Current Biology 21, 12041209, July 26, 2011 2011 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2011.06.044

Report A Secreted Fungal Effector of Glomus intraradices Promotes Symbiotic Biotrophy

Silke Kloppholz,1 Hannah Kuhn,1 and Natalia Requena1,* 1Plant-Microbial Interactions, Botanical Institute, Karlsruhe Institute of Technology, Hertzstrasse 16, D-76187 Karlsruhe, Germany line of plant defenses. Only a few fungal MAMPs have been described, including chitin, ergosterol, and N-glycosylated yeast peptides [1417]. Microbes, in contrast, have learned to cope with plant MAMP-triggered immune responses by delivering effector proteins that counteract defense mechanisms [25]. AM fungi establish long-term biotrophic relationships with their hosts and thus must actively suppress or avoid plant defense responses. We hypothesized this could be at least partly achieved by the delivery of AM fungal effectors into the plant cell. To search for putative effector proteins, and given the lack of an assembled genome, we developed an improved version of the yeast secretion sequence trap method to isolate Glomus intraradices secreted proteins [18]. We hypothesized that secreted proteins containing a nuclear localization signal could be candidate effectors that target the plant nucleus. With this criterion, a small protein family characterized by a conserved domain structure was identied. All members contain a signal peptide, a short sequence without any known motif, a nuclear localization domain (NLS), and several tandem imperfect hydrophilic repeats (Figures 1A and 1B). BLASTp searches showed that SP7 (secreted protein 7) has a low homology (E values from 3e226 to 1e206) to several repeat-containing proteins from Plasmodium spp. expressed during host invasion, as well as to a protein from yeast induced under heat shock and DNA damage conditions. However, only one of those proteins (NCBI GenBank XP_001347627), encoding the S antigen of P. falciparum, contains a signal peptide and is therefore potentially secreted. The other two family members, SP14 and SP31, did not show any signicant homology to proteins from the database, despite the similarity between SP31 and SP7 (48% identity). We decided to study in detail the role of the founder member of the family, SP7. To our surprise, several cDNA varieties of SP7 were identied at different developmental stages, although they all appeared to be encoded by one single gene. Detailed RT-PCR analysis revealed that all cDNA varieties were identical in the 50 and 30 untranslated regions, in the signal peptide, and in the NLS but contained different repeat compositions (Figure 1C; see also Figure S1 available online). We propose that alternative splicing of the longest transcript by an intron-skipping mechanism might produce the different mRNAs. The longest cDNA contains ten of the long repeats (16 aa), with the last repeat incomplete and each separated by four small repeats of eight amino acids (Figure 1C; Figure S1). SP7 RNA accumulated in fungal material that was in contact with the plant (Figure 1D). Southern blot analysis of RT-PCR products to reveal the different mRNA forms showed that the 1.8 kb variety, corresponding to the longest cDNA, was the predominant form during fungal growth in planta (Figure 1E). SP7 Enters the Plant Cell and Localizes within the Nucleus We predicted that SP7, after secretion in the apoplast, enters the plant cell and then moves to the nucleus. We rst investigated whether the nuclear localization domain of SP7 is functional. Given that there is no known stable transformation

Summary Biotrophic fungi interacting with plants establish long-term relationships with their hosts to fulll their life cycles. In contrast to necrotrophs, they need to contend with the defense mechanisms of the plant to develop within the host and feed on living cells [1]. It is generally accepted that microbial pathogens produce and deliver a myriad of effector proteins to hijack the cellular program of their hosts [15]. Arbuscular mycorrhizal (AM) fungi are the most widespread biotrophs of plant roots [6]. We investigated whether AM fungi use effector proteins to short-circuit the plant defense program. Here we show that Glomus intraradices secretes a protein, SP7, that interacts with the pathogenesis-related transcription factor ERF19 in the plant nucleus. ERF19 is highly induced in roots by the fungal pathogen Colletotrichum trifolii as well as by several fungal extracts, but only transiently during mycorrhiza colonization. When constitutively expressed in roots, SP7 leads to higher mycorrhization while reducing the levels of C. trifolii-mediated defense responses. Furthermore, expression of SP7 in the rice blast fungus Magnaporthe oryzae attenuates root decay symptoms. Taken together, these results suggest that SP7 is an effector that contributes to develop the biotrophic status of AM fungi in roots by counteracting the plant immune program. Results and Discussion Do Symbiotic Arbuscular Mycorrhizal Fungi Possess Effector Proteins? Arbuscular mycorrhizal (AM) fungi from the phylum Glomeromycota are soil inhabitants forming the most widespread mutualistic symbiosis with plant roots [6]. They provide plants with mineral nutrients in exchange for photoassimilates. All AM fungi are obliged biotrophs and, in the absence of the host plant, they only produce a tiny mycelium that aborts growth after a few days. In the presence of a compatible host, however, fungus and plant exchange several secreted signals that initiate the symbiotic cellular program [7, 8]. This mutual long-lasting benecial association is possible because AM fungi do not elicit massive plant defense responses. Nevertheless, during an early phase of the symbiosis, the plant reacts to the colonization by AM fungi, inducing transient defense and stress responses [913]. Plants have a defense system trained to detect conserved traits of microbial pathogens called MAMPs (microbial-associated molecular patterns; reviewed in [2]). Perception of these conserved microbial epitopes by pattern recognition receptors induces the rst

