimeter. bTissues were treated first with anti-PLD antibody alone or with either anti-PLD or anti-POC1 a F(ab’)2 antibodies. The tissues were then treated with anti-PLD antibodies. Bound antibodies were detected by treatment with protein A labeled with 10-nm colloidal gold particles. Least significant difference (LSDo.05 = 0.5) for comparing antibody treatments within tissue type. Least significant difference (LSDo,05 = 0.6) for comparing tissue type within a treatment.

DlSCUSSlON

In this study, we present evidence that PLD is located in the plasma membrane of mesophyll cells of rice and that the dis- tribution patterns of the enzyme in the membrane are significantly different between resistant and susceptible inter- actions with X. o. oryzae. The change in distribution of PLD protein in the membranes is consistent with a postulated role for the enzyme in alterations of membrane permeability dur- ing host-pathogen interactions (Plumbley and Pitt, 1979; Cahill et al., 1985; Goodman, 1986; Saini et al., 1988,1990). In addi- tion, we detected three membrane-bound forms of PLD and demonstrated varied changes in these variants between treat- ments. Finally, we report differential expression patterns for PLD transcripts with various treatments.

PLD and PLC were differentially expressed in untreated or water-treated leaves or in leaves undergoing susceptible or resistant interactions within X. o. oryzae over a 5-day interval. lncreases in PLD but not PLC transcript accumulation were observed in water-treated rice leaves or leaves undergoing sus- ceptible or resistant interactions with X. o. oryzae at 1 day after infiltration. This suggests that PLD may function in the mem- branes’ response to changes in the extracellular environment induced by water infiltration or possibly to wounding associated with the infiltration process. The expression pattern of PLD mRNA in susceptible interactions gradually increased over 4 days and is similar to the expression patterns that have been observed during senescence in castor bean leaf discs. These

1084 The Plant Cell

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Figure 4. Immunolocalization of PLD in Rice Leaf Tissue Infiltrated with X. o. oryzae.

Transmission electron microscopy shows PLD location during susceptible (IR24) and resistant (IRBB10) interactions 3 and 24 hr after infiltrationwith X. o. pv oryzae PXO99A(pBUavrXa10.F1).

Phospholipase D in Rice 1085

Figure 5. Freeze-Fracture Scanning Electron Microscopy of Rice LeafTissue Infiltrated with X. o. oryzae.(A) and (B) show rice tissue that was probed with anti-PLD antibodies.(A) Scanning electron microscopy of a rice mesophyll cell surroundedby X. o. oryzae. Bar = 5 urn. b, bacteria; ch, chloroplast; m, mesophyllcell.(B) Inset from (A). The magnified image of the mesophyll cell plasmamembrane shows PLD location during a resistant (IRBB10) interac-tion 24 hr after infiltration with X. o. oryzae PX099A(pBUavrXa10.F1).The arrows indicate gold particles. Bar = 0.5 urn.

findings are consistent with a role for this enzyme in eventsassociated with cell deterioration (Ryu and Wang, 1995). PLDtranscript levels in resistant interactions peaked at 2 days andreached higher overall levels than in susceptible interactions.Transcript levels declined to control levels by day 4. Interest-ingly, the increased accumulation of PLD transcripts was notaccompanied by increases in PLD protein, as determined byimmunoblot analysis (Figure 3B) and immunogold labeling(Figures 6 and 7). One explanation for these results is thatin concert with increased gene expression, the turnover of PLDprotein increased after infection in both susceptible and resis-tant interactions.

