www.sciencemag.org/cgi/content/full/science.aai8898/DC1

Supplementary Materials for

Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance

Yiwen Deng, Keran Zhai, Zhen Xie, Dongyong Yang, Xudong Zhu, Junzhong Liu, Xin Wang, Peng Qin, Yuanzhu Yang, Guomin Zhang, Qun Li, Jianfu Zhang, Shuangqing

Wu, Joëlle Milazzo, Bizeng Mao, Ertao Wang, Huaan Xie, Didier Tharreau, Zuhua He*

*Corresponding author. Email: zhhe@sibs.ac.cn

Published 2 February 2017 on Science First Release DOI: 10.1126/science.aai8898

This PDF file includes:

Materials and Methods Figs. S1 to S17 Tables S1 to S7 References

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Materials and Methods Plant materials and pathogen inoculation

The rice parents, transgenic materials and NILs used in this study were listed in table

S6. For blast resistance evaluation in seedling rice, two-week-old rice seedlings were

spray-inoculated with blast spore suspensions (1 ×105 spores/ml) in a dew growth

chamber for 24 h in darkness at 26°C, and were subsequently kept at 12 h/12 h

(day/night), 26°C and 90% relative humidity for 7 days. Lesion types on leaves were

observed and scored from 0 (resistant) to 5 (susceptible) according to the standard scale

as described (27). Percentages of lesion areas (disease index) were scored using image

analysis with software ImageJ (http://rsbweb.nih.gov/ij/). Relative infection ratio of blast

fungus in rice cells was determined by DNA-based quantitative PCR as described

previously (28). In brief, the rice ubiquitin gene (LOC_Os03g13170) was used to

quantify the rice DNA, the Pot2 transposon sequence used to quantify the fungus, the

infection ratio (N:MgPot2/[N:OsUbq × 100]) was then calculated from the determined

number of target sequences of MgPot2 and OsUbq for each sample. Each inoculation was

assayed with 20 DNA samples.

For bacterial blight resistance assay, rice plants were inoculated with Xanthomonas

oryzae pv oryzae strain PXO99A using the leaf clip method as described (29). Lesion lengths

and bacterial growth were recorded at 14 dpi. For sheath blight resistance assay, plants were

inoculated with Rhizoctonia solani AG1-IA (isolate RH-9) with the toothpick method. In

brief, R. solani colony plugs were transferred to new PDA plates and co-incubated with short

(0.8-1.0 cm) woody toothpicks for 5 d at 28°C. Toothpicks with mycelia were then inserted

into the third leaf sheath of rice plants grown in the paddy field at booting stage. Sheath

blight symptom was recorded at 15 dpi with the 6-score system (30).

Field trial, yield evaluation and statistical analysis

Field resistance to blast was assessed in the Enshi blast nursery (Hubei province, mid-

western China) and the Daweishan blast nursery (Hunan province, southeastern China)

field in the consecutive 3 years (2012-2014), which have been long used to select blast

resistant varieties with high selection pressure of blast disease. The Pigm donor variety

GM4, NIL-Pigm lines and the recurrent parents (9311 and Maratelli) were tested in the

3

blast nurseries. Field test was also conducted for transgenic rice in our Linshui protected

blast nursery (Hainan Island, southern China). In addition, fourteen NILs containing

individual blast resistance gene Pi2, Pi9, Pi11, Pia, Pib, Pik, Pii, Pikp, Piks, Pit, Pita,

Pita2, Piz and Pizt in the genetic background of a high susceptible cultivar

Lijiangxintuanheigu, respectively (31), were also grown for resistance spectrum

comparison. Plants were grown in the nurseries with 3 replications with more than 300

plants each replicate. To ensure uniform infection of blast, a row of susceptible variety

was planted every five rows as well as on the borders. Resistance was evaluated

according to the standard evaluation system of IRRI for leaf lesion types with scores

ranging from 0 to 9 (32). Disease severity of leaf blast was scored by the percentage of

lesion area. Neck (panicle) blast severity was scored as a percentage of panicle infection

as described (33). Yield performance was evaluated in field tests of NIL-Pigm (9311 and

Maratelli) at the Daiweishan blast nursery and the neighbor experimental station (2014),

and the Enshi blast nursery under disease conditions during 2012 and 2014. NIL-Pigm

and parent lines were planted in a completely Randomized Block Design with 6

replicates in a rice paddy with an interplant spacing of 20 × 20 cm. Statistical analysis

was performed by Student’s t-test or Tukey-Kramer test for multiple comparisons.

Construction of rice BAC library and BAC screening and sequencing

The genomic DNA was isolated from the etiolated leaves of GM4 and partially

digested with HindIII and inserted into the BAC cloning vector plndigoBAC-5 to

construct a BAC library with 40,500 clones with an average insert size of 110 kb.

Construction and screening of the BAC library were performed as described (34). The

BACs covering the Pigm locus were isolated using PCR-based markers M35572 and

M80410 that co-segregate with Pigm (table S7). Two positive and overlapping clones

BAC7 and BAC30 were completely sequenced by shot-gun technique using an ABI 3730

sequencer (Applied Biosystems). The genomic sequence of the Pigm locus was annotated

by using the gene prediction program Fgenesh (www.softberry.com) and was manually

edited by a homology search against available databases on GenBank

(www.ncbi.nlm.nih.gov/genbank).

4

Plasmid construction and rice transformation

To construct the plasmids for complementation test, the BAC30 clone containing the

entire Pigm locus was partially digested with Sau3AI, the digested fragments larger than

15-kb in size were purified and ligated into the binary vector pCAMBIA1300 digested by

BamHI. The subclones were verified by PCR and sequencing using specific primers

(table S7). To generate overexpression clones, the entire coding regions (CDS) of PigmR,

PigmS and R4 were amplified using gene-specific primers from leaves or panicles of

GM4, and the PCR products were inserted into the binary vector pCAMBIA1300-

CaMV35S to generate overexpression plasmids 35S::PigmR, 35S::PigmS and 35S::R4.

The CDS of PigmS also was cloned into the binary vector pCAMBIA1300-PigmR with

the PigmR promoter to conduct the chimeric NLR gene PigmR::PigmS. All the constructs

and the empty vector were introduced into susceptible variety NIPB via Agrobacterium-

mediated transformation to generate more than 30 independent lines for each construct,

which were selected by PCR-based gene expression assays. In addition, 35S::PigmS was

also transformed into the resistant NIL-Pigm plants. To construct the clones of PigmS

driven by entire and different truncated promoters, the full CDS of PigmS was first

inserted into the binary vector pCAMBIA1300 to generate plasmid p1300-PigmS. The

different truncated promoters with removal of either MITE1, MITE2 or non-MITE region

were amplified from the PigmS promoter using specific primers and individually inserted

into pCAMBIA1300. All the constructs and empty vector were transformed into NIL-

Pigm to generate more than 30 independent lines for each construct, which were selected

by PCR-based gene expression assays. To generate double transgene plants of

PigmR/PigmS or PigmR/35S::PigmS or PigmR/PigmR::PigmS, transgenic plants PigmR

were crossed with transgenic plants PigmS, 35S::PigmS and PigmR::PigmS respectively,

stable homozygous progenies were selected by PCR-based expression assays.

To generate the PigmR RNAi constructs, the 200-bp 5’ UTR fragment of PigmR was

amplified by RT-PCR using specific primers and inserted as inverted repeats into the

conventional RNAi vector PTCK303 to generate hairpin RNAi constructs. The generate

double RNAi of PigmR and PigmS (PigmRS-RNAi), a 500-bp cDNA fragment of the

PigmR LRR region that has high similarity with the PigmS LRR domain was amplified

by RT-PCR and inserted as inverted repeats into the conventional RNAi vector PTCK303

5

to generate a hairpin RNAi construct. The 300-500bp cDNA fragments of the key genes

involved in the rice RdDM pathway, OsRDR2 (LOC_Os04g39160), OsAGO4a

(LOC_Os01g16870) (16) and OsDCL3a (LOC_Os01g68120) (17), were amplified using

gene-specific primers from the cDNAs of GM4 and individually inserted as inverted

repeats into PTCK303 to generate hairpin RNAi construct respectively. All the resulting

RNAi vectors and the empty vector were introduced into NIL-Pigm via Agrobacterium-

mediated transformation to generate more than 50 independent RNAi lines for each

construct, selected by PCR-based gene expression assays. The OsAGO4a RNAi construct

and the empty vector also were introduced into the susceptible variety NIPB. All primers

used for clone constructions are listed in table S7.

