Open Access

Transcriptional Profiling of Rice Leaves Undergoing a Hypersensitive Response Like Reaction Induced by Xanthomonas oryzae pv. oryzae Cellulase

  • Gopaljee Jha1, 2,
  • Hitendra Kumar Patel1,
  • Madhumita Dasgupta1,
  • Ramesh Palaparthi1 and
  • Ramesh V. Sonti1Email author
Rice20093:9033

https://doi.org/10.1007/s12284-009-9033-z

Received: 14 August 2009

Accepted: 20 November 2009

Published: 22 December 2009

Abstract

A secreted cellulase of Xanthomonas oryzae pv. oryzae induces innate immune responses in rice including a hypersensitive response (HR) like reaction. Microarray analysis was conducted using RNA isolated 12 h following ClsA treatment of leaves. BLAST searches were performed for the 267 (152 up- and 115 downregulated) differentially expressed (≥2-fold) genes. A number of defense and stress-response functions are upregulated while a number of functions involved in metabolism and transport are downregulated following induction of HR. A significant proportion of the differentially expressed genes (41/267) are predicted to encode transcription factors. Co-infiltration of X. oryzae pv. oryzae suppresses ClsA-induced expression of two transcription factors, OsAP2/ethylene response factor (ERF) and OsRERJ1, that are predicted to be involved in the jasmonic-acid-mediated defense pathway. Transient transfer of OsAP2/ERF via Agrobacterium results in the induction of callose deposition, programmed cell death, and resistance against subsequent X. oryzae pv. oryzae infection.

Keywords

Cell wall degrading enzymes Innate immunity Plant defense response Jasmonic acid Hypersensitive response Microarray Programmed cell death

Introduction

Plants have powerful innate immune responses that help them ward off most potential pathogens. These immune responses are triggered following recognition of common microbe-associated molecular pattern molecules (MAMPS) or pathogen-associated molecular pattern molecules (He et al. 2007; Bent and Mackey 2007). For bacterial pathogens, such MAMPS include elicitors such as lipopolysaccharides (Newman et al. 2002), cold shock protein (Felix and Boller 2003), flagellin (Felix et al. 1999), elongation factor Tu (Kunze et al. 2004), an extracellular signaling molecule called AvrXa21 (Lee et al. 2006), etc. The recognition of these signals occurs through receptors at the plant cell surface (Zipfel et al. 2004, 2006; Lee et al. 2006; He et al. 2007; Bent and Mackey 2007). Bacterial pathogens have the capacity to suppress innate immune responses of plants (Palva et al. 1993; Keshavarzi et al. 2004; Li et al. 2005; Jha et al. 2007). This ability of plant pathogenic bacteria to suppress host innate immunity is considered to be a precondition for their ability to cause disease (Grant et al. 2006; Bent and Mackey 2007; Jha et al. 2007; He et al. 2007).

The plant cell wall is a formidable barrier for potential pathogens. Cell wall degrading enzymes are important virulence factors of phytopathogenic bacteria (reviewed in Jha et al. 2005). Conversely, the oligosaccharides that are released following the action of these enzymes on plant cell walls elicit potent innate immune responses (Darvill and Albersheim 1984; Ryan and Farmer 1991). These immune responses are in turn suppressible by plant pathogenic bacteria (Palva et al. 1993; Jha et al. 2007). Prior treatment of tobacco with purified preparations of Erwinia carotovora subsp. carotovora pectate lyase and polygalacturonase induced resistance against subsequent infection by the same pathogen (Palva et al. 1993). These defense responses are mediated by the jasmonic acid (JA)- and ethylene (ET)-dependent pathways but not the salicylic acid (SA) pathway (Norman-Setterblad et al. 2000; Vidal et al. 1998).

