the epiphytic fungus pseudozyma aphidis jasmonic acid ......studies, however, have shown that p....

The Epiphytic Fungus Pseudozyma aphidis InducesJasmonic Acid- and Salicylic Acid/Nonexpressor ofPR1-Independent Local and Systemic Resistance1[C][W]

Kobi Buxdorf2, Ido Rahat2, Aviva Gafni2, and Maggie Levy*

Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food andEnvironment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Pseudozyma spp. are yeast-like fungi, classified in the Ustilaginales, which are mostly epiphytic or saprophytic and are notpathogenic to plants. Several Pseudozyma species have been reported to exhibit biological activity against powdery mildews.However, previous studies have reported that Pseudozyma aphidis, which can colonize plant surfaces, is not associated with thecollapse of powdery mildew colonies. In this report, we describe a novel P. aphidis strain and study its interactions with its planthost and the plant pathogen Botrytis cinerea. This isolate was found to secrete extracellular metabolites that inhibit various fungalpathogens in vitro and significantly reduce B. cinerea infection in vivo. Moreover, P. aphidis sensitized Arabidopsis (Arabidopsisthaliana) plants’ defense machinery via local and systemic induction of PATHOGENESIS-RELATED1 (PR1) and PLANTDEFENSIN1.2 (PDF1.2) expression. P. aphidis also reduced B. cinerea infection, locally and systemically, in Arabidopsis mutantsimpaired in jasmonic acid (JA) or salicylic acid (SA) signaling. Thus, in addition to direct inhibition, P. aphidismay inhibit B. cinereainfection via induced resistance in a manner independent of SA, JA, and Nonexpressor of PR1 (NPR1). P. aphidis primed the plantdefense machinery and induced stronger activation of PDF1.2 after B. cinerea infection. Finally, P. aphidis fully or partiallyreconstituted PR1 and PDF1.2 expression in npr1-1 mutant and in plants with the SA hydroxylase NahG transgene, but not in ajasmonate resistant1-1 mutant, after B. cinerea infection, suggesting that P. aphidis can bypass the SA/NPR1, but not JA, pathway toactivate PR genes. Thus, either partial gene activation is sufficient to induce resistance, or the resistance is not directed solelythrough PR1 and PDF1.2 but probably through other pathogen-resistance genes or pathways as well.

Plants encounter a vast array of pathogens duringtheir growth and development, including viruses, bac-teria, and fungi. To cope with constant attacks by in-vading pathogens, plants have evolved a wide range ofdefense mechanisms, including the use of preexistingphysical and chemical barriers. Pathogens that pass thesefirst two layers of defense may encounter the hyper-sensitive response—the rapid induction of localized celldeath—that prevents their spread beyond local infection(Glazebrook, 2005). Those pathogens that persist andovercome the hypersensitive response still need to chal-lenge the well-orchestrated plant defense responses,which include systemic acquired resistance (SAR; Perfectet al., 1999) or induced systemic resistance (Heil andBostock, 2002; Somssich, 2003; Haas and Défago, 2005;Grant and Lamb, 2006).

Plant-defense responses that are activated in responseto pathogen attack are regulated by different cross-communicating signaling pathways in which signalingmolecules such as salicylic acid (SA), jasmonic acid(JA), and ethylene (ET) play major roles (Dong, 2004;Durrant and Dong, 2004; Beckers and Spoel, 2006;Broekaert et al., 2006; Loake and Grant, 2007). The SA-dependent pathway correlates with the local and SARresponse, especially against biotrophic pathogens. Thisusually results in the induced expression of SAR-marker pathogenesis-related (PR) genes (e.g. PR1, PR2,PR5, and BGL2-encoding genes) in local and systemicplant tissues (van Loon et al., 2006). PR gene inductioncan be either dependent on the NONEXPRESSOR OFPR1/NONINDUCIBLE IMMUNITY1 (NPR1/NIM1)protein or NPR1 independent (Dong, 2004). In the NPR1-dependent pathway, NPR1 “senses” the SA signalthrough changes in redox status in the plant cell, and lowredox potential can lead to translocation of NPR1 intothe cell nucleus (Kinkema et al., 2000; Mou et al., 2003).In the nucleus, NPR1 regulates the expression of PRproteins through interactions with the transcription fac-tor TGA (Zhang et al., 1999; Després et al., 2000; Zhouet al., 2000; Fan and Dong, 2002; Wang et al., 2005).Cytosolic NPR1 is also involved in the cross talk betweenthe SA- and JA-signaling pathways and acts as a negativeregulator in the synergistic effect of SA on the JA re-sponse (Spoel et al., 2003). The defense-signalingpathway that is mediated by JA and ET plays an impor-tant role in plant defense responses against necrotrophic

1 This work was supported by the Hebrew University of Jerusaleminternal fund and U.S.-Israel Binational Agricultural Research andDevelopment Fund.

2 These authors contributed equally to the article.* Corresponding author; 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:Maggie Levy ([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.www.plantphysiol.org/cgi/doi/10.1104/pp.112.212969

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pathogens and herbivorous insects (Penninckx et al.,1996; Penninckx et al., 1998; Thomma et al., 1998).Fungal biocontrol agents have become an important

alternative to the use of chemicals because of environ-mental concerns. Biological control can be achieved byone or a combination of mechanisms, including antibio-sis, mycoparasitism, competition, and induced resistancein the host plant. These mechanisms may hinder patho-gen growth and development, thereby reducing disease.The complex mode of action of biocontrol agents reducesthe likelihood that pathogens will develop a resistance tothem (Elad and Freeman, 2002; Shoresh et al., 2010).Studies indicate that biocontrol agents can protect

plants from diverse pathogens by inducing systemicresistance mechanisms that are often associated withup-regulation of PR genes and/or with accumulationof phytoalexins (Shoresh et al., 2005; Hossain et al.,2007; Stein et al., 2008; Shoresh et al., 2010). Trichodermaasperellum, for example, is an effective biocontrol agentfor a number of soilborne pathogens of cucumber(Cucumis sativus). T. asperellum infection modulates theexpression of genes involved in the JA/ET-signalingpathways of inducing systemic resistance (Shoresh et al.,2005). Similarly, in Arabidopsis (Arabidopsis thaliana), re-sistance to powdery mildew (Golovinomyces orontii) can beconferred by the mycorrhizal fungus Piriformospora indicathrough systemic resistance (Stein et al., 2008). P. indicainduces resistance via JA signaling and NPR1 (Stein et al.,2008), and Penicillium simplicissimum can induce re-sistance in Arabidopsis by activating multiple defensemechanisms, including both SA- and JA/ET-signalingpathways (Hossain et al., 2007). Transcription analysis ofplant interaction with Trichoderma hamatum failed todetect induction of induced systemic resistance markers,and only one marker of SAR (PR5) was up-regulated(Alfano et al., 2007). Therefore, it is likely that differentbiocontrol agents use different mechanisms to induceplant defense.Epiphytic yeasts colonizing different plant surfaces

