glufosinate ammonium-induced pathogen inhibition and ...by magnaporthe grisea and rhizoctonia solani...

Glufosinate Ammonium-Induced Pathogen Inhibitionand Defense Responses Culminate in Disease Protectionin bar-Transgenic Rice1[C]

Il-Pyung Ahn*

National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon 441–100, Korea

Glufosinate ammonium diminished developments of rice (Oryza sativa) blast and brown leaf spot in 35S:bar-transgenic rice.Pre- and postinoculation treatments of this herbicide reduced disease development. Glufosinate ammonium specificallyimpeded appressorium formation of the pathogens Magnaporthe grisea and Cochliobolus miyabeanus on hydrophobic surface andon transgenic rice. In contrast, conidial germination remained unaffected. Glufosinate ammonium diminished mycelial growthof two pathogens; however, this inhibitory effect was attenuated in malnutrition conditions. Glufosinate ammonium causedslight chlorosis and diminished chlorophyll content; however, these alterations were almost completely restored in transgenicrice within 7 d. Glufosinate ammonium triggered transcriptions of PATHOGENESIS-RELATED (PR) genes and hydrogenperoxide accumulation in transgenic rice and PR1 transcription in Arabidopsis (Arabidopsis thaliana) wild-type ecotype Columbiaharboring 35S:bar construct. All transgenic Arabidopsis showed robust hydrogen peroxide accumulation by glufosinateammonium. This herbicide also induced PR1 transcription in etr1 and jar1 expressing bar; however, no expression wasobserved in NahG and npr1. Fungal infection did not alter transcriptions of PR genes and hydrogen peroxide accumulationinduced by glufosinate ammonium. Infiltration of glufosinate ammonium did not affect appressorium formation of M. grisea invivo but inhibited blast disease development. Hydrogen peroxide scavengers nullified blast protection and transcriptions ofPR genes by glufosinate ammonium; however, they did not affect brown leaf spot progression. In sum, both direct inhibition ofpathogen infection and activation of defense systems were responsible for disease protection in bar-transgenic rice.

Rice (Oryza sativa) is one of the most important cropsworldwide. Farmers have applied integrated crop man-agement and governments have implemented envi-ronmental regulations to reduce chemical applicationsto a desirable level; however, synthetic chemicals arestill required for stable cereal production. In addition,development of genetically modified rice plants resis-tant to nonselective herbicides like glufosinate ammo-nium is expected to improve crop productivity (Delannayet al., 1995). Agricultural chemicals frequently induceside effects on their target crops and agroecosystems.For example, some herbicides are known to inducemorphological and physiological alterations in cropsand higher incidence of plant diseases (Campbell andAltman, 1977; Sanogo et al., 2000). Glyphosate occa-sionally suppressed disease defense responses and en-hanced disease development (Keen et al., 1982; Brammalland Higgins, 1988). Interestingly, glufosinate ammo-nium treatment onto transgenic rice expressing bargene controls rice blast and sheath blight progressions

by Magnaporthe grisea and Rhizoctonia solani (Uchimiyaet al., 1993; Tada et al., 1996). This herbicide also in-duced resistance against brown patch and dollar spot,caused by R. solani and Sclerotinia homoeocarpa, in bar-transgenic bentgrass (Agrostis spp.; Higgins et al., 2003).

The bar gene, which codes for the enzyme phos-phinothricin (PPT) acetyl transferase (PAT), is one of themost prevalent selectable markers of genetically mod-ified crops and confers tolerance against glufosinateammonium, an active ingredient of the nonselectiveherbicide Basta. Glufosinate ammonium is an ammo-nium salt of PPT and efficiently kills various kinds ofplants, including rice. PAT inactivates PPT by acety-lating it (Botterman et al., 1991). Glufosinate ammo-nium is a Glu analog that inhibits Gln synthetase byirreversible binding (Manderscheid and Wild, 1986).Gln synthetase catalyzes an ATP-dependent incorpo-ration of ammonium into the amide position of Glu,resulting in the formation of Gln. This is indispensablefor capturing toxic ammonium produced duringphotorespiration and inorganic nitrogen assimilation(Wallsgrove et al., 1983). The inhibition of Gln synthe-tase by glufosinate ammonium results in an accumula-tion of toxic ammonium derived from photorespiration(Martin et al., 1983; Wild et al., 1987; Sechley et al.,1992; Last, 1993). Accumulation of toxic ammoniumdisturbs electron transport systems of both chloroplastsand mitochondria and induces production of hazard-ous free radicals (Krogmann et al., 1959; Puritch andBarker, 1967). Free radicals in turn cause lipid perox-idation (especially on membranes), damage of other

1 This work was supported by the National Institute of Agricul-tural Biotechnology (grant to I.-P.A.).

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

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Il-Pyung Ahn ([email protected]).

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

www.plantphysiol.org/cgi/doi/10.1104/pp.107.105890

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cellular constituents, and eventually, cell death (Hess,2000). Because of this nature, glufosinate ammoniumis often termed a ‘‘pro-oxidative herbicide’’ (Strobel andKuc, 1995). Herbicidal mechanisms and target sites ofglufosinate ammonium are definitive in nontransgenicplants; however, physiological alterations induced bythis chemical in bar gene-harboring plants still remainunclear.

Plant defense activators induce systemic acquired resis-tance and condition the plant in a resistant state. Thesechemicals trigger defense-related responses like aug-mented and accelerated transcription of PATHOGENESIS-RELATED (PR) genes, defense-related materials, andburst of active oxygen species (AOS; Friedrich et al.,1996; Benhamou and Belanger, 1998; Cohen, 2002; Iritiand Faoro, 2003). Systemic resistance-acquired plantsshow broad-spectrum disease resistance against mul-tiple pathogens because of the activation of defensemechanisms including hypersensitive response and sys-temic translocation of resistance via secondary signalmessengers like salicylic acid and jasmonic acid. Tran-scriptional activation of PR genes and rapid accumu-lation of hydrogen peroxide have been recognized asreliable molecular and cellular markers of disease resis-tance in rice and other plants (Ganesan and Thomas,2001; Kachroo et al., 2003; Tsukamoto et al., 2005).Augmented and accelerated mRNA synthesis of PR1has been reported in incompatible interaction betweenrice and avirulent M. grisea and in rice treated withplant defense activators (Kim et al., 2001b; Ahn et al.,2005b). Recently, novel functions of AOS have beeninvestigated. Besides arresting pathogen proliferationin planta, AOS are involved in cell wall reinforcement(Olivain et al., 2003), callose deposition (Huckelhovenet al., 1999), and acts to signal molecules in systemictranslocation of acquired resistance (Bolwell et al., 1995,1998; Tenhaken et al., 1995; Wojtaszek, 1997; Alvarezet al., 1998; Chamnongpol et al., 1998). Similar to pre-viously known plant defense activators, methyl viol-ogen and mercuric chloride promote oxidative damageand chlorosis-induced plant defense responses (vanLoon, 1975; Lund et al., 1993; Strobel and Kuc, 1995).Phosphates and oxalates inducing localized chlorosisand necrosis and AOS accumulation also trigger sys-temic acquired resistance in cucumber (Cucumis sa-tivus; Doubrava et al., 1988; Gottstein and Kuc, 1989;Mucharromah and Kuc, 1991; Orober et al., 2002).