For the β-glucuronidase (GUS) reporter gene constructs, DNA fragments containing

the entire PigmS promoter (-1600 to -1) isolated from BAC30 and the different truncated

PigmS fragments as described above were inserted upstream of the GUS coding sequence

in the binary pCAMBIA1300-GUS vector to make fusion reporters (table S7). All the

resulting constructs were sequenced and introduced into the japonica variety NIPB via

Agrobacterium-mediated transformation to generate more than 20 independent lines that

showed similar expression patterns of the fusion reporters.

GUS histochemical staining

GUS staining was performed following the standard procedure (35). The different

issues including leaves, internodes, roots and young panicles were collected from at least

five independent lines for each construct, submerged in the staining solution and

incubated at 37°C up to 48 h or until blue color became visible. Then the samples were

incubated with 95% ethanol for 24 h to remove the chlorophylls. The GUS-staining

panicles and pollens were taken photos using a microscope.

RNA extraction and quantitative RT-PCR (qPCR) analysis

Total RNAs were extracted from different rice tissues using TRIzol reagent and treated

with RNase-free DNase I according to the manufacturer’s protocol (Invitrogen). The

resulting RNA was reverse-transcribed using the Superscript III RT kit (Invitrogen). For

defense and abiotic stress responses, expression of the defense–related genes, OsPR1

6

(LOC_Os07g03710), OsPR3 (LOC_Os04g41620), OsPR5 (LOC_Os12g43430), OsPBZ1

(LOC_Os12g36880), and the abiotic stress responsive genes, OsHSP70-1

(LOC_Os11g08470), OsHSP70-2 (LOC_Os11g08440) (25), OsDEF48

(LOC_Os11g47120), OsDEF70 (LOC_Os10g20550), OsDEF56 (LOC_Os08g15550)

(26), were analyzed. qRT-PCR was performed using SYBR Green (Takara) with the

Eppendorf AG 22331 cycler following the manufacturer's instructions, each qRT-PCR

assay was replicated at least three times with three independent RNA preparations, and

the rice OsActin1 gene (LOC_Os03g50885) was used as an internal control. The primers

for qRT-PCR are listed in table S7.

Yeast two-hybrid assays

The different domains of PigmR and PigmS including CC, NB-ARC, LRR and full-

length cDNAs, were amplified and inserted into the vectors pDEST22 and pDEST32

(Invitrogen) by the Gateway cloning technology, respectively, using the gene-specific

primers (table S7). The resulting constructs were transformed into yeast strain AH109.

Co-transformants were plated on synthetic medium without uracil, tryptophan, leucine

and histidine, supplemented with 3mM 3-AT and incubated at 28°C for 3 days.

Experimental procedures for screening and plasmid isolation were performed according

to the manufacturer’s user guide (Invitrogen).

Split luciferase complementation assay

For split luciferase complementation assay constructs, the full-length CDS of PigmR

and PigmS were amplified and inserted into the vector pCAMBIA-35S-NLuc and

pCAMBIA-35S-CLuc to generate the constructs CLuc-PigmR, CLuc-PigmS, PigmR-

NLuc, and PigmS-NLuc. Transient expression in Nicotiana benthamiana leaves was

performed by Agrobacterium-infiltration (36). In brief, Agrobacterium strains GV3101

containing the indicated constructs were cultured overnight in LB media. Equal amounts

of Agrobacterium cultures for CLuc and NLuc constructs were mixed to a final

concentration OD600 = 1.0 and collected and resuspended in infiltration buffer (10mM

MES, pH5.6, 10 mM MgCl2 and 150 μM acetosyringone). The mixed suspensions were

incubated at room temperature for ~3 h and then infiltrated into fully expanded young

7

leaves of N. benthamiana plants grown at 25 °C. The infiltrated plants were immediately

covered with plastic bags and placed at 23 °C for 48 h. Plants were then incubated at

28 °C with 16 h light/d for LUC activity measurement, with negative controls.

Coimmunoprecipitation (Co-IP) assays

Co-IP assays were performed according to a published procedure (37) with minor

modifications. Briefly, the CC domains of PigmR and PigmS were amplified and inserted

into the vector pCAMBIA1305-35S-FLAG and pCAMBIA1305-35S-HA to generate the

tagged constructs PigmR-CC-FLAG, PigmS-CC-FLAG, PigmR-CC-HA, and PigmS-CC-

HA. The plasmid GUS-HA was used as a control. All the plasmids were individually

transformed into Agrobacterium strain GV3101. The clones carrying different constructs

were co-infiltrated into N. benthamiana leaves. About 1g infiltrated leaf tissues were

ground into powder in liquid nitrogen and homogenized in 800 μL of extraction buffer

(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 5% glycerol, 2 mM EDTA,

and 1% protease inhibitor cocktail). Immunoblotting and Co-IP experiments were

performed following the Pierce HA Tag IP/Co-IP Kit instruction (Thermo).

Protein site mutagenesis and cell death assay in N. benthamiana

The conserved amino acids in the PigmS CC domain were site-mutated by PCR-based

approach as previously reported (9), and the mutated PigmS proteins were fused with

YFP (PigmS:YFP) or Myc (PigmR:Myc) to determine their expression in the N.

benthamiana. PigmR-triggered cell death in N. benthamiana was investigated as

described (38). In brief, suspensions (OD600 = 0.5) of Agrobacterium GV3101

containing the PigmR:Myc, PigmS:YFP, and mutant PigmS:YFP fusion expression

constructs respectively were infiltrated into fully expanded young leaves of 4-week-old N.

benthamiana plants grown at 25 °C. YFP was expressed as an internal control. The

infiltrated plants were immediately covered with black bags and placed at 23 °C. The

hypersensitive response cell death on infiltrated leaves was recorded and photographed at

48 hpi. Proteins were extracted from the infiltrated N. benthamiana leaves at 40 hpi

(hours post infiltration) and purified by anti-Myc or anti-GFP beads, the

immunoprecipitations were applied to analyze accumulation of the PigmR:Myc,

8

PigmS:YFP and the mutated PigmS:YFP fusion proteins with anti-Myc or anti-GFP

antibody.

Protein competition experiment

For competition of PigmS with PigmR in dimerization, the fusion protein constructs,

PigmR-CC-GST, PigmR-CC-His, and PigmS-CC-MBP, were generated and expressed in

E. coli (DE3) and purified by antibody-conjugated beads, and protein reaction samples

were prepared (0.75 µg/µl), respectively. Competition assay was performed as described

(39). In brief, PigmR-CC-GST and PigmR-CC-His (20 µl) were co-incubated in the anti-

GST beads. Then different amount of PigmS-CC-MBP (0, 5, 15 and 20 µl) was added in

the same mix to compete the homodimerization of PigmR-CC-GST and PigmR-CC-His.

Interacting proteins were pulled down using anti-GST beads and immunoblotted with

different antibodies to determine protein levels. .

Bisulfite sequencing

Genomic DNAs were prepared from leaves and pollens of NIL-Pigm using CTAB

method and DNeasy plant kit according to the manufacturer’s instructions (Qiagene).

Genomic DNA (2 μg) was digested using HindIII, followed by phenol/chloroform

extraction and ethanol precipitation. DNA was treated using the Epitect bisulfite kit

according to the manufacturer’s instructions (Qiagen). The bisulphited DNA was used for

PCR amplification of the PigmS promoter. PCR primers were designed with Methyl

Primer Express software (Applied Biosystems). Amplified PCR fragments were cloned

into pGEM-T easy vector (Promega) and sequenced. Sequencing data was analyzed using

Web-based Kismeth software (40). Primers used for bisulfite sequencing are listed in

table S7.

Small RNA sequencing

Small RNAs were isolated from total RNAs from the leaves of two-week-old NIL-Pigm

and NIPB seedlings. Small RNA libraries were constructed and deep-sequenced with an

Illumina Hiseq 2000 according to the manufacturer’s instructions (Illumina) by BGI.

Small RNAs (21 and 24-nt siRNAs) were mapped to the PigmS promoter sequence using

9

software Bowtie-0.12.7 (41)

.

Small RNA gel blot assay

Small RNA gel blot experiments were performed as previously described (42). Total

RNAs prepared from leaves of NIL-Pigm and the transgenic lines OsAGO4aRNAi,

OsRDR2RNAi and OsDCL3aRNAi were loaded to detect the siRNAs derived from the

MITE1 and MITE2 regions of the PigmS promoter. The probe amplified with specific

primers (table. S7) was labeled with digoxigenin (DIG) using the PCR DIG Probe

Synthesis Kit (Roche). U6 was served as an internal control.