(Last and Price, 1969; Phaff and Starmer, 1987; Puseyet al., 2009; Fernández et al., 2012) are thought to havebiocontrol activity and to provide a natural barrieragainst some plant pathogens (Fokkema et al., 1975;Fokkema and Schippers, 1986; Starmer et al., 1987;Avis and Bélanger, 2001; Urquhart and Punja, 2002;Bleve et al., 2006; Jacobsen, 2006; Robiglio et al., 2011).Raacke et al. (2006) demonstrated that yeasts can ac-tivate systemic acquired resistance in Arabidopsisplants and reduce symptoms of Botrytis cinerea infectionin an SA-dependent, but JA-independent, manner. Incontrast, symptoms of Pseudomonas syringae infectionwere reduced independently of the JA or SA pathways(Raacke et al., 2006).The genus Pseudozyma is a small group of Basidio-

mycota related to the Ustilaginales (Boekhout, 1995).These fungi are mostly epiphytic or saprophytic andare not pathogenic to plants or animals (Avis andBélanger, 2002). Pseudozyma rugulosa and Pseudozymaflocculosa have both been found to exhibit biologicalactivity against the different powdery mildews with

which they are associated (Jarvis et al., 1989; Hajlaouiand Bélanger, 1991, 1993; Bélanger et al., 1994; Hajlaouiet al., 1994; Dik et al., 1998; Hammami et al., 2011).P. flocculosa has been found to secrete an unusual fattyacid that displayed antibiotic activity against severalpathogens (Hajlaoui et al., 1994; Benyagoub et al., 1996;Avis and Bélanger, 2001; Avis et al., 2001). Studies onother Pseudozyma species (e.g. Pseudozyma antarctica andPseudozyma prolifica) have suggested that those specieshold great potential for biocontrol applications andproduction of novel fatty acids that may be useful forcosmetics and other industries (Avis and Bélanger,2001). P. aphidis is a close relative of P. rugulosa (Begerowet al., 2000), which was first isolated from aphid secre-tions (Henninger andWindisch, 1975), but was later alsoidentified on plant surfaces (Allen et al., 2004). Avis et al.(2001) reported that P. aphidis isolated from aphid se-cretions (isolate CBS 517.83) is not associated with col-ony collapse of powdery mildew, and that it does notproduce unique antifungal fatty acids. More recentstudies, however, have shown that P. aphidis is a novelproducer of mannosylerythritol lipids, and while thepossible function of these lipids as antifungal agentsneeds to be determined, their value as a surfactant in thecosmetic industry has been recognized (Rau et al., 2005).

In our laboratory, we isolated a unique strain of P.aphidis (designated isolate L12; Levy and Gafni, 2011).Here we demonstrate that the epiphytic yeast P. aphidisisolated from the surface of strawberry (Fragaria 3ananassa) leaves (isolate L12) has biocontrol abilityagainst fungal phytopathogens with a dual mode ofaction: antibiosis and induced resistance that is in-dependent of JA and SA/NPR1.

RESULTS

Isolation of a Unique P. aphidis Strain

We isolated a strain of P. aphidis (isolate L12) fromstrawberry leaves. The L12 isolate was classified asP. aphidis using specific primers for the entire ribosomalDNA region of the internal transcribed spacer and forthe partial sequence of the mitochondrial large subunitand nuclear small subunit as described by Avis et al.(2001). Sequences showed 100% identity to P. aphidis(Supplemental Fig. S1; Supplemental Materials andMethods S1).

The fungus secreted pinkish metabolites when grownon potato (Solanum tuberosum) dextrose agar (PDA; Fig.1A). Light and scanning electron microscopy (SEM)revealed that P. aphidis isolate L12 is a dimorphic epi-phytic fungus. We demonstrated that the fungus canhave a yeast-like form (Fig. 1, B and E) and synnema-likestructure (Fig. 1C) on PDA, and can also form hyphae(Fig. 1, D and I). The fungus could also grow on andcover both host (tomato [Solanum lycopersicum]) andmodel (Arabidopsis) leaf surfaces (Fig. 1, F and G).

Foliar application of P. aphidis at two different con-centrations (104 and 108 spores mL–1) on tomato andArabidopsis plants and detached leaves showed no

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evidence of pathogenicity or symptoms of plant sen-sitivity up to 4 weeks post application. Similarly, therewas no evidence of pathology associated with tomatoplants after drenching the root system with a suspen-sion of the biocontrol agent.

P. aphidis Secretions Inhibit Fungal Pathogens in Vitro

P. aphidis L12 isolate secretes extracellular pinkish-colored metabolites (Fig. 1A). We tested the growth-

inhibitory effect of P. aphidis extracts on B. cinerea, whichcauses gray mold, and Alternaria brassicicola, which cau-ses dark leaf spot in most Brassica species, by measuringgrowth haloes around filters saturated with hexane ex-tracts of P. aphidis L12 isolate culture-filtrate metabolites.We obtained inhibition haloes of 13 mm for B. cinerea and19 mm for A. brassicicola with P. aphidis secretion extractsand no inhibition with extracts from potato dextrosebroth (PDB) only as a control (Fig. 2A). We next revealedthat PDA containing P. aphidis secretions can completelyinhibit the spore germination of B. cinerea and A. brassi-cicola (Fig. 2B), as well as their mycelium linear growth(Supplemental Fig. S2, A and B). Inhibition of B. cinereaand A. brassicicola mycelium linear growth persistedeven when autoclaved P. aphidis secretions were used(Supplemental Fig. S2C). Since P. aphidis secretions couldinhibit B. cinerea in vitro, we further examined its abilityto inhibit pathogen symptoms on plants.