M. grisea and Cochliobolus miyabeanus, the teleomorphsof Pyricularia grisea and Bipolaris oryzae, are hemibio-trophic and necrotrophic fungal pathogens that causeblast and brown leaf spot, the most devastating dis-eases of rice worldwide. Yield losses due to blast havebeen estimated at 11% to 30%. Moreover, 10% incidenceof neck blast in the field could reduce rice productionby 5% to 6%. Brown leaf spot is one of the most com-mon diseases and is observed at any stage of growth inthe field. The outbreak of rice brown leaf spot causedthe Bengal famine and was the major cause of twomillion fatalities in 1943 (Stuthman, 2002). Prior to in-fection by both pathogenic fungi, a series of infection-

related morphological changes, initiated by spore ad-hesion to the host surface, spore germination, germtube elongation, appressorium formation, and pene-tration by an infection peg, were observed (Goto, 1958;Ou, 1985). Conidial germination and appressorium for-mation have been recognized as important target sitesfor screening and developing novel fungicides (Ohand Lee, 2000). For example, tricyclazole, an inhibitorof melanin biosynthesis prerequisite for turgor gener-ation, effectively inhibits rice blast disease. Inhibitionof appressorium formation by methylglyoxal-bis-guanyl hydrazone (a polyamine biosynthesis inhibi-tor) in C. miyabeanus resulted in a significant reductionof rice brown leaf spot (Ahn and Suh, 2007a).

This research shows the inhibitory effects of glufo-sinate ammonium on the developments of blast andbrown leaf spot in bar gene-expressing rice. Direct andindirect effects of glufosinate ammonium on the dis-ease progressions by pathogens and defense-relatedcellular and molecular responses in rice were examined.

RESULTS

Effects of Glufosinate Ammonium on Rice Blast andBrown Leaf Spot

Rice ‘Dongjin’ (nontransgenic control [NC]) and35S:bar-transgenic ‘Dongjin’ (transgenic) inoculated withM. grisea strain KJ201 exhibited typical rice blast symp-toms and normal disease development (Fig. 1). Water-soaked lesions began to form 3 d postinoculation (dpi).Invasive mycelial growth was apparent within andaround lesions. At 5 dpi, almost one-half of the inoc-ulated leaf surface was covered with blast lesions, andmassive sporulation began to appear in the center ofthe lesion at 10 dpi. When NC and transgenic rice wereinoculated with C. miyabeanus strain HIH-1, approxi-mately 93% of conidia germinated within 12 h postin-oculation (hpi). Visible spot, representing host cell death,was evident around 16 hpi. At 3 dpi, the average sizeof lesions was approximately 0.6 3 3 mm (width 3length), and chlorosis began to appear around themargin of lesions.

The effect of glufosinate ammonium on disease de-velopment was evaluated. Transgenic rice was highlysusceptible to KJ201; however, treatment of 100 mgmL21 glufosinate ammonium 24 h prior to infectiongreatly increased blast protection. The number and sizeof blast lesions were significantly decreased. In addi-tion, their developments were defined within the initialinfection sites or retarded. Sporulation was barelyobserved on any of the lesions on the glufosinateammonium-treated leaves. Moreover, glufosinate am-monium treatment noticeably hampered brown leafspot disease development. The number of lesions wasalso decreased, and symptom development was defineddistinctively.

Glufosinate ammonium treatments 5 d or 1 d priorto (preinoculation treatment) and 12 h or 1 d after

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(postinoculation treatment) inoculation significantlyreduced the developments of both diseases. Protectioneffect against rice blast was retained at the same levelin all testing periods; however, that against brown leafspot reached a maximum level 1 d prior to fungal in-oculation and slightly decreased thereafter (Fig. 2).

Direct Effects of Glufosinate Ammonium onFungal Developments

Glufosinate ammonium treatment reduced the num-ber and size of lesions in transgenic rice plants. Toinvestigate direct, antimicrobial activities of this her-bicide, the effects of glufosinate ammonium on conid-ial germination and appressorium formation in M.grisea and C. miyabeanus were examined on the artifi-cial substratum, hydrophobic surface of GelBond, andon transgenic rice leaves. Appressorium formations inM. grisea and C. miyabeanus on the GelBond were in-hibited by glufosinate ammonium in a dose-dependentmanner (Fig. 3, A and B). Glufosinate ammoniumreduced appressorium formation in M. grisea and C.miyabeanus by 71% and 83% at 100 mg mL21 concen-tration, respectively. In contrast, the same treatmentdid not affect conidial germination of both pathogens.To evaluate the effect of glufosinate ammonium on theprepenetration morphogenesis in vivo, conidial sus-pension along with or without glufosinate ammoniumwas placed on detached NC and transgenic rice leaves.For more precise observation, chlorophyll was removed

from the samples, and fungal cells were stained. Twenty-four hours after placements, most of the water-treatedconidia germinated and formed appressoria. Glufosi-nate ammonium inhibited 61% of appressorium for-mation in M. grisea and 85% in C. miyabeanus. Conidialgermination was reduced by 22% in M. grisea and 13%in C. miyabeanus.

Phytotoxic effects of glufosinate are induced by ac-cumulation of toxic ammonium derived from nitrogenassimilation. To investigate the effects of glufosinateon the pathogen growth in the plant mimic conditions,the inhibition rates of pathogen growth by glufosinateon the nutrient-rich and nutrient-deprived media werecompared. After infection into host tissues, pathogenwas encountered to a malnutrition conditions deficientin nitrogen or carbon source. Transcriptions of sev-eral fungal genes, MPG1 in M. grisea and ccSNF1 inCochliobolus carbonum infecting maize (Zea mays), spe-cifically expressed during in planta ramification, werealso induced in the malnutrition conditions (Beckermanand Ebbole, 1996; Talbot et al., 1996; Tonukari et al.,2000). In addition, the same treatment was expected toattenuate accumulation of toxic ammonia derived fromnitrogen assimilation. Similar results were describedpreviously (Snoeijers et al., 2000). Glufosinate ammo-nium supplementation inhibited mycelial growth ofM. grisea and C. miyabeanus by 91% and 88% on thecomplete media (CM; Fig. 4). In contrast, glufosinateammonium inhibited mycelial growth of M. grisea andC. miyabeanus by about 20% and 8% on the nutrient-

Figure 1. Effects of glufosinate ammonium (G) on thedevelopments of rice blast and brown leaf spotcaused by M. grisea strain KJ201 and C. miyabeanusstrain HIH-1, respectively. Glufosinate ammonium ormock was applied 24 h before fungal inoculation.Disease progression was estimated according to thenumber and size of lesions. A, Disease progression in‘Dongjin’ (2, NC) and bar-transgenic ‘Dongjin’ (1)pretreated with mock (2, 250 mg mL21 Tween 20) orglufosinate ammonium (1, 100 mg mL21) and inoc-ulated with M. grisea strain KJ201 and C. miyabeanusstrain HIH-1. Each data point is mean 6 SE. B, Blastand brown leaf spot developments in ‘Dongjin’ (NC)and bar-transgenic ‘Dongjin’ (bar) pretreated withglufosinate ammonium (1) or mock (2). Photographsdepicting representative symptoms were taken 10 dafter fungal inoculation. Experiments were repeatedmore than three times with three replicates consistingof 15 plants; almost similar tendencies were ob-tained. [See online article for color version of thisfigure.]