Molecular breeding of Pigm

The cultivar GM4 (Pigm) was crossed with varieties 9311(indica), Maratelli (japonica)

and Nipponbare (japonica), and backcrossed four times with the recurrent parents to

generate the BC4F1 plants, which self-crossed three times to generate the BC4F4 lines.

Total of 192 SSR markers covering all 12 rice chromosomes were used to detect the

backgrounds of the selected NILs. The NIL-Pigm lines carrying a single ~500-kb

fragment with the Pigm locus from the donor GM4 were selected in the 9311, Maratelli

and Nipponbare genomes. The NIL-Pigm lines (9311 and Maratelli) were cultivated at

the two different blast nurseries located in Enshi (Hubei province) and Daweishan

(Hunan province) for field evaluation (see above).

Database deposition

The full genomic sequence of the Pigm locus including PigmR and PigmS can be found

in GenBank (accession number KU904633). . The entire small RNA sequencing data can

be obtained from the NCBI Gene Expression Omnibus (GEO) database under series

accession number GSE83522.

10

Fig. S1. Pigm confers durable blast resistance and does not confer resistance to other

rice diseases.

(A) Field resistance phenotype of EnD3 (Pigm) exhibiting high resistance to M. oryzae in

the Enshi blast nursery, compared with the susceptible variety V14B. (B) Field resistance

score records from 1984 to 2015 indicate that EnD3 confers durable resistance to M.

oryzae in the blast nursery for more than 30 years. (C, D) PigmR transgenic plants did

not change susceptibility to bacterial blight (Xanthomonas oryzae pv oryzae). (E, F)

PigmR transgenic plants did not change susceptibility to fungal sheath blight

(Rhizoctonia solani).

11

Fig. S2. Gene structure of the three NLR members of the Pigm locus.

The full-length open reading frame (ORF) was determined for the expressed members,

R4, R6 (named PigmR) and R8 (named PigmS), by RT-PCR and sequencing using

specific primers listed on table S7. Exons are indicated by black rectangle. Introns are

indicated by lines. The 5’-UTR (untranslated region) and 3’-UTR are indicated by white

rectangle. The initiation (ATG) and termination (TGA) codons are also indicated.

12

Fig. S3. Schematic representation of the genomic structure of the Pigm locus in

different rice germplasm.

(A) Comparison of the Pigm cluster with the Pi2 and Pi9 clusters. (B) Copy number

variation of R genes in the Pigm locus from different germplasm. a, O. nivara (AA); b, O.

brachyantha (AA); c, O. rufipogon (AA); d, O. sativa 9311 (AA); e, O. sativa CO39

(AA); f, O. sativa NIPB (AA); g, O. sativa 75-1-127/Pi9 (AA); h, O. sativa C101A51/Pi2

(AA); i, O. sativa GM4/Pigm (AA). R1 to13, NLR genes; NIP: nitrate-induced protein;

PK, protein kinase. The genes in the same colors are close homologues or duplicated

genes.

13

Fig. S4. Identification of the functional Pigm gene.

(A) PCR analysis of the three loss-of-function suppressor mutants (GM4-S1 to S3)

showed deletions in the Pigm locus, with GM4 as a positive control and NIPB as a

negative control. (B) The deletions in the three susceptible mutants. (C) Overlapping

subclones harboring single NLR gene which were transferred to the susceptible rice

cultivar NIPB. (D) Disease resistance of transgenic lines with different NLR gene

inoculated with M. oryzae isolate CH12 virulent to NIPB. The subclone 5-7 contains the

functional R gene (R6, named as PigmR) (see table S3 and fig. S7 for inoculation results

14

with more isolates). (E) PCR analysis of 21 plants from the T2 generation of a PigmR

line indicated that all the transgene positive plants were blast resistant, with NIL-Pigm as

the positive and NIPB as negative controls. R, resistance; S, susceptible (D, E). (F)

Disease resistance of transgenic lines with PigmR, driven by its native promoter (PigmR)

or the 35S promoter (35S::PigmR) in the blast nursery, with the transgenic R4 lines (R4

and 35S::R4) as controls that do not confer blast resistance. Note that the PigmR and

35S::PigmR transgenic plants exhibit complete resistance against M. oryzae. (G) qRT-

PCR detection of PigmR transcript levels in the 4 representative PigmR-RNAi lines in

NIL-Pigm background, the rice OsActin1 was used as a control to normalize expression

levels. (H) Lesion areas of the PigmR-RNAi lines at 7 dpi with CH12. (I) Infection ratio

of the PigmR-RNAi lines at 7 dpi with CH12.

15

Fig. S5. R4 does not confer blast resistance.

(A-C) The transgenic plants of R4 and 35S::R4 were susceptible, similarly to the receipt

parent NIPB (A), with the same lesion area (B) and fungal infection ratio (C) as the NIPB

control. Two-week-old seedlings were inoculated with M. oryzae (isolate CH12), and

resistance was measured at 7 dpi. Bar, 1 cm. (D) Protein sequences and conserved motifs

in PigmR and R4. CC (coiled-coil), P-loop and other conserved motifs are underlined.

The LRR domain consists of 17 imperfect LRR repeats with the consensus

IXX(L)XX(L)XX(L). Note that there are four different amino acids (in red) between

PigmR and R4.

16

Fig. S6. Functional identification of the four amino acids in the PigmR LRR domain.

(A) Substitution of four amino acids within the LRR domain of PigmR with ones of R4:

F858V, S860Y, E909Q and D961Y. (B) Inoculation experiment with different blast

isolates showed that the site-specific substitutions in the 4 amino acids changed resistance

spectrum. MR, middle resistance; MS, middle susceptibility.

17

Fig. S7. Blast resistance of PigmR against representative strains with

complementary avirulence effectors from different countries.

(A) Inoculation of different blast strains with different avirulence spectra indicates that

the 3 representative PigmR transgenic lines are all resistant to the world-selection strains

with diverse predicted effectors, supporting that PigmR confers broad-spectrum

resistance to M. oryzae. The susceptible variety Maratelli and the transgenic parent NIPB

were used as controls. (B) Factorial analysis of genotypic diversity of the M. oryzae

strains collected from worldwide rice-cultivated regions. Red dots are the collection

positions of the strains used in this study, and gray dots indicate positions of M. oryzae

isolates from a worldwide collection (7, 8).

18

Fig. S8. Disease resistance and gene expression analysis of different PigmS and

PigmR transgenic lines.

(A, B) Lesion area (A) and infection ratio (B) of the NIL-Pigm parental and transgenic

plants overexpressing PigmS, driven by the CaMV 35S promoter or PigmR promoter, and

expressing PigmS from its native promoter (see table S6 for details of the rice lines), 7

dpi with isolate CH12, with NIL-Pigm and NIPB as resistant and susceptible parent

controls. (C, D) Lesion area (C) and infection ratio (D) of the NIPB parental and

19

transgenic plants overexpressing PigmS in NIPB and PigmR transgene background at 7

dpi. Data are shown as means ± SD (n = 30). Means labeled with different letters indicate

significant difference at 1% level via Tukey-Kramer test for multiple comparisons (A to

D). (E) PigmS transcript levels in NIL-Pigm transgenic plants ectopically expressing

PigmS by 35S or the PigmR promoter, with the wild-type NIL-Pigm as control. (F)

PigmR expression was not affected in NIL-Pigm transgenic plants overexpressing PigmS

(A). (G) Transcript levels of PigmS and PigmR in transgenic plants overexpressing

PigmS in NIPB or PigmR transgene background (C). Gene expression levels were

detected by qRT-PCR. The rice OsActin1 was used as a control to normalize expression

levels (E to G).

20

Fig. S9. Yeast two-hybrid assay to identify protein interaction.

(A, B) Yeast two-hybrid assay confirmed that the CC domains of PigmR and PigmS are required for pairwise interaction of PigmR and PigmS, confirmed by growth of yeast cells on basal media without supplement of Trp, Leu, Ade and His and supplemented with 3mM 3AT. (C, D) Yeast two-hybrid assay showed that PigmS does not interact with the full length CDS and different domains of Pish that confers race-specific resistance to M. oryzae isolate YN2 in the NIPB genome. (E) PigmS did not decrease resistance to isolate YN2 avirulent to NIPB. Two-week-old seedlings were inoculated, photographs were taken 7 dpi. Bar, 1 cm.