PLD was predominately associated with the microsomal frac-tion in untreated or treated 10- to 15-day-old rice leaves.Previously, PLD has been reported to be associated withmicrosomal membranes of carnation flowers (Paliyath et al.,1987; Brown et al., 1990), bean cotyledons (Paliyath andThompson, 1987), castor bean leaves (Ryu and Wang, 1995),and tomato fruits (Todd et al., 1990; McCormac et al., 1993).Protein gel blot analysis after SDS-PAGE indicated an appar-ent molecular mass of 92 kD for the membrane-bound ricePLDs. In addition, three major forms of PLD in the microsomalfraction in rice were resolved by nondenaturing PAGE. ThesePLD isoforms are catalytically active, and their expression isspecific to different treatments. The identification of the threeisoforms in rice is not surprising, because three PLD isoformsare present in castor bean (Dyer et al., 1994). The roles of thecastor bean isoforms in plant metabolism were suggestedbased on the association of specific PLD variants with certainphysiological stages. For example, castor bean PLD 2 was ob-served to be continually present, suggesting that it is ahousekeeping enyzme. Rice PLD 2 was present in all treat-ments of rice leaves, except after 3 days in susceptibleinteractions, in which all PLD was absent; this is likely becausethe infected rice leaves are blighted and dead. In castor bean,PLD 1 has been suggested as playing a role in membranelipid turnover during the rapid growth of plants (Dyer et al.,1994). Similarly, rice PLD 1 was found only in treated tissues1 day after infiltration with either water or X. o. oryzae cells,suggesting that it may be associated with rapid membrane

Figure 4. (continued).(A) Cross-section of leaf tissue 3 hr after inoculation in IRBB10 (resistant interaction).(B) Cross-section of leaf tissue 24 hr after inoculation in IRBB10 (resistant interaction).(C) Mesophyll cell 3 hr after inoculation in IR24 (susceptible interaction).(D) Mesophyll cell 3 hr after inoculation in IRBB10 (resistant interaction).(E) Mesophyll cell with bacteria adjacent to cell wall 12 hr after inoculation in IRBB10 (resistant interaction).(F) Mesophyll cell with bacteria adjacent to cell wall 24 hr after inoculation in IRBB10 (resistant interaction).(G) Mesophyll cell with bacteria adjacent to cell wall 24 hr after inoculation in IR24 (susceptible interaction).(H) Mesophyll cell with no bacteria adjacent to cell wall 24 hr after inoculation in IRBB10 (resistant interaction).(I) Mesophyll cell with no bacteria adjacent to cell wall 24 hr after inoculation in IR24 (susceptible interaction).In (A) and (B), tissue was stained with uranyl acetate and lead; rice tissue was not probed with antibodies. (C) to (I) show rice tissue that wasprobed with anti-PLD antibodies, b, bacteria; ch, chloroplast; sg, starch granules; xs, extracellular space. Thin arrows indicate the membrane;thick arrows indicate the cell wall. Bars in (A) and (B) = 5 urn. Bars in (C) and (D) = 0.25 |im. Bars in (E) to (I) = 0.5 \im. Note that (G) isat a higher magnification.

1086 The Plant Cell

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Quantitation of the Cellular Location of PLD

An analysis of rice tissues undergoing susceptible and resistant inter- actions at 3, 6, 12, and 24 hr after inoculation with X. o. oryzae PXO99~pBUavrXalO.Fl) is shown. IR24 (susceptible), open bars; IRBBlO (resistant), shaded bars. The mean number of particles per square centimeter for each cellular location indicated are cell wall, cytoplasm, chloroplast, vacuole, and extracellular space around mesophyll cells. Gold particles distributed on the membrane are reported as mean number of gold particles per centimeter of mem- brane. Sections were treated with anti-PLD antibodies, followed by gold-conjugated anti-rabbit antibodies.

turnover in a wounding response or in association with dras- tic changes in the extracellular environment. Interestingly, PLD 3 was present in all treatments of rice leaves, except after 3 days in susceptible and resistant interactions. Castor bean PLD 3 was associated with senescent endosperms and has been speculated to be involved in membrane deterioration and senescence (Dyer et al., 1994). The roles for PLD 3 in rice are at this point unclear. The molecular origin of these variants is under investigation.