Mechanisms of Disease Protection by Glufosinate Ammonium

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deficient media (CM lack of nitrogen and carbonsources).

Indirect Effects of Glufosinate Ammonium onDisease Development

Glufosinate ammonium distinctively inhibited symp-tom development of rice blast and brown leaf spot.Preinoculation treatment also significantly diminisheddisease progression. Further, this chemical did not showsufficient pathogen growth inhibition in the nutrient-deficient condition. These results implied that glufo-sinate might induce other unknown disease-inhibitingmechanisms except its direct, antimicrobial activities.To investigate effects of glufosinate ammonium on hostdisease resistance, the activity of glufosinate ammo-nium on the transcription of PR1 in transgenic rice andArabidopsis (Arabidopsis thaliana) was analyzed. Pre-vious results showed that pathogen infection, plantdefense activators, and environmental stresses inducetranscriptions of the PR genes (Lawton et al., 1996; Kimet al., 2001b). Glufosinate ammonium higher than 10mg mL21 per se induced transcription of PR1 gene intransgenic rice (Fig. 5A). A large amount of PR1 tran-script was accumulated locally and systemically (Fig.5B). Further, fortified PR1 transcription was retained

for more than 15 d after glufosinate spray (Fig. 5C).In NC and transgenic rice infected with KJ201 aftermock (250 mg mL21 Tween 20) treatment, the induc-tions of PR1, PBZ1 (a probenazole-inducible PR gene),and POX22.3 (a gene encoding PEROXIDASE22.3)were significantly delayed (by 1–2 d), and their transcrip-tions reached maximum levels at 3 dpi (Fig. 5D). Thiscoincided with the formation of water-soaked lesions(Fig. 1B). Beyond 3 dpi, the level of transcripts decreasedslightly. Transcriptions of PR1, PBZ1, and POX22.3peaked within 1 dpi with HIH-1 in NC and transgenicrice. Glufosinate ammonium treatment triggered robusttranscriptions of tested marker genes in transgenic rice.Robust transcription remained unaffected and patho-gen infection did not alter this pattern.

To further confirm indirect effects of glufosinateand investigate defense signaling pathways inducedby this herbicide, we investigated the effects of glufo-sinate ammonium on the hydrogen peroxide accu-mulation and PR1/PDF1.2 transcriptions in ecotypeColumbia-0 (Col-0), bacterial NahG-expressing Col-0,and defense-defective mutants like npr1, etr1, and jar1harboring 35S:bar construct. There was no discrete hy-drogen peroxide accumulation in rosette leaves treatedwith mock (Fig. 6A). In contrast, glufosinate ammo-nium on rosette leaves induced robust hydrogen per-oxide accumulation in treated rosette leaves (local) oftransgenic Col-0 and all transgenic plants expressingbar. Further, hydrogen peroxide accumulation was alsoobserved in the cauline leaves (systemic) of all trans-genic lines tested. Although there were some differences,quantitative analyses of hydrogen peroxide productionalso corroborated these phenomena (Fig. 6B). Mockspray induced no PR1 and PDF1.2 transcriptions in alltested lines (Fig. 6C). Discrete PR1 transcription wasobserved in glufosinate-treated local (rosette) and non-treated systemic (cauline) leaves of transgenic Col-0,etr1, and jar1 lines; however, NahG and npr1 did notshow these transcriptions. Salicylic acid spray trig-gered local PR1 transcription in the rosette leaves oftransgenic Col-0. Glufosinate did not induce PDF1.2transcriptions in all transgenic lines tested. However,jasmonic acid treatment triggered local PDF1.2 tran-scriptions in the rosette leaves from transgenic Col-0line. Transgenic Arabidopsis was produced using thefloral dip method and it possessed single PR1:eGFP(enhanced GFP gene) and 35S:bar construct (data notshown). Similar with the above result, strong greenfluorescence was induced on the transgenic Col-0 seed-lings by glufosinate treatment (Fig. 6D). Mock-treatedtransgenic plants did not show detectable fluorescence.Glufosinate triggered eGFP mRNA synthesis in trans-genic plants (Fig. 6E).

Glufosinate ammonium induced severe chlorosisand wilting on NC (Fig. 7A). The transgenic plant washighly resistant to glufosinate ammonium; however,slight chlorosis was evident 1 to 2 d posttreatment (dpt),and this alteration was almost completely restoredwithin 7 to 10 d. Glufosinate ammonium diminishedmaximum photochemical efficiency (Fv/Fm) of PSII by

Figure 2. Rice blast and rice brown leaf spot disease progressions ofglufosinate ammonium-treated bar-transgenic (bar) or NC rice plants.Plants were inoculated by M. grisea strain KJ201 or C. miyabeanusstrain HIH-1. In the meantime, 100 mg mL21 of glufosinate ammoniumwas sprayed 5 d or 1 d prior to (preinoculation treatments; 25 and 21,respectively) fungal inoculation. Treatments were also performed 12 hor 1 d after (postinoculation treatments; 10.5 and 11, respectively)inoculation. In the analysis of preinoculation treatment effects, glufo-sinate ammonium-treated rice leaves were carefully washed severaltimes by spraying distilled water at 24 h after treatment. Diseaseprogression was determined 7 dpi. Each data point is mean 6 SE.Different letters indicate statistically significant differences betweentreatments (Duncan’s multiple range test; P , 0.05). Experiments wererepeated more than three times with three replicates consisting of 15rice plants; almost similar tendencies were observed.

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28% in NC and 26% in transgenic rice at 3 dpt. Fv/Fmvalue of transgenic plants was restored within 7 dpt;however, that within NC decreased continuously andfinally died (Fig. 7B). Hydrogen peroxide accumula-tion and pathogen growth were analyzed in transgenicrice infected by two pathogens. In NC inoculated withM. grisea strain KJ201, hydrogen peroxide was notaccumulated at the infection site 24 hpi and began tobe observed at 72 hpi (Fig. 6C; data not shown). Activefungal ramification was observed at 72 hpi. In trans-genic rice, glufosinate ammonium induced hydrogenperoxide accumulation within 24 h posttreatment; how-ever, the same treatment did not trigger host cell alter-ation. No hydrogen peroxide was observed at 24 hpiand fungal ramification developed normally in thetransgenic rice leaves. In the transgenic rice pretreated

with glufosinate ammonium, most rice cells exhibitedhydrogen peroxide accumulation and KJ201 infectiondid not affect this cellular response.