21

Fig. S10. PR gene induction in different transgenic plants.

Pathogenesis-related (PR) gene expression in transgenic plants PigmR and PigmR/35S::PigmS inoculated with M. oryzae with a time-course analysis of OsPR1 (LOC_Os07g03710) (A), OsPBZ1 (LOC_Os12g36880) (B), OsPR3 (LOC_Os04g41620) (C) and OsPR5 (LOC_Os12g43430) (D). Two-week-old seedlings were spray-inoculated with isolate CH12 and samples were harvested at 0, 8, 12, 24, 48 and 72 hours post inoculation (hpi). Error bars indicate the SD from three biological replicates, and asterisks indicate significant differences at 24 hpi between PigmR and PigmR/35S::PigmS plants (student’s t-test, **P< 0.01). Gene expression was detected by qRT-PCR with the gene-specific primers (table S7). (E) Transcript levels of the PR genes were slightly elevated in the PigmR transgenic plants or NIL-Pigm lines compared with the wild-type NIPB or the recurrent parents 9311 and Maratelli under non-infection condition. The rice OsActin1 was used as a control to normalize expression levels.

22

Fig. S11. Screening of special amino acids of the PigmS CC domain that are critical

to the PigmS-PigmR antagonistic interaction in defense activation.

(A) Conserved amino acids in the CC domains of PigmR, PigmS and MLA10 (9). The

numbers (41, 45 and 48) above sequences indicates three conserved amino acids. (B) Site

mutagenesis screening identified two other amino acids, L45 and M48 of PigmS, critical

to interaction with PigmR. The site mutations on the PigmS protein, pigmsL45E (left) and

pigmsM48E (right) lose the suppression activity of PigmS in PigmR-mediated cell death in

N. benthamiana leaves. YFP transient expression was used as a negative control. Bars, 1

cm. (C) The abundance of fusion proteins was determined by immunoblot with anti-Myc

or anti-GFP antibody in the tobacco transient expression assay.

23

Fig. S12. Expression of GUS fusion reporters driven by full-length and truncated

versions of the PigmS promoter.

(A) Schematic representation of pPigmS::GUS fusion reporter constructs. a, the entire PigmS promoter; b, the truncated PigmS promoter with MITE1 deleted; c, the truncated PigmS promoter with MITE2 deleted; d, the truncated PigmS promoter with MITE1 and MITE2 deleted; e, the truncated PigmS promoter containing only MITE1 and MITE2; f, the truncated PigmS promoter containing only MITE1. (B) GUS activity showed that the all reporter versions except d without both MITEs express in anther. About 20 independent transgenic plants for each construct showed similar expression patterns. Bar, 0.3 cm. (C) Expression of GUS detected by qRT-PCR showed that removal of MITE1 or MITE2 increases GUS expression in leaves. The rice OsActin1 was used as a control to normalize expression levels. Error bars represent SD (n = 3).

24

Fig. S13 PigmS expression is repressed by the RdDM pathway in leaves.

(A) CG (red), CHG (blue) and CHH (green) methylation of the MITE1 and MITE2

regions in leaves and pollen. Each cytosine is shown as a hollow circle (not methylated)

or solid circle (methylated). (B) Accumulation of small RNAs in the MITE regions of the

PigmS promoter revealed by small RNA-seq. Blue peaks indicate abundance of small

RNAs. (C) Transcript abundance of OsDCL3a and PigmS in the four representative

OsDCL3a-RNAi lines was detected by qRT-PCR. (D) DNA methylation level of the

25

MITE sequences in the leaves of the OsDCL3a-RNAi line, detected by bisulfite

sequencing. (E) Transcript abundance of OsRDR2 and PigmS in the four representative

OsRDR2-RNAi lines was detected by qRT-PCR. (F) DNA methylation level of the MITE

sequences in the leaves of the OsRDR2-RNAi line, detected by bisulfite sequencing. Note

that CHH methylation of the MITE region in leaves is decreased in the RNAi lines

compared with the wild-type (NIL-Pigm) (D, F). (G, H) Disease phenotypes of the two

different OsAGO4a-RNAi lines in the NIPB background infected with isolates YN2 (G)

and CH12 (H). Bar, 1 cm. (I, J) Quantitative analysis of lesion area (I) and M. oryzae

growth (J) in the OsAGO4a-RNAi lines infected with isolate CH12 at 7 dpi. The results

indicate that the OsAGO4a does not affect blast resistance in NIPB that does not have the

Pigm locus or PigmS gene. (K) Expression levels of PigmS and PigmR in NIL-Pigm

transgenic lines expressing PigmS from different truncated promoters in leaves. Sa, the

full length PigmS promoter; Sb, the truncated PigmS promoter with MITE1 deleted; c,

the truncated PigmS promoter with MITE2 deleted; d, the truncated PigmS promoter with

MITE1 and MITE2 deleted; e, the truncated PigmS promoter containing only MITE1 and

MITE2; Sf, the truncated PigmS promoter containing only MITE1 (see fig. S12A for

details). Note that removal of the MITE sequence increases PigmS expression in leaves,

similar to the GUS reporter expression (fig. S12, B and C), but does not affect PigmR

expression. Consequently, PigmR-mediated resistance is compromised in the PigmS

transgenic lines (Sb, Sc and Sf). (L, M) Quantitative analysis of lesion area (L) and M.

oryzae growth (M) in the in NIL-Pigm transgenic lines expressing PigmS from different

truncated promoters infected with isolate CH12 at 7 dpi. The rice OsActin1 was used as a

control to normalize expression levels (C, E, K). Error bars represent SD (n = 3) (C, E,

K), (n = 30) (I, J, L, M).

26

Fig. S14 Effect of PigmR and PigmS on grain yield components in transgenic lines

and NIL-Pigm.

(A-C) Comparison of grain yield per plant (A), 1000-grain weight (B) and seed setting

ratio (C) between the NILs (9311 vs 9311-Pigm, Maratelli vs Maratelli-Pigm) under no-

disease conditions. (D) Transcript levels of PigmR in leaves and PigmS in anthers of the

RNAi lines that synchronously knocked down PigmR and PigmS (PigmRS-RNAi), which

were normalized as 1 with OsActin1 in the wild-type NIL-Pigm leaves and anthers,

respectively. Data are shown as means ± SD (n = 3). (E-G) Comparison of seed setting

ratio (E), 1000-grain weight (F) and grain yield per plant (G) between the PigmR RNAi,

PigmRS RNAi transgenic lines and the NIL-Pigm control (NIPB background). (H, I)

27

Other agronomic traits including tiller number (H) and plant height (I) are not affected by

PigmR and PigmS in transgenic plants. Data are shown as means ± SD (n = 60) (A to C,

E to I). Means labeled with different letters indicate significant difference at 5% level via

Tukey-Kramer test for multiple comparisons (E to G), and asterisks represent

significance difference determined by the Student’s t-test at P < 0.05 (A to C).

28

Fig. S15 Expression levels of stress-responsive genes in pollen.

qRT-PCR was performed to detect the transcript levels of the two predicted HSP70 genes

(OsHSP-1, LOC_Os11g08470; OsHSP-2, LOC_Os11g08440), and three defensin genes

(OsDEF48, LOC_Os11g47120;OsDEF70, LOC_Os10g20550; OsDEF56,

LOC_Os08g15550), indicating that the transcription levels of these genes increased in

pollen of PigmS transgenic line and NIL-Pigm in comparison with that of the receipt

parent NIPB. The rice OsActin1 was used as a control to normalize expression levels.

29

Fig. S16 A proposed model for Pigm function and evolution.

The paired NLR receptors PigmR and PigmS exhibit opposing functions with respect to

disease resistance and yield. PigmR is expressed constitutively at low levels in all tissues,

whereas PigmS is expressed highly in pollen, lowly in leaves with epigenetic regulation.

The low expression of PigmS in leaves attenuates competitive homodimerization with

PigmR, which would otherwise strongly compromise PigmR homodimer-mediated

resistance. The high expression of PigmS in pollen likely improves seed setting to

increase grain yield. The elegant regulation of PigmS plays a vital role in coordinating the

equilibrium between broad-spectrum resistance and yield in rice evolution and breeding

selection. Yellow squares, PigmR homodimer; green squares, PigmS homodimer; yellow

and green squares, PigmR/PigmS heterodimer.

30

Fig. S17 Yield performance and field resistance of the new hybrid rice

Longliangyou3189 harboring Pigm in different regions.