Gold-labeled antibodies specific for PLD immunodecorated the' plasma membranes of rice mesophyll cells in both sus- ceptible and resistant interactions. The location of PLD can vary with the age and development of tissues. In young castor bean leaves, PLD is largely associated with the vacuoles, whereas in older, mature leaves, most PLD is associated with the endoplasmic reticulum and plasma membrane (Dyer et al., 1994; Xu et al., 1994). Washing of microsomal fractions with 0.2 M KCI was observed to release .v6O0/0 PLD from cas- tor bean membranes (X. Wang, unpublished data), suggesting that some PLD is peripheral to the membrane. The present observation that microsomal PLD variants of rice leaves were resolved by nondenaturing PAGE without detergent solublili- zation also suggests a peripheral association of most PLD with membranes. This notion is further supported by our scanning electron microscopy studies, which demonstrated that at least

some of the PLD was exposed on the surface of the inner side of the membrane (Figure 5).

Interestingly, in resistant interactions at 12 and 24 hr after infection, clusters of gold-labeled PLD were observed when bacteria were adjacent to the mesophyll cell wall. Concurrently, the amount of PLD decreased in the mesophyll cell membrane away from the site of dose association with the bacteria. Sig- nificant changes in the PLD distribution were not observed in membranes of cells undergoing a susceptible response at 3, 6, 12, or 24 hr or in the resistant interaction at 3 and 6 hr. It is important that the total numbers of gold particles between susceptible and resistant interactions were the same at all time points (Figures 6 and 7, and data not shown).

A model depicting one possible explanation of how the changes in PLD distribution during resistant interactions might occur is shown in Figure 8. Within 24 hr after treatment with either virulent or avirulent strains of bacteria, an increase in PLD transcript accumulation was observed (Figure 1). How- ever, no apparent increase in protein was observed within this time (Figures 3 and 6). Thus, we predict that (1) the results reflect the heterogeneity of the tissue sampled, (2) there is re- duced translation, or (3) there is increased turnover of the enzyme. In susceptible interactions, PLD was evenly distributed in the plasma membranes of cells. In contrast, in the resistant interactions, the particles were clustered at sites in which bac-

Plasma Membrane

I 3 6 9 12 15 18 21 24

Hours

Figure 7. Quantitative Analysis of Distribution of PLD in the Plasma Membrane in Relation to the Proximity of Bacterial Cells during Resis- tant and Susceptible Interactions.

Gold particle distribution was compared in rice tissues undergoing susceptible and resistant interactions at 3,6, 12, and 24 hr after inocu- lation with X. o. oryzae PX099A(pBUavrXa10.F1). Shown are the mean number of gold particles per centimeter (*SE) in membranes of IR24 (susceptible; dashed line) and IRBB10 (resistant; solid line). Filled squares indicate sites in mesophyll cells in which bacteria are adja- cent to the cell wall; open circles indicate sites in the same cell in which bacteria were not adjacent to the wall. Sections were treated with anti- PLD antibodies followed by treatment with gold-conjugated anti-rabbit antibodies. Thin sections of infected tissue were analyzed ata mag- nification of ~30,000.

Phospholipase D in Rice 1087

Susceptible Interaction

Resistant Interaction

bncterium

3 C 3 C

, plaat cell 3 " C

iQ o a . a 9

*'o , O A O

I PLD plasma

behveen : membrane * i 6 a n d 2 4 h r

Figure 8. Model lllustrating the Change in Distribution of PLD in the Plasma Membrane during Resistant Interactions.

In cells undergoing a susceptible response, PLD was uniformly dis- tributed along the plasma membrane at 3, 6, 12, and 24 hr after inoculation. However, between 6 and 24 hr after bacterial challenge in resistant interactions, PLD was clustered preferentially in membranes adjacent to bacterial cells. The amount of PLD protein remained the same a: all sampling times.

teria were associated with the responding cell. Because the increase in particles detected at these sites was concurrent with a decrease in PLD at sites not adjacent to bacteria (Fig- ure 7), it is unlikely that the change in distribution reflects differences in degradation rates of PLD. More likely, this change in distribution occurs under conditions of increased enzyme turnover if a newly translated enzyme was targeted to the sites at which bacteria are associated with the hypersensitively re- sponding plant cells.

