suppression of a phospholipase d gene, ospld 1 activates ...to positively regulate ros generation...

Suppression of a Phospholipase D Gene, OsPLDb1,Activates Defense Responses and Increases DiseaseResistance in Rice1[C][W][OA]

Takeshi Yamaguchi*, Masaharu Kuroda, Hiromoto Yamakawa, Taketo Ashizawa, Kazuyuki Hirayae,Leona Kurimoto, Tomonori Shinya, and Naoto Shibuya

National Agricultural Research Center, Joetsu, Niigata 943–0193, Japan (T.Y., M.K., H.Y., T.A., K.H.); andDepartment of Life Sciences, Faculty of Agriculture, Meiji University, Kawasaki, Kanagawa 214–8571,Japan (L.K., T.S., N.S.)

Phospholipase D (PLD) plays an important role in plants, including responses to abiotic as well as biotic stresses. A survey ofthe rice (Oryza sativa) genome database indicated the presence of 17 PLD genes in the genome, among which OsPLDa1,OsPLDa5, and OsPLDb1 were highly expressed in most tissues studied. To examine the physiological function of PLD in rice,we made knockdown plants for each PLD isoform by introducing gene-specific RNA interference constructs. One of them,OsPLDb1-knockdown plants, showed the accumulation of reactive oxygen species in the absence of pathogen infection.Reverse transcription-polymerase chain reaction and DNA microarray analyses revealed that the knockdown of OsPLDb1resulted in the up-/down-regulation of more than 1,400 genes, including the induction of defense-related genes such aspathogenesis-related protein genes and WRKY/ERF family transcription factor genes. Hypersensitive response-like cell deathand phytoalexin production were also observed at a later phase of growth in the OsPLDb1-knockdown plants. These resultsindicated that the OsPLDb1-knockdown plants spontaneously activated the defense responses in the absence of pathogeninfection. Furthermore, the OsPLDb1-knockdown plants exhibited increased resistance to the infection of major pathogens ofrice, Pyricularia grisea and Xanthomonas oryzae pv oryzae. These results suggested that OsPLDb1 functions as a negativeregulator of defense responses and disease resistance in rice.

Phospholipase D (PLD) is one of the representativephospholipases in plants and hydrolyzes phospho-lipids to generate phosphatidic acid (PA) and a free-head group. In recent years, PLD has been suggestedto be involved in many plant cellular processes, suchas membrane degradation (Ryu and Wang, 1995),membrane tethering (Dhonukshe et al., 2003; Gardineret al., 2003), and signaling for stress and hormoneresponses (Testerink and Munnik, 2005; Wang, 2005;Bargmann and Munnik, 2006; Wang et al., 2006). PA,which can be directly generated by the action of PLD,has been recognized as an important lipid secondmessenger. PA mediates the generation of reactive

oxygen species (ROS; Sang et al., 2001; Yamaguchiet al., 2003) and activates wound-activated mitogen-activated protein kinase (Lee et al., 2001). Several PAtargets have been functionally characterized in plantcells (Testerink and Munnik, 2005).

Plant PLDs are a family of heterologous enzymes(Wang, 2005). In Arabidopsis (Arabidopsis thaliana),12 PLD genes were reported, seven of which weredirectly cloned and the others were predicted bysearching the BLAST database (National Center forBiotechnology Information; http://www.ncbi.nml.nih.gov/). They can be classified into six types,PLDa(3), b(2), g(3), d, «, and z(2), based on their genearchitectures, sequence similarities, domain struc-tures, and biochemical properties (Wang, 2005). Thefunction of each plant PLD has been studied usingknockout, knockdown, and overexpression of corre-sponding genes, resulting in the identification of thephysiological functions of several PLDs. PLDa hasbeen reported to be involved in the signaling forabscisic acid, ethylene, wounding, freezing, drought,and hyperosmotic stress (Testerink and Munnik, 2005;Wang, 2005; Hong et al., 2008). PLDa was also shownto positively regulate ROS generation (Sang et al.,2001) and to be involved in the loss of seed viability byaging (Devaiah et al., 2007). PLDbwas suggested to beinvolved in the regulation of seed germination in rice(Oryza sativa; Li et al., 2007). Involvement of PLDd inthe regulation of various responses such as drought,

1 This work was supported by the Ministry of Agriculture,Forestry, and Fisheries, Japan, by the Ministry of Education, Culture,Sports, Science, and Technology, Japan, to T.Y., and by the Programfor the Promotion of Basic Research Activities for Innovative Bio-science to N.S.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Takeshi Yamaguchi ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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hydrogen peroxide, freezing, and UV irradiation hasbeen reported (Testerink and Munnik, 2005; Wang,2005). PLDd has also been shown to play an importantrole in the reorganization of microtubules as well astheir association with plasma membrane (Dhonuksheet al., 2003; Gardiner et al., 2003). PLDz has beensuggested to regulate root hair growth and patterning(Ohashi et al., 2003), phosphate recycling during phos-phate starvation (Li et al., 2006), vesicle trafficking, andauxin response (Li and Xue, 2007).For defense responses in plants, activation of PLD

was observed by the challenge of a pathogen in riceleaves (Young et al., 1996) and the addition of defenseelicitors in tomato (Solanum lycopersicum) cells (Van derLuit et al., 2000) and rice cells (Yamaguchi et al., 2003).In addition, the pathogen- and elicitor-induced PLDactivation involved the recruitment of PLD to themembrane (Young et al., 1996; Yamaguchi et al., 2005).Concerning the expression of PLD genes in defenseresponses, induction of PLDb genes was reported inthe elicitor treatment of cultured tomato cells (Laxaltet al., 2001) and tobacco (Nicotiana tabacum) cells(Suzuki et al., 2007). De Torres Zabela et al. (2002)reported the induction of Arabidopsis PLDa, -b, and-g genes by the challenge of a pathogen in the leaves.McGee et al. (2003) also showed the induction of ricePLDa genes by the challenge of a pathogen in theleaves. In addition, the pathogen- and elicitor-inducedPLD activation involved the recruitment of PLD to themembrane (Young et al., 1996; Yamaguchi et al., 2005).Concerning the function of the reaction product of

PLD, PA, in defense responses, it has been reportedthat PA can induce ROS generation in Arabidopsis(Sang et al., 2001) and rice (Yamaguchi et al., 2003) andthe expression of defense-related genes (Yamaguchiet al., 2005). On the other hand, 1-butanol, whichinhibits the generation of PA by PLD, suppressed theelicitor-induced ROS generation and phytoalexin pro-duction in rice cells (Yamaguchi et al., 2005). Based onthese findings, it has been expected that PLD posi-

tively regulates defense responses in plants. On thecontrary, Bargmann et al. (2006) reported that xylanaseelicitor-induced ROS generation was increased inPLDb-suppressed tomato cells. Although these findingsindicate that plant PLDs play important roles in defensesignaling, most evidence remains circumstantial andthe potential role of each PLD isoform is still obscure.Moreover, there is no evidence for the involvement ofPLDs in the disease resistance against pathogens.

