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1 Supplementary igure 1. The expression level of PP4 (At4g16860) is diminished in the rpp4 mutant (SK017569). The mna was extracted to analyze the expression level of PP4 using quantitative PC (qpc). Gene expression was normalized against constitutively expressed UBQ5. Error bars represent SD (n = 3). WT, wild-type. WWW NATUE.COM/NATUE 1
2 Supplementary igure 2. Identification of PP4-regulated genes through expression profiling. a, In planta growth of Hpa Emwa1 and plant responses were examined under a microscope following lactophenol trypan blue (LTB) staining. ed arrows indicate H cell death and blue arrows point at hyphae. dpi, days post inoculation. b, Expression levels of the marker gene P1 were measured using qpc and normalized with UBQ5. Error bars represent SD (n = 3 biological replicates). c, Expression profiles of candidate genes that were significantly induced (q-values < 0.01) more than 2-fold in WT at 2 dpi. WWW NATUE.COM/NATUE 2
3 WWW NATUE.COM/NATUE 3
4 Supplementary igure 3-1. T-DNA insertion diminished the expression of candidate genes as measured by qpc. Gene expression levels were normalized against UBQ5. Error bars represent SD (n = 3 biological replicates). ull numbers for the T-DNA insertion mutant lines are shown in Supplementary Table 1. Primers used in this analysis are shown in Supplementary Table 2. WWW NATUE.COM/NATUE 4
5 WWW NATUE.COM/NATUE 5
6 Supplementary igure 3-2. T-DNA insertion diminished the expression of candidate genes as measured by qpc. Gene expression levels were normalized against UBQ5. Error bars represent SD (n = 3 biological replicates). ull numbers for the T-DNA insertion mutant lines are shown in Supplementary Table 1. Primers used in this analysis are shown in Supplementary Table 2. WWW NATUE.COM/NATUE 6
7 Supplementary igure 4. Characterization of mutants in PP4-mediated resistance against Hpa Emwa1. a, The seven phenotypes scored in WT, rpp4, and 22 mutants 7 dpi with Hpa Emwa1. SPP, Sporangiophore; TN, Trailing necrosis; OOS, Oospore; H, ree hypha; HI, ree hyphal intermediate; EXH, Expanding H; DIH, Discrete H. b, Bar graphs showing distributions of phenotype (PT) scores (% of occurrence in 40 leaves/genotype) of the mutants (black bars) compared with WT (blue bar) and rpp4 (red bar). Mutants are ranked from the highest to the lowest with no correspondence from panel to panel. The error bars, 95% confidence intervals based on binomial distribution. WWW NATUE.COM/NATUE 7
8 Supplementary igure 5. Phenotypic clustering analysis using data from 3 biological replicates. Hierarchical clustering performed using the mean phenotype scores of the three biological replicates with bootstrap (100,000 times) showed that mutants were divided into 2 groups similar to ig. 1b (Mutants were coloured according to ig. 1b). Distance was measured by the standard correlation coefficient (average linkage; scale 0-1). WWW NATUE.COM/NATUE 8
9 Supplementary igure 6. Defects in gene-mediated resistance in Group 2 mutants. a, Phenolic compound accumulation at 7 dpi with Hpa Emwa1. Arrows point at sites of spore penetration. b, Callose deposition 7 dpi with Hpa Emwa1. ed arrows indicate callose produced by plants (WT) and along the hyphae of the pathogen (mutants). WWW NATUE.COM/NATUE 9