(A) De novo molecular breeding of the hybrid parent 9311-Pigm through backcrossing

and maker-aided selection (MAS) using 192 SSR markers covering all 12 rice

chromosomes, which has identical genomic background to the recurrent variety 9311

except the Pigm region (~500 kb). (B, C) Field resistance of the new elite hybrid rice

Longliangyou3189 (Longke638S/9311-Pigm) is greatly improved at seedling stage (B)

and heading stage (C) in comparison to the control Longliangyou9311

(Longke638S/9311). Note that many panicles of the control Longliangyou9311 are white

empty due to panicle blast (C). (D) Yield performance of Longliangyou3189 is

significantly improved compared to the control variety Longliangyou9311in the field

tests at two rice cultivation regions during 2014. Disease resistance scores were evaluated

at the neighbor blast nurseries in the same year. Asterisk indicates significance difference

in comparison with the control variety determined by the Student’s t-test at P < 0.05 (n =

6).

31

Pi gene isolate

Pib Pia Pikh Pit Pikp Piks Pikm Pizt Piz Pi9 Pi2 Pigm Pish

CH101 R S S S S S S S R R S R S CH108 R S S S R S S R R R S R S CH12 R R S S R S S S S S S R S CH16 R S S S S S S S R S S R S CH43 R S S S R S S R R R R R S CH45 S S R S S S R R R R R R S CH46 MS S R S S S R S S R R R S CH49 R R R R R R R R R R R R R CH51 R S R R R S R S R R R R S CH52 S S R R R S R S R R R R S CH53 S S R S R R R R R R R R S CH63 R S R R S S R R S R R R S CH72 R R R S S R R R R R R R S CH77 MS S R S R S R R R R R R S CH93 S S R S S S R R R R R R S CH97 S S R S R S R R R R R R S CH102 R S R R R S R R R R S R S CH109 S S R S S S R R R R R R S CH131 S S R S S S S S S R S R S CH155 S S R S S S R R R S S R S CH174 R S R S R S R R R R R R S CH184 MS MS R R R S R R R S S R S CH188 R S R S S S S S R R S R S CH193 R S R R S R S R R R R R S CH199 R S S S S S R S S S S R S CH209 R S R S R S R R R R R R S CH227 S S R S S S R R S S R R S CH255 R MS R S S S R R R R R R S CH260 R S R R S S S R R R R R S CH333 R R R S S S R R R R R R S

Table S1. Resistance of different known blast R genes to 30 M. oryzae isolates collected from different rice regions in China. Two-week-old seedlings were spray-inoculated with 30 blast fungal isolates collected in

China. Disease resistance was scored at 7 dpi. R: resistant, S: susceptible, MS: middle

susceptible, as determined by the evaluation system (27).

32

Variety Origin Release year GuanfengA Fujian 2002 GufengA Fujian 2002 ChuanguA Sichuan 2005 Bing1A Hunan 2009 FuyiA Fujian 1995 AnfengA Fujian 2011 Fuyou58 Hubei 1990 Gunong13 Guangxi ND Digu Fujian 1992 XiafengA Fujian 2002 Endao3 Hubei 1994 Shuangchao25 Guangdong 1985 ZhongguA Zhejiang 2010 Peiyou2 Chongqing 2008 Guangkang13A Fujian 2003 Guangyou2643 Fujian 2013 Chuanguyou538 Sichuan 2012 Guyou428 Fujian 2014 Guangyou66 Sichuan 2014 Guangyou673 Sichuan 2014 Guyou527 Fujian 2007 Quanyou527 Fujian 2010 Quanyou77 Fujian 2007 Quanyou94 Fujian 2007 Leyou94 Fujian 2007 ChangfengA Fujian 2011 LianfengA Fujian 2002 LefengA Fujian 2005 FufengA Fujian 2005 JiefengA Fujian 2005 ChengfengA Fujian 2010 Shenyou9734 Guangdong 2012 Shenyou9752 Guangdong 2012 Shen97A Guangdong 2007 N7A Sichuan 2003 Jun1A Hubei 2010 N5B Sichuan 2009 Fuhui9801 Chongqing 2001 803B Chongqing 2002 Fuhui9802 Chongqing 2010 Lingyou2 Chongqing 2008

Table S2. Commercial blast resistant rice varieties harboring Pigm.

A nationwide collection of commercial rice varieties with high blast resistance was

33

screened for the Pigm locus with Pigm-specific primers (table S7). These varieties

containing Pigm are mostly indica, some have been long grown. ND, not determined.

34

Isolate PigmR 35S::PigmR R4 35S::R4 PigmS R2 R3 R5 R7 R9 R10 NIPB NIL-Pigm CH231 R R S S S S S S S S S S R CH27 R R S S S S S S S S S S R CH104 R R S S S S S S S S S S R CH148 R R S S S S S S S S S S R CH18 R R S S S S S S S S S S R CH299 R R S S S S S S S S S S R CH31 R R S S S S S S S S S S R CH921 R R S S S S S S S S S S R CH97 R R S S S S S S S S S S R CH127 CH286

R R

R R

S S

S S

S S

S S

S S

S S

S S

S S

S S

S S

R R

CH12 R R S S S S S S S S S S R CH990 R R S S S S S S S S S S R CH72 R R S S S S S S S S S S R CH993 R R S S S S S S S S S S R CH397 R R S S S S S S S S S S R CH502 R R S S S S S S S S S S R CH913 R R S S S S S S S S S S R CH901 R R S S S S S S S S S S R CH118 R R S S S S S S S S S S R CH520 R R S S S S S S S S S S R CH301 R R S S S S S S S S S S R CH133 R R S S S S S S S S S S R CH982 R R S S S S S S S S S S R CH311 R R S S S S S S S S S S R CH141 R R S S S S S S S S S S R CH341 R R S S S S S S S S S S R CH102 R R S S S S S S S S S S R CH63 R R S S S S S S S S S S R CH174 R R S S S S S S S S S S R CH916 R R S S S S S S S S S S R CH617 R R S S S S S S S S S S R CH109 R R S S S S S S S S S S R CH131 R R S S S S S S S S S S R CH918 R R S S S S S S S S S S R CH155 R R S S S S S S S S S S R CH851 R R S S S S S S S S S S R CH709 R R S S S S S S S S S S R CH649 R R S S S S S S S S S S R CH199 R R S S S S S S S S S S R CH188 CH233

R R

R R

S S

S S

S S

S S

S S

S S

S S

S S

S S

S S

R R

CH14 R R S S S S S S S S S S R CH66 R R S S S S S S S S S S R TH16 R R S S S S S S S S S S R TH12 R R S S S S S S S S S S R K110 R R S S S S S S S S S S R K210 R R S S S S S S S S S S R

35

K102 GUY11

R R

R R

S S

S S

S S

S S

S S

S S

S S

S S

S S

S S

R R

Table S3. Blast resistance evaluation of transgenic lines with different R members of

the Pigm cluster to 50 M. oryzae isolates.

Two-week-old seedlings of the stable transgenic lines were spray-inoculated with a set of

blast collection (50 isolates). Disease resistance was scored at 7 dpi. NIPB is the wild-

type control, and NIL-Pigm is the donor control. Note that only PigmR expressed with its

native promoter (PigmR) or the 35S promoter (35S::PigmR) conferred resistance to all

isolates tested as the donor NIL-Pigm.

36

Line Leaf blast score

Neck blast score

Resistance evaluation

NIL-Pigm Nipponbare

3 7

3 9

R S

5-7 (PigmR) 3 3 R 35S::PigmR 3 3 R 74 (R4) 7 9 S 35S::R4 7 9 S 2-33 (PigmS) 7 9 S 35S::PigmS 9 9 HS 2-8 (R2) 3-17 (R3) 2-20 (R5) 3-18 (R7) 2-36 (R9) 2-56 (R10)

7 7 7 7 7 7

9 9 9 9 9 9

S S S S S S

IRBLa-A (Pia) IRBLi-F5 (Pii) IRBL-F (Piks) IRBL-Ka (Pik) IRBL-K60 (Pikp) IRBL21 (Pi9) IRBLz-Fu (Piz) IRBLz5-CA (Pi2) IRBLzt-T (Pizt) IRBLta-K1 (Pita) IRBLb-B (Pib) IRBLt-K59 (Pit) IRBLta2 (Pita2) IRBL11-Zh (Pi11)

9 9 7 5 7 5 5 5 7 7 5 9 5 5

9 9 9 7 9 7 7 7 9 9 7 9 7 7

S S S MS S MR MR MR S S MS S MR MS

Lijiangxintuanheigu 9 9 HS

Table S4. Field resistance evaluation of the transgenic plants containing individual

NLR genes and NILs with other Pi genes in blast nursery.