In this study, we generated a series of knockdownplants for each PLD isoform in rice and analyzed theirphenotypes for defense responses. We report here thatthe knockdown of a single gene, OsPLDb1, resulted inROS generation and expression of defense genes,followed by spontaneous lesion formation and phyto-alexin production in the absence of pathogen infection.Furthermore, the knockdown transgenic plant showeda marked increase in the disease resistance to the blastfungus, Pyricularia grisea, and the bacterial blight,Xanthomonas oryzae pv oryzae (Xoo). These resultsclearly indicate that OsPLDb1 functions as a negativeregulator of defense responses and is involved in thedisease resistance in rice.

RESULTS

Analysis of the Rice PLD Gene Family

Multisequence alignment analysis was carried outto examine the phylogenetic relationship of the 17 PLDgenes from rice and 12 genes from Arabidopsis. Thephylogeny is shown as a rooted UPGMA tree in Figure1. In Figure 1, we basically followed the terminologyfor PLD family members proposed by Wang (2005) forArabidopsis PLDs to facilitate the comparison ofcorresponding genes in these two model plants. RicePLDs can be grouped into eight types, PLDa, -b, -d, -«,-z, -h, -u, and -i (Fig. 1), based on their predictedsequence homology with Arabidopsis PLDs (Fig. 1).The 16 PLD genes except OsPLDh2 have two HKD

Figure 1. A phylogenetic tree of PLDs in rice andArabidopsis. The phylogenetic tree was constructedfrom the matrix of amino acid sequence similar-ities calculated with the UPGMA program of theWisconsin University Genetics Computer Group.Rice PLDswere classified based on their similarityto Arabidopsis PLDs. The numbers following ricePLD genes indicate The Institute for GenomicResearch locus identifiers and GenBank acces-sion numbers of the corresponding genes. [Seeonline article for color version of this figure.]

Activation of Defense Responses by PLD Knockdown in Rice

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motifs, which constitute the highly conserved domainin the PLD family and are used to define the PLDsuperfamily (Qin and Wang, 2002). Fourteen PLDgenes except OsPLDz1, OsPLDz2, and OsPLDi containa C2 domain that is a Ca2+- and phospholipid-bindingdomain. OsPLDz1 and OsPLDz2 contain the PX andPH domains, which are present in mammalian PLDsas well (Wang, 2005). The remaining one, OsPLDi,contains two HKD motifs but does not contain C2 orPH-PX domains. Homologues of OsPLDi are presentin the genomes of Phytophthora species (Meijer andGovers, 2006), Vitis vinifera, and Medicago truncatula.The phylogenetic relationship of rice PLDs in Figure1 was partially different from the result recentlyreported by Li et al. (2007). In our analysis, OsPLDh1and OsPLDh2 or OsPLD« and AtPLD« were groupedinto the same class, which corresponds to the resultsreported by Elias et al. (2002).

To characterize the expression patterns of PLDgenes in rice, expression of the 16 rice PLD genesexcept OsPLDi was analyzed using cultured cells,roots, leaf sheaths, leaf blades, and immature seedsunder normal growth conditions. The accumulation oftheir mRNA in each tissue was evaluated using quan-titative reverse transcription (RT)-PCR. The expressionof 16 PLD genes was detected in most tissues analyzed(Supplemental Table S1). OsPLDb1 was highly ex-pressed in all tissues analyzed (Fig. 2). OsPLDa1 andOsPLDa5 were also highly expressed in most tissues,although a higher expression of OsPLDa1 mRNA inthe cultured cells was observed. Higher expression ofOsPLDa4 and OsPLDd1 was detected in roots and leafsheaths, respectively. These results demonstrated thatthe PLD isoforms have overlapping but distinct pat-terns of expression in the different tissues/organsunder normal growth conditions. On the other hand,the expression levels of OsPLD«, OsPLDd2, OsPLDd3,OsPLDh1, and OsPLDu were very low in all tissuesstudied (Supplemental Table S1).

Similar studies in Arabidopsis and tomato showedthat AtPLDa1 and AtPLDg1 in Arabidopsis andLePLDa2 and LePLDa3 in tomato were all highlyexpressed in most tissues. In contrast to the ratherubiquitous expression of OsPLDb1 in rice, the expres-sion of both AtPLDb1 and LePLDb1 was very lowunder normal growth conditions (Fan et al., 1999;Laxalt et al., 2001). This may relate to the fact that ricelacks g-type PLD in the genome, which was exten-sively expressed in Arabidopsis and tomato. For ricePLDs, Li et al. (2007) reported similar results withthose reported here, although the expression profilewas significantly different for some PLD isoforms,probably reflecting the differences in the plant mate-rials such as cultivar and growth conditions.

