Supplemental Data

Supplemental Figure 1. Gene structures of PBL genes and location of T-DNA

insertions.

PBL structures were drawn using GSDS (Gene Structure Display Server). Inverted

triangles indicate the position of T-DNA insertions. Red arrows indicate gene direction

on the chromosome. Black arrows indicate RT-qPCR primer positions.

31

8

Supplemental Figure 2. CRISPR-Cas9-mediated mutations of PBL6, PBL17, and 9

PBL25. 10

A, CRISPR-Cas9-mediated mutations of PBL6 in the rlck vii-1 quintuple mutant. B, 11

Mutations of PBL17 in the rlck vii-6 triple mutant were generated using the 12

CRISPR-Cas9 system. C, CRISPR-Cas9-mediated mutations of PBL25 in the rlck vii-2 13

triple mutant. The line numbers (#) refer to alleles created by CRISPR-Cas9. The 14

positive and negative numbers in parentheses indicate frame shifts in the mutant lines. 15

Dots indicate nucleotides not shown in the alignments. Dashed lines indicate sequence 16

deletions. 17

18

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19

Supplemental Figure 3. Expression profiles of RLCK VII members in response to 20

different molecular patterns. 21

The microarray data reveal several RLCK VII members that are transcriptionally 22

regulated in response to different patterns. Heat map displays the log2 transformed 23

ratio of pattern-triggered expression relative to the basal expression of each RLCK VII 24

gene. 25

26

27

28

29

30

31

33

32

Supplemental Figure 4. Replicate ROS production data for Figure 2. 33

Pattern-triggered ROS production in all higher order mutants. Leaf strips were treated 34

with flg22 (A), elf18 (B), and chitin (C), and H2O2 production was measured. 35

Significant differences relative to WT are indicated by * (P<0.05), ** (P<0.01), and 36

*** (P<0.001) (Student’s t-test). 37

38

34

39

Supplemental Figure 5. Chitin-triggered ROS production in single mutants. 40

ROS production triggered by chitin in single mutants of RLCK VII-4 (A), RLCK VII-5 41

(B), RLCK VII-7 (C), and RLCK VII-8 (D). Values are means of the sum of 42

luminescence units collected from all 20 time points over 30 min following the 43

addition of chitin ± SD, n≥6. Different letters denote significant differences at P ≤ 44

0.05 (Student’s t-test). 45

46

35

47

Supplemental Figure 6. Replicate blots for Figure 4. 48

Mutants of RLCK VII-4 are compromised in chitin-triggered MAPK activation. 49

Seedlings of the indicated genotypes were treated with chitin or flg22 and harvested at 50

the indicated time points. MAPK activation was detected by immunoblot analysis 51

using anti-pERK antibody. Equal loading is demonstrated by Ponceau staining of 52

Rubisco (below). 53

54

36

55

56

57

58

59

Supplemental Figure 7. RLCK VII-4 members are not required for elf18- and 60

Pep2-triggered MAPK activation. 61

Eight-day-old seedlings of the indicated genotypes were treated with elf18 or Pep2 and 62

harvested at the indicated time points. MAPK activation was detected by immunoblot 63

analysis using anti-pERK antibody. Equal loading is shown by Ponceau staining. 64

65

37

66

Supplemental Figure 8. MAPK activation of RLCK VII-8 mutants. 67

MAPK activation triggered by different patterns was analyzed in indicated plants. 68

MAPK activation was analyzed by immunoblots using anti-pERK antibody. Equal 69

loading is shown by Ponceau staining. 70

71

72

38

73

Supplemental Figure 9. MAPK activation triggered by flg22 and chitin. 74

Eight-day-old seedlings of the indicated genotypes were treated with flg22 or chitin and 75

collected at the indicated time points. An anti-pERK antibody was used to detect 76

MAPK activation by immunoblot analysis. The line numbers (#) refer to alleles created 77

by CRISPR-Cas9. The experiments were repeated three times with similar results. 78

79

Supplemental Table 1. List of mutant lines and higher order mutants.

a: mutations generated by the CRISPR-Cas9 system.

Genes Subgroups Mutant lines Higher order mutants

PBL5 1 SALK_045613

PBL6 1 a

PBS1 1 pbs1-1PBL7 1 SALK_114130

PBL27 1 GK_088H03

PBL41 1 SALK_150918

PBL23 1 SALK_112111

PBL42(LIP2) 2 SALK_009528

PBL43(LIP1) 2 SALK_055909

PBL26 2 SALK_023374

PBL25 2 a

PBL24 2 SALK_072589

PBL21 3 SALK_025049

PBL22 3 SALK_045159

CDG1

PBL28 SALK_120599

PBL20 4 SALK_049965

PBL19 4 SALK_021064

PBL37 4 GK_090A05

PBL38 4 SAIL_416_A03

PBL39(PCRK1) 4 SALK_145629

PBL40(PCRK2) 4 SAIL_129_D02

PBL36 5 SAIL_885_B03

PBL35 5 SALK_039402

PBL34 5 SALK_067743

PBL17 6 a

PBL16 6 SALK_201102

PBL8 6 GK_625H05

PBL15 6 SALK_055095

PBL33 6

PBL12 6 SALK_017105

PBL14(RIPK) 6 ripkPBL13 6 GK_586B09

PBL30(CST) 7 SAIL_296_A06

PBL31 7 SAIL_273_C01

PBL32 7 SALK_113804

PBL10 8 SALK_001115

PBL9 8 GK_430G06

PBL11 8 SALK_046795

BIK1 8 SALK_005291

PBL1 8 SAIL_1236_D07

PBL29 SALK_050111

PBL2 9 SALK_149140

PBL3 9 SALK_039503

PBL18 9 SALK_097486

PBL4 9 SALK_097999

pbl5,6,7,27 pbs1-1

(rlck vii-1)

pbl24,25,26

(rlck vii-2)

pbl21,22

(rlck vii-3)

pbl19,20,37,38,39,40

(rlck vii-4)

bik1 pbl1,9,11

(rlck vii-8)

pbl2,3,4,18

(rlck vii-9)

pbl30,31,32

(rlck vii-7)

pbl8,16,17

(rlck vii-6)

pbl34,35,36

(rlck vii-5)