The clustering of PLD in the plasma membrane is consis- tent with a role of PLD in membrane damage, that is, in making the membrane more permeable, specifically at sites adjacent to bacteria. Although we observed very little obvious mem- brane degradation or perturbation at 12 hr after infiltration in the rice-bacterial blight interactions, evidence for plasma mem- brane changes was observed as early as 2 to 3 hr after inoculation in resistant and sometimes susceptible interactions between bacterial pathogens and dicot hosts (Brown et al., 1993,1995; Goodman and Novacky, 1994). For example, con- volution of the pepper plasma membrane adjacent to X. campestris pv vesicatoria cells was observed as early as 2 to 3 hr after inoculation, but in both resistant and susceptible in- teractions (Brown et al., 1993). Differences in the ultrastructure between these two interactions in the pepper system were not apparent until 20 hr after inoculation, when regions of cytoplasm in mesophyll cells next to avirulent bacteria (resis- tant interaction) became vesiculated, indicative of localized membrane dysfunction. Decompartmentation and cytoplas- mic collapse followed convolution and vesiculation at 48 hr.

Whether the differences observed in timing of ultrastruc- tural events between rice-Xanthomonas and pepper- Xanthomonas interactions reflect differences between the host plants (monocots and dicots) or in ways these bacteria inter- act with their host plant (vascular wilt versus intercellular leaf spot diseases) is unclear. Another early manifestation of mem- brane alterations in plant tissues undergoing the HR is an increased leakage of electrolytes 2 to 3 hr after inoculation (Cook and Stall, 1968; Goodman, 1968; Lyon and Wood, 1976); such studies have not been performed with rice tissues ex- posed to X. o. oryzae.

The increased mRNA expression, the presence of different isoforms, and the change in distribution ,of PLD observed in the resistant interactions raise many qufstions. For example, what is the signal that initiates the change in distribution of PLD, why is the PLD clustered at the site at which the bacteria are adjacent to the plant cell wall, which isoforms of PLD are at the site of clustering, what is thsfunction of each isoform in resistance, and what is the biochemical basis for the iso- forms? In addition, in light of recentdindings on the role of PLD in signal transduction, are PLD activity and location related to the signaling processes that mediate resistance in plants? Future work on PLD will address these questions and lead to a better understanding of the role of this enzyme in host-pathogen interactions.

METHODS

Plant Material, Bacterial Strains, and, Media

Rice (Oryza sativa) cultivar IRBBlOJcontaiirs,the Xa70 gene for bac- teria1 blight resistance. IR24 is near4sogenic;to IRBB10 but lacks the XalO gene and is susceptible to all strainsofiXanthomonas oryzae pv oryzae used in this study. Seedlingswamgpwn in a growth chamber, as described previously (Reimers and! Ileaeh:. 1991).

X. o. oryzae strains were maintainedi at 28OC on WF-P medium (Karganilla et al., 1973). For plant inaalations, X. o. oryzae cultures were grown in nutrient broth (Difco Lahorataries, Detroit, MI) on a ro- tary shaker (250 rpm) at 28OC;. Whenl required, media were supplemented with antibiotics at the following concentrations (pg/mL): carbenicillin (100) and kanamycin. (lQ0). lnoculum (5 x 109 colony- forming units per mL) was prepamd andt infiltrated into multiple sites on the second fully expanded leaf'of 10-day-old rice plants, as described previously (Reimers and Leach, 1991; Reirners et al., 1992).

DNA Gel Blot Analysis

Agarose gel electrophoresis, DNA restriction digests, and DNA gel blot transfers were performed using standard procedures (Maniatis et al., 1982). Genomic DNA was isolated from 10-day-old rice seedlings, as described by Murray and Thompson (1980), and digested with enzymes. The digests were separated by electrophoresis in a 0.8% agarose gel. DNA gel blot transfers were performed using MagnaGraph nylon mern- branes purchased frorn Micron Separations Inc. (Westboro, MA). Conditions for prehybridizations, hybridizations, and high-stringency posthybridization washes were as described previously (Bender et al.,

1088 The Plant Cell

1991). The phospholipase D (PLD) probe was a 2.8-kb EcoRI-Kpnl frag- ment from a PLD cDNA isolated from castor bean (Wang et a1.,1994). The phospholipase C (PLC) probe was a 2.1-kb EcoRl-Xhol fragment from a PLC cDNA from soybean (Shi et al., 1995). Rapid, small-scale plasmid isolations from Escherichia coli were performed by the boil- ing method 2 of Crouse et al. (1983). DNA probes were labeled with phosphorus-32 by the random primer labeling technique (Feinberg and Vogelstein, 1983).

