Supplemental data. Uppalapati et al. (2012) Plant Cell /tpc
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1 PAL Control P. emaculata OPR Control P. emaculata CHS PR3 CHR PR5 CHI PR10 IFS IFR Supplemental Figure 1. Expression profiles for selected genes in phenylpropanoid pathway (PAL, CHS, CHR, CHI, IFS, and IFR) and pathogenesis-related genes (PR3 and PR10) in wild type () at 0, 8, 24 and 48 hours postinoculation with P. emaculata urediniospores.
2 12 plants/tnt1 line (23 lines/week) Spray inoculation of detached trifoliate leaves with P. emaculata urediospores (1 X 10 5 spores/ml, 0.001% Tween 20) Dew chamber-24 h (24 C; 24D) and further incubation in growth chamber (24 C/22 C; 16L:8D) for 5 days Enhanced susceptibility or resistance phenotype 1. ROS localization (2 dpi) 2. WGA staining initial events (3 dpi) 3. Penetration and colonization (5 dpi) Characterization of interesting mutants Supplemental Figure 2. A schematic showing the sequence of events for conducting the forward genetic screens to identify M. truncatula genes involved in nonhost resistance against P. emaculata.
3 A irg1-1 PAL P. pachyrhizi P. pachyrhizi CHS CHR CHI IFS IFR PR3 PR10 B Percentage of urediniospores Ge Gt Ap C Percentage of urediniospores Ge Gt 0 Buffer Con irg1-1 irg1-2 0 Buffer Con irg1-1 irg1-2 Supplemental Figure 3. Expression profiles for selected defense-related genes and antimicrobial activity of protein extracts on fungal differentiation. (A) RNA gel blot analyses of selected genes from phenylpropanoid pathway (PAL, CHS, CHR, CHI, IFS, and IFR) and pathogenesis-related genes (PR3 and PR10) in wild type () and irg1-1 at 0, 8, 24 and 48 hours post-inoculation with P. pachyrhizi. (B, C) Effect of total proteins (at 10 mg/ ml concentration) from wild-type and irg1 mutant alleles on differentiation of P. pachyrhizi (B) and P. emaculata (C) urediospores on artificial plastic (hydrophobic) surfaces. The percentage germination (Ge) of urediniospores and germ-tubes with no appressoria (Gt) and germ-tubes with differentiated appressoria (Ap) were evaluated and the average of five observations is presented.
4 A irg1-2/palm1-4 (line NF 1271) irg1-1/palm1-5 (line NF0227) irg1-5/palm1-6 (line NF5022) IRG1/PALM1 (Medtr5g ) B 200 bp irg1-1/palm1-5 irg1-3 (line NF1432) irg1-4 (line NF4045) irg1-2/palm1-6 irg1-3 irg1-4 irg1-5/palm1-6 C A17 palm1-1/palm1-1 irg1-1/palm1-5 irg1-2/palm1-4 irg1-5/palm µm 100 µm Supplemental Figure 4. Rust germ-tube differentiation on multiple mutant alleles of irg1 (A) A schematic showing the location of Tnt1 insertion in the PALM1/IRG1 exon of the different mutant alleles and corresponding line numbers used in this study. (B) Multiple alleles of Medicago truncatula irg1 mutant lines inhibit differentiation of pre-infection structure formation of Phakopsora pachyrhizi on abaxial surface of leaves. (C) Multiple alleles of Medicago irg1 mutant inhibit differentiation of pre-infection structure formation of Puccinia emaculata on abaxial surface of leaves. Scale bar= 100 µm.
5 A irg1-1 B irg1-1 Supplemental Figure 5. Leaf phenotype of four-week old wild-type and irg1-1 plants of M. truncatula. The adaxial (A) and abaxial (B) leaves of M. truncatula and irg1. rer
6 Acyl Reduction Pathway CER4 aldehyde C18 C26 C28 C30 C32 Fatty acid Elongation CER6/CER10 Decarbonylation Pathway Aldehydes CER1/CER2/CER3? 1 alchohol Alkanes 2 alchohol Ketones Supplemental Figure 6. A simplified wax biosynthesis pathway and some CER genes implicated in wax biosynthesis in Arabidopsis (Modified from Kunst and Samuels, 2003; Samuels et al., 2008).
7 ng/cm 2 A 5, , , , Ad irg1-1-ad irg1-2-ad ng/cm 2 ng/cm 2 ng/cm 2 B 1, , , , , , , C D C16 C18 C20 C24 C26 C27 C28 C29 C30 C32 -Ab irg1-1-ab irg1-2-ab C16 C18 C20 C24 C26 C27 C28 C29 C30 C32 -Ad irg1-1-ad irg1-2-ad C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 -Ab irg1-1-ab irg1-2-ab C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 Supplementary Figure 7. Composition of adaxial alcohols (A), abaxial alcohols (B), adaxial alkanes (C), and abaxial alkanes (D) of wild-type and irg1 mutant alleles (irg1-1 and irg1-2) of Medicago truncatula. Means ±SE of five replications are presented for each data point.