*Correspondence: natalia.requena@kit.edu

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Figure 1. The SP7 Family of Small Secreted Proteins (A) Domain structure of the SP7 protein family, consisting of a signal peptide (SP, red), a conserved region of unknown function (yellow), a nuclear localization signal (NLS, green), and a series of hydrophilic tandem repeats (blue). (B) ClustalW alignment of the amino acid sequences of SP7, SP31, and SP14 as found in the secretion library. The SP, the conserved region of unknown function, and the NLS are indicated with red, yellow, and green bars, respectively. (C) Repeat organization of different SP7 cDNA varieties. Repeat subunits with similar sequence are represented in the same colors. Asterisks indicate repeat subunits of reduced length. (D) Transcript accumulation of SP7 in fungal tissues and infected roots. Expression data in germinated spores or extraradical mycelium (ERM) noninduced or induced by coincubation with roots and in mycorrhizal roots 12 or 25 days postinfection (dpi) are given relative to GiTEF. Error bars represent standard deviations of three biological replicates. *p < 0.05; **p < 0.01 versus expression in spores. (E) Analysis of SP7 varieties in different developmental stages. SP7 varieties were amplied from total cDNA using primers located in the 50 and 30 untranslated regions common to all SP7 mRNAs. PCR products were blotted and detected using the SP7 variety 0.9 kb as a probe. See also Figure S1.

system for G. intraradices, we expressed SP7 without the signal peptide (SP7DSP-GFP) in the lamentous fungus Aspergillus nidulans. Indeed, upon promoter induction, SP7 localized to the cell nucleus (Figure 2A; Figure S2A). To investigate whether SP7 is able to enter the plant cell, SP7-GFP was expressed transiently in Nicotiana benthamiana leaves or stably in Medicago truncatula hairy roots. In both cases, the protein was localized to the plant cell nucleus (Figures 2B and 2D; Figure S2B). The better suitability of N. benthamiana leaves for microscopic studies allowed detection, in addition to the nuclear localization, of SP7-GFP in small vesicles (Figure 2D). In contrast, GFP fused to SP7 signal peptide showed uorescence only in vesicles and the apoplast (Figures S2B and S2C), but not in the nucleus. This localization was identical to that of GFP fused to the signal peptide of the secreted plant PR1 (pathogenesis-related) protein (Figure S2B). The DsRed1 control protein, as expected, localized in the cytoplasm, freely diffusing into the nucleus (Figures 2C and 2E). These results indicate that SP7-GFP localization in the nucleus was solely due to secreted SP7 that crossed the plant cell membrane