Effects of Hydrogen Peroxide Scavengers on the DiseaseResistance Induced by Glufosinate Ammonium

Hydrogen peroxide scavengers, ascorbic acid orcatalase, were infiltrated 24 h after glufosinate ammo-nium spray and treated transgenic plants were inoc-ulated with M. grisea or C. miyabeanus. Both hydrogenperoxide scavengers significantly diminished blast dis-ease protection induced by glufosinate ammonium;however, the same treatment did not affect the inci-dence of rice brown leaf spot (Fig. 8, A and B). Further,ascorbic acid or catalase alone did not affect rice brown

Figure 3. Effects of glufosinate am-monium on conidial germinationand appressorium formation in M.grisea strain KJ201 and C. miyabea-nus strain HIH-1. Conidial germi-nation and appressorium formationwere estimated microscopically24 h after placement onto the testedsurface. A, Effects of glufosinateammonium on prepenetration de-velopment of M. grisea and C.miyabeanus on the hydrophobicsurface of GelBond. Each datapoint is mean 6 SE. B, Effects ofglufosinate ammonium (100 mgmL21) on conidial germination(white bar) and appressorium for-mation (black bar) of M. grisea andC. miyabeanus on rice leaves frombar-transgenic plants. Differentletters indicate statistically signifi-cant differences between treatments(Duncan’s multiple range test; P ,0.05). C, Prepenetration develop-ments of M. grisea and C. miyabea-nus on the hydrophobic surface ofGelBond (artificial surface) or trans-genic (bar) rice leaves in the pres-ence (1) or absence (2) of 100 mgmL21 glufosinate ammonium. a,Appressorium; c, conidium; g, germtube. Bars 5 50 mm. Experimentswere repeated more than threetimes with three replicates; almostsimilar tendencies were obtained.[See online article for color versionof this figure.]



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leaf spot developments (data not shown). Infiltrationof glufosinate ammonium did not affect appressoriumformation in M. grisea and C. miyabeanus (Fig. 8B). In-filtration of glufosinate ammonium induced robusthydrogen peroxide accumulation; however, catalase orascorbic acid almost completely inhibited hydrogenperoxide production induced by glufosinate ammo-nium (Fig. 8C; data not shown). In addition, glufosi-nate ammonium spray and catalase infiltration resultedin the vigorous in planta M. grisea ramification at 72 hpi(Fig. 8D) and abolishment of PR1, PBZ1, and POX22.3transcriptions (Fig. 8E).

DISCUSSION

Glufosinate Ammonium Confers Disease Protection

Glufosinate ammonium treatment protected 35S:bar-transgenic rice from blast and brown leaf spot. Num-ber and size of lesions were significantly lower thanthe mock-treated plants. Effects of glufosinate ammo-nium on disease development in bar-expressing cropshave been investigated previously. Similar with ourresults, glufosinate ammonium treatment protected bar-transgenic rice from blast and sheath blight caused byR. solani (Uchimiya et al., 1993; Tada et al., 1996). Sim-ilar disease-protecting ability against R. solani andS. homoeocarpa was observed in bar-transgenic bent-grass (Liu et al., 1998; Higgins et al., 2003; Wang et al.,2003). Hence, appropriate application of glufosinateammonium could be helpful in inhibiting disease aswell as in controlling weeds.

Glufosinate Ammonium InhibitsPrepenetration Morphogenesis

Disease protection by glufosinate ammonium is aresult of direct inhibition of pathogens or indirect in-hibition via plant-mediated responses. We examineddirect effects of glufosinate ammonium on the conidialgermination and appressorium formation in M. griseaand C. miyabeanus on the hydrophobic surface of Gel-Bond (Fig. 3). The hydrophobic side of GelBond isknown to be suitable to examine the chemical effectson the prepenetration development of both fungi invitro (Lee and Dean, 1994; Ahn and Suh, 2007b). Ourresults showed that glufosinate ammonium (100 mgmL21) effectively inhibited appressorium formation ofboth fungi, while conidial germination remained un-affected. Similar results were generated for M. grisea invitro (Tada et al., 1996). Further, this specific inhibitionwas also observed in the leaves of transgenic rice. In-hibition of appressorium formation is one of the strongpresumptions why glufosinate ammonium diminishes

Figure 4. Effects of nutrient starvation and/or glufosinate ammoniumon the fungal growth. Prior to colony contact on the CM, colonymorphologies of M. grisea (A) and C. miyabeanus (B) on the CMcontaining carbon and nitrogen sources (1) or mock (2) and supple-mented with glufosinate (1) or mock (2) were photographed andcolony areas were measured (C). Different letters indicate statistically

significant differences between treatments (Duncan’s multiple rangetest; P , 0.05). Experiments were repeated more than three times withthree replicates; almost similar results were obtained. [See onlinearticle for color version of this figure.]

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both diseases. In contrast, bean (Phaseolus vulgaris) rootstreated with a low dose of glufosinate ammonium en-hanced sporangial germination of Pythium ultimum (Liuet al., 1997). These variable effects on development offungal species might be due more to the alternatives ofbiochemical signaling pathway(s) than to the struc-tural differences of Gln synthetase in these fungi. Thisis supported by the fact that Gln synthetase genes arehighly conserved in their amino acid sequences inseveral plant pathogenic fungi and play a crucial rolein ammonium assimilation and Glu biosynthesis in

prokaryotes and eukaryotes, including fungi (Fileticiet al., 1996; Stephenson et al., 1997). To confirm the roleof inhibition of appressorium formation on the diseaseprotection, glufosinate ammonium was washed 24 hprior to inoculation and the remaining chemicals werecompletely removed 1 h prior to inoculation. In spiteof the normal appressorium formation, blast andbrown leaf spot diseases were distinctively decreased(Fig. 7). These results suggested that glufosinate am-monium exerts disease-controlling activity via multipleroutes, including inhibition of appressorium forma-tion in M. grisea and C. miyabeanus.