P. aphidis Inhibits B. cinerea Infection on Tomato Plants

Detached tomato leaves were sprayed with P. aphidis(104 or 108 spores mL–1) 3 d before inoculation with B.cinerea. Infection was significantly reduced, by 70% to80% and by almost 100%, when leaves were sprayedwith 104 or 108 spores mL–1 of P. aphidis, respectively, ascompared with leaves sprayed with water (control; Fig.3, A and B). Application of 108 P. aphidis spores mL–1 on

Figure 1. P. aphidis growth on PDA plate and on plants. A, P. aphidisafter 10-d growth on PDA media; arrow indicates secretions. B, Yeast-likegrowth shape on PDA (light microscopy from above). C, Synnema-likeappearance on PDA (light microscopy, profile). D, P. aphidis mycelium/yeast-like form on PDA (SEM, profile). E, P. aphidis yeast-like form on PDA(SEM, from above). F, P. aphidis after 3-d growth on Arabidopsis leaf(SEM). G, P. aphidis mycelium-like form on tomato (light microscopy,profile). H, Spore shape on PDB. I, Spore shape on yeast-malt-peptone-dextrose (YMPD) liquid medium (hemicytometer, light microscopy).

Figure 2. In vitro inhibition of fungal phytopathogens by P. aphidissecretions. A, Extracts of P. aphidis culture filtrate fraction (PA) or PDBfraction (Control) were used for fungal-inhibition halo assay. B, PDA(Control) and PDA containing P. aphidis secreted fraction (PA) wereused for spore germination-inhibition assays. Percentage germinationinhibition by PA relative to control is shown. [See online article forcolor version of this figure.]

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intact tomato plants in the greenhouse 3 d before inoc-ulation with B. cinerea also reduced infection of B. cinereaby 60% to 75% (Fig. 3C). Autoclaving P. aphidis abolishedthe inhibitory effects on B. cinerea infection (Fig. 3C). Theability of live P. aphidis to inhibit B. cinerea symptoms onplants could be complex, involving antibiosis combinedwith some other direct mode of action such as compe-tition or parasitism, or an indirect mode of action such asinduced resistance. To verify what other modes of action,

if any, are used by P. aphidis, we further monitoredP. aphidis-B. cinerea interactions on the plant and theability of P. aphidis to induce resistance.

P. aphidis-B. cinerea Interaction in Planta

The direct interaction of P. aphidiswith B. cinereawasstudied on Arabidopsis leaves by SEM 24 h after in-oculation with B. cinerea. We observed a higher pro-liferation or accumulation of P. aphidis in the lesion area(Fig. 4, A and D), forming many more hyphae than yeast-like morphs in the interaction area (Fig. 4B), as comparedwith the uninfected area (Fig. 4C). P. aphidis cells seemedto adhere to the B. cinerea hyphae and spores (Fig. 4B).

P. aphidis Application Activates Plant-Induced Resistanceand Priming

We studied the effect of P. aphidis on the activation ofthe plant defense responses in three Arabidopsis mu-tants. When Arabidopsis mutants impaired in JA sig-naling (jasmonate resistant1-1 [jar1-1]; Staswick et al.,1992) and in SA accumulation and signaling (the salic-ylate hydroxylase-expressing transgenic plant NahG;Gaffney et al., 1993), and npr1-1 (Cao et al., 1994),were treated with P. aphidis and then inoculated withB. cinerea, local disease inhibition was as strong asin wild-type plants (Fig. 5, A and B). By applyingP. aphidis to only one of the Arabidopsis rosette leavesfor 24 h and then inoculating the other leaves withB. cinerea, disease inhibition in systemic leaves wasdemonstrated in the wild type as well as in NahG,npr1-1, and jar1-1 mutants (Fig. 5C). PR1 expressionwas up-regulated 4,000-fold, and PLANT DEFEN-SIN1.2 (PDF1.2) expression was up-regulated 2.5-foldrelative to that in untreated wild-type Arabidopsisplants 3 d after application of the live culture ofP. aphidis, but not after application of autoclaved cul-ture (Fig. 6A). While induction of PR1 in NahG andnpr1-1 plants was not significantly induced 3 d posttreatment with P. aphidis, it was up-regulated in thejar1-1 mutant, albeit 1,000-fold less than in the P.aphidis-treated wild type (Fig. 6A). PDF1.2 expression,which was not as markedly affected as PR1 in the wildtype, was similarly induced in NahG and npr1-1 plants,but it was not up-regulated at all in the jar1-1 mutants(Fig. 6A). PR1 and PDF1.2 gene up-regulation wasdemonstrated locally and systemically in wild-typeplants as early as 24 h after treatment with P. aphi-dis (Supplemental Fig. S3). PDF1.2 gene expressionwas induced 7.5-fold both locally and systemically;PR1 expression was systemically induced 21-fold,but only 2.5-fold locally as compared with the un-treated wild type (Supplemental Fig. S3). When geneexpression was monitored in P. aphidis-treated ver-sus untreated plants 24 h post infection with B. ci-nerea, we observed up-regulation of PDF1.2 and PR1by 17-fold and 72-fold, respectively, in P. aphidis-treated versus untreated wild-type plants (Fig. 6B),

Figure 3. In vivo biocontrol ability of P. aphidis against B. cinerea. A,Photographs of plants and detached leaves treated with P. aphidis (PA) orwith water (Control) and inoculated with B. cinerea. B and C, Detachedtomato leaves (B) were sprayed with P. aphidis spores (104 or 108 sporesmL–1) and whole tomato plants (C) were sprayed with P. aphidis spores(108 or 108 autoclaved [108 D] spores mL–1) or with water (Control) 3 dbefore inoculation with B. cinerea (7,500 spores per leaflet), and in-fection was scored 5 d post inoculation. Averages of infection scores of30 leaves or 15 plants and SEs are presented. Different letters above thecolumns indicate statistically significant differences, using one-wayANOVA for whole plants and Kruskal-Wallis one-way ANOVA on Ranksfor detached leaves (P , 0.001).



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while the expression of PR1 was 60-fold less thanthat observed in P. aphidis-treated uninfected wild-type plants (Fig. 6A). In npr1-1 plants, the expressionof both genes was only partially recovered; in NahGplants, PDF1.2 expression was similar to that in thewild type whereas PR1 expression was partially re-constituted, and in jar1-1 plants neither gene wasexpressed after infection (Fig. 6B).