and suggest that SP7 is able to enter the plant cell without the assistance of any other fungal protein. A motif search using SP7 repeats revealed a weak similarity to the hemolysin-type calcium binding repeat motif also found in NodO from Rhizobium leguminosarum. NodO is a secreted repeat-containing protein that forms pores in the membrane of legume plants and assists in the perception of Nod factors [1921]. Entry into plant cells by SP7 could be mediated by the ability of the repeats to integrate in the plant plasma membrane. SP7 Interacts with the Pathogenesis-Related Transcription Factor ERF19 within the Plant Nucleus To investigate the role of SP7 during symbiosis, a yeast twohybrid analysis was carried out using a cDNA library of M. truncatula mycorrhizal roots. A small set of interacting plant proteins was found (Table S1). Among those, a protein with the signature of the AP2-EREBP transcription factor family showed the strongest interaction in the yeast assay (Figure S3A). We showed that M. truncatula ERF19 localizes to the nucleus (Figure S3B) and is indeed able to interact with SP7 at that location, as conrmed in planta by bimolecular uorescence complementation (Figure S3C). The most closely characterized homolog of this transcription factor is the protein ERF19 from Lotus japonicus (NCBI GenBank BAG50065) (Figure S3D). LjERF19 has been shown to be mainly expressed in roots and induced during nodulation as well as in response to a combined application of ethylene

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Figure 2. SP7 Localizes to the Nucleus (A) Nuclear localization of SP7 minus signal peptide (SP7DSP-GFP) in a germinated conidium of Aspergillus nidulans expressed under derepressing (glycerol) conditions and examined by epiuorescence microscopy. The picture is the overlay of the bright-eld and the GFP channel. SP7 was exclusively localized in nuclei. (B) Single confocal plane of a representative transgenic Medicago truncatula hairy root line constitutively expressing SP7-GFP. The fusion protein localizes to the nucleus of cortical root cells. (C) In contrast, constitutive DsRed1 as a control in the same root as in (B) localized to the nucleus and the cytoplasm. (D) Single confocal plane of a representative Nicotiana benthamiana leaf expressing SP7GFP. Leaves were observed 3 days after inltration with Agrobacterium tumefaciens. SP7 was visible in the plant nucleus, in small vesicles located in the cytoplasm, and in the vicinity of the plasma membrane (showed by a dashed line in F). (E) Control DsRed1 uorescence in the same cell as in (D) was only observed in the nucleus and in the cytoplasm, but not in vesicles. (F) Overlay of (D) and (E). Scale bars represent 10 mm. Filled arrowheads indicate nuclei; empty arrowheads indicate vesicles; dashed lines indicate plasma membrane. See also Figure S2.

and jasmonic acid [22]. However, in a time course of symbiosis with G. intraradices, MtERF19 was only transient and slightly induced at very early stages (Figure 3A). ERF19 belongs to the novel class IV of ERF transcription factors, characterized by a conserved amino acid stretch (MCGGAII/L) at the amino terminus [23]. Members of this family have been shown to be induced in response to pathogen infection, mechanical wounding, and abiotic stresses as well as specically by ethylene, jasmonic acid, and/or salicylic acid [2326]. They are known to activate expression of target defense proteins by binding to the GCC box in the promoter of the corresponding genes. In agreement with this, MtERF19 was highly induced by the legume pathogen Colletotrichum trifolii as well as by application of crude extracts from several different fungi, including nonplant pathogens (Figures 3B and 3C). These results indicated that MtERF19 expression could be triggered by the presence of a common fungal MAMP. We therefore tested whether a crude extract of G. intraradices spores would also be able to induce MtERF19. Interestingly, the Glomus extract induced MtERF19 to an extent similar to the other fungal extracts (Figure 3C), indicating that the mild activation of MtERF19 during mycorrhization could be the result of SP7 protein counteracting the plant defense program that involves ERF19. SP7 Counteracts the Defense Response that Involves ERF19 Overexpression of some class IV ERF transcription factors has been shown to increase resistance to pathogen attack as well as to several abiotic stresses [2426]. This increased resistance correlated with induced expression of various defense-related genes, including PR1, PR2, PR4, Osmotin, and SAR8.2 [24, 26]. To ascertain whether SP7 could interfere with the defense program of ERF19 to support mycorrhizal colonization, we used hairy root lines where SP7 was constitutively expressed. Indeed, mycorrhizal colonization in SP7-expressing lines was enhanced (Figure 3D; Figure S3E). An even more striking result was that C. trifolii-mediated MtERF19