RNA lsolation and Analysis

Transcript accumulation by RNA gel blot analysis in IRBBIO rice leaves that were not treated or were infiltrated with water orX. o. ofyzae strains PX09gA or PX086 was monitored. Rice leaf tissue was harvested daily from O to 5 days after treatment and ground to a fine powder in liquid N2. Approximately six times more tissue undergoing suscep- tible interactions was harvested at day 5 because the tissue was desiccated. Total RNA was isolated from frozen plant cells by using theTRlzol Reagent (LifeTechnologies, Inc., Gaithersburg, MD). RNA (15 pg) from each sample was added to 100 pL of 6 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate) and 7.5% formaldehyde, dena- tured at 65OC for 20 min, and blotted (slot blot analysis) to Hybond N membranes (Amersham). The membranes were hybridized with DNA probes of the castor bean PLD cDNA (Wang et al., 1994), the PLC cDNA (Shi et al., 1995), or the constitutive actin gene (McElroy et al., 1990). The DNA probes were labeled by random priming by using the ECL nucleotide labeling kit (Amersham), and hybridization was per- formed according to manufacturer’s specifications. Autoradiograms were scanned with a densitometer (Interactive Technologies Interna- tional, St. Petersburg, FL) and quantified relative to the signal obtained after reprobing with an actin cDNA. These experiments were performed three times.

Tissue Fractionation of PLD

lnoculated rice leaves (two leaves per sample) were harvested at O, 24, 48,72,96, and 120 hr after infection and ground to a fine powder in liquid N2 with a mortar and pestle. The following steps were per- formed on ice unless stated otherwise. Proteins were extracted in one equal volume of buffer A (Wang et al., 1993) consisting of 50 mM Tris- HCI, pH 8.0,l mM EDTA, 10 mM KCI, 2 mM DTT, 0.5 mM phenylmethyl- sulfonyl fluoride, and 0.5 M sucrose (Wang et al., 1993). The homogenate was filtered through six to nine layers of cheesecloth and centrifuged at 60009 for 20 min, and the supernatant was centrifuged at 110,OOOg for 1 hr. The supernatant (cytosolic fraction) and pellet (microsomal fraction) were suspended in buffer B (the same as buffer A, except that sucrose was omitted). To enrich PLD concentrations, cytosolic pro- teins were precipitated by slowly adding an equal volume of -2OOC acetone with vigorous stirring (Wang et al., 1993). The precipitated proteins were pelleted at 14,OOOg for 10 min. Pelleted proteins were dissolved in buffer B. Protein concentrations were determined colorimetrically (Bio-Rad).

Extracellular proteins were extracted from rice leaves, as described previously (Reimers et al., 1992). except that 1 mM EDTA, 2 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride were added to the extrac- tion buffer. Extracellular proteins were precipitated by slowly adding an equal volume of -2OOC acetone, pelleted at 14,OOOg for 10 min, and dissolved in buffer 6.

lmmunodetection and PLD Activity

Cytosolic or microsomal proteins were separated either on 10% SDS-polyacrylamide gels as previously described (Laemmli, 1970) or on a nondenaturing 10% polyacrylamide gel (Dyer et al., 1994). Pro- teins were electrophoretically transferred to polyvinylidene difluoride membranes (Schleicher i% Schuell) by using Towbin buffer (Towbin et al., 1979) that had a final methanol concentration of 5%. The poly- vinylidene difluoride membranes were probed with anti-PLD polyclonal antibodies raised to purified PLD from castor bean (Wang et al., 1993). Antigen-antibody complexes were visualized by alkaline phospha- tase-conjugated antibodies.