The rice lines were grown at the natural blast nursery. The 14 NILs with different Pi

genes in the background of Lijiangxintuanheigu (a high susceptible cultivar). Disease

resistance was recorded for both leaf and neck/panicle blast during the growth season.

HS: high susceptible, MS: middle susceptible, MR: middle resistant as determined by the

evaluation system (30). The result indicated that PigmR conferred high blast resistance in

the field.

37

Line/Variety Leaf blast

Neck blast Neck blast (%)

Grain yield loss (%)

Resistance score

Year 2012 Gumei4 (Pigm) Nipponbare

3 7

3 7

16 90

5.3 70

R S

Nipponbare-Pigm 3 5 23 5.3 R Maratelli 9 9 100 91 HS Maratelli-Pigm 3 3 29 6.7 R 9311 7 7 90 60 S 9311-Pigm 1 3 19 4.3 R IRBLa-A (Pia) IRBLi-F5 (Pii) IRBLks-F (Piks) IRBLk-Ka (Pik) IRBLkp-K60 (Pikp) IRBL21 (Pi9) IRBLz-Fu (Piz) IRBLz5-CA (Pi2) IRBLzt-T (Pizt) IRBLta-K1 (Pita) IRBLb-B (Pib) IRBLt-K59 (Pit) IRBLta2 (Pita2) IRBL11-Zh (Pi11)

9 8 7 7 8 5 5 5 8 7 7 9 8 7

9 9 9 9 9 7 7 7 9 9 9 9 9 9

90 100 70 60 80 55 50 40 70 90 80 100 80 70

80 90 60 55 70 25 25 25 70 80 80 90 80 50.0

S HS S S S MS MS MS S S S S S S

Lijiangxintuanheigu 9 9 100 100 HS Year 2013 Gumei4 (Pigm) Nipponbare

1 8

3 7

8 82

3.5 66

R S

Nipponbare-Pigm 1 3 25 3.2 R Maratelli 9 9 100 80 HS Maratelli-Pigm 1 3 21 4.5 R 9311 7 7 85 46 S 9311-Pigm 1 3 12 2.8 R IRBLa-A (Pia) IRBLi-F5 (Pii) IRBLks-F (Piks) IRBLk-Ka (Pik) IRBLkp-K60 (Pikp) IRBL21 (Pi9) IRBLz-Fu (Piz) IRBLz5-CA (Pi2) IRBLzt-T (Pizt) IRBLta-K1 (Pita) IRBLb-B (Pib) IRBLt-K59 (Pit) IRBLta2 (Pita2) IRBL11-Zh (Pi11)

9 8 7 7 8 5 5 5 8 7 7 9 8 7

9 9 9 9 9 7 7 9 9 9 9 9 9 9

90 90 70 50 60 50 40 30 50 60 70 90 50 70

80 90 60 55 70 30 35 30 50 40 40 70 30 50.0

HS HS S S S MS MS MS S S S HS MS S

Lijiangxintuanheigu 9 9 100 90 HS Year 2014 Gumei4 (Pigm) 3 5 22 4.5 R

38

Nipponbare 9 9 75 65 HS Nipponbare-Pigm 3 5 25 9.5 R Maratelli 9 9 100 95 HS Maratelli-Pigm 3 5 29 11.5 R 9311 7 9 60 50 S 9311-Pigm 1 3 10 5 R IRBLa-A (Pia) IRBLi-F5 (Pii) IRBLks-F (Piks) IRBLk-Ka (Pik) IRBLkp-K60 (Pikp) IRBL21 (Pi9) IRBLz-Fu (Piz) IRBLz5-CA (Pi2) IRBLzt-T (Pizt) IRBLta-K1 (Pita) IRBLb-B (Pib) IRBLt-K59 (Pit) IRBLta2 (Pita2) IRBL11-Zh (Pi11)

9 8 7 7 7 5 5 5 5 7 7 9 5 5

9 9 9 9 9 7 7 7 9 9 9 9 9 9

80 100 60 40 60 45 50 40 60 70 70 90 40 50

70 90 40 25 40 20 15 15 40 50 50 80 25 45

HS HS S MS S MS MR MR S S S HS MS S

Lijiangxintuanheigu 9 9 100 90 HS Table S5. Resistance and yield loss evaluation of lines harboring different R gene in

the natural blast nursery (years 2012 to 2014).

Field blast resistance performance was assessed for the 3 pairs of NILs (Nipponbare

and Nipponbare-Pigm, 9311 and 9311-Pigm, Maratelli and Maratelli-Pigm) in the Enshi

blast nursery (Hubei province, mid-western China) in 3 consecutive years (2012-2014).

Gumei4 and 14 NILs containing individual blast resistance genes in the background of

the susceptible cultivar Lijiangxintuanheigu were also grown as controls. Field yield loss

was evaluated accordingly. Note that the Pigm lines displayed high leaf and neck/panicle

resistance to blast in the field tests. HS, high susceptible; MS, middle susceptible; MR,

middle resistant as determined by the blast score system.

39

Variety/line MAS/transgene Purpose GM4 Pigm donor Gene cloning Maratelli Recurrent parent NIL development 9311 Recurrent parent NIL development Nipponbare (NIPB) Susceptible variety Transgene receipt and

NIL development NIL-Pigm Near-isogenic lines harboring Pigm in

Nipponbare Transgene receipt variety and resistance analysis

9311-Pigm Near-sogenic lines harboring Pigm in 9311

Resistance and grain yield analysis

Maratelli-Pigm Near-isogenic lines harboring Pigm in Maratelli

Resistance and grain yield analysis

PigmR PigmR transgenic lines in Nipponbare Resistance and grain yield analysis

35S::PigmR PigmR overexpressing transgenic lines in Nipponbare

Resistance and grain yield analysis

PigmS PigmS transgenic lines in Nipponbare Resistance and grain yield analysis

35S::PigmS PigmS overexpressing transgenic lines in Nipponbare

Resistance and grain yield analysis

PigmR::PigmS Chimeric PigmS transgenic lines driven by the PigmR promoter based in Nipponbare

Resistance and grain yield analysis

PigmR/PigmR::PigmS Double transgenic lines in Nipponbare developed by crossing

Resistance and grain yield analysis

PigmR/35S::PigmS Double transgenic lines in Nipponbare developed by crossing

Resistance and grain yield analysis

PigmR/PigmS NIL-Pigm/PigmR::PigmS

Double transgenic lines in Nipponbare developed by crossing Chimeric PigmS transgenic lines driven by the PigmR promoter based in NIL-Pigm

Resistance and grain yield analysis Resistance and grain yield analysis

NIL-Pigm/PigmS PigmS transgenic lines in NIL-Pigm Resistance and grain yield analysis

NIL-Pigm/35S::PigmS PigmS overexpressing transgenic lines in NIL-Pigm

Resistance and grain yield analysis

R4 R4 transgenic lines in Nipponbare Resistance analysis 35S::R4 R4 overexpression transgenic lines in

Nipponbare Resistance analysis

R2 R2 transgenic lines in Nipponbare Resistance analysis R3 R3 transgenic lines in Nipponbare Resistance analysis R5 R5 transgenic lines in Nipponbare Resistance analysis R7 R7 gDNA based on Nipponbare Resistance analysis R9 R9 transgenic lines in Nipponbare Resistance analysis R10 R10 transgenic lines in Nipponbare Resistance analysis PigmR-RNAi PigmR-RNAi lines in NIL-Pigm Resistance and grain

yield analysis PigmRS-RNAi Simultaneous PigmR and PigmS RNAi

lines in NIL-Pigm Resistance and grain yield analysis

4a-1 OsAGO4a-RNAi lines in NIL-Pigm DNA methylation and

40

resistance analysis 3a-1 OsDCL3a-RNAi lines in NIL-Pigm DNA methylation R2-1 OsRDR2-RNAi lines in NIL-Pigm DNA methylation N1 OsAGO4a-RNAi lines in Nipponbare Resistance analysis Sa PigmS CDS transgenic lines driven by

the full PigmS promoter in NIL-Pigm Resistance analysis

Sb PigmS CDS transgenic lines driven by the truncated promoter (deletion of MITE1) in NIL-Pigm

Resistance analysis

Sc PigmS CDS transgenic lines driven by the PigmS truncated promoter (deletion of MITE2) in NIL-Pigm