DISCUSSION

In this article we examined the ability of the epi-phytic fungus P. aphidis (isolate L12) to control B. ci-nerea disease and explored its mode of action. Wedemonstrated P. aphidis colonization and establish-ment on plant surfaces (Figs. 1 and 4) as previouslydescribed (Allen et al., 2004). The protective effect of P.aphidis against plant pathogens was demonstrated byboth direct antagonistic interactions and induced re-sistance. In contrast to Avis et al. (2001), likely dueto the use of different isolates, the P. aphidis isolateused in this work secreted metabolites that have in-hibitory effects on several phytopathogenic fungi invitro (Fig. 2; Supplemental Fig. S2) and also inhibitgray mold and other plant diseases in planta (Fig. 3;A. Gafni, I. Rahat, and M. Levy, unpublished data).Secreted metabolites inhibited both spore and linearhyphal growth of fungal pathogens in vitro (Fig. 2;Supplemental Fig. S2). Application of P. aphidis beforepathogen inoculation significantly reduced the graymold-causing agent B. cinerea on tomato plants inthe greenhouse, and disease inhibition was abolishedwhen P. aphidis was not vital (Fig. 3). The application

of a biocontrol agent before plants are exposed to apathogen can sometimes improve biocontrol activity(Filonow et al., 1996), as demonstrated in this study.However, in other studies, no significant effects wereobserved (Chalutz and Wilson, 1990; Cook et al., 1997).

Metabolite secretion was not the only mechanism ofP. aphidis biocontrol; we also observed local and sys-temic activation of inducible defense mechanisms inplants treated with P. aphidis (Fig. 5). Activation ofinducible mechanisms in plants is potentially suitablefor the enhancement of plant resistance to pathogens(Shoresh et al., 2010). We found that spraying Arabi-dopsis plants with live culture of P. aphidis leads to ac-tivation of PR genes from the SA and JA pathways (Fig.6A; Supplemental Fig. S3), indicating that P. aphidismediates induced resistance via both pathways. Furtheranalysis with Arabidopsis SA-pathway mutants NahGand npr1-1 and JA pathway mutant jar1-1 showed thatP. aphidis can reduce B. cinerea infection locally but alsosystemically in Arabidopsis mutants impaired in JA orSA signaling, i.e. jar-1-1, NahG, and npr1-1 (Fig. 5C). Thedata suggest that beyond the direct local antagonisticinhibition, P. aphidis inhibits B. cinerea infection sys-temically via induced resistance in a SA/NPR1- andJA-independent manner. Defensive resistance can belocally induced at the site of inoculation or systemi-cally throughout the whole plant; our results withhormonal-defective mutants suggest that both modesare activated by P. aphidis.

B. cinerea activated PDF1.2 expression 17-fold andPR1 expression 72-fold in plants treated with P. aphidisversus untreated plants (Fig. 6B). However, PR1 ex-pression was actually antagonized by B. cinerea, while

Figure 4. P. aphidis-B. cinerea in-teractions in planta. SEM of Arabi-dopsis leaves treated with P. aphidisand infected with B. cinerea. A,Lesion area (to the left of dashedline) full of B. cinerea myceliumand white P. aphidis cells 48 hpost infection with B. cinerea. B,Closer look at the interaction ofP. aphidis and B. cinerea myce-lium and spores inside the lesion.C and D, Trichome covered withP. aphidis outside (C) and inside(D) the lesion. B. cinerea myce-lium and spores are indicated withwhite arrows and P. aphidis cellswith dashed arrows.


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PDF1.2 was enhanced as compared with uninfectedplants treated with P. aphidis (Fig. 6A). A proposedscenario in wild-type plants, which has also beensuggested by Tucci et al. (2011) for Trichoderma spp.,might be that in the absence of pathogen, preventativetreatment with P. aphidis activates expression of PRgenes from both SA and JA pathways, in order toprime the plant defense reaction. However, whenthese treated plants are inoculated with B. cinerea, theJA-dependent response is enhanced as a consequenceof the priming effect, eventually resulting in the ob-served increase in local and systemic resistance. Sincewe found that P. aphidis activates induced resistance in

a JA- and SA-independent manner, it was reasonableto suggest that this antagonistic effect is connected todifferent or unknown defense pathways, and not tothe known antagonistic effect between SA and JApathways. We further found that P. aphidis treatmentcould partially or fully recover PR1 and PDF1.2 ex-pression in npr1-1 and NahG mutants but not in jar1-1mutants (Fig. 6). This suggests that P. aphidis can by-pass the SA/NPR1 pathway, but not the JA pathway,to activate pathogenesis genes. Thus, either partialgene activation is sufficient to induce resistance, orthe resistance is not directed solely through PR1 andPDF1.2 but also through other putative pathogen-resistance genes and/or pathways. This suggests thatother hormone-independent pathways or PR genes arealso involved in the resistance induced by P. aphidis or,perhaps as yet unknown, partially SA/NPR1- and JA-dependent pathways. The combination of hormone-dependent and -independent pathways as a mode ofaction for induced resistance has been demonstrated inplants treated with Trichoderma spp. or yeast (Raackeet al., 2006; Korolev et al., 2008).

Figure 5. P. aphidis controls B. cinerea on Arabidopsis mutants impairedin SA or JA signaling. A, Representative B. cinerea-infected leaves with (PA)or without (Control) P. aphidis treatment. B, B. cinerea lesion area wasmeasured 72 h after inoculation with hormonemutantsNahG, jar1-1,npr1-1,and wild type (Control), and compared with lesions on their counterpartssprayed with P. aphidis (PA). C, Lesion area was monitored on systemicleaves of treated (PA) and untreated (Control) plants 72 h after infection withB. cinerea. Asterisks denote significant differences (P, 0.05) as determinedby Student’s t test. Statistics for wild type, npr1-1, and jar1-1 in B weredetermined using Mann-Whitney’s rank sum test. WT, Wild type.

Figure 6. Induced resistance by P. aphidis. Arabidopsis plants weresprayed with 108 spores mL–1 of P. aphidis, and PR1 and PDF1.2 geneexpression was monitored using quantitative reverse transcription PCRin hormone mutantsNahG, jar1-1, and npr1-1, and in the wild type. A,Three days after application (+PA) relative to untreated plants (–PA). B,Twenty-four hours after B. cinerea infection of treated or untreatedplants (+PA/–PA). Relative gene expression was calculated by thecomparative cycle threshold method. WT, Wild type.