Accumulation of ROS in the OsPLDb1-KnockdownTransgenic Rice

As an initial step to characterize the function of eachPLD isoform in rice, we made knockdown plants for

eight PLD genes in rice,OsPLDa1, -a3, -a4, -a5, -«, -b1,-b2, and -d2, by introducing gene-specific RNA inter-ference (RNAi) constructs. More than 10 lines wereestablished for each gene, and at least five indepen-dent lines were used for the analysis of the phenotypesof the T1 transgenic plants grown under normal con-ditions in a greenhouse. Among them, OsPLDb1-knockdown plants showed a marked enhancementin the generation of ROS. The amount of hydrogenperoxide (H2O2) in the leaves of OsPLDb1-knockdownplants (20 or 30 d after seeding) was approximately2-fold higher than that in the vector control plants (Fig.3A). Quantitative analysis of OsPLDb1 mRNA in theleaves of the plant confirmed a clear suppression of

Figure 2. Expression analysis of rice PLD genes. Wild-type rice plantswere grown in the greenhouse under normal growth conditions for 50d, and total RNA was extracted from the roots, leaf sheaths, and leafblades. Immature seeds were harvested from plants at 10 d afterflowering. The values were standardized to the expression level of arice polyubiquitin gene (RUBIQ1). The values represent averages oftriplicate experiments, and the error bars indicate SD. [See online articlefor color version of this figure.]

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OsPLDb1 expression (Fig. 3B). Five independentknockdown lines gave similar results (SupplementalFig. S1). No difference in the appearance of the leaveswas observed between the OsPLDb1-knockdownplants and the vector control plants at this stage(data not shown).

The analysis of PLD activity in the leaf extractsshowed that the PLD activity of the OsPLDb1-knock-down plants was decreased to half of the vectorcontrol level. However, this decrease of PLD activitywas observed only for phosphatidylcholine-specific(PC-) PLD activity but not for phosphatidylethanolamine-specific (PE-) PLD activity (Fig. 3C). These resultsindicate that the knockdown of OsPLDb1 really re-flects the change of in planta PLD activity and that thein vivo substrate of OsPLDb1 is PC.

To examine whether the higher accumulation ofH2O2 in the OsPLDb1-knockdown plants is caused bythe down-regulation of scavenging activity of ROS, weanalyzed the catalase- and peroxidase-like activities inthe leaf extracts of the OsPLDb1-knockdown andvector control plants. No difference in the enzymeactivities was observed between these plants (data notshown), indicating that the knockdown of OsPLDb1probably affected ROS generating rather than scav-enging activity.

Up-Regulation of Defense-Related Genes in theOsPLDb1-Knockdown Plants

We also analyzed whether the expression of defense-related genes was up-regulated in these OsPLDb1-knockdown plants. Quantitative RT-PCR analysis ofthree typical defense-related genes, probenazole-inducible protein1, chitinase, and thaumatin-like pro-tein, in the leaves of 30-d-old plants (i.e. 30 d afterseeding) showed that the expression of these geneswas dramatically increased in theOsPLDb1-knockdownplants, concomitant with the decrease of OsPLDb1expression (Fig. 4A). Global changes in the gene ex-pression induced by OsPLDb1 knockdown were fur-ther analyzed for the leaves from 30-d-old plants usinga rice 44K DNAmicroarray. Figure 4B summarizes theup- and down-regulation of those genes in each cat-egory, whose expression was changed more than threetimes compared with that of the vector control plants.Approximately 1,400 genes changed their expressionsignificantly in theOsPLDb1-knockdown plants undersuch conditions (Supplemental Table S2). More than600 genes with the annotated functions, notably thoseassociated with defense responses, signal transduc-tion, and a number of transcription factors, were up-regulated in the OsPLDb1-knockdown plants. Thesedefense-related genes included those for PR-1,b-glucanase, chitinase, PR-4, thaumatin-like protein,and probenazol-inducible proteins. In addition, anumber of transcription factor genes belonging to theWRKYand ERF families were also up-regulated. Thesegenes are well known to be up-regulated during the

Figure 3. The phenotype of theOsPLDb1-knockdown plants grown for30 d under normal growth conditions. A, Accumulation of H2O2 in theleaves of 20- and 30-d-old knockdown (#7; shaded columns) andvector control (white columns) plants. H2O2 was analyzed using aquantitative H2O2 assay kit. The values represent averages of triplicateexperiments, and the error bars indicate SD. B, Accumulation ofOsPLDb1 mRNA in the leaves of the knockdown (#7; RNAi; shadedcolumn) and vector control (Control; white column) plants was deter-mined by quantitative RT-PCR. The values were standardized to theexpression level of a rice polyubiquitin gene (RUBIQ1). The valuesrepresent averages of triplicate experiments, and the error bars indicateSD. C, PLD activity in the leaf extracts of the knockdown (#7; RNAi;shaded columns) and vector control (Cont; white columns) plants wasdetermined using NBD-PC (PC-PLD) or NBD-PE (PE-PLD) as a substratein the presence of 1-butanol (final concentration, 0.1% [v/v]) asdescribed in “Materials and Methods.” The values represent averagesof triplicate experiments, and the error bars indicate SD. [See onlinearticle for color version of this figure.]



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defense responses (Van Loon and Van Strien, 1999;Maleck et al., 2001). On the other hand, many cellcycle-related genes, such as a- and b-expansin genes,were down-regulated (Fig. 4B; Supplemental TableS2). These results are similar to the previous obser-vations on the elicitor-induced gene responses in ricecells (Akimoto-Tomiyama et al., 2003; Desaki et al.,2006). These data, therefore, indicate that the knock-down of OsPLDb1 resulted in the induction ofdefense-related genes in the absence of pathogeninfections, a novel observation that, to our knowl-edge, has not been reported for any plant PLD.Recently, Li et al. (2007) reported that OsPLDb1plays a positive role in the abscisic acid responseand that the suppression of OsPLDb1 resulted in theup-regulation of GAmyb (X98355) and a-amylase(AK101744) genes during seed germination. How-ever, the up-regulation of the GAmyb gene as well asthe a-amylase gene was not observed in either theseedlings or leaves of OsPLDb1-knockdown plantsby DNA microarray analysis (data not shown), prob-ably reflecting the differences in the experimentalsystems.

It was also observed that the expression ofOsPLDb2, a closely related PLD isoform in rice, wassignificantly up-regulated in the OsPLDb1-knock-down plants (Supplemental Fig. S2). The exact biolog-ical significance of this phenomenon is not clear, but itmight reflect the cellular response to compensate forthe knockdown ofOsPLDb1. In spite of the up-regulationof OsPLDb2 expression, PC-PLD activity of the knock-down plant was clearly suppressed (Fig. 3C), indi-cating that OsPLDb1 is the major PLD isoformcontributing to this activity.

