A Secreted Fungal Effector of Glomus intraradices Promotes Symbiotic Biotrophy  Silke Kloppholz, Hannah Kuhn, Natalia Requena  Current Biology  Volume 21, Issue 14, Pages 1204-1209 (July 2011) DOI: 10.1016/j.cub.2011.06.044 Copyright © 2011 Elsevier Ltd Terms and Conditions

Current Biology 2011 21, 1204-1209DOI: (10.1016/j.cub.2011.06.044) Copyright © 2011 Elsevier Ltd Terms and Conditions

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 amplified from total cDNA using primers located in the 5′ and 3′ 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. Current Biology 2011 21, 1204-1209DOI: (10.1016/j.cub.2011.06.044) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 SP7 Localizes to the Nucleus (A) Nuclear localization of SP7 minus signal peptide (SP7ΔSP-GFP) in a germinated conidium of Aspergillus nidulans expressed under derepressing (glycerol) conditions and examined by epifluorescence microscopy. The picture is the overlay of the bright-field 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 SP7-GFP. Leaves were observed 3 days after infiltration 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 fluorescence 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 μm. Filled arrowheads indicate nuclei; empty arrowheads indicate vesicles; dashed lines indicate plasma membrane. See also Figure S2. Current Biology 2011 21, 1204-1209DOI: (10.1016/j.cub.2011.06.044) Copyright © 2011 Elsevier Ltd Terms and Conditions

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. Significance 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. Significance is versus the noninoculated control. (D) Estimations of mycorrhization levels in SP7ΔSP-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 SP7ΔSP-GFP or SP7-GFP. Significance 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 SP7ΔSP-GFP- and SP7-GFP-expressing lines are compared. Significance 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 SP7ΔSP-GFP- and SP7-GFP-expressing lines are compared. Significance 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. Significance 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. Current Biology 2011 21, 1204-1209DOI: (10.1016/j.cub.2011.06.044) Copyright © 2011 Elsevier Ltd Terms and Conditions

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 μm. (B) Rice roots 10 days postinoculation with SP7-mRFP-expressing M. oryzae strains (Guy11-GFP/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. Current Biology 2011 21, 1204-1209DOI: (10.1016/j.cub.2011.06.044) Copyright © 2011 Elsevier Ltd Terms and Conditions