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Resistance to B. cinerea can result from a combinationof several mechanisms, including plant response, anti-fungal metabolite secretion (as indicated in our in vitroexperiments; Fig. 2), and attachment of P. aphidis toB. cinerea spores, preventing their germination (as indi-cated by our SEM observations; Fig. 4; Allen et al., 2004).Another direct effect could be delayed spore germinationdue to decreased hydrophobicity of the leaf surface. Theabundance of P. aphidis in B. cinerea-infected tissue andthe close relationship between the two organisms (Fig. 4)might suggest parasitism, although we could not dem-onstrate this in vitro (Supplemental Fig. S4); however, itmight also suggest competition for nutrients because co-nidial germination in many B. cinerea isolates is nutrientdependent and P. aphidis outcompeting B. cinerea conidiafor available nutrients would reduce disease severity(Bashi and Fokkema, 1977; Fokkema et al., 1979; Diket al., 1991, 1992; Filonow et al., 1996; Barnett et al., 2000).

Although activation of an inducible defense mecha-nism in plants is potentially sufficient to enhance plantresistance against pathogens, it should be noted thatdifferent pathogens induce different, mostly complex setsof responses, including accumulation of JA, SA, andsecondary metabolites, and sometimes these responsesdiffer only in their kinetics or magnitude, which arecrucial for the outcome of the interactions. Furthermore,external factors (e.g. plant genotype, environmentalconditions, etc.) can easily increase this complexity,also influencing the outcome.

In conclusion, our results demonstrate that P. aphidishas the potential for use as a biocontrol agent of fungalpathogens. The mechanisms of action involved inP. aphidis control are antifungal metabolite secretionand induced resistance, which are independent of orpartially dependent on the JA and SA/NPR1 pathways.However, to increase our knowledge of P. aphidis mech-anisms of action, it would be useful to further characterizethe interaction with the plant and determine whetherthere are other as yet unknown pathways involved in theactivation of the observed induced resistance.

MATERIALS AND METHODS

Pseudozyma aphidis Culture

Pseudozyma aphidis isolate L12 was maintained in solid culture on PDA(Difco) at 26°C and transferred to fresh medium monthly. Liquid cultureswere maintained in PDB (Difco) for 7 to 10 d at 26°C on a rotary shaker at 150rpm. We obtained 108 cells mL–1 after 5 to 10 d in liquid culture.

Propagation of Plants and Pathogens

Botrytis cinerea (B05.10) and Alternaria brassicicola were grown on PDAmedium at 22°C to 27°C under 12-h daily illumination.

Tomato (Solanum lycopersicum ‘870’) plants were grown at 25°C and 40%relative humidity in the greenhouse. Arabidopsis (Arabidopsis thaliana) acces-sion Columbia and its mutants jar1-1, NahG, and npr1-1 were used. All seedswere obtained from the Arabidopsis Biological Resource Center (Columbus,OH). All Arabidopsis seeds were vernalized on moist soil at 4°C for 2 to3 d before placing them in a growth chamber. Arabidopsis plants were grownat 22°C and 60% relative humidity under illumination with fluorescent andincandescent light at a photofluency rate of approximately 120 mmol m–2 s;day length was 12 h unless otherwise specified.

RNA Isolation and Quantitative Reverse TranscriptionPCR Analysis

Total RNAwas isolated from untreated Arabidopsis plants and from plants24 to 72 h post treatment with 108 P. aphidis spores mL–1 with Qiagen RNeasykit (Invitrogen) according to the manufacturer’s instructions or using theLogSpin method (Yaffe et al., 2012). DNase treatment was performed onQiagen columns according to the manufacturer’s instructions (Invitrogen).Total RNA (1 mg) was reverse transcribed with EZ-first strand cDNA synthesiskit (Biological Industries). Quantitative reverse transcription PCR was per-formed with the SYBR master mix and StepOne real-time PCR machine(Applied Biosystems). The thermal cycling program was as follows: 95°C for20 s and 40 cycles of 95°C for 3 s and 60°C for 30 s. Relative fold change ingene expression normalized to AtPTB1F in samples from inoculated versusuninoculated Arabidopsis leaves was calculated by the comparative cyclethreshold method. The primer sequences were as follows: AtPTB1F,59-GATCTGAATGTTAAGGCTTTTAGCG-39; AtPTB1R, 59-GGCTTAGATCAG-GAAGTGTATAGTCTCTG-39 (Czechowski et al., 2005); AtPR1F, 59-GCCT-TACGGGGAAAACTTA-39; AtPR1R, 59-CTTTGGCACATCCGAGTCT-39;AtPDF1.2F, 59-CTGCTCTTGTTCTCTTTGCT-39, and AtPDF1.2R, 59-GT-GTGCTGGGAAGACATA-39 (Conn et al., 2008).

Isolation of P. aphidis-Secreted Fraction for in VitroInhibition Assays

Secreted metabolites were extracted from PDB or PDB culture filtrate of P.aphidis using ethyl acetate followed by hexane. We grew P. aphidis in PDBmedium at 26°C for 10 d in Erlenmeyer flasks at a constant agitation of 150rpm. We then spun down the fungal cells (20 min at 10,000 rpm). The su-pernatant, consisting of culture filtrate, was titrated to pH 2.0 using 1 N HCland extracted with an equivalent volume of ethyl acetate using separatingfunnels. The ethyl-acetate fraction was collected, reextracted with hexane, andevaporated in a rotor evaporator (Buchi) at 42°C (Paz et al., 2007). The dryfraction was reconstituted in methanol and used for in vitro experiments afterapplication on Whatman paper discs (6 mm diameter). The discs were placedin the center of the PDA plates embedded with the pathogen spores.

In Vitro Inhibition Assays usingP. aphidis-Secreted Fraction

P. aphidis was placed on PDA covered with dialysis tubing and incubatedat 26°C for 10 d. We then removed the tubing with the fungi and used theplates with the P. aphidis-secreted fraction for inhibition assays with differentfungal pathogens. Alternatively, we grew P. aphidis directly on PDA and usedthe bottom side of the PDA for inhibition assays. We inoculated control PDAplates and PDA plates containing the P. aphidis secretions with the differentpathogens, incubated them at their optimum temperatures, and estimatedtheir spore germination or hyphal growth.