Induction of Hypersensitive Response-Like Reactions in

the OsPLDb1-Knockdown Plants

When the knockdown plants for eight PLD geneswere grown further in a greenhouse, small reddishbrown lesions became visible over the surface of theleaves only in the OsPLDb1-knockdown plants ap-proximately 40 d after seeding, although such a phe-notype was not visible for the 30-d-old plants, wherethe up-regulation of various defense genes and theH2O2 accumulation were already started (Fig. 5A).Similar results were observed for 51 of 55 independentOsPLDb1-knockdown lines, although the density ofthe lesions was different among them. Quantitativeanalysis ofOsPLDb1mRNA in the leaves ofOsPLDb1-knockdown lines, all of which developed the lesions,showed a clear suppression ofOsPLDb1 expression, asshown in Figure 5B (RNAi). In contrast, the vectorcontrol plants did not show the development of lesionsat all (Fig. 5A, Control; 22 lines were analyzed andgave similar results). These results indicate that the de-velopment of the lesions in the OsPLDb1-knockdownplants is due to the down-regulation of OsPLDb1,directly or indirectly.

Figure 4. Expression of defense-related genes (A) and changes of geneexpression (B) in the OsPLDb1-knockdown plants grown for 30 d afterseeding under normal growth conditions. A, Total RNA was preparedfrom the leaves of knockdown (#7; RNAi) and vector control (Control)plants grown for 30 d, and the expression levels of probenazol-inducibleprotein (PBZ1), chitinase (RCC), and thaumatin-like protein (TLP) geneswere analyzed by quantitative RT-PCR. The values were standardized tothe expression level of a rice polyubiquitin gene (RUBIQ1). The valuesrepresent averages of triplicate experiments, and the error bars indicateSD. B, Classification of up- and down-regulated genes in the knockdownplants (#7) obtained from the DNA microarray analysis. Total RNA wasprepared from the leaves of knockdown and vector control plants grownfor 30 d and used for the microarray analysis. The duplicate experimentswere conducted using two independent plants. Genes for which expres-sion in the knockdown plants was increased or decreased more thanthree times compared with that in the vector control plants wereclassified according to their annotations. Data represent the sums ofup-regulated (shaded columns) and down-regulated (white columns)genes. The numbers are averages of duplicate experiments. [See onlinearticle for color version of this figure.]

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Suppression of OsPLDb1 InducesPhytoalexin Production

To examine whether phytoalexin production isup-regulated in the OsPLDb1-knockdown plants, weanalyzed the amount of momilactone A, a majorphytoalexin of rice (Cartwright et al., 1977), in the leavesof 50-d-old OsPLDb1-knockdown plants and vectorcontrol plants. As shown in Figure 6 (RNAi b), momi-lactone A concentration in the OsPLDb1-knockdownplants with visible lesions was approximately 100times higher compared with the vector control plants.On the other hand, the amount of momilactone A inthe leaves of the OsPLDb1-knockdown plants withoutthe lesions was only slightly increased comparedwith the vector control plants (RNAi a). These re-sults indicate that the phytoalexin production is up-regulated in the OsPLDb1-knockdown plants butstarts mostly after the development of the lesions.

Suppression of OsPLDb1 Enhances Disease Resistance

against Rice Bacterial Blight as Well as Rice Blast

Susceptibility of the OsPLDb1-knockdown riceplants to a virulent blast fungus was evaluated to seewhether the activation of defense responses inducedby the OsPLDb1 knockdown resulted in an increase of

disease resistance. TheOsPLDb1-knockdown and vec-tor control plants (at the three- to six-leaf stage) wereinoculated with P. grisea (teleomorph Magnaporthegrisea) race 007, a major fungal pathogen and compat-ible to the rice variety used in this experiment, and thedisease symptoms were evaluated 7 d later. Contraryto the development of extensive lesions in the leaves ofthe vector control plants, the OsPLDb1-knockdownplants displayed remarkably decreased lesion forma-tion, irrespective of the age of the tested plants (20 or30 d old; Fig. 7, A and C). Quantification of the lesionarea indicated that the development of susceptible-type lesions in the OsPLDb1-knockdown plants wasdecreased more than 7-fold compared with the vec-tor control plants (Fig. 7, B and D), indicating an evi-dent increase of disease resistance against rice blastinfection. Similar results were obtained with otherOsPLDb1-knockdown lines, where the degree of dis-ease resistance seemed to correlate with the degree ofOsPLDb1 knockdown (Supplemental Fig. S3; cor-relation coefficient = 0.904). To examine whether theOsPLDb1-knockdown plants also show disease resis-tance against other pathogens, we tested their re-sponse to rice bacterial blight infection, Xoo, usingthe uppermost fully extended leaves of 50-d-old plants(Fig. 7, E and F). Similar to the case of blast infection,lesion formation caused by the bacterial blight was

Figure 5. The phenotype of theOsPLDb1-knockdown plants grown for50 d under normal growth conditions. A, The phenotypes of the leavesof the knockdown (#7; RNAi) and vector control (Control) plants. B,Accumulation ofOsPLDb1mRNA in the leaves of the knockdown (#7;RNAi; shaded column) and vector control (Control; white column)plants was determined by quantitative RT-PCR. The values werestandardized to the expression level of a rice polyubiquitin gene(RUBIQ1). The values represent averages of triplicate experiments, andthe error bars indicate SD.

Figure 6. Accumulation of phytoalexin in the OsPLDb1-knockdownplants grown for 50 d under normal growth conditions. The phytoalexinfraction was prepared from the leaves of these plants and analyzed forthe major rice phytoalexin, momilactone A, by liquid chromatography-mass spectrometry as described in “Materials and Methods.” Forknockdown plants (#7; RNAi), a indicates the leaves with very fewlesions and b indicates the leaves with numerous lesions. The valuesrepresent averages of triplicate experiments, and the error bars indicateSD. [See online article for color version of this figure.]



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dramatically decreased (4.4-fold decrease) in theOsPLDb1-knockdown plants (Fig. 7E). In addition,the population of Xoo in the OsPLDb1-knockdownplants was decreased more than 10-fold comparedwith the vector control plants at 15 d after Xoo inoc-ulation (Supplemental Fig. S4). These results clearlyshow that the suppression of OsPLDb1 enhanceddisease resistance against a broad range of pathogens,including bacteria and oomycetes. It is noteworthy

that these experiments were performed with the up-permost fully extended leaves, where the hypersensi-tive response (HR)-like cell death and the phytoalexinaccumulation were not yet visible.