induction was reduced to half or less in roots expressing SP7 (Figure 3E). Furthermore, upregulation of PR10-1, which encodes a known pathogenesis-related protein activated by C. trifolii in M. truncatula leaves [27], was also attenuated in roots expressing SP7 (Figure 3F). The results above imply that an overexpression of ERF19 would result in an impaired mycorrhizal symbiosis, whereas inactivation of ERF19 should mimic SP7 expression in planta. This was corroborated by RNA interference (RNAi) inactivation of MtERF19 that phenocopied the effect of constitutive SP7 expression in planta, accelerating mycorrhizal colonization (Figure 3G; Figures S3F and S3G). In addition, overexpression of MtERF19 significantly impaired root colonization by G. intraradices (Figure 3H; Figures S3H and S3I). SP7 Increases the Biotrophic Phase in Roots of the Pathogen Magnaporthe oryzae The goal of biotrophic microbes such as AM fungi is to keep their hosts alive so that the microbe can feed on living cells. In contrast, hemibiotrophs live for a short time as biotrophs, after which they switch to the necrotrophic modus, killing the host cell [1]. To investigate the possibility that SP7 could work as a universal effector to promote the biotrophic status of a fungus inside of the plant, we expressed SP7 in Magnaporthe oryzae. M. oryzae is a hemibiotrophic pathogen that can infect both leaves and roots of host plants [28, 29]. We rst showed that SP7-mRFP localized to the plant nucleus after secretion by M. oryzae in a modied onion epidermis assay (Figure 4A; Figures S4A and S4B). This corroborated our hypothesis that SP7 can translocate into plant cells and enter the nucleus. After a biotrophic phase, colonization of roots by M. oryzae induces necrosis symptoms and abnormal root development [29]. Here we found that root decay symptoms in rice infected with M. oryzae expressing SP7-mRFP were strongly attenuated (Figure 4B). Roots infected with the wild-type strain were much shorter than mock-inoculated roots and were poorly developed (Figure 4B; Figure S4C). In contrast, roots infected with M. oryzae expressing

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Figure 3. SP7 Counteracts the ERF19 Defense Response and Enhances Mycorrhizal Colonization (A) Time-course analysis of MtERF19 expression in mycorrhizal roots 1, 12, and 25 days postinoculation (dpi) with Glomus intraradices. (B) Analysis of MtERF19 expression after infection of stably transformed M. truncatula hairy root lines with Colletotrichum trifolii. VC indicates vector control line. Signicance is versus the uninfected control. (C) Transcript accumulation of MtERF19 after 24 hr induction of M. truncatula roots with crude extracts of spores of different fungi, including G. intraradices. Signicance is versus the noninoculated control. (D) Estimations of mycorrhization levels in SP7DSP-GFP and SP7-GFP hairy root lines in comparison to a control line (VC) 12 dpi with G. intraradices. Parameters measured are frequency (F%) and intensity (M%) of mycorrhiza and abundance of intraradical hyphae (I%), arbuscule (A%) and appressoria (App%). The data are the average of three biological replicates of one representative line expressing SP7DSP-GFP or SP7-GFP. Signicance is versus mycorrhization parameters of the line transformed with the vector control. (E) Analysis of MtERF19 expression after infection of stably transformed M. truncatula hairy root lines with C. trifolii. Vector control line and SP7DSP-GFPand SP7-GFP-expressing lines are compared. Signicance is versus the infected vector control. (F) Transcript accumulation of MtPR10-1 after infection of stably transformed M. truncatula hairy root lines with C. trifolii. Vector control line and SP7DSPGFP- and SP7-GFP-expressing lines are compared. Signicance is versus the infected vector control. (G) Estimation of mycorrhization level of a M. truncatula MtERF19-RNAi line compared to a control line 12 dpi with G. intraradices. Data are the average of three biological replicates of the line ERF19-RNAi2. Signicance is versus mycorrhization parameters of the line expressing the vector control. (H) Estimations of mycorrhization levels 12 dpi with G. intraradices in transgenic M. truncatula hairy root lines expressing MtERF19 under control of the CMV 35S promoter. Data are the average of three biological replicates of the line ERF19-OE6. All data are the average of three biological replicates. Error bars represent standard deviations. *p < 0.05; **p < 0.01. See also Figure S3.