Resistance analysis

Sd PigmS CDS transgenic lines driven by the PigmS truncated promoter (deletion of MITE1 and MITE2) in NIL-Pigm

Resistance analysis

Se PigmS CDS transgenic lines driven by PigmS truncated promoter (only with MITE1 and MITE2) in NIL-Pigm

Resistance analysis

Sf PigmS CDS transgenic lines driven by PigmS truncated promoter (only containing MITE1) in NIL-Pigm

Resistance analysis

a GUS fusion reporter transgenic lines driven by the full PigmS promoter in Nipponbare

GUS staining analysis

b GUS fusion reporter transgenic lines driven by the PigmS truncated promoter (deletion of MITE1) in Nipponbare

GUS staining analysis

c GUS fusion reporter transgenic lines driven by the PigmS truncated promoter (deletion of MITE2) in Nipponbare

GUS staining analysis

d GUS fusion reporter transgenic lines driven by the PigmS truncated promoter deletion of MITE1 and MITE2) in Nipponbare

GUS staining analysis

e GUS fusion reporter transgenic lines driven by the PigmS truncated promoter (containing MITE1 and MITE2) in Nipponbare

GUS staining analysis

f GUS fusion reporter transgenic lines driven by the PigmS truncated promoter (containing only MITE1) in Nipponbare

GUS staining analysis

IRBLa-A (Pia) IRBLi-F5 (Pii) IRBLks-F (Piks) IRBLk-Ka (Pik) IRBLkp-K60 (Pikp) IRBL21 (Pi9) IRBLz-Fu (Piz) IRBLz5-CA (Pi2) IRBLzt-T (Pizt) IRBLta-K1 (Pita) IRBLb-B (Pib)

The near-isogenic lines with individual Pi genes in Lijiangxintuanheigu for resistance tests

Blast nursery test

41

IRBLt-K59 (Pit) IRBLta2 (Pita2) IRBL11-Zh (Pi11) Lijiangxintuanheigu Table S6. Rice materials developed and used in this study.

All transgenic plants were developed in the Nipponbare background, and the Pigm NILs were developed by backcrossing with the recurrent varieties in this study. The IRBL NILs are world-collection with Pi genes.

42

Gene/clone primer 5'-3' Purpose C5483 TTAGGCTGCTTGTCTTGGG Mapping and genotyping GGGAGGAGGAATGGTAGGAA C24 ACCTCCAGGCTCTAGTCA CCTCTGTTGTTAATCTTCG C29742 CAGTGAAACGAACGCTATG AATAGGAAGGGTTGATGTTG P18849 AACCCTTACCTTGTTACTATCCTCT AAAGATGTAACTATTGCTACTGTCCA S26205 GTTCTCCACTTCACCTCCAT TTGCTCTACCCAAACCTTTA M80375 GACGAGTAAACGAGAAGTCACG GGACAACCATATTCCCCTAAGA M80410 GGATTGTCTTGTCTCTCTCGC CAGGACTTAGGGTTTCTCTCTTT S48596 GTAGTCATCAAGGTCGTAGTCTCG GCCACTGCTTTGCGGTAC C680 TAACTAACACATTATGCCTGCC CGTTTTGAATACTAGCTTCTCC C0428 AAGGTTCTCGTGGTTTCA TCCCCATTGTTTATAGCAG M35572 TCGCTATCCGTATCCACAAC ACTTCTCCGCAAGATCAACA S2 GTTGAAGAAGTGAGTAGCAGGAA Screening the subclone AGTAGCAATGTTATGGCATCGT S3 TATACAGACAAGCAACGCAGTC CATACCGACAACACATACAACCT S4 CGCTACTCAACTGCCTGATG CACTCCAAGATGTTACTGTCCTC S5 GTTACAACTACTTACCGTCTCATCT TCCTTCTAATATCCACTACCTCCAA S6 ATGGTCTTGTTGTAGGTCTGGTA AAGGTTGTGGATACGGATAGCA S7 TCTCACCTCCATTCTGTTCTCC ACTCTTGAATTGCTCTGCTGTC S8 TGGCAATGGCAATAATCGGTTA TGAGGTTGTGGATACGGATAGC S9 TTGTCGGAGTTGCTGGTCTT GGAGAACAGAATGGAGGTGAGA S10 GGTAGCAAGCAGTGGTGGTA ACATTCTAGCGACTCCTTACATAC

43

R4 CGCTGGTCGCCTAGTGCTATCT cDNA amplification CTCTGCCCTTTCTGTTCGCTGA PigmR CGCTGGTCGCCTAGTGCTATCT CTCTGCCCTTTCTGTTCGCTGA PigmS GCTGCTTGCTGTTCTGAGCG AGCCCTGCCCTATCTGTTCG PigmR TTCCTTTCTCTTTATCATAATT qRT-PCR detection AGATTCCAACCTGCACTTGCCT PigmS GTCCTTGATCTTTATCAGAAAG TGGTTCCAACCTGCAGAATCTG OsActin1 GTCCTCTTCCAGCCTTCCTT TACCACCACTGAGAACGATGT OsAGO4a GAGGACCCTTCAATGCGACG TCAGCAAGAACACAGAGCAGAAA OsRDR2 TGGACTACACAGCAACAAGGC TTTCAGTGGCGAACGGTC OsDCL3a GTCTGGAAATCTGTCGCCTG GAGTAGCACTTATCCTGCGGAA OsPR1 GTGTCGGAGAAGCAGTGGTA CGAGTAGTTGCAGGTGATGAAG OsPR3 GTCACCGAGGCGTTCTTCA GCTTGGAGTCGTCGTTGGT OsPR5 GCAGCCAGGACTTCTACGA TGTGTCTTGGTGTTGTCTTCG OsPBZ1 CGCCGCAAGTCATGTCCTAA CCACGATGTCCTTCTCCTTCTC OsHSP70-1 GGACGCCAAGATGGACAAGA CTGGAAGAAGTCGTGGAGCAT OsHSP70-2 AACAACAACAGCAGCGACAG GGTTGCTCCAGATGCCTTCT OsDEF48 CTTGATTGTGCGACTGCTGAT GCCTTCAACTTGGATTCCTTCT OsDEF70 GCCGTTTGTTTCCTCCTAGTT CACAGGTACTTGATGCAGAACA OsDEF56 CTGAACAATGGTGCTGGTATGA GTGAGACTGGACTTTGGCTTT GUS GCGTGGTGATGTGGAGTATTGC TCGCTGATGGTATCGGTGTGAG R8PS1557 GTGTGTTAGTGTGTATAGTGATGTGAA DNA methylation CAACTCTACTCAACTAAATCTCCCTTC R8P1226 AAATAAAATGAGGTATAAATTGTAAAATT

44

ATAATTCACATCACTATACACACTAACA R8PS778 AGTAAATGGAGTAAAATAAATTTTATTA TATTAAATAAATTTTACAATTTATACCT R8pros363 TTTAGGAATTATAGAATTAAGTAAAGTATTT TTCTAAAATTAATTTTATAATATTTTTCAT R8PS525 TTTAGTAGTGGGGATGATTTGTATAGATATATAGG TCCATCTATACCCTATATCCAAATTCCAATCTCA 35S::PigmR CAATGTCGACATGGCGGAGACGGTGCTGAG Transgene constructs ATCAACTAGTTCAGCCAGCTTGAGCTGTG 35S::PigmS CAATGTCGACATGGCGGAGACGGTGCTGAG ATCAACTAGTTCAGCCAGCTTGAGCTGTG PigmR::PigmS ATGCAAGCTTTGGCGTCTTGAGGAGTCGTA ATGCCTGCAGGGACTCGTTCGACTCTCCCT CAATGTCGACATGGCGGAGACGGTGCTGAG ATCAACTAGTTCAGCCAGCTTGAGCTGTG 35S::R4 CAATGTCGACATGGGCGGAGACGGTGCTGA AATACTGCAGTCAGCCAGCTTGAGCTTGAG Sa GTCAAGCTTTTATCTGGCCCAATTTTATATTG CAGTCGACCGACTCTGCTCGACTAGATCTC Sb GTCAAGCTTTTATCTGGCCCAATTTTATATTG AGAGGATTATCATTATAAGAGCAAGTTCAATAGTA TTGAACTTGCTCTTATAATGATAATCCTCTCTGCT CAGTCGACCGACTCTGCTCGACTAGATCTC Sc GTCAAGCTTTTATCTGGCCCAATTTTATATTG GTCCACTATTGTACCTGCTCCAATATTGTCCATCTG