The expression of OsPLDb1 itself was induced bythe inoculation of P. grisea to the wild-type rice leaves(Fig. 7G). The expression of OsPLDb1 reached amaximum (3.6-fold increase) at 1 d after inoculation,preceding the susceptible lesion formation.

Figure 7. Increased disease resistance of theOsPLDb1-knockdown plants to pathogen infec-tions and the expression patterns of OsPLDb1 inresponse to rice blast infection. A and C, Typicaldisease symptoms on the extended leaves of 20-d-old (A) and 30-d-old (C) knockdown (#7; RNAi)and vector control (Control) plants at 1 week afterrice blast inoculation. B and D, Quantification ofthe lesion area in the leaves of the knockdown(RNAi; shaded columns) and vector control (Con-trol; white columns) plants shown in A and C,respectively. The values represent the percentageof lesion area to the whole leaf area. The valuesrepresent averages of four (B) or five (D) replicateexperiments, and the error bars indicate SD. E,Typical disease symptoms on the uppermost fullyextended leaves of the 50-d-old knockdown (#7;RNAi) and vector control (Control) plants at 2weeks after rice bacterial blight inoculation. F,Quantification of the lesion area in the leaves ofthe knockdown (RNAi; shaded column) and vec-tor control (Control; white column) plants shownin E. The values represent averages of four repli-cate experiments, and the error bars indicate SD.G, The temporal expression patterns of OsPLDb1in 30-d-old wild-type rice plants treated with P.grisea. Accumulation of OsPLDb1 mRNA in theextended leaves treated with (shaded columns) orwithout (white columns) rice blast inoculation forthe indicated days was determined by quantitativeRT-PCR. The values represent averages of tripli-cate experiments, and the error bars indicate SD.

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DISCUSSION

Knockdown of OsPLDb1 Activates Defense Responsesand Increases Disease Resistance in Rice

In this paper, we show that the knockdown of a ricePLD gene, OsPLDb1, resulted in the activation ofdefense responses and increased disease resistance inrice. We establish the knockdown plants for eight PLDgenes in rice, OsPLDa1, -a3, -a4, -a5, -«, -b1, -b2, and-d2, which were listed in the database when we startedthis work, using RNAi. Among them, only OsPLDb1-knockdown plants showed the activation of typicaldefense responses such as ROS generation and defensegene expression in the absence of pathogen infection,followed by the development of HR-like lesions andphytoalexin production in a later growth stage. Thefact that the induction of ROS generation as well asdefense gene expression in the OsPLDb1-knockdownplants preceded the HR-like lesion formation indicatesthat the knockdown of OsPLDb1 directly affected theupstream part of these defense responses, rather thanthrough HR cell death. Concerning the induction ofHR-like lesion formation, various mutants or trans-genic plants that exhibit a lesion-mimic phenotypehave been reported, providing a goodmodel system tostudy the molecular mechanisms of HR in plants(Lorrain et al., 2003). However, it has not been reportedthat the knockdown or knockout of the genes involvedin phospholipid signaling leads to the lesion-mimicphenotype associated with defense responses.The OsPLDb1-knockdown plants exhibited in-

creased disease resistance to major pathogens ofrice, P. grisea and Xoo, before the formation of visiblespontaneous lesions (Fig. 7). In this respect, theOsPLDb1-knockdown plants are similar to the barley(Hordeum vulgare) mlo (Wolter et al., 1993) and rice cdr3(Takahashi et al., 1999) mutants, which exhibit resis-tance to fungal pathogens before the formation ofvisible lesions. Interestingly, the up-regulated genes inthe OsPLDb1-knockdown plants included those genesfor transcription factors that have been reported to beinduced by the pathogen infection/elicitor treatmentand play an important role for defense signaling, suchas WRKY45 and WRKY71. Shimono et al. (2007) re-cently showed that the overexpression of WRKY45enhanced blast resistance, and Liu et al. (2007) showedthat the overexpression of WRKY71 enhanced resis-tance to virulent bacterial pathogens. The expressionlevels of OsWRKY71 and OsWRKY45 genes in theOsPLDb1-knockdown plants increased 8.7- and 3.8-fold compared with that in the vector control plant,respectively (Supplemental Table S2), indicating thatthe up-regulation of these genes may contribute to theinduction of defense-related genes and the enhance-ment of blast resistance.No significant difference was observed between the

growth of the vector control and OsPLDb1-knock-down plants, such as plant height, number of leaves,and heading date after seeding, although the seed

weight of the OsPLDb1-knockdown plants seemed tobe slightly decreased compared with that of the vectorcontrol plants (Supplemental Table S3). These resultssuggested that the suppression of OsPLDb1 increaseddisease resistance of rice without a significant effect ongrowth and development.

Concerning the up-regulation of OsPLDb1 by the in-fection of a compatible pathogen (Fig. 7G), it is worthremembering that some negative regulator genes fordefense responses, such as rice spl11 and ArabidopsisWRKY48, were up-regulated in response to pathogeninfection (Zeng et al., 2004; Xing et al., 2008). As theinduction of defense reactions such as HR potentiallyhas an adverse effect on normal growth, these re-sponses must be induced when plants are infectedwith pathogens and also be down-regulated properly.Thus, the induction of negative regulators such asOsPLDb1 by pathogen infection may constitute part ofsuch a negative feedback loop.

OsPLDb1, a Negative Regulator of Defense Responses?

