The N-end rule pathway regulates pathogen responses. in plants
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1 SUPPLEMENTARY INFORMATION The N-end rule pathway regulates pathogen responses in plants Rémi de Marchi 1,2, Maud Sorel 1, Brian Mooney 1, Isabelle Fudal 3, Kevin Goslin 1, Kamila Kwaśniewska 4, Patrick T. Ryan 4, Marina Pfalz 5, Juergen Kroymann 5, Stephan Pollmann 6, Angela Feechan 7, Frank Wellmer 4, Susana Rivas 2*, and Emmanuelle Graciet 1* 1
2 Supplementary figures Supplementary Figure 1: Comparison of transcriptomics datasets obtained with ate1 ate2 seedlings. a, Overlap between our transcriptomics dataset obtained and the results of a microarray analysis using seedlings grown under normal oxygen conditions 1. For the latter dataset, DEGs with log 2 (fold change) > 0.5 and adjusted P-value < 0.05 were considered. b, Overlap between our transcriptomics dataset and core hypoxia response genes defined by Mustroph et al. 2. 2
3 Supplementary Figure 2: Results of glucosinolate measurements. a, Results of glucosinolate measurements. The biosynthesis pathways of methionine-derived aliphatic glucosinolates and tryptophan-derived indolic glucosinolates (inset) are shown. The biosynthesis pathway of aliphatic glucosinolates includes reactions that occur in the chloroplast (represented by a green rectangle). The number of methylene groups in the chain of different precursors is indicated (2C, 3C, etc ). The level of the different glucosinolates in wild-type and ate1 ate2 seedlings is represented by blue and yellow 3
4 circles, respectively. The diameter of the circles is proportional to the quantity of the different glucosinolates measured in nine independent sets of samples. Only protein names encoded by genes that were found to be differentially expressed in ate1 ate2 are indicated, with black and red fonts corresponding to genes that were down and upregulated in the mutant, respectively. n.d.: not detected. b, Estimated total levels of aliphatic glucosinolates. c, Estimated total levels of indole glucosinolates. Estimated minimum and maximum are indicated for each genotype. 3BZOP (aliphatic glucosinolate) and 1MOI3M (indole glucosinolate) could not be separated, hence minimum and maximum levels of the two types of glucosinolates were calculated using ratios between 4BZOB and 3BZOP of 1.27:1 and 1.92:1, respectively 3-5 (see Methods for details). 4
5 Supplementary Figure 3: Response of JA and SA related genes in ate1 ate2 and prt6-5 mutant seedlings. The regulation of selected JA (a-c) and SA response genes (d-f) in ate1 ate2, prt6-5 and wild-type seedlings was monitored after treatment with 20 µm MeJA (a, d), 0.5 mm SA (b, e) and 10 µm ABA (c, f). Solid and dashed bars represent mock- or hormone-treated samples, respectively. Error bars correspond to SEM of at least four independent biological replicates. The results of Student s t-tests are shown with a indicating P-values < 0.05 and b P-values between 0.05 and 0.1. VSP1: VEGETATIVE STORAGE PROTEIN1; VSP2: VEGETATIVE STORAGE PROTEIN2; LOX2: LIPOXYGENASE2; AOS: ALLENE OXIDE SYNTHASE; PR1: PATHOGENESIS- RELATED1; NDR1: NON RACE-SPECIFIC DISEASE RESISTANCE1; PAD4: PHYTOALEXIN DEFICIENT4; EDS1: ENHANCED DISEASE SUSCEPTIBILITY1; ABI5: ABA INSENSITIVE5. 5
6 Supplementary Figure 4: Representative symptoms exhibited following inoculation with S. sclerotiorum. Wild-type and N-end rule mutant plants were photographed 7 days following inoculation with S. sclerotiorum. The Shahdara (Sha) and Rubezhnoe (Rbz) accessions were used as susceptible and resistant control plants, respectively. 6