The rice pathogen, Xanthomonas oryzae pv. oryzae, uses a bacterial Type 2 secretion system (T2S) to secrete several cell-wall-degrading enzymes including two cellulases (ClsA and EglXoB), cellobiosidase (CbsA), lipase/esterase (LipA), and xylanase (Rajeshwari et al. 2005; Jha et al. 2007; Hu et al. 2007). The EglXoB protein is expressed only during in planta growth, but all five enzymes are important for X. oryzae pv. oryzae virulence. Conversely, treatment with either ClsA or CbsA or LipA induces innate immune responses such as HR like reaction and callose deposition in rice (Jha et al. 2007). These defense responses are so potent that prior treatment with these cell-wall-degrading enzymes induces resistance against subsequent infection by X. oryzae pv. oryzae. It appears that soluble elicitors that are released by the action of these enzymes on rice walls are the actual elicitors of innate immune responses. These defense responses are suppressed by X. oryzae pv. oryzae using proteins that are secreted through the type 3 secretion system (T3S). Prior treatment with a T3S X. oryzae pv. oryzae mutant induces resistance against subsequent infection by the wild-type pathogen indicating that defense suppression is crucial for the ability of X. oryzae pv. oryzae to cause disease. A T2S T3S double mutant of X. oryzae pv. oryzae is very much compromised in the ability to elicit rice innate immune responses indicating that a functional T2S and presumably the cell-wall-degrading enzymes that are secreted through this system play an important role in elicitation of innate immunity during growth within the plant (Jha et al. 2007).

Secreted ClsA is a 48-kDa protein that exhibits endoglucanase as well as exoglucanase activities. In order to understand the molecular events associated with ClsA-induced innate immune responses in rice, we have performed transcriptome analysis following treatment with the ClsA protein at concentrations that would induce HR. Our results indicated that a number of functions that are related to defense and stress are upregulated while those that are related to metabolism and transport are downregulated following ClsA treatment. A number of transcription factor genes are differentially expressed during cellulase-induced HR. Transient transfer of one of these transcription factor genes, OsAP2/ethylene response factor (ERF), via Agrobacterium, results in induction of callose deposition in rice leaves, programmed cell death (PCD) in rice roots, and enhanced resistance against subsequent X. oryzae pv. oryzae infection.

Results

Microarray analysis of ClsA-induced transcriptome of rice

In order to understand gene expression changes in rice leaves undergoing a ClsA-induced HR, we performed microarray analysis using the Affymetrix GeneChip rice genome array. Each array contains probe sets for 51,279 rice transcripts; out of which, around 48,564 are from japonica and 1,260 from indica cultivars. The adaxial surfaces of leaves of 15 days old Taichung Native-1 (TN-1) rice seedlings were infiltrated using a needleless syringe with either X. oryzae pv. oryzae ClsA (500 µg/ml) or buffer. Approximately, 10 μl of the solution is infiltrated into each leaf. The zone of infiltration is approximately 1 cm2. This means that 5 µg of ClsA is infiltrated into an area of approximately 1 cm2. Total RNA was isolated, 12 h after treatment, from leaf tissues encompassing the zone of infiltration. The samples from 20–30 infiltrated leaves were pooled together to obtain enough RNA for the analysis. Following a series of steps (as described in the “Methods”), the RNA was converted into biotin-labeled cRNA and hybridized to the rice gene chip. Separate gene chips were hybridized for the ClsA and buffer-treated samples, and the experiments were repeated with three independent biological replicates. All the raw data files (CEL files; three for ClsA and three for buffer treatment) obtained from GeneChip Operating Software (GCOS; Affymetrix) were subjected to AVADIS™ Software (Strand Life Science, Bangalore, India) for further analysis. The Probe Logarithmic Intensity Error (PLIER; Affymetrix Inc 2005) and Robust Multichip Average (RMA; Irizarry et al. 2003a, b) algorithms used in the analysis picked different numbers of genes at p < 0.05. PLIER identified 1,582 genes as differentially expressed with ≥1.5-fold change and 427 genes with ≥2.0-fold change at p < 0.05 whereas RMA picked 1,004 and 302 genes with 1.5- and 2.0-fold change, respectively, at p < 0.05. In order to reduce the possibility of false discovery, we adopted a stringent criterion wherein we considered only those genes as differentially expressed, which were selected by both the algorithms. A total of 862 genes were common to both lists at 1.5-fold change and p < 0.05. Q values were calculated from p values of all 862 genes, and q-value-based significance analysis suggested that the expected false positives are only four genes out of this list. The gene ontology (GO) enrichment analysis of ≥1.5-fold change rice genes differentially expressed upon ClsA treatment have revealed that genes with GO terms associated with response to stress are overrepresented among upregulated genes while those associated with primary and secondary metabolism, photosynthesis, chloroplast, and plastids are overrepresented among downregulated genes. Genes with GO terms associated with calcium and magnesium ion binding are also overrepresented in upregulated genes while the genes involved in iron ion binding, transporter activity, lipid metabolism, and fatty acid biosynthesis are overrepresented among downregulated genes.