TAC

GTACAGATGGACAATATTGGAGCAGGTACAATAGTGGAC

CAGTCGACCGACTCTGCTCGACTAGATCTC Sd GTCAAGCTTTTATCTGGCCCAATTTTATATT CAAGCAGAGAGGATTATCACGTTAGCACTTAGCT

GGCGC

GCGCCAGCTAAGTGCTAACGTGATAATCCTCTCTGCTTG

CAGTCGACCGACTCTGCTCGACTAGATCTC Se GCTCAAGCTTTTAGAGCAGGTACAATAGCAGG CTGTCGACCGACTCTGCTCGACTAGATCTC Sf GCAAGCTTGAGCAGGTACAATAGTGGACTA CTGTCGACCGACTCTGCTCGACTAGATCTC PigmR-RNAi ATGGTACCGGACTAGTTGGAAAATAATGG

AGGAC

CTGGATCCGTGAGCTCGCAACCAATCTCACGACTG

PigmRS-RNAi TTGGTACCGGACTAGTGAGGATGTTACGG

GTCTTG

GCTGGATCCGTGAGCTCTACTACTGGTCCGCTTG

OsAGO4aRNAi ATTGGTACCGGACTAGTGAGTTCACGGTA

GTTCTGGA

CTGGATCCGTGAGCTCCAGTATTTGCTGGACTTGTT

45

OsDCL3aRNAi ATTGGTACCGGACTAGTTGTGGACTTCTTGAGTTGGATG GCTGGATCCGTGAGCTCAGTAGTGGCTGTGAGGGGTG OsRDR2RNAi ATTGGTACCGGACTAGTTCAGGGCACTTGATTTTACAC GCTGGATCCGTGAGCTCAGCAAAACATTATGGCTTCTC CLuc-PigmR ATCAACTAGTATGGCGGAGACGGTGCTGAG Luciferase CCGTGTCGACTCCGCCAGCTTGAGCTGTG complementation PigmR-NLuc ATCAACTAGTATGGCGGAGACGGTGCTGAG CCGTGTCGACTCCGCCAGCTTGAGCTGTG CLuc-PigmS ATCAACTAGTATGGCGGAGACGGTGCTGAG CCGTGTCGACTCCGCCAGCTTGAGCTGTG PigmS-NLuc ATCAACTAGTATGGCGGAGACGGTGCTGAG CCGTGTCGACTCCGCCAGCTTGAGCTGTG PigmR-CC-HA ATCAACTAGTATGGCGGAGACGGTGCTGAG Constructs for co-IP CGTGTCGACTTTGGCCGGACCATCATTAGC and pull down PigmR-CC-FLAG ATCAACTAGTATGGCGGAGACGGTGCTGAG CGTGTCGACTTTGGCCGGACCATCATTAGC PigmS-CC-HA ATCAACTAGTATGGCGGAGACGGTGCTGAG CGTGTCGACTTTGGCCGGACCATCATTAGC PigmS-CC-FLAG ATCAACTAGTATGGCGGAGACGGTGCTGAG CGTGTCGACTTTGGCCGGACCATCATTAGC PigmR-CC-His GTGGTCGACCTTGGCCGGACCATCATTAGC CGTGGTCGACATGGCGGAGACGGTGCTGAG PigmR-CC-GST ACCACTAGTCTTGGCCGGACCATCATTAGC TGGTCGACTGATGGCGGAGACGGTGCTGAG PigmS-CC-MBP GGCGGCCGCCTTGGCCGGACCATCATTAGC TGGCGGCCGCATGGCGGAGACGGTGCTGAG GUS-HA CACTAGTATGTTACGTCCTGTAGAAACC GCGTCGACTCCTTGTTTGCCTCCCTGCTG PigmR-CDS CACCATGGCGGAGACGGTGCTGAG Constructs for TCAGCCAGCTTGAGCTGTG yeast-two-hybrid PigmR-CC CACCATGGCGGAGACGGTGCTGAG TCATTTGGCCGGACCATCATTAGC PigmR-NBS CACCATGATCTGTGTTGTTGGGATG TCAGCAGTTTGTCCAGATCAATTG PigmR-LRR CACCATGAGGATGTTACGGGTCTTGGA TCAGCCAGCTTGAGCTGTG PigmS-CDS CACCATGGCGGAGACGGTGCTGAG TCAGCCAGCTTGAGCTGTG PigmS-CC CACCATGGCGGAGACGGTGCTGAG TCATTTGGCCGGACCATCATTAGC PigmS-NBS CACCATGATCTGTGTTGTTGGGATG

46

TCAGCAGTTTGTCCAGATCAATTG PigmS-LRR CACCATGAGGATGTTACGGGTCTTGGA TCAGCCAGCTTGAGCTGTG Pish-CDS CACCATGGCGGAGGTGGTGTTGGCTGGC TCATCTGAATTCCTTCCAGCGG Pish-CC CACCATGGCGGAGGTGGTGTTGGCTGGC TCAGGGACCCTCAAGACTATTACC Pish-NBS CACCATGAGTGTACCAACAATTGTTGTTC TCATAGCTGATTAAAAATATCATCTC Pish-LRR CACCATGTTGAAGAATCTGAAGAAGCTAC TCATCTGAATTCCTTCCAGCGG a (full PigmS GCAAGCTTTTATCTGGCCCAATTTTATATTG Constructs for promoter) CATGGATCCCGACTCTGCTCGACTAGATCTC GUS reporters b GTCAAGCTTTTATCTGGCCCAATTTTATATTG AGAGGATTATCATTATAAGAGCAAGTTCAATAGTA TTGAACTTGCTCTTATAATGATAATCCTCTCTGCT CAGGATCCCGACTCTGCTCGACTAGATCTC c GTCAAGCTTTTATCTGGCCCAATTTTATATTG TCCACTATTGTACCTGCTCCAATATTGTCCATCTGTAC GTACAGATGGACAATATTGGAGCAGGTACAATAGTGGA CAGGATCCCGACTCTGCTCGACTAGATCTC d GTCAAGCTTTTATCTGGCCCAATTTTATATT AAGCAGAGAGGATTATCACGTTAGCACTTAGCTGGCGC GCGCCAGCTAAGTGCTAACGTGATAATCCTCTCTGCTT CAGGATCCCGACTCTGCTCGACTAGATCTC e GCTCAAGCTTTTAGAGCAGGTACAATAGCAGG CTGGATCCCGACTCTGCTCGACTAGATCTC f GTCAAGCTTGAGCAGGTACAATAGTGGACT CATGGATCCCGACTCTGCTCGACTAGATCTC PigmR:Myc ACGCGTGGTCGACATGGCGGAGACGGTGCTGAG Constructs for TCCAGACCACTAGTTCCGCCAGCTTGAGCTGTGCC cell death assay PigmS:YFP ACGCGTGGTCGACATGGCGGAGACGGTGCTGAG TCCAGACCACTAGTTCCGCCAGCTTGAGCTGTGCC pigmsI41E:YFP GAAAGACATCTGGTATGAGAAAGATGAGCTAAAAAC GTTTTTAGCTCATCTTTCTCATACCAGATGTCTTTC pigmsL45E:YFP GTATATCAAAGATGAGGAGAAAACGATGCAAGCATT AATGCTTGCATCGTTTTCTCCTCATCTTTGATATAC pigmsM48E:YFP GATGAGCTAAAAACGGAGCAAGCATTCCTTAGAGC GCTCTAAGGAATGCTTGCTCCGTTTTTAGCTCATC MgPot2 ACGACCCGTCTTTACTTATTTGG Analysis of blast AAGTAGCGTTGGTTTTGTTGGAT infection

47

OsUbq GACGGACGCACCCTGGCTGACTAC TGCCAATTACCATATACCACGAC M12 GCCAGCTAAGTGCTAACGTTAG MITE sRNA gel CGACTCTGCTCGACTAGATCTC blot assay

Table S7. Primers used for plasmid construction , PCR detection and site mutation.

48

Author Contribution

Y.D., Y.Y., E.W., H.X., D.T., and Z.H. designed experiments; Y.D., K.Z., Z.X., X.D.,

D.Y, J.L., X.W., P.Q., G. Z., Q.L., J.Z., B.M. D.T., Z.H. performed experiments and data

analysis. Y.D., E.W., and Z.H. wrote the manuscript. All authors have read, edited and

approved the content of the manuscript.

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