It has been suggested that the activation of PLDand the resulting PA generation during pathogeninfection/elicitor treatment play a positive role in themobilization of defense responses. We previouslyreported that the elicitor treatment of rice cells acti-vated PC-PLD and induced PA generation (Yamaguchiet al., 2003, 2005). The addition of PA itself directlyinduced ROS generation and expression of defense-related genes in the absence of the elicitor treatment.Sang et al. (2001) also reported that the suppression ofAtPLDa1 decreased the level of ROS generation andthat the addition of PA recovered the ROS generationin Arabidopsis leaves. Our results described here,however, indicate that OsPLDb1 negatively regulatesdefense responses, probably at the upstream part ofthe defense signaling cascade. Bargmann et al. (2006)also reported that the knockdown of a tomato PLDbgene, LePLDb1, resulted in the up-regulation of ROSgeneration, although in the presence of xylanase elic-itor. Although the contribution of other isoforms ofrice PLDs, such as OsPLDb2, in the up-regulation ofdefense responses in the OsPLDb1-knockdown ricecannot be excluded, these results seem to indicate theinvolvement of PLDb1 in the negative regulation ofdefense responses in plants. Our results also indicatethat OsPLDb1 seems to suppress defense responses inrice even under normal growth conditions; however, itis not clear whether this is the case for tomato as well.As already described, the expression level ofOsPLDb1was very high in most tissues studied (Fig. 2), whereasthat of LePLDb1 was very low (Laxalt et al., 2001).These differences in the expression levels of PLDb1genes might cause the differences in their regulation ofdefense responses in the corresponding plants undernormal growth conditions.

These results indicate that plant PLDs are involvedin the positive and negative regulation of defenseresponses, probably depending on the corresponding



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isoforms. Similar observations have been reported inthe responses against freezing or oxidative stress inArabidopsis. It was reported that the knockdown ofPLDa1 in Arabidopsis enhanced freezing tolerance,whereas the knockout of PLDd weakened freezingtolerance (Wang, 2005). Wang (2005) also indicatedthat AtPLDa1 is involved in ROS generation, whereasAtPLDd is involved in the suppression of ROS-promoted programmed cell death. Which isoforms ofPLDs are involved in the opposite regulation of thesedefense responses should be clarified further in futurestudies.

Possible Mechanisms for the Different Functions ofPLD Isoforms

If plant PLDs play opposite roles in plant defensesignaling, as discussed above, what can be the expla-nation for such mechanisms? One possibility is thedifferent localization of each PLD isoform, whichenables the regulation of different target proteinsdownstream of each PLD. Several papers have indi-cated that the different PLD isoforms localize in dif-ferent organelles/membranes in plant cells. In rice,OsPLDa1 was detected in the cell wall, membranes,and chloroplasts, whereas OsPLDa4 and OsPLDa5were predominantly detected in the chloroplasts of theleaves (McGee et al., 2003). AtPLDa1 in Arabidopsiswas detected in the plasma membrane, intracellularmembranes, mitochondria, and clathrin-coated vesi-cles (Fan et al., 1999). AtPLDb1 was reported to bindactin in vitro, which is a major component of a micro-filament in the cells (Kusner et al., 2003). Bargmannet al. (2006) showed that the fusion protein of tomatoLePLDb1 and GFP was detected in the cytosol ofcultured cells, whereas treatment with xylanase elici-tor induced the relocalization to punctate structureswithin the cytosol. They proposed a possibility thatplant PLDb1may form a tether between vesicular mem-branes and the actin cytoskeleton (Bargmann et al.,2006). These results suggest that the cellular localiza-tion is different between PLDa and PLDb, indicatingthe different physiological functions of these PLDs.

In addition to the different localization of PLDisoforms, different modes of regulation of each PLDmay be a factor controlling the function of each PLD.For example, it is well known that the Ca2+ depen-dence of activation is very different among PLD iso-forms (Qin andWang, 2002). Similarly, PLDb1, PLDg1,and PLDz1 require phosphatidylinositol 4,5-bisP forthe activation, whereas other isoforms do not (Qin andWang, 2002). These results indicate that different PLDsare involved in the different physiological responsesdownstream of these second messengers.

Different molecular species of PA may also be gen-erated by different PLD isoforms, as differences in thesubstrate preference among PLD isoforms have beenreported (Qin and Wang, 2002). More recently, Honget al. (2008) reported that the molecular species of PA

generated by the action of AtPLDa3 are clearly differ-ent from those by AtPLDa1 in Arabidopsis. Thisobservation suggests that the differences in the mo-lecular species of PA generated by these PLDs mightreflect differences in the functions of the PLD isoforms.Furthermore, different phospholipid compositions oforganelle membranes might also contribute to thegeneration of different PAs by PLD isoforms, resultingin the differences in PA target proteins.

Although the results obtained in this study withknockdown transgenic plants indicate that OsPLDb1negatively regulates defense responses in rice, it is notclear whether OsPLDb1 is actually involved in thedefense regulation under physiological conditions,and this should be clarified in future studies. Thefact that the knockdown of OsPLDb1 resulted in theactivation of various defense responses, includingthe up-/down-regulation of numerous genes, ROSgeneration, followed by HR cell death and phytoalexinproduction, indicates that OsPLDb1 regulates the de-fense responses at the upstream part of the signaltransduction pathways. The analysis of the mecha-nism of negative regulation of defense responses byOsPLDb1, including the survey of downstream com-ponents in OsPLDb1 signaling, should be the subjectof future studies as well.

MATERIALS AND METHODS

Plant and Fungal Materials

The rice (Oryza sativa japonica ‘Nipponbare’, blast resistance gene: Pik-s)

was used in this study. The sterilized transgenic or wild-type rice seeds were

germinated for 2 weeks on Murashige and Skoog (MS) agar medium with or

without 50 mg L21 hygromycin, respectively. The resulting plants were

transferred to soil and grown in a greenhouse (25�C/27�C, solar radiation).A strain of Pyricularia grisea (teleomorph Magnaporthe grisea, ‘Ina 86-137’,

race 007) and Xanthomonas oryzae pv oryzae (‘T-7133’, Japanese race IIIa) were

used as fungal and bacterial pathogens, respectively. Both Ina 86-137 and

T-7133 are compatible with Nipponbare.

DNA Database Search and Computer Analysis

To identify the members of the PLD gene family in rice, we searched the

following databases: DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp),

National Center for Biotechnology Information (http://www.ncbi.nml.nih.

gov/blast/Genome), and National Institute of Agrobiological Sciences

(http://cdna01.dna.affrc.go.jp/cDNA/). Multisequence alignment analysis

for the deduced amino acid sequences was carried out using Genetyx

(Software Development).