All the six raw CEL files and the corresponding Avadis™ processed RMA and PLIER normalized data files have been deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE8216.

Rice metabolic pathway analysis

In order to understand the changes associated with metabolic pathways during ClsA-induced HR, pathway analysis was performed using RiceCYC 1.2 Software (http://dev.gramene.org/pathway). For this analysis, we selected genes that were identified as being differentially expressed at ≥1.5-fold and p ≤ 0.05 by both PLIER and RMA algorithms. Out of the 862 genes deemed to be differentially expressed (363 upregulated and 499 downregulated), we could retrieve the requisite locus information for only 709 genes from the TIGR rice genome database. Out of these, 197 genes could be associated with different metabolic pathways using the RiceCYC 1.2. Interestingly, two paralogous genes encoding putative 12-oxo phytodienoate reductases that are predicted to be involved in JA biosynthesis as well as four different glutathione-S-transferases were upregulated following ClsA treatment (Supplementary Table S1). A number of plant glutathione-S-transferases have been reported to be upregulated following pathogen infection (Marrs 1996), and silencing of the gene for a glutathione-S-transferase of Nicotiana benthamiana has been shown to lead to enhanced susceptibility to Colletotrichum orbiculare (Dean et al. 2005). Five putative lipases that are predicted to be involved in triacylglycerol degradation were downregulated. Several lipase-like functions have been shown to be associated with the SA-mediated defense pathway (Falk et al. 1999; Jirage et al. 1999; Kumar and Klessig 2003).

Functional categorization of the differentially expressed genes

A majority of the differentially expressed rice genes that were identified from above were not functionally annotated in public databases. We performed BLASTX-based homology searches for 267 genes that were identified as being differentially expressed using both PLIER and RMA at ≥2.0-fold change and p < 0.05 at the NCBI server (Altschul et al. 1997) and the function of the protein with which they demonstrated significant homology (E ≥ 10−7) was ascribed to them. We used the 2.0-fold change cutoff for this analysis as each gene was individually analyzed and the smaller number of genes (267 with 2.0-fold cutoff as opposed to 862 obtained with 1.5-fold cutoff) was easier for us to handle. By this method, we could assign a putative function to nearly 80% (215/267) of the differentially expressed genes. Of these 215 genes, an E > 10−10 was used to assign a function for 205 genes, and a value in the range of 10−7 to 10−10 had to be used to assign a function for about 10 genes. For the remaining genes, a function could not be assigned even if we considered less stringent E values, and these genes were put in the category of “others.” Based upon the assigned function, the differentially expressed genes were categorized into 14 different functional categories (Fig. 1; Tables 1 and 2). The functional categories of Bevan et al. (1998) were used with some minor changes as described in “Methods.” A large number of putative transcription factors were present among the upregulated (26/152; 17%) as well as downregulated (15/115; 13%) genes. In comparison, approximately 6% of the total genes in the rice genome are predicted to encode transcription factors (Gao et al. 2006). Interestingly, disease/defense and stress-related genes constituted 14% and 9% of the upregulated genes, respectively, whereas each of them constituted 6% of the ClsA-downregulated genes. The genes under the categories of metabolism and transport constituted 19% and 10% of the downregulated genes, respectively. These categories constituted 7% and 2% of the upregulated genes. Furthermore, genes involved in protein synthesis/turnover constituted 4% of the ClsA-upregulated genes and ~1% of the downregulated genes. Several metallothioneins and stress responsive proteins like heat shock proteins as well as a peroxidase and glutathione S-transferase were significantly upregulated (Table 1). The gene for rice ortholog of Arabidopsis GUN4, a positive regulator of chlorophyll synthesis (Larkin et al. 2003) as well as several genes encoding chlorophyll A/B binding proteins, glycine rich proteins, cytokinin dehydrogenases, and auxin responsive proteins were downregulated during ClsA-induced HR (Table 2). An important host susceptibility factor (Os8N3), whose expression is highly upregulated during X. oryzae pv. oryzae pathogenesis in rice (Yang et al. 2006), was ~2.6-fold downregulated during ClsA-induced HR. Also downregulated were several condensing enzymes (Os.7669.1.S1_at, Os.27713.1.A1_at, Os.9724.1.S1_at) and a ß-ketoacyl reductase (Os.12839.1.S1_at; Table 2) that are predicted to function in long chain fatty acid synthesis.
Fig. 1

Functional categorization of differentially expressed rice genes. The functions of differentially expressed genes (≥2-fold change) were classified into 14 different categories based upon BLASTX analysis. The number of up- and downregulated genes for each functional category is shown in the histogram. Percentage representations of the number of genes present per individual category with respect to total up- or downregulated genes, respectively, are shown in the pie charts. The functional categories are: a cell growth/division; b cell structure; c disease/defense; d energy; e intracellular traffic; f metabolism; g other; h protein synthesis/turnover; i secondary metabolism; j signal transduction; k stress; l transcription; m transporter; n transposons.