For assaying PLD activity after electrophoresis, the gel was sliced and placed directly in reaction mixtures. The reactions were initiated by adding dipalmitoyl-glycer0-3-P-methyl-~H-choline (Amersham) as substrate. Tritiated phosphotidylcholine (2.5 pCi of 500 pCi/mmol) was mixed with 20 pmol of cold phosphotidyl (egg yolk; Sigma) in chloro- form, and the mixture was dried under a stream of NP. The lipid was emulsified in 1 mL of H20 by sonication at room temperature. A stan- dard enzyme assay mixture contained 100 mM Mes-NaOH, pH 6.5, 25 mM CaCI,, 0.5 mM SDS, 20 pL of substrate (0.4 pmol), and 20 pL of enzyme solution in a total volume of 200 pL. The assay conditions and reaction product separation and quantitation were as previously described (Wang et al., 1993). Briefly, the reaction was initiated by ad- dition of substrate and incubated in a 5OoC water bath with shaking (100 rpm). The reaction was stopped by the addition of 1 mL of chloro- form-methanol(2% [vh]). After vigorous vortexing and centrifugation, an aliquot (100 pL) of the aqueous phase was mixed with scintillation counting fluid, and the release of 3H-choline was measured by stan- dard scintillation counting.

Electron Microscopy and lmmunocytochemistry

Tissue samples were prepared for electron microscopy as described by Young et al. (1994). Anti-PLD antibodies were purified on a protein A column and used at a dilution of 150 for all labeling experiments, as described by Lutkenhaus and Bi (1991). Control reactions were treated with antibodies from preimmune serum at a dilution of 150. In competitive binding studies, grids containing sections were treated as described by Lutkenhaus and Bi (1991), except that grids were in- cubated first with either anti-PLD or anti-POCla F(ab’)2 fragments diluted 150 in 50 mM Tris-HCI, pH 7.4, 150 mM NaCI, and 0.1% BSA (TBS buffer). Anti-POCla F(ab’)* fragments were prepared as de- scribed previously (Harlow and Lane, 1988). Grids were then treated with anti-PLD antibodies diluted 150 in TBS buffer and finally in pro- tein A labeled with 10-nm colloidal gold particles (Sigma) diluted 150 in TBS buffer.

Scanning Electron Microscopy

Rice tissue samples were immersed in liquid nitrogen and freeze frac- tured, fixed in paraformaldehyde, probed with anti-PLD antibodies as described above, dehydrated in an ethanol series, and critical point dried. The freeze-dried samples were mounted on specimen stubs and sputter-coated with gold to a thickness of 200 A.

Phospholipase D in Rice 1089

Quantitation of lmmunogold Particles

Thin sections of infected leaf tissue were analyzed at a magnification of ~30,000. For each treatment with antibodies, two grids, with two sec- tions per grid, two fields per section, and 3 cm2 or 3 cm per organelle per field were examined for gold particles. Each treatment consisted of two replicates. The gold particles in and around the different plant organelles were counted on prints with the aid of a l-cmZ grid. Values are reported as mean numbers plus or minus the standard error of the mean. For binding studies, least significant difference values were calculated and compared between antibody treatments within a tis- sue type and also between tissue type within an antibody treatment. The experiment was a split plot design.

ACKNOWLEDGMENTS

We thank Dr. Madan Bhattacharyya for sending the PLC cDNA clone in advance of publication. We thank Sue Brown and Emily Skinner for excellent technical assistance, Tim Todd for assistance with statistical analyses, Lloyd Willard for helpful discussions and assistance in elec- tron microscopy work, and the Pathology Microbiology Department in the College of Veterinary Medicine at Kansas State University for use of their facilities. This work is supported by the U.S. Department of Agriculture-National Research lnitiative Grant No. 524532 to J.E.L. and Frank White. This is contribution No. 96-162-J from the Kansas Agricultura1 Experiment Station.

Received February 20, 1996; accepted April 5, 1996.

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DOI 10.1105/tpc.8.6.1079 1996;8;1079-1090Plant Cell

S. A. Young, X. Wang and J. E. LeachInteractions with Xanthomonas oryzae pv oryzae.

Changes in the Plasma Membrane Distribution of Rice Phospholipase D during Resistant

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