10 Supplementary igure 7. Some of the mutants compromised in PP4-mediated resistance against Hpa Emwa1 are also defective in immune responses against Pseudomonas. a, Mutants 2, 6, 8 and 12 are compromised in PS2-mediated resistance against Psm ES4326/Avrpt2. Error bars represent 95% confidence interval (n = 8). cfu, colony-forming unit. This experiment was performed three times with similar results. Asterisks indicate p-values < b, Mutants 2, 8, and 12 showed enhanced susceptibility to Psm ES4326. WWW NATUE.COM/NATUE 10
11 WWW NATUE.COM/NATUE 11
12 Supplementary igure 8. Luciferase-based diurnal and free running tests. a, Luciferase measurement of CCA1:LUC under the conditions used for the ASL-seq experiment (ig. 3c, Col CK). Two biological replicates were included. The white bars, the day; black bar, the night. b, ree running test of CCA1:LUC and LHY:LUC expression with/without Hpa Emwa1 treatment. CK, control; Emwa1, Hpa Emwa1 treated; ZT, zeitgeber time. Error bars represent SEM (n=18). This experiment has been repeated three times with similar results. WWW NATUE.COM/NATUE 12
13 Supplementary igure 9. Determination of the cluster number used in nonnegative matrix factorization (NM). A range of cluster numbers from 2 to 22 were implemented in NM with 300 runs (10,000 iterations/run) and the corresponding cophenetic correlation coefficients were calculated. When cluster number equalled 2, the highest cophenetic correlation coefficient was achieved. WWW NATUE.COM/NATUE 13
14 Supplementary igure 10. Convergence of NM. 300 runs (10,000 iterations/run) were performed using 2 clusters and the corresponding costs were calculated for each run. The NM algorithm converged with fewer than 50 runs. WWW NATUE.COM/NATUE 14
15 WWW NATUE.COM/NATUE 15
16 Supplementary igure 11. Membership distance for Cluster 1 and Cluster 2 genes determined by their expression patterns. a, The two expression clusters overlap with the phenotypic grouping (as in ig. 1b; Group 1 genes in red and Group 2 in blue). or each gene, its membership distance in the expression cluster is represented by the radius of the circle. The calculation is detailed in Methods. b, Timecourse expression of the NM Cluster 2 genes with/without Hpa Emwa1 infection. The white bars, the day; black bar, the night. CK, control; EMWA1, Hpa Emwa1 inoculated. WWW NATUE.COM/NATUE 16
17 Supplementary igure 12. Enhanced disease resistance to Hpa Noco2 observed in the CCA1 OE transgenic line based on sporangiospore count 7 dpi. Error bars, SD (n = 3, One-way ANOVA and Bonferroni's Multiple Comparison Test); **, p- values < 0.01, *** p-values < This experiment has been repeated twice with similar results. WWW NATUE.COM/NATUE 17
18 WWW NATUE.COM/NATUE 18
19 WWW NATUE.COM/NATUE 19
20 Supplementary Table 1. A list of all mutant alleles and their phenotypes. SK, Salk line; CS, Sail line; S, susceptible;, resistant; +, WT level of callose or phenolic deposition observed; +/-, lower than WT level of callose observed. NT, not tested; EDS, enhanced disease symptoms; Ins, insensitive. Vir, virulent; MAMP, microbial-associated molecular pattern; W/T, fresh weight assay and root length assay. Note (*) that the infection phenotype against Hpa Emwa1 was determined based on microscopic observations of the symptoms; not all alleles were quantitatively measured as in ig. 1a. WWW NATUE.COM/NATUE 20