Table 1

Rice Genes Upregulated (≥2 Fold) Following Cellulase Treatment

Probe set IDa

Public IDb

FCAc

Gene descriptiond

Homolog (E score)e

Cell growth/division

 Os.10388.1.S1_at

AK120374

2.74

MEE59 (maternal effect embryo arrest 59)

NP_195447 (3e-22)

 OsAffx.7876.1.S1_s_at

9640.m04245

2.46

Os12g0626500 (Seed maturation protein domain containing protein)

NP_001067326 (2e-35)

 Os.38249.2.S1_at

CB638262

2.39

Putative early flowering 3

BAA83571 (6e-28)

Cell structure

 OsAffx.22303.1.S1_at

9635.m03076

5.06

Expansin-like B1 precursor (OsEXLB1) (Expansin-related 1)

Q850K7 (3e-147)

 Os.37723.1.S1_at

AK072531

2.27

Putative tonoplast membrane integral protein

AAS98488 (3e-95)

 Os.5754.1.S1_at

AK099590

2.23

Putative plastid developmental DAG protein

BAD03018 (8e-91)

 Os.36975.3.S1_x_at

NM_189551

2.17

Putative MAR binding filament-like protein 1

BAD68082 (0.0)

 Os.24307.1.S1_at

AK066849

2.01

TMP14 (THYLAKOID MEMBRANE PHOSPHOPROTEIN OF 14 KDA)

NP_566086 (1e-24)

Disease/defense

 Os.53444.1.S1_at

AK072661

4.61

Putative pathogen-induced protein 2–4

BAD08038 (7e-17)

 Os.11575.3.S1_x_at

AK065363

3.04

Ankyrin-repeat protein/OsNPR3

ABE11617 (0.0)

 Os.10546.1.S1_s_at

AU183565

2.92

Putative UDP-glucose:salicylic acid glucosyltransferase

BAD34356 (2e-33)

 OsAffx.30138.1.S1_at

9637.m02954

2.9

Putative UDP-glucose:salicylic acid glucosyltransferase

BAD34356 (0.0)

 Os.8760.1.S1_a_at

AK099869

2.6

Putative nodulin-like protein

BAD34227 (0.0)

 Os.36969.1.S1_at

AK120328

2.43

NB-ARC domain containing protein

EAY99786 (3e-25)

 Os.11179.1.S1_at

CB647801

2.37

Ankyrin repeat family protein, putative, expressed

ABF95250 (1e-43)

 Os.25266.1.S1_at

AK058891

2.34

Putative beta 1,3-glucanase

BAB63855 (2e-167)

 OsAffx.23277.2.S1_at

9629.m01656

2.3

Ribosome inactivating protein, expressed

ABF98182 (1e-08)

 Os.38249.1.S1_at

AK065173

2.23

Nematode responsive protein-like

BAD45081 (0.0)

 Os.4327.1.S1_at

AF384030

2.21

Seven transmembrane protein MLO2

AF384030_1 (0.0)

 Os.11831.1.S1_at

AK062130

2.12

Putative benzothiadiazole-induced S-adenosyl-L-methionine:salicylic acid Carboxyl methyltransferase1

BAD45797 (0.0)

 Os.11575.1.S1_at

AK065952

2.09

Ankyrin-repeat protein/OsNPR3

ABE11617 (0.0)

 Os.15355.1.S1_x_at

AK067000

2.07

Putative bacterial-induced peroxidase precursor

BAD28871 (2e-166)

Energy

 OsAffx.12379.1.S1_at

9630.m03520

3.26

Putative oxygen-evolving enhancer protein 3-2, chloroplast precursor (OEE3)

BAD29563 (8e-61)

 OsAffx.32230.1.A1_at

X15901

2.96

ATP synthase CF1 epsilon subunit

NP_039389 (4e-65)

 Os.52588.1.S1_at

AK068191

2.85

2Fe-2S iron-sulfur cluster protein-like/ferredoxin-related

BAC80058 (5e-86)

 Os.19061.1.S1_s_at

AK099499

2.77

4Fe-4S ferredoxin, iron-sulfur binding; Heat shock protein DnaJ

ABP02426 (2e-21)

 Os.26744.1.A1_at

CA762807

2.66

Chloroplast photosystem I assembly protein Ycf3

BAD81968 (7e-16)

 Os.44475.1.S1_x_at

AK121213

2.66

Putative NADH dehydrogenase (ubiquinone oxidoreductase)

BAC15811 (0.0)

 Os.29056.1.S2_at

AK110782

2.54

Apocytochrome f precursor, putative

AAP53263.2 (4e-54)

 Os.27830.1.S1_at

AK068377

2.54

PsbQ domain protein family/oxygen evolving enhancer 3

BAC10375 (4e-105)

 OsAffx.32225.1.A1_at

X15901

2.5

Photosystem I assembly protein Ycf3

NP_039384 (3e-95)

 Os.3406.1.S1_at

AB004865

2.48

Alternative oxidase

BAA28771 (0.0)

 Os.11570.1.S1_s_at

AK101484

2.22

Putative glycerol-3-phosphate dehydrogenase

AAU44049 (0.0)

Intracellular traffic

 Os.8700.1.S1_at

AK066218

2.74

Dynein light chain type 1-like

BAD28635 (2e-36)

 Os.31464.1.S1_a_at

AK101489

2.6

Os04g0527400

NP_001053367 (0.0)

 Os.40002.1.S1_s_at

CF315096

2.17

Got1-like family

NP_001060166 (1e-07)

Metabolism

 Os.18490.1.S1_x_at

AK120238

3.16

Thiamine biosynthesis protein ThiC, putative, expressed

ABF98201 (0.0)

 Os.56682.1.S1_at

AK110292

3.1

GDSL-lipase-like

BAD61697 (0.0)

 Os.23154.2.S1_at

AK111360

2.68

Putative proline-rich protein APG (GDSL-lipase-like)

BAD15755 (1e-61)

 Os.52414.1.S1_at

AK066972

2.41

Hydrolase, alpha/beta fold family protein-like

BAD23358 (8e-145)

 OsAffx.27752.1.S1_s_at

9634.m02096

2.41

Putative aldose 1-epimerase

BAD05401 (4e-149)

 Os.50951.1.S1_at

AK059666

2.21

Metallo-beta-lactamase protein-like

BAD28843 (6e-136)

 OsAffx.32255.1.A1_at

X15901

2.19

Acetyl-coa carboxylase beta subunit

NP_039394 (5e-56)

 Os.8957.1.S1_at

AK071523

2.19

Phosphatidic acid phosphatase-related/PAP2-related

NP_190970 (6e-128)

 Os.47381.2.S1_at

AK120916

2.12

Putative 3-oxoacyl-[acyl-carrier-protein] synthase I, chloroplast precursor

BAD35225 (0.0)

 Os.8957.1.S1_a_at

AK071523

2.06

Phosphatidic acid phosphatase-related/PAP2-related

NP_190970 (3e-128)

Otherf

 OsAffx.5746.1.S1_s_at

9636.m00746

8.72

Hypothetical protein OsJ_025189

EAZ41706 (3e-20)

 Os.4618.1.S1_at

AK062310

4.71

Hypothetical protein OsI_002101

EAY74254 (2e-50)

 Os.40007.1.S1_x_at

CF318727

3.86

Os03g0120600

NP_001048790 (4e-18)

 Os.6288.1.S1_at

AK106356

3.58

Unknown protein/Os08g0412700

NP_001061796 (2e-169)

 Os.20548.1.S1_at

AK066753

3.58

Hypothetical protein OsI_016689

EAY95456 (9e-34)

 Os.30528.1.S1_at

AK108716

3.45

Unknown protein/Os08g0412800

NP_001061797 (0.0)

 Os.46544.1.A1_at

AK066066

3.38

Expressed protein/Os10g0389500

NP_001064501 (3e-113)

 Os.21260.1.S1_at

AK067400

3.38

Os03g0184100

NP_001049187 (2e-120)

 Os.6516.1.S1_at

CF303902

3.37

No significant similarity found

 

 Os.52004.1.S1_at

AK064520

3.31

Hypothetical protein LOC_Os12g17920

ABA97088 (3e-06)

