Arabidopsis ROP1 and ROP6 Influence Germination Time, Root Morphology, the Formation of F-Actin Bundles, and Symbiotic Fungal Interactions  Yvonne Venus, Ralf Oelmüller  Molecular Plant  Volume 6, Issue 3, Pages 872-886 (May 2013) DOI: 10.1093/mp/sss101 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 ROP Expression in Wild-Type, Knockout, and Overexpressor Lines. (A) RT–PCR for ROP1 and ROP6 in roots of rop1, rop6, and rop1rop6 mutants and in the wild-type (WT). ACTIN2 and A. dest. were used as control. (B, C) RT–PCR for ROP1 (B) and ROP6 (C) in 35S::ROP1 and 35S::ROP6 overexpressors. Multiple individuals (35S::ROP1a–d, 35S::ROP6a–c) and WT were analyzed. GAPC was used as control. PCR conditions were described in the Methods section. Molecular Plant 2013 6, 872-886DOI: (10.1093/mp/sss101) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 ROP1 and ROP6 Influence Seed Germination. Percent of germinated seeds within 24 h after stratification. Wild-type and transgenic seeds were planted on Petri dishes as described in the Methods section and kept at 4°C for 48 h for stratification before transfer to 20°C for 24 h under continuous illumination with white light (100 µmol m–2 sec–1). Three independent experiments with 50 seeds each. Error bars represent SEs. Bars marked with one (p < 0.05) or two (p < 0.01) asterisks are significantly different compared to wild-type (t-test). Molecular Plant 2013 6, 872-886DOI: (10.1093/mp/sss101) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Influence of ROP1 and ROP6 on the Root Morphology. (A) Lengths of the primary roots of wild-type, mutant, and overexpressor lines. Based on at least 20 independent measurements of independent seedlings. Bars represent SEs. The difference is caused by differences in the germination time. (B) Number of root hairs/mm of lateral root (i), lengths of root hairs (ii), and number of branched root hairs (iii) of wild-type and transgenic seedlings. The root hair analysis was performed in a 1-mm section, 2.5 mm away from the root tip. Data are based on at least five independent experiments with 10 roots each; bars represent SEs. Bars marked with one (p < 0.05) or two (p < 0.01) asterisks are significantly different compared to wild-type (t-test). (iv) Pictures showing branched root hairs for 35S::ROP1 and 35S::ROP6 seedlings. Molecular Plant 2013 6, 872-886DOI: (10.1093/mp/sss101) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Growth Promotion (Measured as Percent Increase in Fresh Weight of Roots and Shoots) by P. indica of Wild-Type and Transgenic Seedlings. Fresh weights were determined 14 d after co-cultivation of the two symbionts on agar plates. Data are based on five independent experiments with 12 plants for each treatment. Bars represent SEs. Comparable results were obtained with younger seedlings (6 d of co-cultivation). Molecular Plant 2013 6, 872-886DOI: (10.1093/mp/sss101) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Five-Week-Old Wild-Type (WT), rop1 rop6, and ric4 Plants on Soil either Without (– P. indica) or With (+ P. indica) the Fungus. Representative plants from four independent experiments (20 plants per treatment per experiment). In the presence of P. indica, the fresh weight of WT plants increased by 37% ± 8%, that of rop1 rop6 decreased by 43% ± 9%, and that of ric4 decreased by 29% ± 7% (n = 20 plants). Molecular Plant 2013 6, 872-886DOI: (10.1093/mp/sss101) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Visualization of F-Actin in the Roots of Arabidopsis Wild-Type and Transgenic Seedlings, Using the GFP–FABD2 Construct (Voigt et al., 2005) Expressed in the Wild-Type (A, B), rop1, rop6, and rop1 rop6 Background (B).The seedlings were either co-cultivated with P. indica (+ P. indica) or mock-treated (– P. indica) for 6 d on agar plates. Errors in (A) show F-actin bundling in the presence of fungal hyphae. Representative pictures of >20 independent experiments. Molecular Plant 2013 6, 872-886DOI: (10.1093/mp/sss101) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Cytoplasmic Ca2+ elevation and stimulation of mRNA levels by P. indica.(A) Cytoplasmic Ca2+ elevation in the roots of wild-type (WT) and rop1 rop6 mutants in response to the application of a cell wall extract from P. indica. WT, rop1, and rop6 plants were transformed with the plasmid pMAQ2 and the mutants were crossed to obtain homozygote rop1 rop6 mutants in the apoaequorin (http://en.wikipedia.org/wiki/Aequorin) background. Changes in the intracellular Ca2+ levels were followed for 20 min after the application of 50 µl of the cell wall extract. The Ca2+ levels were measured every 5 s as relative light units (RLU). The graph shows typical kinetics from four experiments with four roots each. As control, water was used instead of the cell wall extract. (B) Stimulation of the mRNA levels for the trypsin inhibitor ATT1 (At2g43510), chitinase (At2g43570), the Ca2+/calmodulin-binding protein Cbp60g (At5g26920), and glutathione-S-transferase (At1g02930) in the roots of Arabidopsis thaliana, co-cultivated with P. indica for 2 d. Fold induction = mRNA level from roots co-cultivated with P. indica/mRNA level from roots without P. indica. Based on three independent real-time PCR experiments with RNA from the roots of WT, rop1, rop6, and rop1 rop6 seedlings. The Arabidopsis actin mRNA level was used as control and was not regulated. Errors represent SEs. Molecular Plant 2013 6, 872-886DOI: (10.1093/mp/sss101) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions