Supporting Information · technology). The PCR primers for ACT7, PR1,andPR2 are listed in Table S1....
6
Supporting Information Wang et al. 10.1073/pnas.1005978107 SI Materials and Methods Plant Material and Transformation. All Arabidopsis plants used in this study were of the Columbia (Col-0) ecotype. The brca2b mutant (SALK_037617) was obtained from the Arabidopsis Bi- ological Resource Center. The rad51 mutant is GABI_134A01 (1). Arabidopsis transformation was performed as described previously (2). Mutant Screen. Genetic screening for ssn3 mutants was carried out as described previously (3). Through complementation analysis, five ssn alleles—6C-1, 9F-1, 13G-1, 13F-1, and 21B-1—were grouped into the SSN3 locus. Tiling Array-Based Cloning. WT (Col-0) and three sni1 ssn3 mutant alleles (9F-1, 13F-1, and 13G-1) were used for tiling array–based cloning. To eliminate unlinked deletions, eight sni1 ssn3 homo- zygous progeny were pooled from each segregating F2 population after backcrossing with the sni1 parent. DNA was extracted using the Qiagen DNeasy Plant Mini Kit and labeled by the Invitrogen BioPrime DNA Labeling System. Hybridization with the Gen- eChip Arabidopsis Tiling 1.0R Array (Affymetrix), washing, and scanning were performed by Expression Analysis, Inc. The chip data were analyzed using GeneChip Tiling Analysis Software (Affymetrix). The thresholds of P values, calculated by comparing hybridization signals between WT and mutant to each probe, were tested from 0.001 to 0.1 and finally set at 0.1 (−10 × log P value = 10) for the interval analysis. In the graph, intervals con- taining at least two consecutive probes with P values over the threshold are highlighted as regions possibly containing deletions. GUS Reporter Analysis. The BGL2::GUS reporter was used for detecting SA response (4). The Gateway vector of pMDC163 (5) was used for analyzing RAD51, BRCA2A, and BRCA2B expres- sion patterns. Translational fusions of GUS to the carboxyl ter- minus of the gene (genomic DNA) were constructed using the following primers: RAD51-P-F and RAD51-P-R for RAD51, BRCA2A-P-F and BRCA2A-P-R for BRCA2A, and BRCA2B-P- F and BRCA2B-P-R for BRCA2B (Table S1). The histochemical GUS assay was performed as described previously (6). Samples were stored in 70% ethanol before microscopic observation. HR Measurement. The frequency of somatic recombination was analyzed using a reporter containing overlapping segments of the GUS gene in inverted orientation (line 1445), as described pre- viously (3, 7). Pseudomonas Infection. Pseudomonas infection was carried out as described previously (3). Leaf discs were collected from 5-wk-old plants 3 d after infiltration with P. syringae pv. maculicola (Psm) ES 4326 (OD 600 = 0.0001), and in planta bacterial growth was measured. To infect the sterile brca2a, brca2b, and rad51 mu- tants, homozygous plants were identified among the segregating progeny of heterozygous parents. For the SAR test, plants were treated with 1 mM SA for 24 h before being infiltrated with Psm ES 4326 (OD 600 = 0.001). Treatment of Genotoxic Substances. Plants were grown on plates containing MS media plus mitomycin C (0, 10, 20, or 40 μM) or bleomycin (0, 5, 10, or 20 μM). The degree of DNA damage was quantified by counting the percentage of plants with no true leaves at 12 d after germination. qPCR and RT-PCR. Arabidopsis RNA was extracted using TRIzol reagent (Invitrogen), and cDNA was synthesized using the Su- perScript III cDNA Synthesis Kit (Invitrogen). qPCR was per- formed using the QuantiTect SYBR Green PCR Kit (Qiagen) in a LightCycler Real-Time PCR System (Roche Applied Science). The primers used for qPCR and RT-PCR are listed in Table S1. Expression of Arabidopsis BRCA2A and BRCA2B was de- tected by RT-PCR using the following primers: for BRCA2A-N (amino terminal), BRCA2A-RT-NF and BRCA2A-RT-NR; for BRCA2A-C (carboxyl terminal), BRCA2A-RT-CF and BRCA2A- RT-CR; for BRCA2B, BRCA2B-RT-F and BRCA2B-RT-R; for UBQ5 (control), UBQ5-RT–F and UBQ5-RT-R (Table S1). Protein Analysis. Plant tissues were homogenized in liquid ni- trogen. Total protein was extracted using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.2% Nonidet P-40, 1 mM PMSF, and 1% Pro- tease Inhibitor Mixture (P9599; Sigma-Aldrich). The extract was centrifuged at 16,000× g for 10 min at 4 °C. The superna- tant was then transferred to a new tube and centrifuged twice more before SDS/PAGE analysis. Western blot analysis was performed as described previously (8). Arabidopsis α-tubulin (anti–α-tubulin, T5168; Sigma-Aldrich) was used as an internal loading control. For co-IP, 10 mg of total protein was mixed with 20 μL of IgG Sepharose 6 Fast Flow (GE Healthcare) and kept on rotator at 4 °C for 2 h. The beads were then transferred into Spin Cups- Cellulose Acetate Filter (Pierce) and washed with a stringent buffer containing 50 mM Tris HCl (pH 7.5), 500 mM sodium chloride, 1% BSA, and 0.5% Nonidet P-40. The protein was eluted with 0.5 M acetic acid (pH 3.4). Microarray Analysis. After treatment of 3-wk-old plants with 0.5 mM SA for 16 h, total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen). The MessageAmp Premier RNA Amplifica- tion Kit (Ambion) was used for RNA labeling. Hybridization with the GeneChip Arabidopsis ATH1 Genome Array (Affymetrix), washing, and scanning were performed at the Duke Microarray Facility. Experiments were performed in triplicate. The microarray data values were first normalized using Gene- Spring GX Software (RMA algorithm; Agilent). Subtractions between the specified samples were used for the scatterplot and linear regression analysis. The genes with significant interactions between genotypes and SA treatments (P < 0.01) were identified by two-way ANOVA. The Student t test was used to find the SA- induced genes in npr1 sni1 (P < 0.01) among the genes with sig- nificant interactions. The statistical significance (P < 0.01) of the BRCA2A-dependent or -independent genes induced by SA in npr1sni1 was determined based on hypergeometric distribution. Whether or not a gene was related to plant defense was de- termined by GO at the Arabidopsis Information Resource (www. arabidopsis.org). ChIP. ChIP was carried out as described previously (9). In brief, 4- wk-old Arabidopsis plants were sprayed with water or 0.5 mM SA. Samples were collected 16 h after treatment. Formaldehyde was added at 1% of final concentration for cross-linking of proteins to DNA, and 1 mM PMSF and 1% Protease Inhibitor Mixture were supplemented in extraction buffer and nuclei lysis buffer. The chromatin was sonicated at 40% duty for 4 × 10 s in a Branson Digital Sonifier 250 to generate fragments ranging from 0.5 kb to 1.0 kb. IP was performed using 10 μg of a polyclonal antibody Wang et al. www.pnas.org/cgi/content/short/1005978107 1 of 6
Transcript of Supporting Information · technology). The PCR primers for ACT7, PR1,andPR2 are listed in Table S1....
Supporting InformationWang et al. 10.1073/pnas.1005978107SI Materials and MethodsPlant Material and Transformation. All Arabidopsis plants used inthis study were of the Columbia (Col-0) ecotype. The brca2bmutant (SALK_037617) was obtained from the Arabidopsis Bi-ological Resource Center. The rad51 mutant is GABI_134A01(1). Arabidopsis transformation was performed as describedpreviously (2).
Mutant Screen.Genetic screening for ssn3mutants was carried outas described previously (3). Through complementation analysis,five ssn alleles—6C-1, 9F-1, 13G-1, 13F-1, and 21B-1—weregrouped into the SSN3 locus.
Tiling Array-Based Cloning. WT (Col-0) and three sni1 ssn3mutantalleles (9F-1, 13F-1, and 13G-1) were used for tiling array–basedcloning. To eliminate unlinked deletions, eight sni1 ssn3 homo-zygous progeny were pooled from each segregating F2 populationafter backcrossing with the sni1 parent. DNA was extracted usingthe Qiagen DNeasy Plant Mini Kit and labeled by the InvitrogenBioPrime DNA Labeling System. Hybridization with the Gen-eChip Arabidopsis Tiling 1.0R Array (Affymetrix), washing, andscanning were performed by Expression Analysis, Inc. The chipdata were analyzed using GeneChip Tiling Analysis Software(Affymetrix). The thresholds of P values, calculated by comparinghybridization signals between WT and mutant to each probe,were tested from 0.001 to 0.1 and finally set at 0.1 (−10 × log Pvalue = 10) for the interval analysis. In the graph, intervals con-taining at least two consecutive probes with P values over thethreshold are highlighted as regions possibly containing deletions.
GUS Reporter Analysis. The BGL2::GUS reporter was used fordetecting SA response (4). The Gateway vector of pMDC163 (5)was used for analyzing RAD51, BRCA2A, and BRCA2B expres-sion patterns. Translational fusions of GUS to the carboxyl ter-minus of the gene (genomic DNA) were constructed using thefollowing primers: RAD51-P-F and RAD51-P-R for RAD51,BRCA2A-P-F and BRCA2A-P-R for BRCA2A, and BRCA2B-P-F and BRCA2B-P-R for BRCA2B (Table S1). The histochemicalGUS assay was performed as described previously (6). Sampleswere stored in 70% ethanol before microscopic observation.
HR Measurement. The frequency of somatic recombination wasanalyzed using a reporter containing overlapping segments of theGUS gene in inverted orientation (line 1445), as described pre-viously (3, 7).
Pseudomonas Infection. Pseudomonas infection was carried out asdescribed previously (3). Leaf discs were collected from 5-wk-oldplants 3 d after infiltration with P. syringae pv. maculicola (Psm)ES 4326 (OD600 = 0.0001), and in planta bacterial growth wasmeasured. To infect the sterile brca2a, brca2b, and rad51 mu-tants, homozygous plants were identified among the segregatingprogeny of heterozygous parents. For the SAR test, plants weretreated with 1 mM SA for 24 h before being infiltrated with PsmES 4326 (OD600 = 0.001).
Treatment of Genotoxic Substances. Plants were grown on platescontaining MS media plus mitomycin C (0, 10, 20, or 40 μM) orbleomycin (0, 5, 10, or 20 μM). The degree of DNA damage wasquantified by counting the percentage of plants with no trueleaves at 12 d after germination.
qPCR and RT-PCR. Arabidopsis RNA was extracted using TRIzolreagent (Invitrogen), and cDNA was synthesized using the Su-perScript III cDNA Synthesis Kit (Invitrogen). qPCR was per-formed using the QuantiTect SYBR Green PCR Kit (Qiagen) ina LightCycler Real-Time PCR System (Roche Applied Science).The primers used for qPCR and RT-PCR are listed in Table S1.Expression of Arabidopsis BRCA2A and BRCA2B was de-
tected by RT-PCR using the following primers: for BRCA2A-N(amino terminal), BRCA2A-RT-NF and BRCA2A-RT-NR; forBRCA2A-C (carboxyl terminal), BRCA2A-RT-CFandBRCA2A-RT-CR; for BRCA2B, BRCA2B-RT-F and BRCA2B-RT-R; forUBQ5 (control), UBQ5-RT–F and UBQ5-RT-R (Table S1).
Protein Analysis. Plant tissues were homogenized in liquid ni-trogen. Total protein was extracted using a buffer containing50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.1%Triton X-100, 0.2% Nonidet P-40, 1 mM PMSF, and 1% Pro-tease Inhibitor Mixture (P9599; Sigma-Aldrich). The extractwas centrifuged at 16,000× g for 10 min at 4 °C. The superna-tant was then transferred to a new tube and centrifuged twicemore before SDS/PAGE analysis. Western blot analysis wasperformed as described previously (8). Arabidopsis α-tubulin(anti–α-tubulin, T5168; Sigma-Aldrich) was used as an internalloading control.For co-IP, 10 mg of total protein was mixed with 20 μL of IgG
Sepharose 6 Fast Flow (GE Healthcare) and kept on rotator at4 °C for 2 h. The beads were then transferred into Spin Cups-Cellulose Acetate Filter (Pierce) and washed with a stringentbuffer containing 50 mM Tris HCl (pH 7.5), 500 mM sodiumchloride, 1% BSA, and 0.5% Nonidet P-40. The protein waseluted with 0.5 M acetic acid (pH 3.4).
Microarray Analysis. After treatment of 3-wk-old plants with 0.5mM SA for 16 h, total RNA was extracted using the RNeasy PlantMini Kit (Qiagen). The MessageAmp Premier RNA Amplifica-tion Kit (Ambion) was used for RNA labeling. Hybridization withthe GeneChip Arabidopsis ATH1 Genome Array (Affymetrix),washing, and scanning were performed at the Duke MicroarrayFacility. Experiments were performed in triplicate.The microarray data values were first normalized using Gene-
Spring GX Software (RMA algorithm; Agilent). Subtractionsbetween the specified samples were used for the scatterplot andlinear regression analysis. The genes with significant interactionsbetween genotypes and SA treatments (P < 0.01) were identifiedby two-way ANOVA. The Student t test was used to find the SA-induced genes in npr1 sni1 (P < 0.01) among the genes with sig-nificant interactions. The statistical significance (P < 0.01) of theBRCA2A-dependent or -independent genes induced by SA innpr1sni1 was determined based on hypergeometric distribution.Whether or not a gene was related to plant defense was de-termined by GO at the Arabidopsis Information Resource (www.arabidopsis.org).
ChIP. ChIP was carried out as described previously (9). In brief, 4-wk-old Arabidopsis plants were sprayed with water or 0.5 mM SA.Samples were collected 16 h after treatment. Formaldehyde wasadded at 1% of final concentration for cross-linking of proteins toDNA, and 1 mM PMSF and 1% Protease Inhibitor Mixture weresupplemented in extraction buffer and nuclei lysis buffer. Thechromatin was sonicated at 40% duty for 4 × 10 s in a BransonDigital Sonifier 250 to generate fragments ranging from 0.5 kb to1.0 kb. IP was performed using 10 μg of a polyclonal antibody
Wang et al. www.pnas.org/cgi/content/short/1005978107 1 of 6
against the full length of human RAD51 (ab48981; Abcam) and40 μL of Protein A Agarose/Salmon Sperm DNA (Upstate Bio-
technology). The PCR primers for ACT7, PR1, and PR2 are listedin Table S1. PCR was performed for 35 cycles.
1. Li WX, et al. (2004) The Arabidopsis AtRAD51 gene is dispensable for vegetativedevelopment but required for meiosis. Proc Natl Acad Sci USA 101:10596–10601.
2. Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-mediatedtransformation of Arabidopsis thaliana. Plant J 16:735–743.
3. Durrant WE, Wang S, Dong X (2007) Arabidopsis SNI1 and RAD51D regulate both genetranscription and DNA recombination during the defense response. Proc Natl Acad SciUSA 104:4223–4227.
4. Cao H, Glazebrook J, Clarke JD, Volko S, Dong X (1997) The Arabidopsis NPR1 genethat controls systemic acquired resistance encodes a novel protein containing ankyrinrepeats. Cell 88:57–63.
5. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughputfunctional analysis of genes in planta. Plant Physiol 133:462–469.
6. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: Beta-glucuronidase asa sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907.
7. Lucht JM, et al. (2002) Pathogen stress increases somatic recombination frequency inArabidopsis. Nat Genet 30:311–314.
8. Mou Z, Fan W, Dong X (2003) Inducers of plant systemic acquired resistance regulateNPR1 function through redox changes. Cell 113:935–944.
9. Gendrel AV, Lippman Z, Yordan C, Colot V, Martienssen RA (2002) Dependence ofheterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1.Science 297:1871–1873.
Col-0 sn
i1 6C
-1 9F
-1 13
F-1
13G-1
21B-1
sni1 brca2a
BRCA2A-N
BRCA2A-C
BRCA2B
UBQ5
Fig. S1. Expression of Arabidopsis BRCA2A and BRCA2B in Col-0, sni1, and sni1 brca2a (6C-1, 9F-1, 13G-1, 13F-1, and 21B-1) was detected by RT-PCR. BRCA2A-Nand BRCA2A-C (Table S1) represent the transcript segment coding for amino-terminal and carboxyl-terminal parts of the protein, respectively. RT-PCR wasperformed for 30 cycles, with UBQ5 used as a loading control.
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Fig. S2. Plant BRCA2 proteins. (A) Amino acid sequence alignment between Arabidopsis BRCA2A and BRCA2B. The differences in amino acid sequences arehighlighted in red. The sequences in bold type indicate the putative BRC repeats and the cyclin-binding site. (B) Alignment of the plant BRCA2 proteins byClustalX. The sequences of the last 83 amino acids in the BRCA2A carboxyl-terminal region were used. A putative cyclin-binding site is given in the red box.
BRCA2A
BRCA2B
UBQ5
- + - + SA
Col-0 sni1BRCA2A::BRCA2A-GUS BRCA2B::BRCA2B-GUS
A B
Fig. S3. Expression patterns of BRCA2 genes. (A) Expression of Arabidopsis BRCA2 genes in Col-0, with 10-d-old seedlings containing the BRCA2A::BRCA2A-GUS and BRCA2B::BRCA2B-GUS transgenes stained for GUS activity. (B) Expression of Arabidopsis BRCA2 genes in Col-0 and sni1. Plants were sprayed withwater (−) or 0.5 mM SA (+). RT-PCR was performed for 30 cycles, with UBQ5 used as a loading control.
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0
40 M
itom
yoci
n C
(μM
)
Col-0 brca2a brca2b
Fig. S4. Response of Col-0, brca2a, and brca2b to mitomycin C treatment. The experiment was performed as described in Fig. 3D.
Col-0 brca2a rad51 npr1
Water SA
5
6
7
8
9
log
cfu
/ lea
f dis
c
Fig. S5. SAR testing of brca2a and rad51 mutants. WT (Col-0) and npr1 were used as controls. Plants were treated with water or 1 mM SA for 24 h and theninfiltrated with Psm ES 4326 (OD600 = 0.001). The error bars represent 95% confidence intervals of log-transformed data. cfu, colony-forming unit. Duplicateexperiments were conducted, with similar results.
80 77
160 157
240 237
320 317
342 339
1 1
81 78
161 158
241 238
321 318
AtRAD51 HsRAD51
AtRAD51 HsRAD51
AtRAD51 HsRAD51
AtRAD51 HsRAD51
AtRAD51 HsRAD51
Fig. S6. Alignment of Arabidopsis (At) and human (Hs) RAD51 proteins by ClustalX.
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Double- +
Triple- +
Double- +
Triple- +
Double- +
Triple- +SA
PR1 PR2 PR5
NIMIN1 EDS1 NDR1
PAD4 NSL1 ERF1
Def
ense
gen
e/UBQ5
Def
ense
gen
e/UBQ5
Def
ense
gen
e/UBQ5
Fig. S7. qPCR of some defense genes found to be both SA- and BRCA2A-dependent through microarray analysis (Fig. 5B), including PR1 (PATHOGENESIS-RELATED GENE 1), PR2, PR5, NIMIN1 (NIM1-INTERACTING 1), EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1), NDR1 (NON–RACE-SPECIFIC DISEASE RESISTANCE 1),PAD4 (PHYTOALEXIN DEFICIENT 4), NSL1 (NECROTIC SPOTTED LESIONS 1), and ERF1 (ETHYLENE RESPONSE FACTOR 1). Double indicates npr1 sni1; triple, npr1sni1 brca2a. Treatment was done with water (−) and SA (+). UBQ5 was used as an internal control.
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5SA
Input Mock (-Ab) IP (+Ab)
-
+
-
+
-
+
ACT7
PR2
PR1
ACT7
PR1
PR2
1 2 3 4 5
1 2 3 4 5
1 2 3 4 51Kb
A
B
- + - + - +Col-0 brca2a brca2b
α-RAD51
α-TUB
C
Fig. S8. Analysis of SA-induced RAD51 binding to independent regions of the PR genes inWT (Col-0) plants. (A) Schematic diagram of five independent regions(labeled 1, 2, 3, 4, and 5) of ACT7, PR1, and PR2 genes for the ChIP analysis. The third regions of PR1 and PR2 (3) were chosen based on the literature (1, 2). Thedistances from region 3 to the adjacent regions (2 and 4) and the distal regions (1 and 5) are ∼500 bp and ∼3,000 bp, respectively. The open boxes represent thecoding regions. The arrow indicates the transcriptional direction. (Scale bar: 1 kb.) (B) For ChIP analysis, 4-wk-oldWT (Col-0) plants were treated with water (−) or0.5 mM of SA (+) for 16 h. The chromatin precleared by the protein A agarose beads and the IP products without antibody served as input and mock (−Ab),respectively. The immunoprecipitated DNA fragments were subject to PCR using primer pairs for five independent regions of ACT7, PR1, and PR2, as shown in A.The PCR products were examined using ethidium bromide staining after agarose gel electrophoresis. (C) Protein blot of RAD51 inWT (Col-0), brca2a, and brca2btreated with water (−) or SA (+). Arabidopsis α-tubulin was used as an internal loading control.
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1. Lebel E, et al. (1998) Functional analysis of regulatory sequences controlling PR-1 gene expression in Arabidopsis. Plant J 16:223–233.2. Cao H, Bowling SA, Gordon AS, Dong X (1994) Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell 6:1583–1592.
Table S1. Primer sequences used for the confirmation of the 9F-1 allele (sni1 ssn3), RT-PCR, and qPCR
Table S1 (DOCX)
Table S2. Genes induced by SA in the npr1 sni1 double mutant
Table S2 (DOCX)
These genes were identified as demonstrating significant interactions between genotypes (npr1 sni1 and npr1 sni1 brca2a) and treatments (water and SA)by two-way ANOVA (P < 0.01). The genes were first grouped by whether or not they were defined by GO at the Arabidopsis Information Resource (www.arabidopsis.org) as defense-related, and were then sorted by their dependency on BRCA2A (P values from low to high).
Other Supporting Information Files
Wang et al. www.pnas.org/cgi/content/short/1005978107 6 of 6