21 # Description Mutant Sequence 1 L-kinase SK SK GGCTCACGCTTTATCG TGTCCCCGTGGTTCTA GGCTCACGCTTTATCG TGTCCCCGTGGTTCTA 2 Calcineurin-like phosphoesterase SK SK CCCAACTCAGGATCGT ACGGTGAGCGAGGAATA CCCAACTCAGGATCGT ACGGTGAGCGAGGAATA 3 AD1-like 1 (AD1-L1) CS SK TCTCGTGGCACTACCT CAACGTCGGTATCGCA TCTCGTGGCACTACCT CAACGTCGGTATCGCA 4 Aspartic protease 38 (ATASP38) SK UDP-glucosyl transferase 72E1 (UGT72E1) SK Ankyrin (ANK) SK GTACTCAGTTTACTTTCCTGC GTTCTGCTGATGATGATGTC TGCAGTGCATATTTATCCG GCCCTCGTAATCACTCC AGGTGACGTAGACCTT AAAACCGTGTTGCCTC 7 Disease resistance protein-related SK SK ATTGTGTTTGATGATGTCC AGCCAACAGTCTAGCA ACGAATGGCTAGGGAC CACCGTATTCTCCGACA 8 Protein kinase family protein SK SK CAGAACATCGCCCTAC GTGAGGCATTAGTGGTTT CAGAACATCGCCCTAC GTGAGGCATTAGTGGTTT 9 ABC transporter family protein SK SK TGCCCCAAATTACATTACG TGGAGGCTAAGCACGA CCGATGCTACTGCTCT GGCGAACATACCGAAT 10 ATPase-like domain-containing protein CS SK TTCGTACCGCTATCGAC CTCCCATAACACCATTGC TTCGTACCGCTATCGAC CTCCCATAACACCATTGC 11 Ankyrin repeat family protein SK TTGGGAATTAACAGCC GCAACCGTATTACCTC WWW NATUE.COM/NATUE 21
22 SK GCACTGTTCACGTCCT AGTACGGTCAACGCTC 12 Lipase class 3 family protein SK CS ACTCAATAGGCTCCGC CTCTGCTCCCCTCGAA ACTCAATAGGCTCCGC CTCTGCTCCCCTCGAA 13 Constitutive shade-avoidance 1 (CSA1) SK AACTGTAAAAGTCATGCC CATTCCTTGGGGATGGAAC 14 Cyclic nucleotide gated channel 3 (CNGC3) SK CS AGGACATTAAACGCCATC ATAGGGAATTGGGAACTG AGGACATTAAACGCCATC ATAGGGAATTGGGAACTG 15 Lipid transfer family protein (LTP) SK CS ATGGTCTCTGCTCAGT CTGGTACGGTTTTCGAT ATGGTCTCTGCTCAGT CTGGTACGGTTTTCGAT 16 Tyrosine aminotransferase 3 (TAT3) SK SK AATACCGTCGCAATGG CTTGAACTACCCCTGTAGAT AATACCGTCGCAATGG CTTGAACTACCCCTGTAGAT 17 Glycoside hydrolase SK CS GATCGAGTAGTTGGGA GCCAAAGGTATTGTCA GGAGTGGAAGTCAGGT GTAGGTGCTCGCCAAA 18 Cold-responsive protein (CO15B) SK TCGTTGATATACGCCG CTCTTCTGCTTTACCCTC 19 Aspartyl protease family protein SK CS TAGCGCCATGTACTGC AGGGACTAGATTGGCTG TGCACTACGCCAATGT GCTCTAAACCCTCGTCC 20 BON association protein 1 (BAP1) SK SK CTGAGATGATGGCGGT GCAATTGAAGTAGCAGAAACA CTGAGATGATGGCGGT GCAATTGAAGTAGCAGAAACA 21 L-kinase SK SK TGCTGGTACTTACGGC AGAAAAGGCAGTGGAG TGAAAGGTGTGGCTCAT GCCGTAAGTACCAGCA WWW NATUE.COM/NATUE 22
23 22 Ser/Thr protein kinase family protein SK SK GTGGCTTCGGTACTGT CGGGTTGACCAGCAGA GTGGCTTCGGTACTGT CGGGTTGACCAGCAGA ecognition of Peronospora parasitica 4 (PP4) SK GCATTTGAAGTTGCCGAGC GGGCATCCAGTGAGATTGAGG Supplementary Table 2. Primers used in qpc characterization of gene expression in T- DNA insertion mutants in Supplementary ig. 3. SK, Salk line; CS, Sail line. WWW NATUE.COM/NATUE 23
24 Description Name Sequence 1 2 L-kinase Calcineurin-like phosphoesterase family protein 3 AD1-like 1 (AD1-L1) 4 Aspartic protease 38 (ATASP38) 5 UDP-glucosyl transferase 72E1 (UGT72E1) 6 Ankyrin (ANK) 7 Disease resistance protein-related 8 Protein kinase family protein 9 ABC transporter family protein 10 ATPase-like domain-containing protein 11 Ankyrin repeat family protein 12 Lipase class 3 family protein Constitutive shade-avoidance 1 (CSA1) Cyclic nucleotide gated channel 3 (CNGC3) 15 Lipid transfer family protein (LTP) 16 Tyrosine aminotransferase 3 (TAT3) 17 Glycoside hydrolase family 1G35710AD1 1G35710pA1 1G13750AD1 1G13750pA1 4G33300AD1 4G33300pA1 5G02190AD2 5G02190pA2 3G50740AD1 3G50740pA1 5G54610AD2 5G54610pA2 5G45440AD1 5G45440pA1 1G16260AD1 1G16260pA1 5G52860AD1 5G52860pA1 4G36290AD1 4G36290pA1 1G10340AD2 1G10340pA2 3G48080AD1 3G48080pA1 5G17880AD2 5G17880pA2 2G46430AD1 2G46430pA1 3G22600AD2 3G22600pA2 2G24850AD2 2G24850pA2 3G54440AD2 3G54440pA2 CTGGTGCAACGTAGCCGTAGCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATCTTCATCGTGTAAGCAAACT CTGGTAAACCGGGCATGTTCCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATTCATGACTCGTGCATACACT CAGTATGAATAGAGACAATCCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATTTGCATTTCATTCATTTCCC TGATGATGTCCAATCACGTACCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATCGATCCACATGTTCTGCTGA TGACACGCTCCAAAAGATGCCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATGGCACCACGTGCCATGCACC CTCCTCTACCACGAATACGACCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATAAGATGCAATGGTGTCATAC CTTCTGAATTGGGGTTGTCGCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATCAGAGGTTTAGTTTTACTCT TTAAAATGGCTGGCCAAGTTCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATTCCAGAGCTTAGAGTTCCAC CAACACACGATAAAGTACGACCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATCTAAGGAGAGCAAGAAAACA CGTCTGTTCTACTTCATTCTCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATTCTTTTTCTAAGCTCTTTAC TAGTGGATCCACTTGATATGCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATCAGTATAGTCCCTACGATAC CATTCCAAGGTCGTCGAGAGCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATCACTGTCTGGCTCTTGTGCT CTCATCGCATCGTTTAAGCGCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATAAAATCTCAGGAACACAACT CTGAACATGTGTCGGAGTTGCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATGCCATTGCACTGAATAAAAC CGGTATTACAGCGACTGACACCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATAGAACCACCACCACCACCAC CCATAAATAAGAACAAGATTCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATATTGATGTGTCAAGCTTCAA CTTCCAACCTTCAATAGTCCCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATATAAGGAGAACTCCTTGAAT WWW NATUE.COM/NATUE 24
25 18 Cold-responsive protein (CO15B) 19 Aspartyl protease family protein 20 BON association protein 1 (BAP1) 21 L-kinase 22 Ser/Thr protein kinase family protein Ubiquitin 5 (UBQ5) Late elongated hypocotyl (LHY) Circadian clock associated 1 (CCA1) 2G42530AD1 2G42530pA1 3G51330AD1 3G51330pA1 3G61190AD1 3G61190pA1 4G08850AD1 4G08850pA1 1G66880AD1 1G66880pA1 3G62250AD1 3G62250pA1 1G01060AD2 1G01060pA2 2G46830AD1 2G46830pA1 GTGGCTTCGTTGAGGTCATCCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATCGAAATCAGAAGCTTTCTTT AATCAGACCGTTTCCAACCCCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATCAAGCTTTCATCTTCAAAAC CCCAAACCGGAACTCCGGTACCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATAAATTGGCGTTGATACAGAC CTGGAGCAACATAGCCGTAACCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATTTTCATCGCATAAGCTAGTT CTTTTATGTGGAGGAATGATCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATGATGTCTCTGTGTATGATTC GAACCTTTCCAGATCCATCGCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATGCACTCCTTCCTCAAACGCT TTTCGTTGGTAAGGGATACTCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATTCAATGTCGCCACTTACCTG CTCGTCAGACACAGACTTCCCCTGTGGTCGTAGCATCAGC AATGATACGGCGACCACCGAGATGCTTGGAAGGCAATTCGACC Supplementary Table 3. Primers used in ASL-seq in ig. 3c. WWW NATUE.COM/NATUE 25
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Open Flower. Juvenile leaf Flowerbud. Carpel 35 NA NA NA NA 61 NA 95 NA NA 15 NA 41 3 NA
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NC bp. b 1481 bp
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VO (ml kg - min - ) VCO (ml kg - min - ) Respiratory exchange ratio Energy expenditure (cal kg - min - ) Locomotor activity (x count) Body temperature ( C) Relative mrna expression TA Sol EDL PT Heart
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