7 Supplementary Figure 5: Expression of N-end rule genes following inoculation with Pst AvrRpm1. Four-week-old plants were inoculated with Pst AvrRpm1 (5x10 7 cfu/ml) and tissue was collected at the indicated time points. Gene expression was assessed using RT-qPCRs and oligonucleotides described in Supplementary Table 3. Gene expression levels were first normalized using the MON1 reference gene. Results are presented relative to the expression level of a given gene at the t0 time point. 7
8 Supplementary Figure 6: Comparison of transcriptomics datasets obtained following inoculation with Pst AvrRpm1. a, Overlap between our transcriptomics datasets obtained with wild-type (Col-0) plants following inoculation with Pst AvrRpm1 and the dataset presented by Truman et al. 6. The latter was generated using wild-type Col-0 plants inoculated with Pst AvrRpm1 and total RNA isolated at 4 hpi. b, Overlap between our transcriptomics datasets obtained with ate1 ate2 mutant plants following inoculation with Pst AvrRpm1 and the dataset presented by Truman et al. 6. 8
9 Supplementary Figure 7: Expression profile of genes associated with GO categories enriched in both the wild type and the ate1 ate2 mutant. Gene expression changes in wild type and ate1 ate2 plants following inoculation with Pst AvrRpm1 were extracted from the microarray datasets. ICS1 and PAD4 are SA-related genes; AOS and VSP2 are JA-response genes; MYB30 and MPK3 are known regulators of Arabidopsis response to Pst AvrRpm1. 9
10 Supplementary Figure 8: Expression profile of genes associated with GO categories that are only enriched among DEGs identified in the wild type. Gene expression changes in wild type and ate1 ate2 plants following inoculation with Pst AvrRpm1 were extracted from the microarray datasets. MAM3 and FMO-GSox1 are glucosinolate-related genes; ADH1 (ALCOHOL DEHYDROGENASE 1) and GLB1 (HEMOGLOBIN 1) are hypoxia-related genes; VPS45 (VACUOLAR PROTEIN SORTING 45) and RABA6b (RAB GTPase HOMOLOG A6B) are involved in intracellular protein movement. 10
11 Supplementary Figure 9: Gene expression changes of selected genes in prt6-5 mutant plants inoculated with Pst AvrRpm1. Gene expression changes of JA (VSP1 and VSP2) and SA (ICS1 and PR1) response genes, as well as of known regulators of the response to Pst AvrRpm1 (MPK3 and MYB30), were monitored following inoculation of wild type (Col-0) and prt6-5 plants with Pst AvrRpm1 at a density of 5x10 7 cfu/ml. Expression levels relative to those of the REF1 reference gene are presented. Error bars indicate SEM of four independent experiments. The results of Student s t-tests are shown with a indicating P-values < 0.05 and b P-values between 0.05 and
12 Supplementary Table 3: List of oligonucleotides used for this study, including gene name and AGI. Oligonucleotide name and gene AGI REF1 fwd (AT1G13320) REF1 rev (AT1G13320) REF2 fwd (AT4G34270) REF2 rev (AT4G34270) MON1-F (AT2G28390) MON1-R (AT2G28390) DNA contamination control fwd DNA contamination control rev EG_qPCR143 (MYB34; AT5G60890) EG_qPCR144 (MYB34; AT5G60890) EG_qPCR147 (CYP79F2; AT1G16400) EG_qPCR148 (CYP79F1 & CYP79F2; AT1G16410 & AT1G16400) EG_qPCR149 (CYP79F1; AT1G16410) EG_qPCR38 (MAM3; AT5G23020) EG_qPCR39 (MAM3; AT5G23020) EG_qPCR122 (FMO GS-ox1; AT1G65860) EG_qPCR123 (FMO GS-ox1; AT1G65860) EG_qPCR124 (BCAT4; AT3G19710) EG_qPCR125 (BCAT4; AT3G19710) PR1 fwd (AT2G14610) PR1 rev (AT2G14610) WRKY70 fwd (AT3G56400) WRKY70 rev (AT3G56400) NDR1 rev (AT3G20600) NDR1 fwd AT3G20600) PAD4 rev (AT3G52430) PAD4 fwd (AT3G52430) EDS1 rev (AT3G48090) EDS1 fwd (AT3G48090) Oligonucleotide sequence (5 to 3 ) AAGCGGTTGTGGAGAACATGATACG TGGAGAGCTTGATTTGCGAAATACCG ATCTGCGAAAGGGTATCCAGTTGAC TGGAAGCCTCTGACTGATGGAGC AACTCTATGCAGCATTTGATCCACT TGATTGCATATCTTTATCGCCATC TCATCAGCGGTAGATGGAACTCTGAG ATGACGTAGAAGCATCATCCTGCG CATGCTCTTAAGGGTAACAAGTGGGC CCTAGCGGAACCGGATGAATATACCG CATCGTAGAGTCCATTGGAGACAATTACAA CCGACCGTTGATACATCGAGTGAATG TCATCATGGAGACAATCGGAGACAATTACA TGGTGAACGGTGCTGAAATCTCATC AATGTCTTCCCAAACTTATACAACAGCGG GGTTCATTCCGAGATAGACTTCGCC AGACATGCTTGTATAGAGGTTCGACGC CGGCTACCAGGTCGAGGAAC CTAGTCACAATGGAAGCAGTGCCAG GAGAAGGCTAACTACAACTACGCTGC TTGGCACATCCGAGTCTCACTGA TACTTGAGGACGCATTTTCTTGGAGG GGACTTGCTTTGTTGCCTTGCAC CTGGTTGTTTAGCGGCTTTACTTGACC CACCATCAACACGACCAAGATCAATTCATC TTCCGAGCAGAGGAGAGCCAA CCTCCGATGAACCTCTACCTATGGTC ACCTCTCTTGCTCGATCACCTGA GAGGCAGACAGTACGTTCAAGCT 12
13 VSP1 fwd (AT5G24780) VSP1 rev (AT5G24780) VSP2 rev (AT5G24770) VSP2 fwd (AT5G24770) LOX2 fwd (AT3G45140) LOX2 rev (AT3G45140) AOS fwd (AT5G42650) AOS rev (AT5G42650) ABI5 rev (AT2G36270) ABI5 fwd (AT2G36270) MYC2 rev (AT1G32640) MYC2 fwd (AT1G32640) ICS1 rev (AT1G74710) ICS1 fwd (AT1G74710) MPK3 fwd (AT3G45640) MPK3 rev (AT3G45640) MYB30 fwd (AT3G28910) MYB30 rev (AT3G28910) RCA fwd (AT2G39730) RCA rev (AT2G39730) ATE1 fwd (AT5G05700) ATE1 rev (AT5G05700) ATE2 fwd (AT3G11240) ATE2 rev (AT3G11240) NTAQ1 fwd (AT2G41760) NTAQ1 rev (AT2G41760) PRT1 fwd (AT3G24800) PRT1 rev (AT3G24800) PRT6 fwd (AT5G02310) PRT6 rev (AT5G02310) GACCTAGACGACACTCTCCTCTCTAGT ACTCTAACCACGACCAGTACGCC CACGAGACTCTTCCTCACCTTTGACT AAGCTGCTGGCGTGACCTAC ACGGAGGTGGAATCATTGAGACTTGTT CGGTCTTATCTTCCTCAGCCAACC TTGGTGGCGAGGTTGTTTGTGAT ACAGATGGACTACACAGGTGCGA CAATGTCCGCAATCTCCCGTTCG CGG AGT TGG AGA GGA AGA GGA AGC CCATCTTCACCGTCGCTTGTTGA GAGGTTGATGTCGGCGTTGATGG AGATGGGTCACTTCCAGCTACTATCC CGAGGAGAGTGAATTTGCAGTCGG CTGTTGAACAAGCTCTGAATCACCAGT GTGCTATGGCTTCTTGGTAGATCATCTC AAGGGAGTTCAAGATCATAATGGTGAGG GCCCAGTATTGGTAGGAACAGCTC AGAGAGTCCAACTTGCCGAGACC TTGCTGGGCTCCTTTTCCGTAGAA TACAAGGAAATGCGGCAGATC TGGAGCTTGGTATTGTTGAAGC TGTATAACTCTGATGAAGACTCAGACTC CGGTATATCCTTGTATCGAAGTCGA TTCGCAGAGTACCAGAGGTTCTTCC GGTGGAGGTTGAGCAGTCCAAC TCAAAGCTATCTCAAAGAAGGCC ATGATTGGATACACCCCACAAGA CCTTGTTGCAGAGAAAGTGGTT CCAGGTAAGGAGATGGCCA 13
14 Supplementary references 1. Gibbs, D.J. et al. Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature 479, (2011). 2. Mustroph, A. et al. Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. Proc Natl Acad Sci U S A 106, (2009). 3. Kliebenstein, D.J. et al. Genetic control of natural variation in Arabidopsis glucosinolate accumulation. Plant Physiol 126, (2001). 4. Petersen, B.L., Chen, S., Hansen, C.H., Olsen, C.E. & Halkier, B.A. Composition and content of glucosinolates in developing Arabidopsis thaliana. Planta 214, (2002). 5. Brown, P.D., Tokuhisa, J.G., Reichelt, M. & Gershenzon, J. Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry 62, (2003). 6. Truman, W., de Zabala, M.T. & Grant, M. Type III effectors orchestrate a complex interplay between transcriptional networks to modify basal defence responses during pathogenesis and resistance. Plant J 46, (2006). 14
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