RNA Extraction and Real-Time PCR

Total RNAs for first-strand cDNA synthesis were isolated from rice tissues

by the SDS-phenol method. The isolated RNA (5 mg) was reverse transcribed

(SuperScript II; Invitrogen) using an oligo(dT)13 primer. An aliquot of the first-

strand cDNA mixture corresponding to 10 ng of the total RNAwas used as a

template for real-time PCR analysis. The reaction was carried out on the Smart

Cycler System (Cepheid) with the SYBER premix ExTaq (Takara Bio) accord-

ing to the manufacturer’s instructions. The gene-specific primers designed for

each gene are listed in Supplemental Table S4. The amplified bands were

cloned directly into pGEM-T vector (Promega) and sequenced to confirm that

they were indeed the fragments of the intended genes. A calibration curve for

each gene was obtained by performing real-time PCRwith several dilutions of

the cloned cDNA fragment. The specificity of the individual PCR amplifica-

Yamaguchi et al.


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tion was checked using a heat dissociation protocol from 65�C to 95�Cfollowing the final cycle of PCR. The results obtained for the different cDNAs

were standardized to the expression level of a rice polyubiquitin gene

(RUBIQ1; Wang et al., 2000).

DNA Constructs and Rice Transformation

The OsPLDb1-RNAi vector was constructed by generating an inverted

hairpin loop into the pZH2Bik binary vector, which was modified to contain

the rice ubiquitin promoter (OsmUbiP), the intron of a rice aspartic protease

gene (RAP intron; Asakura et al., 1995), and the Nos terminator (T-Nos), in the

sequence from pPZP202 (Hajdukiewicz et al., 1994). A 343-bp fragment

corresponding to nucleotides 2,323 to 2,665 of OsPLDb1, which does not

overlap with the amplified OsPLDb1-specific fragment (1,973–2,314) in the

real-time PCR experiments described in Supplemental Table S4, was amplified

using the following oligonucleotides: 5#-GAATGCATGAGACGAGTTCGC-3#(forward) and 5#-CCTTTTCTTGTCATGTCCTACT-3# (reverse). The PCR

fragment was ligated in the reverse orientation into pZH2bik vector between

OsmUbiP and RAP intron and between RAP intron and T-Nos. The resulting

vector contains the RNAi intermediate sequence downstream of the rice

ubiquitin promoter. The empty vector, pZH2Bik, was also used to generate

vector control plants. The other rice PLD gene-specific RNAi vectors were also

constructed according to the same methods described above. To confirm the

gene specificity, part of the cDNA including the 3# noncoding region, the

sequence of which is different for each PLD gene, was amplified using each

primer set (Supplemental Table S5) and inserted into the vector.

Agrobacterium tumefaciens EHA105 carrying the above construct was used

to transform Nipponbare rice. The Agrobacterium-mediated transformation

was performed according to the method of Toki (1997) using vigorously

growing callus derived frommature embryos. Transformed calli were selected

on MS agar medium containing 50 mg L21 hygromycin. Regenerated plants

were grown in a greenhouse, and T0 transgenic seeds were harvested 40 d

after flowering. T1 transgenic plants carrying the transgene were selected by

germinating seeds on MS agar medium containing 50 mg L21 hygromycin for

2 weeks, and then selected plants were transferred to soil and grown in a

greenhouse (25�C–30�C, solar radiation).

Determination of H2O2 Accumulation and

Scavenging Activity

A 50-mg sample of leaf tissue harvested from 30- to 50-d-old plants was cut

into small pieces (5 3 5 mm) and immediately homogenized with 5% (w/v)

aqueous trichloroacetic acid containing EGTA (10 mM) on ice. The extract was

centrifuged at 10,000g at 4�C for 10 min, and the amount of H2O2 in the

supernatant was analyzed using a quantitative H2O2 assay kit (Bioxytech

H2O2-560; Oxis International).

ROS-scavenging activity was determined using 10 mM H2O2 as a substrate.

A 20-mg sample of leaf tissue harvested from 30- to 50-d-old plants was cut

into small pieces (53 5 mm) and immediately homogenized with 0.5 mL of 25

mM Tris-HCl buffer (pH 7.5) containing 10 mM EGTA, 1 mM dithiothreitol,

1 mM phenylmethylsulfonyl fluoride, and 0.3 M Suc for 1 min on ice. The

homogenate was centrifuged at 10,000g at 4�C for 10 min, and the supernatant

was stored as the cellular extract fraction at 220�C until analysis. Protein

content in each fraction was determined according to Bradford (1976). The

standard reactionmixture contained 1.0 to 1.5 mg of cellular protein and 10mM

H2O2 in a final volume of 50 mL. Reactions were performed at 37�C for 10 min,

and the amount of H2O2 in the supernatant was analyzed using a quantitative

H2O2 assay kit.

Measurement of in Vitro PLD Activity

PLD activity in the cellular extract fraction described above was de-

termined using fluorescent lipids (Avanti Polar Lipids) as a substrate as

described previously (Yamaguchi et al., 2004) with a slightmodification. 7-Nitro-

2-1,3-benzoxadiazol-4-yl amino (NBD)-PC (14:0 12:0) and NBD-PE (14:0 12:0)

were stored at 220�C in chloroform. Prior to use, they were dried and

emulsified by sonication in 5 mM sodium cholate. The standard reaction

mixture contained 25 mM HEPES (pH 7.0), 10 to 15 mg of cellular protein, 0.1

mM NBD-PC or NBD-PE, and 1-butanol (final concentration, 0.1% [v/v]) in a

final volume of 50 mL. Reactions were performed at 37�C for 30 min and then

stopped by the addition of 0.25 mL of CHCl3:methanol:HCl (100:100:0.6, v/v).

After 0.1 mL of 1 N HCl containing 5 mM EGTA was added, the mixture was

vortexed and centrifuged at 10,000g for 1 min. The solvent layer was then

recovered and dried under vacuum. The samples were applied to a thin-layer

chromatography plate (20 3 10 cm) and developed with the organic upper

phase of ethylacetate:iso-octane:acetic acid:water (12:2:3:10, v/v). Fluores-

cently labeled phosphatidylbutanol (PtdBut) was visualized using a UV

transilluminator, and the regions corresponding to PtdBut were marked. The

marked spots were scraped from the plates and placed in 0.5 mL of CHCl3:

methanol:water (5:5:1, v/v), vortexed, and centrifuged for 5 min at 10,000g.

The supernatant was dried under vacuum and emulsified by sonication in 5

mM sodium cholate. The fluorescence (excitation, 460 nm; emission, 534 nm)

from the eluted PtdBut was measured with a fluorescence spectrophotometer

(Versa Fluor; Bio-Rad). The spots of PtdBut were identified by comparison

with the standards (Avanti Polar Lipids) visualized by iodine vapor. A

standard curve was constructed using a range of NBD-PC concentrations.

Microarray Analysis

Microarray analyses were performed using a 60-mer rice oligomicroarray

containing 44K features (Agilent Technologies). The total RNA used for the

analysis was prepared from leaves of 30-d-old plants by the SDS-phenol

method. The RNA extracts were fluorescently labeled and hybridized to the

microarray slides according to the manufacturer’s protocol. The slides were

then scanned by an Agilent scanner. To assess reproducibility, two indepen-

dent experiments were conducted using two independent plants. Data

indicating a less than 3-fold increase in fluorescence between Cy5 and Cy3

in each experiment were excluded from further analysis. Genes whose

expression in the knockdown plants was increased or decreased more than

three times compared with that in the vector control plants are listed in

Supplemental Table S2.

Determination of Phytoalexin

A 50-mg sample of leaf tissue harvested from 50-d-old plants was cut into

small pieces (5 3 5 mm) and extracted with 0.5 mL of 70% (v/v) methanol at

room temperature for 24 h. After centrifugation at 10,000g for 10 min, the

supernatant was analyzed with a liquid chromatography-mass spectrometry

system. An HP 1100 HPLC apparatus (Hewlett-Packard) equipped with an

Inertsil ODS 3 column (4.63 250 mm; GL Science) was used. Elution with 80%

(v/v) aqueous acetonitrile (containing 0.1% [v/v] formic acid) was carried out

at a flow rate of 1.0 mL min21. Mass spectrometry was performed using a

Mariner electrospray ionization-time of flight mass spectrometer (Applied

Biosystems) in the positive-ion mode.

Inoculation of P. grisea and Xoo on Rice Plants

Inoculation of blast fungus on rice plants was performed according to the

methods of Ashizawa et al. (2005). The isolate of P. grisea (Ina 86-137) was

transferred to oatmeal agar plates and incubated at 25�C for 14 d in the dark.

The aerial hyphae on these plates were removed by gently brushing the agar

surface, and then the plates were incubated for 72 h at 25�C under white

fluorescent light. To prepare the conidial suspension, the conidia formed on

the surface were collected by adding distilled water containing 0.02% (w/v)

Tween 20. TheOsPLDb1-knockdown and vector control plants grown for 20 or

30 d after seeding (the three- to six-leaf stage) under normal growth conditions

were sprayed with 10 mL of a conidial suspension per plant (104 conidia mL21

in distilled water containing 0.02% Tween 20) using an atomizer with a fine

nozzle. The sprayed plants were kept in a moist chamber at 100% relative

humidity in the dark at 25�C for 20 h and then moved to a greenhouse (25�C/27�C, solar radiation). The disease symptoms were observed for 1 to 2 weeks

after inoculation, and the area of diseased leaf lesions was measured.

Inoculation of bacterial blight was performed according to the methods of

Kauffman et al. (1973). The isolate of Xoo (T-7133) was grown on PPGA

medium (Nishiyama and Ezuka, 1977). The uppermost fully extended leaves

of OsPLDb1-knockdown and vector control plants grown for 50 d after

seeding under normal growth conditions were inoculated with a bacterial

suspension (108 cells mL21) by the leaf-clipping method. The disease symp-

toms were observed for 2 weeks after the inoculation, and the area of the

diseased leaf lesions was measured. To determine the bacterial populations,

the inoculated leaves were harvested at 0, 5, and 15 d after Xoo inoculation and

surface sterilized with 70% (v/v) ethanol and 3% (v/v) sodium hypochlorite

solution. The leaves were cut into small pieces and resuspended in 10 mL of

water to harvest bacteria separately. Diluted extract (0.05 mL) was incubated



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on PSAmedium (5 g L21 Bacto peptone, 15 g L21 Suc, 5 g L21 soluble starch, 0.5

g L21 calcium nitrate, and 15 g L21 agar, pH 7.0) at 28�C for 4 to 6 d, and the

number of colonies formed on the agar plate was counted.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers listed in Supplemental Tables S4 and S5.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Accumulation of H2O2 and OsPLDb1 mRNA in

the leaves of 30-d-old OsPLDb1-knockdown plants.

Supplemental Figure S2. Accumulation of OsPLDb1 and OsPLDb2

mRNA in the OsPLDb1-knockdown plants.

Supplemental Figure S3. Comparison between blast resistance and

mRNA levels of OsPLDb1 in the OsPLDb1-knockdown plants.

Supplemental Figure S4. Growth curve of Xoo in the OsPLDb1-knock-

down and vector control plants.

Supplemental Table S1. The expression analysis of rice PLD genes.

Supplemental Table S2. DNA microarray analysis of OsPLDb1-knock-

down plants.

Supplemental Table S3. Growth and development parameters of the

vector control and OsPLDb1-knockdown plants.

Supplemental Table S4. Gene-specific PCR primers used for quantitative

RT-PCR amplification.

Supplemental Table S5. Gene-specific PCR primers used for the construc-

tion of RNAi vectors.

ACKNOWLEDGMENTS

We thank Drs. Yoshiaki Nagamura (National Institute of Agrobiological

Sciences), Morifumi Hasegawa (Ibaraki University), and Tatsuro Hirose

(National Agricultural Research Center) for their useful advice on microarray

analysis, phytoalexin analysis, and quantitative RT-PCR, respectively. Ken

Nagata (Meiji University) contributed to the DNA database search and

computer analysis.

Received November 25, 2008; accepted March 11, 2009; published March 13,

2009.

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ber 2021

suppression of a phospholipase d gene, ospld 1 activates ...to positively regulate ros generation...

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