 Os.27218.1.A1_at

CB649864

3.23

Os05g0575000

NP_001056395 (5e-33)

 Os.57343.1.S1_at

AK111335

3.06

Os02g0733900

NP_001048037 (2e-36)

 Os.22703.1.S1_at

AK066033

3.03

Unknown protein /Os07g0618700

BAC79871 (1e-112)

 Os.27764.1.S1_at

CB629248

3

Os05g0464200

NP_001055778 (4e-04)

 Os.51529.1.S1_at

AK063010

2.99

Os12g0174200

NP_001066286 (1e-16)

 Os.39942.2.S1_x_at

CF321166

2.81

Osjnbb0058j09.9

CAD39870 (9e-06)

 Os.7944.1.S1_at

AK120788

2.76

No significant similarity found

 

 Os.5823.1.S1_at

AK109216

2.75

Os02g0718200

NP_001047938 (0.003)

 Os.51299.1.S1_at

AK062470

2.75

Os05g0464300

NP_001055779 (2e-14)

 Os.4773.1.S1_at

CF325704

2.67

Os06g0133500

NP_001056709 (3e-30)

 Os.57191.1.S1_at

AK111114

2.63

Os06g0147300

NP_001056802 (2e-50)

 Os.18712.1.S1_at

AK108985

2.63

Hypothetical protein OsI_028900

EAZ07668 (7e-127)

 Os.2426.1.A1_at

CA759372

2.49

Expressed protein

ABA92028 (2e-21)

 Os.12342.1.S2_at

CB668460

2.43

No significant similarity found

 

 Os.39725.1.S1_at

CA763989

2.35

No significant similarity found

 

 Os.7130.2.S1_x_at

AU032629

2.27

No significant similarity found

 

 OsAffx.7530.1.S1_s_at

9640.m00831

2.26

Expressed protein

ABA96042 (6e-24)

 Os.38992.1.A1_x_at

D43281

2.23

No significant similarity found

 

 OsAffx.27741.1.S1_s_at

9634.m02034

2.23

Hypothetical protein

BAD62179 (2e-21)

 Os.50952.1.S1_at

AK059668

2.19

No Significant similarity

 

 Os.10823.2.S1_at

AK064162

2.07

Os05g0487300/unknown protein

NP_001055881 (1e-05)

 Os.55407.1.S1_at

AK108023

2.02

Os03g0799300

NP_001051576 (3e-26)

Protein synthesis/Turnover

    

 Os.55628.1.S1_at

AK108443

3.69

Eukaryotic translation initiation factor SUI1, putative

NP_175831 (3e-30)

 OsAffx.26671.1.S1_x_at

9632.m05851

3.34

Eukaryotic translation initiation factor SUI1, putative

NP_175831 (4e-30)

 Os.8724.1.S1_at

AK072982

3.02

Cyclin-like-F-box family protein

NP_567629 (5e-09)

 OsAffx.24862.1.S1_at

9630.m05377

2.43

F-box domain containing protein

AAP54337.2 (1e-27)

 Os.46325.1.A1_at

AK120658

2.32

Zinc finger (C3HC4-type RING finger) family protein

NP_568096 (3e-146)

 Os.27569.1.S2_at

AK121706

2.26

Calcium-binding EF-hand family protein-like

BAD08916 (8e-49)

Secondary metabolism

 Os.50053.1.A1_at

AK119780

3.9

9-cis-epoxycarotenoid dioxygenase 4

AAW21320 (0.0)

 Os.6376.1.S1_at

AK121461

3.41

Chalcone-flavanone isomerase

NP_001057086 (0.0)

 Os.27507.1.S1_at

AK109673

2.97

Reticuline oxidase precursor, putative/berberine bridge enzyme-like protein

ABA93766 (1e-132)

 OsAffx.32219.1.A1_x_at

X15901

2.61

Terpene synthase family, metal binding domain containing protein

ABF96083 (0.0)

Signal transduction

 Os.1606.1.S1_at

AK111969

5.05

Putative receptor-like protein kinase

AAL87185 (8e-137)

 Os.15247.1.S1_s_at

AB125310

3.15

Serine/threonine-protein kinase SAPK9

BAD18005 (0.0)

 Os.10254.1.S1_at

AK066150

3.12

Copine I-like

BAD10026 (0.0)

 Os.9585.1.S1_at

AB060552

3.04

Calmodulin-2

BAB69673 (8e-66)

 Os.26226.1.S1_at

AK103704

2.96

Putative serine/threonine protein kinase

BAD08131 (0.0)

 Os.9585.1.S1_s_at

AB060552

2.62

Calmodulin, putative, expressed

ABA99816 (5e-66)

 Os.35013.1.S1_at

AK069274

2.16

Putative Serine/threonine phosphatases

AAP06902 (0.0)

 OsAffx.11760.1.S1_at

9629.m06614

2

Putative S-receptor kinase

BAD82381 (0.0)

Stress

 Os.51546.1.S1_at

AK063042

10.2

Senescence-associated protein-related

NP_197570 (1e-08)

 OsAffx.32080.1.S1_at

9640.m03722

7.34

Metallothionein-like protein 1, putative, expressed

ABA99660 (8e-30)

 Os.37783.1.S1_a_at

AK105219

4.52

Metallothionein-like protein 4C (OsMT-I-4c)

Q2QNC3 (2e-22)

 OsAffx.7826.2.S1_at

9640.m03694

4.02

Metallothionein-like protein 1, putative, expressed

ABA99599 (1e-10)

 Os.37773.1.S1_at

AU165294

3.82

Low molecular mass heat shock protein

AAC78392 (2e-37)

 Os.37783.2.S1_x_at

AU070898

3.73

Metallothionein-like protein 1, putative, expressed

ABA99639 (4e-24)

 Os.14105.1.S1_at

AK106404

3.63

Cytochrome P450

AAX92767 (0.0)

 Os.4671.1.S1_a_at

AF435970

3.47

Jacalin-related lectin like /Protein mannose-binding lectin

P24120 (2e-78)

 Os.37773.1.S1_x_at

AU165294

3.46

Alpha-crystallin-Hsps/17.4 kda class I heat shock protein, putative

AAP06883 (6e-38)

 Os.6092.1.S1_at

AB120515

3.17

Trehalose-6-phosphate phosphatase

BAD12596 (0.0)

 Os.7756.1.S1_at

AK060639

2.7

Putative stress-responsive protein (pectinesterase inhibitor domain containing)

AAM94917 (2e-83)

 Os.15428.1.S1_at

AK065414

2.61

Putative wound inducive gene

BAD81783 (2e-174)

 Os.8741.1.S1_at

AK102303

2.57

RADICAL-INDUCED CELL DEATH1

ABF94777 (0.0)

 Os.33675.1.S1_at

AK099569

2.5

Rbcx protein having a possible chaperonin-like function

NP_001061837 (2e-82)

 Os.8774.1.S1_at

AK071331

2.43

Peroxidase-like protein

BAD82471 (7e-127)

 Os.5363.1.S1_at

AU174652

2.4

Putative cadmium-induced protein

BAC19956 (1e-29)

 Os.53236.1.S1_at

AK071507

2.28

Putative zinc finger protein/Salt tolerance-like protein

BAD25819 (7e-95)

 Os.49030.1.A1_s_at

CR292984

2.27

Putative glutathione S-transferase OsGSTU5

AF309377_1 (3e-09)

Transcription

 OsAffx.17366.1.S1_at

9636.m03709

6.54

Ethylene-binding protein-AP2 domain containing

BAD38371 (4e-41)

 Os.6043.1.S1_at

AB040744

5.3

RERJ1(Helix-loop-helix domain containing transcriptin factor)

AB040744 (8e-111)

 OsAffx.27442.1.S1_at

NM_185471

5.23

Transcription factor CBF1

BAA90812 (3e-73)

 Os.4463.1.S1_s_at

AY327040

4.57

Transcription factor CBF1

BAA90812 (3e-73)

 Os.15849.1.S1_s_at

AK062882

4.34

AP2 domain-containing transcription factor-like

BAD17116 (4e-15)

 Os.49746.1.S1_at

AY581256

3.87

Induced protein mgi1(MYB-like DNA-binding domain)

AAS90600 (1e-76)

 Os.55259.1.S1_at

AK107750

3.77

ZIM motif family protein, expressed

ABF94311 (1e-07)

 Os.46849.1.S1_at

AK107854

3.46

ZIM motif family protein, expressed

AAP53563 (7e-23)

 Os.54501.1.S1_at

AK105440

3.44

PHD-finger, putative

AAX94898 (0.0)