Inhibition of B. cinerea on Tomato Plants

To examine inhibition of B. cinerea on detached tomato leaves and wholeplants, we sprayed the leaves/plants with different concentrations (104 and108 spores mL–1) of P. aphidis to flowing, allowed it to become established for3 d on the leaves/plants, inoculated them with the pathogen (total of 7,500spores per leaflet), and monitored disease symptoms relative to plants sprayedwith water.

Electron Microscopy

SEM of P. aphidis-B. cinerea interactions was performed using a standardprotocol (Weigel and Glazebrook, 2002). Arabidopsis leaves were sprayedwith P. aphidis and 3 d later were inoculated with B. cinerea. After 24 h, leafsamples were fixed with glutaraldehyde, critical-point dried (BAL-TEC),mounted on aluminum stubs, sputter coated with gold, and examined bySEM at an accelerating voltage of 20 kV.

Determination of Local and Induced Resistance

We monitored PR gene expression levels in Arabidopsis plants 3 d afterfoliar application of live culture of P. aphidis (108 cells mL–1) relative to plants


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treated with water (control). We also monitored gene expression 24 h afterinfection with B. cinerea of untreated and P. aphidis-treated plants. Plantsshowing local resistance were inoculated with B. cinerea 3 d post P. aphidisapplication, and lesion size was monitored 72 h post infection. For systemicresistance to B. cinerea, lesion formation was assayed 3 d after infection insystemic leaves. In this experiment, infection was performed 24 h post ap-plication of P. aphidis to the bottom leaf and not 72 h post application as in thefirst experiment, so as to avoid spreading of P. aphidis to the upper leaves as isknown to happen after 24 h.

Statistical Analysis

Student’s t tests were performed only when data were normally distributedand the sample variances were equal. Otherwise Mann-Whitney’s rank sumtest was performed. Significance was accepted at P , 0.05 and is noted in thetext or figure captions. All experiments shown here are representative of atleast three independent experiments with the same pattern of results.

Supplemental Data

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

Supplemental Figure S1. Identification of L12 isolate.

Supplemental Figure S2. In vitro inhibition of fungal phytopathogens byP. aphidis secretions.

Supplemental Figure S3. Local and systemic induced resistance byP. aphidis.

Supplemental Figure S4. In vitro parasitism assay.

Supplemental Materials and Methods S1.

ACKNOWLEDGMENTS

M.L. and K.B. designed the experiments. K.B., I.R., and A.G. performed theexperiments. M.L. and K.B. analyzed the results and wrote the manuscript. Allauthors read and approved the final manuscript.

Received December 23, 2012; accepted February 2, 2013; published February 6,2013.

LITERATURE CITED

Alfano G, Ivey MLL, Cakir C, Bos JIB, Miller SA, Madden LV, KamounS, Hoitink HAJ (2007) Systemic modulation of gene expression in to-mato by Trichoderma hamatum 382. Phytopathology 97: 429–437

Allen TW, Quayyum HA, Burpee LL, Buck JW (2004) Effect of foliardisease on the epiphytic yeast communities of creeping bentgrass andtall fescue. Can J Microbiol 50: 853–860

Avis TJ, Bélanger RR (2001) Specificity and mode of action of the anti-fungal fatty acid cis-9-heptadecenoic acid produced by Pseudozymaflocculosa. Appl Environ Microbiol 67: 956–960

Avis TJ, Bélanger RR (2002) Mechanisms and means of detection of bio-control activity of Pseudozyma yeasts against plant-pathogenic fungi.FEMS Yeast Res 2: 5–8

Avis TJ, Caron SJ, Boekhout T, Hamelin RC, Bélanger RR (2001) Molec-ular and physiological analysis of the powdery mildew antagonistPseudozyma flocculosa and related fungi. Phytopathology 91: 249–254

Barnett JA, Payne RW, Yarrow D (2000) Yeasts: Characteristics and Iden-tification, Ed 3. Cambridge University Press, Cambridge, UK

Bashi E, Fokkema NJ (1977) Environmental factors limiting growth ofSporobolomyces roseus, an antagonist of Cochliobolus sativus, on wheatleaves. Trans Br Mycol Soc 68: 17–25

Beckers GJ, Spoel SH (2006) Fine-Tuning Plant Defence Signalling: Salic-ylate versus Jasmonate. Plant Biol (Stuttg) 8: 1–10

Begerow D, Bauer R, Boekhout T (2000) Phylogenetic placements of us-tilaginomycetous anamorphs as deduced from nuclear LSU rDNA se-quences. Mycol Res 104: 53–60

Bélanger RR, Labbé C, Jarvis WR (1994) Commercial-scale control of rosepowdery mildew with a fungal antagonist. Plant Dis 78: 420–424

Benyagoub M, Willemot C, Bélanger RR (1996) Influence of a subinhibi-tory dose of antifungal fatty acids from Sporothrix flocculosa on cellularlipid composition in fungi. Lipids 31: 1077–1082

Bleve G, Grieco F, Cozzi G, Logrieco A, Visconti A (2006) Isolation ofepiphytic yeasts with potential for biocontrol of Aspergillus carbonariusand A. niger on grape. Int J Food Microbiol 108: 204–209

Boekhout T (1995) Pseudozyma Bandoni emend. Boekhout, a genus foryeast-like anamorphs of Ustilaginales. J Gen Appl Microbiol 41: 359–366

Broekaert WF, Delauré SL, De Bolle MF, Cammue BP (2006) The roleof ethylene in host-pathogen interactions. Annu Rev Phytopathol 44:393–416

Cao H, Bowling SA, Gordon AS, Dong X (1994) Characterization of anArabidopsis mutant that is nonresponsive to inducers of systemic ac-quired resistance. Plant Cell 6: 1583–1592

Chalutz E, Wilson CL (1990) Postharvest biocontrol of green and bluemold and sour rot of citrus fruit by Debaryomyces hansenii. Plant Dis 74:134–137

Conn VM, Walker AR, Franco CMM (2008) Endophytic actinobacteriainduce defense pathways in Arabidopsis thaliana. Mol Plant Microbe In-teract 21: 208–218

Cook DWM, Long PG, Ganesh S, Cheah LH (1997) Attachment microbesantagonistic against Botrytis cinerea—biological control and scanningelectron microscope studies in vivo. Ann Appl Biol 131: 503–518

Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005)Genome-wide identification and testing of superior reference genes fortranscript normalization in Arabidopsis. Plant Physiol 139: 5–17

Després C, DeLong C, Glaze S, Liu E, Fobert PR (2000) The ArabidopsisNPR1/NIM1 protein enhances the DNA binding activity of a subgroupof the TGA family of bZIP transcription factors. Plant Cell 12: 279–290

Dik AJ, Fokkema NJ, Van Pelt JA (1991) Consumption of aphid honeydew,a wheat yield reduction factor, by phyllosphere yeasts under fieldconditions. Eur J Plant Pathol 97: 209–232

Dik AJ, Fokkema NJ, Van Pelt JA (1992) Influence of climatic and nutri-tional factors on yeast population dynamics in the phyllosphere ofwheat. Microb Ecol 23: 41–52

Dik AJ, Verhaar MA, Bélanger RR (1998) Comparison of three biologicalcontrol agents against cucumber powdery mildew (Sphaerotheca fuligi-nea) in semi-commercial-scale glasshouse trials. Eur J Plant Pathol 104:413–423

Dong X (2004) NPR1, all things considered. Curr Opin Plant Biol 7: 547–552Durrant WE, Dong X (2004) Systemic acquired resistance. Annu Rev

Phytopathol 42: 185–209Elad Y, Freeman S (2002) Biological control of fungal plant pathogens. In F

Kempken, ed, The Mycota: A Comprehensive Treatise on Fungi as Ex-perimental Systems for Basic and Applied Research. XI. AgriculturalApplications. Springer, Heidelberg, Germany, pp 93–109

Fan W, Dong X (2002) In vivo interaction between NPR1 and transcriptionfactor TGA2 leads to salicylic acid-mediated gene activation in Arabi-dopsis. Plant Cell 14: 1377–1389

Fernández NV, Mestre MC, Marchelli P, Fontenla SB (2012) Yeast andyeast-like fungi associated with dry indehiscent fruits of Nothofagusnervosa in Patagonia, Argentina. FEMS Microbiol Ecol 80: 179–192

Filonow AB, Vishniac HS, Anderson JA, Janisiewicz WJ (1996) BiologicalControl of Botrytis cinerea in apple by yeasts from various habitats andtheir putative mechanisms of antagonism. Biol Control 7: 212–220

Fokkema NJ, den Houter JG, Kosterman YJC, Nelis AL (1979) Manipulationof yeasts on field-grown wheat leaves and their antagonistic effect onCochliobolus sativus and Septoria nodorum. Trans Br Mycol Soc 72: 19–29

Fokkema NJ, Schippers B (1986) Phyllosphere versus Rhizosphere as En-vironments for Saprophytic Colonization. Cambridge University Press,Cambridge, UK

Fokkema NJ, van de Laar JAJ, Nellis-Blomberg AL, Schippers B (1975)The buffering capacity of the natural mycoflora of rye leaves to infectionby Cochliobolus sativus and its susceptibility to benomyl. Neth J PlantPathol 81: 176–186

Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E,Kessmann H, Ryals J (1993) Requirement of salicylic Acid for the in-duction of systemic acquired resistance. Science 261: 754–756

Glazebrook J (2005) Contrasting mechanisms of defense against biotrophicand necrotrophic pathogens. Annu Rev Phytopathol 43: 205–227

Grant M, Lamb C (2006) Systemic immunity. Curr Opin Plant Biol 9: 414–420Haas D, Défago G (2005) Biological control of soil-borne pathogens by

fluorescent pseudomonads. Nat Rev Microbiol 3: 307–319



Dow



http://www.plantphysiol.org/cgi/content/full/pp.112.212969/DC1http://www.plantphysiol.org/cgi/content/full/pp.112.212969/DC1http://www.plantphysiol.org/cgi/content/full/pp.112.212969/DC1http://www.plantphysiol.org/cgi/content/full/pp.112.212969/DC1http://www.plantphysiol.org/cgi/content/full/pp.112.212969/DC1

Hajlaoui MR, Bélanger RR (1991) Comparative effects of temperature andhumidity on the activity of three potential antagonists of rose powderymildew. Neth J Plant Pathol 97: 203–208

Hajlaoui MR, Bélanger RR (1993) Antagonism of the yeast-like phyllo-plane fungus Sporothrix flocculosa against Erysiphe graminis var tritici.Biocontrol Sci Technol 3: 427–434

Hajlaoui MR, Traquair JA, Jarvis WR, Bélanger RR (1994) Antifungalactivity of extracellular metabolites produced by Sporothrix flocculosa.Biocontrol Sci Technol 4: 229–237

Hammami W, Castro CQ, Rémus-Borel W, Labbé C, Bélanger RR (2011)Ecological basis of the interaction between Pseudozyma flocculosa andpowdery mildew fungi. Appl Environ Microbiol 77: 926–933

Heil M, Bostock RM (2002) Induced systemic resistance (ISR) againstpathogens in the context of induced plant defences. Ann Bot (Lond) 89:503–512

Henninger W, Windisch S (1975) [Pichia lindnerii sp. n., a new methanolassimilating yeast from soil]. Arch Microbiol 105: 47–48

Hossain MM, Sultana F, Kubota M, Koyama H, Hyakumachi M (2007)The plant growth-promoting fungus Penicillium simplicissimum GP17-2induces resistance in Arabidopsis thaliana by activation of multipledefense signals. Plant Cell Physiol 48: 1724–1736

Jacobsen B (2006) Biological control of plant disease by phyllosphere ap-plied biological control agents. In M Bailey, A Lilley, T Timms-Wilson, PSpencer-Philips, eds, Microbial Ecology of Aerial Plant Surfaces. Centrefor Agricultural Bioscience International, London, pp 133–147

Jarvis WR, Shaw LA, Traquair JA (1989) Factors affecting antagonism ofcucumber powdery mildew by Stephanoascus flocculosus and S. rugulosus.Mycol Res 92: 162–165

Kinkema M, Fan W, Dong X (2000) Nuclear localization of NPR1 is re-quired for activation of PR gene expression. Plant Cell 12: 2339–2350

Korolev N, Rav David D, Elad Y (2008) The role of phytohormones in basalresistance and Trichoderma-induced systemic resistance to Botrytis ci-nerea in Arabidopsis thaliana. BioControl 53: 667–683