SP7-mRFP were as long as mock-inoculated roots and were similarly developed (Figure 4B; Figure S4C). This phenotypic observation was accompanied by reduced expression of two rice genes encoding PR10 proteins (Figure 4C) usually induced

during M. oryzae leaf and root colonization [30, 31]. Our results support the hypothesis that SP7 is an effector protein that plays a role in establishing and/or maintaining the biotrophic status by generally attenuating immune defense responses

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Figure 4. Biological Relevance of SP7 (A) SP7-mRFP is secreted by a Magnaporthe oryzae GFP-expressing strain, translocated into the onion cell, and localized to the nucleus (left and middle panels). No red nuclei were detected when the infection assay was performed using the GFP control strain (Guy11-GFP, right panel). Images show maximal projections of several confocal planes. Scale bars represent 10 mm. (B) Rice roots 10 days postinoculation with SP7mRFP-expressing M. oryzae strains (Guy11GFP/SP7-mRFP) show reduced root decay symptoms and no shortening of lateral roots in comparison to roots infected with the control Guy11-GFP strain. Three biological replicates with four plants each were used. One representative biological replicate of each treatment is shown. (C) PR10a and PR10b transcript accumulation in rice roots 4 and 10 days postinfection with M. oryzae. Strains expressing SP7 (Guy11-SP7) show a reduced induction compared to colonization by the control strain (Guy11). Error bars represent standard deviation of three biological replicates. **p < 0.01 versus PR10 levels induced by wild-type Guy11 strain. See also Figure S4.

in planta. Its expression in M. oryzae extends the length of the biotrophic phase, delaying the root decay that characterizes the necrotrophic phase. Conclusions We show here that the symbiotic AM fungus G. intraradices secretes effector proteins that play a role in managing the accommodation process of the fungus within plant roots. This is a remarkable nding, because it dispels the previously held view that the ancient symbiotic glomeromycotan fungi had not adopted any mechanisms used by pathogens when interacting with plants. We believe that this is the beginning of an era of discoveries toward the understanding of how benecial obligate microbes modulate the cellular program of their host plants (see also Plett et al. [32] in this issue of Current Biology). This knowledge will pave the way toward comprehending and controlling other nonbenecial plantfungus interactions.
Experimental Procedures Supplemental Experimental Procedures contain the details of isolation and cloning of SP7 and MtERF19, molecular biology, transgenic analysis, RNAi, and overexpression. Mycorrhization of plants and expression analyses were carried out as described previously [33]. Table S2 lists the information for all primers used. Details concerning M. truncatula treatment with fungal extracts or C. trifolii and concerning O. sativa infection with M. oryzae are described in Supplemental Experimental Procedures. Supplemental Information Supplemental Information includes four gures, two tables, and Supplemental Experimental Procedures and can be found with this article online at doi:10.1016/j.cub.2011.06.044.

Acknowledgments Funding for this research was provided by the Deutsche Forschungsgemeinschaft (DFG; Re1556/7-1). N.R. holds a Heisenberg Professorship from the DFG and the Karlsruhe Institute of Technology. We are thankful to our Karlsruhe colleagues for useful scientic discussions and to H. Slater for comments on the manuscript. We thank J. Rose for donation of the YSST vectors and R. OConnell and A. Sesma for the C. trifolii and M. oryzae GFP strains, respectively. Received: April 15, 2011 Revised: May 26, 2011 Accepted: June 20, 2011 Published online: July 14, 2011 References 1. Valent, B., and Khang, C.H. (2010). Recent advances in rice blast effector research. Curr. Opin. Plant Biol. 13, 434441. 2. Boller, T., and Felix, G. (2009). A renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by patternrecognition receptors. Annu. Rev. Plant Biol. 60, 379406. 3. Abramovitch, R.B., Anderson, J.C., and Martin, G.B. (2006). Bacterial elicitation and evasion of plant innate immunity. Nat. Rev. Mol. Cell Biol. 7, 601611. 4. Kamoun, S. (2007). Groovy times: Filamentous pathogen effectors revealed. Curr. Opin. Plant Biol. 10, 358365. 5. Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A., and Bonas, U. (2009). Breaking the code of DNA binding specicity of TAL-type III effectors. Science 326, 1509 1512. 6. Smith, S.E., and Read, D.J. (1997). Mycorrhizal Symbiosis (Cambridge: Academic Press). 7. Bonfante, P., and Genre, A. (2010). Mechanisms underlying benecial plant-fungus interactions in mycorrhizal symbiosis. Nat. Commun. 1, 48. 8. Bonfante, P., and Requena, N. (2011). Dating in the dark: How roots respond to fungal signals to establish arbuscular mycorrhizal

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