Effects of Glufosinate Ammonium in Vitro and in Vivo

Glufosinate ammonium almost completely inhib-ited mycelial growth of both fungal pathogens on theCM containing carbon and nitrogen sources (Fig. 4).Similar with this result, glufosinate ammonium sig-nificantly inhibited mycelial growth of R. solani and S.homoeocarpa infecting bentgrass (Wang et al., 2003).Glufosinate also inhibited soil fungi and bacteria ben-eficial for plant growth (Ismail et al., 1995). Glufosinatereduced Pythium blight caused by Pythium aphanider-matum in transgenic bentgrass expressing bar. How-ever, it did not alter mycelial growth in vitro (Liu et al.,1998). The inhibitory effects of glufosinate on fungalmycelial growth were significantly diminished on themedium deficient in carbon and nitrogen sources.Glufosinate did not affect germ tube elongation, an-other form of mycelial growth, in the absence of nu-trient supplementation (Fig. 3). Our results suggest thatnitrogen or carbon starvation could mimic the malnu-trition conditions of host plants. Therefore, direct in-hibitory effects of glufosinate on the fungal growthcould be diminished or abolished in the host cells ortissues. In spite of the similarities in nutrient deficiency,glufosinate distinctively inhibited fungal growth invivo (Fig. 7C). These results imply that glufosinateammonium might induce multiple disease-protectingmechanisms, including inhibition of appressorium for-mation in M. grisea and C. miyabeanus.

Glufosinate Ammonium Induces Plant

Defense Responses

Glufosinate ammonium significantly diminished thesize of lesions of rice blast and brown leaf spot ontransgenic rice. Other reports also showed that glufo-sinate ammonium treatment confers resistance againstrice sheath blight on bar-transgenic rice (Uchimiyaet al., 1993). Our results and this disease protectionsuggest that inhibition of appressorium formation isnot the only mechanism to protect plants from diseaseas conferred by glufosinate ammonium and alterationof plant defense status. To examine this speculation,we compared the effects of pre- and postinoculationtreatment on the rice blast and brown leaf spot devel-opments. In addition, glufosinate ammonium remain-ing on rice leaves was washed carefully 24 h after

Figure 5. Transcriptions of PR genes induced by glufosinate ammo-nium (100 mg mL21) treatment and/or pathogen inoculation at varyingdpt and dpi. Pathogen inoculation and glufosinate ammonium treat-ment were performed as described in Figure 1A. A, PR1 gene tran-scription induced by varying concentrations of glufosinate ammoniumtreatment at 1 dpt. B, Transcription of PR1 gene induced by glufosinateammonium in bar-transgenic rice. Glufosinate ammonium was sprayedon second leaves. Leaves sprayed with glufosinate ammonium (L, local)or left untreated (S, systemic) were harvested from the same plants1 dpt. Transgenic rice leaves treated with mock (C) were also harvestedat the same time. C, PR1 gene expression was retained up to 15 d afterglufosinate treatment. D, Transcriptions of PR1, PBZ1, and POX22.3 inNC and transgenic (bar) rice challenged with M. grisea strain KJ201 orC. miyabeanus strain HIH-1 1 d after mock or 100 mg mL21 glufosinateammonium. Total RNA was extracted from the leaves of five rice plantsrecovered 0, 1, 2, 3, and 4 dpi. Experiments were repeated more thanthree times; almost similar results were obtained.



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spray to rule out its direct effects on the pathogen in-fection. As shown in Figure 2, preinoculation treat-ment also significantly inhibited progressions of bothdiseases. The same results were observed in the bar-transgenic rice (Tada et al., 1996). Further, the mostdistinctive rice brown leaf spot protection was accom-plished by the treatment 1 d prior to C. miyabeanus in-oculation. These results imply that indirect effects likemodulation of host defense responses might be re-sponsible for the disease protection by glufosinateammonium.

We analyzed molecular and cellular defense-relatedresponses in transgenic rice. Glufosinate ammoniumtreatment induced transcriptions of PR1, PBZ1, andPOX22.3 in transgenic rice and PR1 transcription in

transgenic Arabidopsis. This transcription was dosedependent, translocated systemically, and retained fora long period. Both M. grisea and C. miyabeanus did notalter the transcriptions of PR genes triggered by glufo-sinate ammonium. Similar results were also observedin transgenic Col-0 containing PR1:eGFP and 35S:bar.The role of PR1 remains unclear; however, transcriptaccumulation of PR1 has been recognized as a molec-ular marker to determine whether the plant is in aresistant state or not. Constitutive transcriptional acti-vation of PR genes and distinctive disease protectionwere observed in the benzothiadiazole-treated to-bacco (Nicotiana tabacum; Friedrich et al., 1996), Pto-overexpressing tomato (Solanum lycopersicum) plants(Tang et al., 1999), and Arabidopsis cpr and dnd mutants

Figure 6. Effects of glufosinate ammonium on the accumulation of hydrogen peroxide and transcriptions of PR1 and PDF1.2 in35S:bar-transgenic Arabidopsis Col-0 and its mutants. Glufosinate (1, 100 mg mL21) was sprayed onto rosette leaves and treatedrosette and nontreated cauline leaves were harvested 24 h later. A, Hydrogen peroxide accumulation in treated rosette (local)and nontreated cauline (systemic) leaves of 35S:bar-harboring transgenic Arabidopsis Col-0, NahG, npr1, etr1, and jar1 linesexposed to glufosinate. Harvested leaves were stained with DAB (0.1%, w/v). B, Quantification of hydrogen peroxideaccumulation in treated rosette (local) and nontreated (systemic) cauline leaves of transgenic Arabidopsis Col-0, NahG, npr1,etr1, and jar1 lines exposed to glufosinate. Each bar represents the mean 6 SE. Different letters indicate statistically significantdifferences between treatments (Duncan’s multiple range test; P , 0.05). C, Transcriptions of PR1 and PDF1.2 in treated rosette(L; local) and nontreated cauline (S; systemic) leaves of transgenic Arabidopsis Col-0, NahG, npr1, etr1, and jar1. Data were fromArabidopsis sprayed with glufosinate (1) or 250 mg mL21 Tween 20 only (2, mock). In addition, leaves of transgenic Col-0 wereharvested 1 d after salicylic acid (SA, 500 mM) or jasmonic acid (JA, 100 mM) spray. D, Expression of eGFP by glufosinate treatmentin transgenic Arabidopsis containing PR1:eGFP and 35S:bar gene construct. Bar 5 1 mm. E, Transcription of eGFP transcriptunder the control of PR1 promoter by glufosinate treatment in transgenic Arabidopsis. Experiments were repeated more thanthree times; almost similar results were obtained.

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(Bowling et al., 1997; Clough et al., 2000; van Hultenet al., 2006). These results imply that glufosinate am-monium could induce resistance in the transgenic riceand Arabidopsis. Similar to our results, treatment withlactofen, a herbicide belonging to the diphenyletherclass, induced robust transcriptions of PR-1a, PR-5,and PR-10 in soybean (Glycine max) leaves (Graham,2005). In addition, glufosinate ammonium induced sys-temic transcriptions of three PR genes within the leavesof transgenic plants. Therefore, the effect of glufosinateammonium mobilized in other parts of the plant. Sys-temic translocation of defense-related signaling hasbeen described in tobacco, rice, and Arabidopsis treatedwith plant defense activators like benzothiadiazole,probenazole, and salicylic acid (Friedrich et al., 1996;Midoh and Iwata, 1997; Maleck et al., 2000).

We investigated the effects of glufosinate on the hy-drogen peroxide accumulation and PR1 and PDF1.2transcriptions in Arabidopsis Col-0, bacterial NahG-expressing Col-0, and three mutants harboring 35S:barconstruct (Fig. 6). These lines are unable to metabolizesalicylic acid, synthesize mRNAs of PR genes, or per-ceive jasmonic acid or ethylene-dependent signaling.

Glufosinate ammonium triggered robust accumulationof hydrogen peroxide in all tested lines. Glufosinate-treated transgenic Col-0 showed high PR1 transcrip-tion; however, no PDF1.2 transcription was observed.Glufosinate did not trigger PR1 transcription in theNahG and npr1 lines. These results strongly suggestthat glufosinate exerts its effects through the hydrogenperoxide and salicylic acid-dependent signaling path-ways and there is no tight correlation between hydro-gen peroxide accumulation by glufosinate and jasmonicacid-dependent signaling pathways. Similar depen-dencies on hydrogen peroxide and salicylic acid andtight correlation between them were also observed inb-aminobutyric acid-treated tobacco and Arabidopsis(Siegrist et al., 2000; Zimmerli et al., 2000; Ganesan andThomas, 2001; Orozco-Cardenas et al., 2001; Zimmerliet al., 2001; Kachroo et al., 2003). In addition, correla-tion between resistance against rice blast and hydro-gen peroxide and/or salicylic acid has been described(Ganesan and Thomas, 2001; Uchimiya et al., 2002;Agrawal et al., 2003).

Glufosinate ammonium triggered slight chlorosisand reduction of chlorophyll content (Fv/Fm levels) in

Figure 7. Effects of glufosinate ammoniumand M. grisea strain KJ201 on hydrogen per-oxide accumulation and fungal ramification inbar-transgenic and nontransgenic rice. Theplants were sprayed with glufosinate ammo-nium (1) in 250 mg mL21 Tween 20 or mockonly (2). One day after treatment, rice plantswere inoculated with virulent M. grisea strainKJ201. A, Effects of glufosinate ammonium onthe NC and transgenic (bar) rice plants. The4-week-old plants were sprayed with 100 mgmL21 glufosinate ammonium (1) or mock (2).Representative leaves were photographed 10dpt. B, Effects of glufosinate ammonium on theFv/Fm in NC and transgenic (bar) rice. Eachdata point is mean 6 SE. Effects of glufosinateammonium on hydrogen peroxide accumula-tion (C) and fungal ramification (D). Micro-scopic observation of hydrogen peroxide andin planta growth was performed on leavesrecovered at 24 and 72 hpi. *, Hydrogenperoxide accumulation. a, Appressorium; c,conidium; m, invasive mycelia. Bars 5 50 mm.Experiments were repeated more than threetimes; almost similar tendencies were obtained.



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transgenic plants (Fig. 7, A and B). Although trans-genic plants contain PAT, detoxifying glufosinate, theabove phenomena imply that glufosinate induces tem-porary phytotoxicity on bar-transgenic rice. Similar phy-totoxic responses were observed on other transgenicrice plants expressing the bar gene as the selectablemarker (data not shown). Glufosinate ammonium

induced hydrogen peroxide accumulation and patho-gen inoculation did not affect this defense-related cel-lular response (Fig. 7C). Therefore, there should becorrelation between AOS production by glufosinateand activation of disease defense responses. Hydrogenperoxide is involved in cell wall reinforcement, path-ogen abolishment, cell death, and modulates plant

Figure 8. Effects of ascorbic acid and catalase on rice blast and rice brown leaf spot diseases inhibited by glufosinate ammoniumon bar-transgenic rice and transcriptions of PR genes. A, Transgenic rice was sprayed with 100 mg mL21 glufosinate ammonium(1) in 250 mg mL21 Tween 20 or mock only (2, 250 mg mL21 Tween 20) and then mock (2, distilled water), ascorbic acid (1), orcatalase (1) was infiltrated into the carefully washed leaves 24 h after herbicide treatment. M. grisea or C. miyabeanus wasinoculated 3 h after treatment with hydrogen peroxide scavengers. Photographs were taken 7 dpi. B, Quantification of rice blast(white bar) and rice brown leaf spot (black bar) disease developments and effects of catalase and ascorbic acid on theappressorium formation in M. grisea (white bar) and C. miyabeanus (black bar). Different letters indicate statistically significantdifferences between treatments (Duncan’s multiple range test; P , 0.05). C, Effects of catalase (1) on hydrogen peroxideaccumulation in glufosinate ammonium-treated plants. *, Hydrogen peroxide accumulation. a, Appressorium; c, conidium.Bars 5 50 mm. D, Invasive mycelial growth in 35S:bar-transgenic rice pretreated with 100 mg mL21 glufosinate ammonium (1)and infiltrated with 5,000 units mL21 catalase (1). M. grisea was inoculated 24 h after final treatment. Photograph depictingrepresentative infection hyphae in aniline blue staining 72 hpi. m, Invasive mycelial growth. Bars 5 50 mm. E, Analyses of PR1,PBZ1, and POX22.3 transcriptions in 35S:bar-transgenic rice leaves sprayed with glufosinate ammonium (1) and/or infiltratedwith catalase (2). Total RNA was prepared from five plants 24 h after infiltration, separated using denaturing gel electrophoresis,and transferred to nylon membrane. The blots were hybridized with radiolabeled PR1, PBZ1, and POX22.3 probes. Allexperiments were done at least three times; almost similar results were obtained.

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hypersensitive disease resistance (Levine et al., 1994).Because of its relatively long half-life and good per-meability, hydrogen peroxide is generally accepted asthe major AOS messenger (Bowler and Fluhr, 2000).

To confirm the role of hydrogen peroxide, the effectsof hydrogen peroxide scavengers on disease resistancetriggered by glufosinate ammonium were investigated(Fig. 8, A and B). Both hydrogen peroxide scavengerssignificantly attenuated resistance against blast in-duced by glufosinate ammonium; however, the sametreatment did not affect resistance against brown leafspot. These results indicate that hydrogen peroxide ac-cumulation, indispensable for blast disease resistance,is not necessary for expression of resistance againstrice brown leaf spot. In addition, inhibition of appres-sorium formation is not the only mechanism of pro-tection against rice blast and brown leaf spot, becauseglufosinate ammonium infiltration successfully in-hibited this disease in bar-expressing rice in spite of thevigorous appressorium formation by M. grisea and C.miyabeanus (Fig. 8, B and C). Microscopic observationof M. grisea growth in planta also corroborates theseresults (Fig. 8D). Infiltration of catalase with glufosi-nate abolished inhibitory effects of glufosinate ammo-nium on fungal ramification. Further, the same treatmentnullified PR1, PBZ1, and POX22.3 transcriptions in-duced by infiltration with glufosinate (Fig. 8E). Glufo-sinate ammonium-induced disease protection againstM. grisea and C. miyabeanus is due to the induction ofmultiple defense responses and some of them are de-

