Volume 9, Issue 4, Pages 514-527 (April 2016) Polarized Defense Against Fungal Pathogens Is Mediated by the Jacalin-Related Lectin Domain of Modular Poaceae-Specific Proteins  Denise Weidenbach, Lara Esch, Claudia Möller, Goetz Hensel, Jochen Kumlehn, Caroline Höfle, Ralph Hückelhoven, Ulrich Schaffrath  Molecular Plant  Volume 9, Issue 4, Pages 514-527 (April 2016) DOI: 10.1016/j.molp.2015.12.009 Copyright © 2016 The Author Terms and Conditions

Figure 1 OsJAC1 Transcript Accumulation in Response to Different Treatments. Expression of OsJAC1 was analyzed by RT-qPCR in leaves of 2-week-old plants from rice cv. Nipponbare. (A and B) Application of chemical resistance inducers (A) DCINA (100 ppm) or (B) BTH (250 ppm). Controls were treated with the corresponding formulation. (C) Leaves were wounded with a rough forceps at regular distances (0.5 cm). Untreated plants served as a control. (D–F) Plants were challenged with (D) the compatible fungal pathogen M. oryzae FR13 (250 000 spores ml−1), (E) the bacterial pathogen Xoo PXO86 (OD600 = 0.5), or (F) the fungal nonhost pathogen Bgh (15–20 spores mm−2). Control plants were mock treated. All experiments were repeated twice, with a similar result. (G) Characterization of OsJAC1 T-DNA insertion lines Osjac1-1 and Osjac1-2. RT-qPCR analysis of OsJAC1 transcript accumulation in ZmUBI1::OsJAC1 and T-DNA insertion lines Osjac1-1 and Osjac1-2 12 h after wounding. Corresponding wild-type cultivars and untreated plants served as references. The experiment was repeated once, with a similar result. Bgh, Blumeria graminis f.sp. hordei; BTH, benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester; DCINA, 2,6-dichloroisonicotinic acid; M. oryzae, Magnaporthe oryzae; ppm, parts per million; RT-qPCR, quantitative real-time PCR; WG, wettable granulate; WP, wettable powder; Xoo, Xanthomonas oryzae pv. oryzae. Molecular Plant 2016 9, 514-527DOI: (10.1016/j.molp.2015.12.009) Copyright © 2016 The Author Terms and Conditions

Figure 2 Overexpression of OsJAC1 Enhances Broad-Spectrum Resistance against Major Rice Pathogens. Disease development on transgenic rice overexpressing OsJAC1 (ZmUBI1::OsJAC1) and on a T-DNA-insertion line (Osjac1-1) compared with appropriate controls is documented on the left-hand or right-hand side, respectively. (A and E) Development of the blast fungus Magnaporthe oryzae. Blast symptoms per leaf area were counted (5 dpi) and pictures were taken (7 dpi). Data are given as the mean (n = 8) ± SD. Asterisk indicates significant difference (P = 0.01) determined by Student t-test. (B and F) Detached leaf assay for growth monitoring of Rhizoctonia solani (4th leaf of each genotype was used, pictures taken at 3 dpi). (C and G) Rice roots infected by Pythium graminicola (7 dpi). Arrows indicate decayed lateral roots. (D and H) Proliferation of Xanthomonas oryzae pv. oryzae was quantified by re-isolation of bacteria at given time points, and pictures were taken at 10 dpi (left) or 6 dpi (right). Graphs represent the logarithm of cfu per cm leaf material as mean value of three technical replicates with five leaf-tips per sample ± SD. Asterisks indicate significant differences (P ≤ 0.029) determined by a Student t-test. Symptoms are indicated by arrows. All experiments were repeated twice, with a similar result. cfu, colony-forming unit. Molecular Plant 2016 9, 514-527DOI: (10.1016/j.molp.2015.12.009) Copyright © 2016 The Author Terms and Conditions

Figure 3 Modular Proteins Containing a Dirigent and a Jacalin-Related Lectin Domain Are Highly Conserved. (A) Representation of the OsJAC1 and TaJA1 protein sequence. Amino acid positions of protein domains are indicated. (B) Alignment of the OsJAC1 and TaJA1 protein sequence (CLUSTAL multiple sequence alignment by MUSCLE 3.8). Gray shading indicates identical amino acids. The GPWGG-motif invariant in jacalin-related lectins and GG-carbohydrate binding site are framed (Song et al., 2014). Molecular Plant 2016 9, 514-527DOI: (10.1016/j.molp.2015.12.009) Copyright © 2016 The Author Terms and Conditions

Figure 4 Chromosomal Location of Monocot JRLs in Selected Crop Plants. Gene annotations are given in Supplemental Table 1, and explanation of symbols in Supplemental Table 2. Identical symbols indicate synteny of corresponding loci. Graphic was designed using the Comparison Genomics tool of the EnsemblPlants database (Kinsella et al., 2011). OsJAC1 and its functional orthologs are marked red; ambiguous loci are gray. Molecular Plant 2016 9, 514-527DOI: (10.1016/j.molp.2015.12.009) Copyright © 2016 The Author Terms and Conditions