Last FT, Price D (1969) Yeasts Associated with Living Plants and TheirEnvirons, Ed 1, Vol 1. Academic Press, New York and London

Levy M, Gafni A, inventors (2011) Pseudozyma aphidis as a biocontrol agentagainst various plant pathogens. Patent Cooperation Treaty PatentPublication No. WO 2011/151819

Loake G, Grant M (2007) Salicylic acid in plant defence—the players andprotagonists. Curr Opin Plant Biol 10: 466–472

Mou Z, Fan W, Dong X (2003) Inducers of plant systemic acquired resis-tance regulate NPR1 function through redox changes. Cell 113: 935–944

Paz Z, Burdman S, Gerson U, Sztejnberg A (2007) Antagonistic effects ofthe endophytic fungus Meira geulakonigii on the citrus rust mite Phyllo-coptruta oleivora. J Appl Microbiol 103: 2570–2579

Penninckx IA, Eggermont K, Terras FR, Thomma BP, De Samblanx GW,Buchala A, Métraux JP, Manners JM, Broekaert WF (1996) Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis fol-lows a salicylic acid-independent pathway. Plant Cell 8: 2309–2323

Penninckx IA, Thomma BP, Buchala A, Métraux JP, Broekaert WF (1998)Concomitant activation of jasmonate and ethylene response pathways isrequired for induction of a plant defensin gene in Arabidopsis. Plant Cell10: 2103–2113

Perfect SE, Hughes HB, O’Connell RJ, Green JR (1999) Colletotrichum: Amodel genus for studies on pathology and fungal-plant interactions.Fungal Genet Biol 27: 186–198

Phaff HJ, Starmer WT (1987) Yeasts Associated with Plants, Insects andSoil, Ed 2, Vol 1. Academic Press, London

Pusey PL, Stockwell VO, Mazzola M (2009) Epiphytic bacteria and yeastson apple blossoms and their potential as antagonists of Erwinia amylo-vora. Phytopathology 99: 571–581

Raacke IC, von Rad U, Mueller MJ, Berger S (2006) Yeast increases re-sistance in Arabidopsis against Pseudomonas syringae and Botrytis cinerea

by salicylic acid-dependent as well as -independent mechanisms. MolPlant Microbe Interact 19: 1138–1146

Rau U, Nguyen LA, Schulz S, Wray V, Nimtz M, Roeper H, Koch H, LangS (2005) Formation and analysis of mannosylerythritol lipids secreted byPseudozyma aphidis. Appl Microbiol Biotechnol 66: 551–559

Robiglio A, Sosa MC, Lutz MC, Lopes CA, Sangorrín MP (2011) Yeastbiocontrol of fungal spoilage of pears stored at low temperature. Int JFood Microbiol 147: 211–216

Shoresh M, Harman GE, Mastouri F (2010) Induced systemic resistanceand plant responses to fungal biocontrol agents. Annu Rev Phytopathol48: 21–43

Shoresh M, Yedidia I, Chet I (2005) Involvement of jasmonic acid/ethylenesignaling pathway in the systemic resistance induced in cucumber byTrichoderma asperellum T203. Phytopathology 95: 76–84

Somssich IE (2003) Closing another gap in the plant SAR puzzle. Cell 113:815–816

Spoel SH, Koornneef A, Claessens SM, Korzelius JP, Van Pelt JA, Mueller MJ,Buchala AJ, Métraux JP, Brown R, Kazan K, et al (2003) NPR1 modulatescross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol. Plant Cell 15: 760–770

Starmer WT, Ganter PF, Aberdeen V, Lachance MA, Phaff HJ (1987) Theecological role of killer yeasts in natural communities of yeasts. Can JMicrobiol 33: 783–796

Staswick PE, Su W, Howell SH (1992) Methyl jasmonate inhibition of rootgrowth and induction of a leaf protein are decreased in an Arabidopsisthaliana mutant. Proc Natl Acad Sci USA 89: 6837–6840

Stein E, Molitor A, Kogel KH, Waller F (2008) Systemic resistance inArabidopsis conferred by the mycorrhizal fungus Piriformospora indicarequires jasmonic acid signaling and the cytoplasmic function of NPR1.Plant Cell Physiol 49: 1747–1751

Thomma BP, Eggermont K, Penninckx IA, Mauch-Mani B, Vogelsang R,Cammue BP, Broekaert WF (1998) Separate jasmonate-dependent andsalicylate-dependent defense-response pathways in Arabidopsis areessential for resistance to distinct microbial pathogens. Proc Natl AcadSci USA 95: 15107–15111

Tucci M, Ruocco M, De Masi L, De Palma M, Lorito M (2011) The bene-ficial effect of Trichoderma spp. on tomato is modulated by the plantgenotype. Mol Plant Pathol 12: 341–354

Urquhart EJ, Punja ZK (2002) Hydrolytic enzymes and antifungal com-pounds produced by Tilletiopsis species, phyllosphere yeasts that areantagonists of powdery mildew fungi. Can J Microbiol 48: 219–229

van Loon LC, Rep M, Pieterse CMJ (2006) Significance of inducibledefense-related proteins in infected plants. Annu Rev Phytopathol 44:135–162

Wang D, Weaver ND, Kesarwani M, Dong X (2005) Induction of proteinsecretory pathway is required for systemic acquired resistance. Science308: 1036–1040

Weigel D, Glazebrook J (2002) Arabidopsis: A Laboratory Manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY

Yaffe H, Buxdorf K, Shapira I, Ein-Gedi S, Moyal-Ben Zvi M, Fridman E,Moshelion M, Levy M (2012) LogSpin: a simple, economical and fastmethod for RNA isolation from infected or healthy plants and othereukaryotic tissues. BMC Res Notes 5: 45

Zhang Y, Fan W, Kinkema M, Li X, Dong X (1999) Interaction of NPR1with basic leucine zipper protein transcription factors that bind se-quences required for salicylic acid induction of the PR-1 gene. Proc NatlAcad Sci USA 96: 6523–6528

Zhou JM, Trifa Y, Silva H, Pontier D, Lam E, Shah J, Klessig DF (2000)NPR1 differentially interacts with members of the TGA/OBF family oftranscription factors that bind an element of the PR-1 gene required forinduction by salicylic acid. Mol Plant Microbe Interact 13: 191–202


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