pendent on hydrogen peroxide accumulation (againstM. grisea), while others are not (against C. miyabeanus).As described above, hydrogen peroxide is one of thegeneral AOS messengers and its accumulation is ob-served in the host resistant for infected pathogens(Chamnongpol et al., 1998). They are also observed onplants pretreated with plant defense activators orresistance-promoting rhizobacteria and inoculated withcompatible pathogens (Park et al., 2000; Ahn et al.,2007b). This defense-related response effectively fendsoff pathogen invasion and in planta pathogen growthas hydrogen peroxide accumulation is often culmi-nated in host cell death. In contrast, hydrogen perox-ide accumulation did not affect infection and diseasedevelopment by C. miyabeanus. This finding is consis-tent with one of the most representative characteristicsof disease progression by necrotrophic pathogens,such as Botrytis cinerea and Pectobacterium carotovorum(Govrin and Levine, 2000; Govrin et al., 2006). Thereshould be other unknown defense mechanisms trig-gered by glufosinate, which is independent from hy-drogen peroxide accumulation or host cell death andeffective in inhibition of rice brown leaf spot.

In Figure 9, an overall proposed model is presentedto show possible mechanisms of glufosinate ammo-nium in the bar-transgenic rice. This model is based onprevious literature and findings from this study. Oneof the most important observations is distinctive ac-cumulation of hydrogen peroxide by glufosinate am-monium in the transgenic rice and Arabidopsis. Freeradical production by exogenous stimuli resulted inthe burst of AOS like hydrogen peroxide (Rao et al.,1996; Karpinski et al., 1999; Fryer et al., 2002). Hydro-gen peroxide acts as a signaling messenger in thesystemic translocation of defense responses, andpart of them effectively fends off M. grisea infection(Chamnongpol et al., 1998). All resistance mechanismsby glufosinate are not fully dependent on hydrogenperoxide, because glufosinate-induced resistance againstC. miyabeanus was retained in the absence of hydrogenperoxide. In addition, the importance of direct effectsof glufosinate ammonium on the inhibition of prepen-etration development in both pathogens should notbe ruled out in the disease protection caused by thisherbicide.

MATERIALS AND METHODS

Fungal Isolates, Plants, and Chemicals

Magnaporthe grisea strain KJ201 and Cochliobolus miyabeanus isolate HIH-1,

virulent on rice (Oryza sativa) ‘Dongjin’, were obtained from the National

Institute of Agricultural Science and Technology, Rural Development Admin-

istration and recovered from rice leaves showing typical symptoms of brown

leaf spot, respectively. Conidia of M. grisea were harvested from 10- to 12-d

colonies grown on oatmeal agar (Ahn et al., 2005a). Conidia of C. miyabeanus

were harvested from 7-d-old cultures grown on Suc Pro agar (Dhingra and

Sinclair, 1985) at 22�C under continuous fluorescent light. Nontransgenic andtransgenic ‘Dongjin’ harboring bar gene under the regulation of 35S promoter

were also obtained from the National Institute of Agricultural Biotechnology,

Rural Development Administration. Rice seeds were surface sterilized by

immersion in a 100 mg mL21 solution of thiophanate methylthiram for 16 h

Figure 9. Proposed model for glufosinate-induced disease resistance.Glufosinate initiates accumulation of free radicals by irreversiblebinding with and inactivation of Gln synthetase. Toxic ammoniaderived from photorespiration or nitrogen assimilation is increasedwithin the cell and disturbed electron transport system within chloro-plast. Free radicals were produced and in turn, this molecule triggersdisease resistance against M. grisea and C. miyabeanus.



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and sown in a commercial soil mixture at a density of three plants per 5- 3 5-cmpot. Rice plants were propagated in the greenhouse for 4 weeks. Glufosinate

ammonium (99% pure) was purchased from Riedel-de Haen and resuspended

in distilled water. Arabidopsis (Arabidopsis thaliana) wild-type Col-0, trans-

genic Col-0 expressing bacterial NahG gene, and mutants (npr1, etr1, and jar1)

from this line were obtained from The Arabidopsis Information Resource.

Arabidopsis was grown in a growth chamber at 22�C, 65% to 70% relativehumidity, and 16 h of illumination daily. Four-week-old Arabidopsis was used

for chemical treatment.

Generation of Transgenic Arabidopsis and Treatments

To examine the effects of glufosinate ammonium on the PR1 transcription

in Arabidopsis, the 2,000-bp upstream region of PR1 in Arabidopsis Col-0

genome was amplified by PCR using forward primer (5#-ggggacaagtttgta-caaaaaagcaggctATCTCATTTTATCCGTTCGC-3#) and reverse primer (5#-ggg-gaccactttgtacaagaaagctgggtTTTTCTAAGTTGATAATGGT-3#). This fragmentwas introduced into destination vector pBGWFS7 (Karimi et al., 2002), har-

boring 35S:bar, by GATEWAY cloning system according to the manufacturer’s

recommendations (Invitrogen). The resulting expression vector contained eGFP

and GUS gene adjacent to the introduced PR1 promoter and was introduced

into Agrobacterium tumefaciens strain GV3101. This bacterial strain was used for

transformation of Arabidopsis wild-type Col-0 via floral dip (Clough and

Bent, 1998). In addition, pBGWFS7 was transformed into the same bacterial

strain and used for transformation of Arabidopsis wild-type Col-0, NahG

(transgenic Col-0 line expressing the bacterial salicylate hydroxylase), npr1 (a

mutant that does not accumulate PR1 in response to salicylic acid), etr1 (an

altered perception of ethylene mutant), and jar1 (a mutant that displays reduced

sensitivity to methyl jasmonate). T3 seeds from independent transgenic lines

were surface sterilized with 70% ethanol and 5% sodium hypochlorite and

were grown on Murashige-Skoog medium. After incubation for 7 d at 22�C,the seedlings were submerged in distilled water (mock) or 100 mg mL21

glufosinate ammonium for 3 h and washed three times with distilled water.

Green fluorescence was observed using Confocal Laser Microscope (Olympus,

Fluoview TM 300) 6 h after treatment. In addition, total RNA was extracted

from the transgenic Col-0 plants and PR1 activity was assayed by northern

analysis as described later.

Conidial Germination and Appressorium Formation

in Vitro and in Vivo

To examine the effects of glufosinate ammonium on the prepenetration

development on artificial surface and on rice leaves, conidia of both fungi

were harvested and dropped with or without glufosinate ammonium, as

described previously (Oh and Lee, 2000; Ahn and Suh, 2007a). Percentages of

germinated and germinating conidia to form appressoria were determined

from at least 100 conidia with three replicates per treatment. Experiments

were done independently at least three times.