Figure 5 Cross-Species Functionality of Monocot JRLs Shown by Expression of OsJAC1 and TaJA1 in Barley. (A) Development of the nonhost fungus Bgh on rice cv. Dongjin and the OsJAC1-T-DNA-insertion line Osjac1-1 (4 dpi). Aniline blue staining was applied to evaluate the plant reaction, and epiphytic fungal structures were stained with ink-acetic acid. Columns represent mean values (n = 3–4) ± SD with 100 interaction sites inspected per genotype and per leaf. Different characters over same-color columns indicate significant differences (P ≤ 0.029) determined by a Student t-test. (B) Barley leaves (cv. IngridMLO) were co-bombarded with the respective expression constructs as indicated, and the impact on the penetration success of Bgh was evaluated 48 h post inoculation. The host cell entry was calculated as the ratio of transformed cells enabling haustorium formation to the total number of attacked transformed cells; the empty vector control was taken as baseline for graphical representation. Columns represent mean values of at least three bombardments with 80–100 interaction sites inspected per expression construct over at least three leaves (n = 3–4) ± SD. Significant differences (P ≤ 0.001) determined by a Student t-test are indicated by different characters. (C) Barley cv. Golden Promise was transformed with OsJAC1 (ZmUBI1::OsJAC1). Seven-day-old primary leaves from three independent transformation events (homozygous T2 material) were inoculated with Bgh (left), Magnaporthe oryzae (middle), or Puccinia hordei (right). Azygous sibling material served as control. Powdery mildew pustules (5 dpi) and blast symptoms (7 dpi) were counted and the leaf area determined using Adobe Photoshop. Rust infection (9 dpi) was determined with APS Assess 2.0 software. Data are given as the mean of three independent transformation events (n = 5–6 each) ± SD. Asterisk indicates significant difference to azygous controls (P ≤ 0.001) determined by a Student t-test. Experiments were repeated once (A) or twice (C) with a similar result. app, appressorium; elong. hy, secondary elongated hyphae; i. hau, haustorial initial; sp, spore. Molecular Plant 2016 9, 514-527DOI: (10.1016/j.molp.2015.12.009) Copyright © 2016 The Author Terms and Conditions

Figure 6 Interaction of OsJAC1 Protein Domains in FRET Analysis. Quantitative analysis of FRET efficiencies. Columns represent mean values of three experiments (10 cells evaluated per co-bombardment and experiment) ± SD. Asterisk indicates significant differences (P ≤ 0.001) determined by a Student t-test. CFP, CFP-empty-vector; CFP-JAC, OsJAC1 jacalin-related lectin domain with N-terminal CFP tag; FRET, Förster resonance energy transfer; YFP, YFP-empty-vector; YFP-DIR, OsJAC1 dirigent domain with N-terminal YFP tag. Molecular Plant 2016 9, 514-527DOI: (10.1016/j.molp.2015.12.009) Copyright © 2016 The Author Terms and Conditions

Figure 7 Both Domains of OsJAC1 and Re-localization to Fungal Infection Sites Are Required to Confer Resistance. (A) Barley leaves (cv. IngridMLO) were co-bombarded with the respective expression constructs as indicated (dirigent domain [orange], jacalin-related lectin domain [green], eGFP [dark green], amino acid substitutions [black]) and the impact on the penetration success of Bgh was evaluated 48 h post inoculation. The host cell entry was calculated as the ratio of transformed cells enabling haustorium formation to the total number of attacked transformed cells; the empty vector control was taken as baseline for graphical representation. Columns represent mean values of at least three bombardments with 100 interaction sites inspected per expression construct on at least three leaves each (n = 3–5) ± SD. Significant differences (P ≤ 0.032) determined by a Student t-test are indicated by different characters. (B) Localization of YFP-OsJAC1 (yellow) in transiently transformed barley epidermal cells without (left, 3D projection of z-stack) and after Bgh inoculation (right, scale bar represents 10 μm). Cells were co-transformed with marker gene eGFP (green). The fungal spore is digitally stained (cyan) for better representation. (C) Accumulation of OsJAC1-GFP (green) at penetration sites of Bgh and Bgt on stable transformed barley (background cv. Golden Promise) or wheat (background cv. Bobwhite) plants. OsJAC1-GFP was constitutively expressed by the UBIQUITIN1 promoter of Zea mays. Overlays of transmitted light channel and GFP channel are shown (scale bar represents 10 μm). Hence the absence of the GFP signal in susceptible cells without active defense, allowing establishment of a haustorial initial (bottom right). (D) Accumulation of functional RFP-OsJAC1 or nonfunctional RFP-OsJAC1 with mutagenized putative mannose-binding sites beneath Bgh appressoria was evaluated (right). Columns represent mean values of three experiments (n = 3) ± SD with 14–25 cells evaluated per construct and experiment. Asterisk indicates a significant difference (P = 0.05) determined by a Student t-test. The nonfunctional RFP-GA175/GA293/IL296 construct did not accumulate at the penetration site, as shown by fluorescence intensities of fluorophores across the green line (left, scale bar represents 20 μm). In (B–D), cells were evaluated about 24 h after Bgh inoculation. Autofluorescence of plant cells was subtracted by Channel Dye Separation. app, appressorium; ASS_DIR, substitution of the conserved amino acid interval WAIVGGTGQFAMATGVI to YGVAAASATLGLGSAAV in the dirigent domain of OsJAC1; ASS_JAC, substitution of the conserved amino acid interval ITSINFITNKQTYGPFG to VSAVSLVSSTSSFASLA in the jacalin-related lectin domain of OsJAC1; Bgh, Blumeria graminis f.sp. hordei; Bgt, Blumeria graminis f.sp. tritici; DIR, dirigent domain of OsJAC1; eGFP, emerald GFP; GA175/GA293/IL296, OsJAC1 with mutagenized putative mannose-binding sites; i. hau, haustorial initial; JAC, jacalin-related lectin domain of OsJAC1; OsJAC1-GFP, OsJAC1 with C-terminal GFP tag; pg, primary germ tube; sp, spore; YFP-OsJAC1, OsJAC1 with N-terminal YFP tag. Molecular Plant 2016 9, 514-527DOI: (10.1016/j.molp.2015.12.009) Copyright © 2016 The Author Terms and Conditions