8 Supplemental Table 1. Fold changes in expression of corresponding Medicago orthologs of Arabidopsis wax biosynthesis related genes using RT-qPCR and microarray analysis. Gene Gene Description At Gene ID Mt TC ID Affy ID Fold change (irg1/) CER1 Unknown AT1G02205 MtCER1-1 (TC130292) Mtr S1_at 2.88 CER2 Unknown AT4G24510 MtCER2 (TC115187) Mtr S1_at 7.33 CER3/WAX2 Unknown AT5G57800 MtCER3 (TC125004) Mtr S1_at 1.04 CER5 ABC transporter AT1G51500 MtCER5-1 (TC145141) Mtr S1_x_at 1.29 CER6 β-keto acyl-coa synthase (KCS) AT1G68530 MtCER6-4 (TC113573) Mtr S1_s_at 0.33 CER8 Long chain acyl-coa synthase (LACS) AT2G47240 MtCER8-1 (TC140235) Mtr S1_at 0.70 CER10 Enoyl-CoA reductase (ECR) AT3G55360 MtCER10 (TC125186) Mtr S1_at 0.86 KCR1 β-keto acyl-coa reductase (KCR) AT1G67730 MtKCR1-2 (TC113588) Mtr S1_at 0.78 WSD1 Wax synthase AT5G37300 MtWSD1 (TC145247) Mtr S1_at 0.82 FATB Fatty acyl-acp thioesterase B (FATB) AT1G08510 MtFATB (TC148248) Mtr S1_at 0.82 MYB30 MYB Transcription factor AT3G28910 MtMYB30 (TC142663) Mtr S1_s_at 1.52
9 Supplemental Table 2. List of genes and corresponding primers used for qrt-pcr Gene GENEID Forward Primer Reverse Primer CER1 MtCER1-1 (TC130292) GCTTCTACGATAATGGCATCAAGGC TGCTGTGAATCACCCAAGGAGCTA CER2 MtCER2 (TC115187) AAGTGTGGTGGGATTTCATTGGGC TCAACCCGTTTAACTGTAGCCGGA CER3/WAX2 MtCER3 (TC125004) TCTGCGATCCGCTTCTTCATTTGC TACTGAAAGCCACATCGCGGTACA CER4 MtCER4-1 (TC122585) TGGGTTGAGGGTCTCAGAACCATT ATTTGCATGAGCCACCATAGCCAC MtCER4-2 (TC160800) TTTCCCTGGTTGGGTTGAAGGAGT ACCACCATATCAGCAGGGATCACA CER6 MtCER6-1 (TC116151) AGCAGCAGTTCTCCTCTCCAACAA TCTTGAAAGACGCAGCCGTAGGAT MtCER6-2 (TC125487) CACCTGTTACATGTCGTGTCCCTT CCTCTTGCTGATTCCATGGTTGGT MtCER6-3 (TC121408) TGATCCACACCGTCCGAACACATA CTCCGGCAACCGCCATTAAATCTT MtCER6-4 (TC113573) TCCTCTGATCGAACCCGTTCCAAA CAAAGCATCTCCTGCAACAGCCAT CER8 MtCER8-1 (TC140235) AAGCAAGGCTAGGTGGACGTGTTA ATGGCGGAGTTCCAAGAGGATTGT CER10 MtCER10 (TC125186) GGGCTTCAACATTGCAACGCAAAC TCACTTCAATGGCTTGGGCTCCTA PAS2 MtPAS2 (GE348322) TGCATCAGGATGCCGAATACATGG CTTTGCTTTGGCGAGGGCTTTCTT KCR1 MtKCR1 (TC113588) AGAAGCCCTCTTAAGCTTGAGGCA TAAGGATAAGCCACACCAGCACCA KCR2 MtKCR2 (TC145787) TGGGATTGATGTGCAGTGTCAGGT AAGGGTATGTGGCCAGTATGGTGT WSD1 MtWSD1 (TC145247) TGTTCAAGTGAAGGTGGTGGTGAG CTTGCTGGTGAACCTAGGATGCTT FATB MtFATB (TC148248) ATTGGCTGGATTCTGGAGAGTGCT GTCGAAGCAAATGCTGGCACTCAA MYB30 MtMYB30 (TC142663) AGTCTTTGTCACCAGACGCAACGA TGAGCATGATCACCACCACAACCT MYB96 Medtr4g135250/Medtr8g CGCAGCTGCCGAATAAAGGTCAAT GCAAAGTACCAAGGGTTGTAGGGT Ubiquitin MtUbq CTGACAGCCCACTGAATTGTGA TTTTGGCATTGCTGCAAGC
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