In Vivo Effect of Glufosinate Ammonium on RiceBlast and Brown Leaf Spot

Wild-type and transgenic rice plants were grown in commercial soil mix in

plastic pots (5 cm in diameter) for 4 weeks in the greenhouse. Mock (250 mg

mL21 Tween 20) or glufosinate ammonium (100 mg mL21 in 250 mg mL21

Tween 20) was sprayed on 10 rice plants 5 d or 1 d prior to and 12 h and 1 d

after M. grisea (2 3 105 conidia mL21) or C. miyabeanus (1 3 105 conidia mL21)inoculation. Rice leaves were sprayed with distilled water 24 h after glufo-

sinate ammonium treatment. The inoculated rice plants were placed in a dew

chamber (25�C, 100% relative humidity) for 16 to 24 h and transferred to thegreenhouse. Progression of rice blast was assessed 10 d after inoculation and

brown leaf spot was assessed 7 d after inoculation. The diseases were esti-

mated according to the method developed by the International Rice Research

Institute (1988). In vivo assays were done independently more than three times.

Nutrient Deficiency on the Pathogen Growth Inhibitedby Glufosinate

Mycelial blocks (6 mm in diameter) from actively growing colony edges of

M. grisea and C. miyabeanus were placed on CM or CM without nitrogen and

carbon sources and supplemented with mock (distilled water) or 100 mg/mL

glufosinate ammonium (Yang et al., 1994; Talbot et al., 1996). The colony area

was estimated after incubation at 25�C in the dark for 7 d.

Hydrogen Peroxide Accumulation and InvasiveMycelial Growth

To investigate the effect of glufosinate ammonium on hydrogen peroxide

accumulation, 10 transgenic rice plants were applied with 100 mg mL21

glufosinate ammonium. In addition, the same chemical was sprayed onto

rosette leaves of Arabidopsis transformed with pBGWFS7 containing 35S:bar.

All tested plants were grown on the soil. Rice plants were infected 1 d later

with virulent KJ201. Histochemical detection of hydrogen peroxide was per-

formed as described previously (Wohlgemuth et al., 2002) with minor modifi-

cation. To determine the effects of glufosinate ammonium on the accumulation

of hydrogen peroxide, rice leaves were recovered at 24 hpi and stained with

0.1% (w/v) diaminobenzidine (DAB; Sigma). In addition, rosette and cauline

leaves of transgenic Arabidopsis treated with glufosinate were stained with

DAB. Stained plant leaves were cleared with 96% (v/v) ethanol, preserved in

50% (v/v) ethanol, and observed under the light microscope. DAB staining

was a red-brown color under the light microscope. Quantitative determina-

tion of hydrogen peroxide within transgenic Arabidopsis was performed as

described (Ahn et al., 2007a). Briefly, debris was removed by perchloric acid

extraction, purified using AG1-X8 resin (Bio-Rad Laboratories), and hydrogen

levels were determined using Autolumat LB953 luminometer (EG & G Derthod).

To observe fungal growth within rice plants, leaves were recovered 96 hpi,

fixed with lactophenol, stained with 0.1% (w/v) aniline blue, and mycelial

growth was observed under the light microscope (Peng et al., 1986). More than

15 leaves from five randomly selected plants were observed in each exper-

iment. These experiments were done independently at least three times.

Estimation of Chlorophyll Fluorescence

Rice was grown as described above. Green parts of 10 seedlings were cut

by scissors and floated on 100 mg mL21 glufosinate ammonium under con-

tinuous fluorescent light of 150 mmol m22 s21. Fv/Fm value was measured using

CF-1000 chlorophyll fluorescence measurement system (Morgan Scientific) as

previously described (Artus et al., 1996; Jang et al., 2003).

Effects of Catalase and Ascorbic Acid on theHydrogen Peroxide Accumulation Induced by

Glufosinate Ammonium

Approximately 4-week-old transgenic rice was sprayed with mock (250 mg

mL21 Tween 20) or 100 mg mL21 glufosinate ammonium. Twenty-four hours

after treatment, mock (distilled water), 5,000 units mL21 catalase, or 10 mM

ascorbic acid were treated via vacuum infiltrated at 710 mmHg for 10 min

after complete washing with distilled water. After complete washing, infil-

trated rice plants were inoculated 3 h later with M. grisea strain KJ201 or

C. miyabeanus strain HIH-1. Inoculation and estimation of disease develop-

ment were performed as described above.

RNA Preparation and Transcription Analyses

Rice leaves were harvested for RNA isolation at 0, 1, 2, 3, and 4 d after

fungal inoculation or 1 d after glufosinate ammonium spray. Total RNA was

also extracted from 4-week-old transgenic Arabidopsis plants. To determine

whether the effects of glufosinate ammonium on PR1/PDF1.2 transcriptions

could be translocated systemically, glufosinate ammonium was sprayed on

rosette leaves of the 35S:bar-transgenic Arabidopsis and rosette and cauline

leaves were harvested. In addition, salicylic acid- (500 mM) or jasmonic acid

(100 mM)-treated rosette leaves were harvested from transgenic Col-0 24 h after

treatment. Harvested plant materials were preserved at 270�C. Total RNA wasextracted using a lithium chloride-precipitation method (Davis and Ausubel,

1989). For hybridization analyses, 5 mg of total RNA per lane was separated

electrophoretically in denaturing formaldehyde-agarose gel (8% formaldehyde,

0.53 MOPS, 1.5% agarose) and blotted to Hybond-N1 membrane (AmershamPharmacia Biotech) by capillary transfer. Uniform sample loading was con-

firmed by staining ribosomal RNA bands in the gel with ethidium bromide.

DNA probes were labeled with [a-32P] dCTP using a random primer labeling

kit (Boeringer-Mannheim). The PR1, PBZ1, and POX22.3 probes were derived

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from cDNA clones (Kim et al., 2001a; Ahn et al., 2005a). The 379-bp eGFP

probe was prepared by PCR using forward primer 5#-cacatgaagcagcacgactt-3#and reverse primer 5#-tgctcaggtagtggttgtcg-3#. Analyses of Arabidopsis PR1and PDF1.2 gene transcriptions were performed using the reverse transcription-

PCR as described (Pieterse et al., 1998) with some modifications. First-strand

cDNA was synthesized from 50 ng of total RNA of the leaves using the

Reverse-iT first-strand synthesis kit and anchored oligo(dT) as indicated by

the manufacturer’s instructions (AB gene). Independent PCR using equal

aliquots (0.5 mL) of cDNA samples was performed using PR1/PDF1.2-specific

primers as described (Vieira Dos Santos et al., 2003). The TUBULIN gene was

amplified as a quantitative control (Lee et al., 2000).

ACKNOWLEDGMENT

I deeply thank Dr. Maria Excelsis M. Orden for editing this article.

Received July 28, 2007; accepted October 16, 2007; published November 2,

2007.

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