 Os.5335.1.S1_at

AK101209

3.19

Putative MYB transcription factor

BAD61826 (1e-33)

 Os.3400.1.S1_s_at

AB001888

3.05

Zinc finger protein

BAA33206 (0.0)

 Os.12977.1.S1_at

AK072192

2.96

Zinc finger protein/COL9 (CONSTANS-LIKE 9); transcription factor

BAA33206 (4e-141)

 Os.55550.1.S1_at

AK108297

2.88

Transcription factor GT-3b

AAP13348 (3e-04)

 Os.9923.1.S1_s_at

AK120087

2.58

ZIM motif family protein, expressed

ABF94311 (9e-57)

 Os.35681.1.S1_at

AY344493

2.54

Putative heat shock transcription factor 8

BAB68070 (2e-124)

 Os.35343.1.A1_at

AK067922

2.51

No apical meristem protein (NAC domain containing)

ABA95706 (2e-116)

 Os.10411.1.S1_at

AY077725

2.45

C2H2 zinc finger protein

BAD81699 (2e-72)

 Os.23778.1.S1_at

AK101934

2.44

Putative heat stress transcription factor

BAD25410 (0.0)

 Os.7130.1.S1_at

AK073475

2.42

Transcription factor jumonji, putative, expressed

ABB48025 (0.0)

 Os.8031.1.S1_at

AK111775

2.38

Putative ethylene response factor 2/AP2 domain-containing transcription factor

BAB92777 (1e-73)

 Os.49787.1.S1_at

AK111571

2.36

Putative MYB-like transcription factor

AAT69605 (6e-50)

 Os.20750.1.S1_at

CB635910

2.33

bZIP transcription factor-like

BAB89012 (5e-54)

 Os.19899.1.S1_x_at

AK106830

2.12

Transcription factor PCF8

BAC01119 (4e-38)

 Os.11046.1.S1_at

AK119729

2.1

Putative ethylene-responsive transcriptional coactivator

BAD32863 (8e-60)

 Os.10356.1.S1_at

AK073589

2.09

ZIM motif family protein, expressed

ABF94310 (2e-82)

 OsAffx.3006.1.S1_at

9630.m04817

2.08

MYB-like transcription factor-like

BAD13038 (1e-15)

Transporter

 Os.47946.1.S1_s_at

AK070124

4.22

Putative glucose-6-phosphate/phosphate- translocator precursor

BAC57673 (2e-90)

 Os.11800.1.S1_s_at

AK108373

3.37

MDR-like ABC transporter

CAD59587 (3e-141)

 Os.11800.1.S1_at

AK108373

3.13

MDR-like ABC transporter

CAD59587 (4e-140)

Transposons

 Os.18078.1.S1_at

C97547

3.91

Rtac1

AAX95969 (0.17)

 Os.39994.1.S1_at

CF319063

3.8

Rtac1

AAL86017 (1e-10)

 Os.6901.1.S1_at

AK102892

2.72

Os10g0378500/retrotransposon protein, putative

NP_001064482 (2e-31)

aAffymetrix Probe set ID of upregulated gene

bThe public representative ID of these genes obtained from Affymetrix NetAffx analysis center

cPLIER normalized absolute fold change value for upregulation

dThe putative function of the gene based on BLASTX analysis

eAccession ID of the homologous protein based upon which the function has been assigned. The corresponding BLASTX E score is shown in brackets

fGenes encoding hypothetical proteins or those with no significant matches in the databases are kept in this category

Table 2

Rice Genes Downregulated (≥2-Fold) Following Cellulase Treatment

Probe set IDa

Public IDb

FCAc

Gene descriptiond

Homolog (E score)e

Cell growth/division

 Os.27224.2.A1_at

AK103510

3.66

Putative cytokinin dehydrogenase

BAD09964 (8e-59)

 OsAffx.29543.1.S1_x_at

9636.m03606

3.05

Putative cytokinin dehydrogenase

BAD09964 (0.0)

 Os.43596.1.S1_at

CB620528

2.62

Cytokinin oxidase/dehydrogenase

BAE16612 (3e-39)

 Os.9392.1.S1_at

AK071762

2.16

Auxin/aluminum-responsive protein putative

NP_197415 (3e-86)

 Os.55455.1.S1_at

AK108118

2.06

Auxin-responsive protein-like

BAD25785 (2e-26)

 Os.12201.1.S1_at

AK065039

2.05

Putative 1-aminocyclopropane-1-carboxylate oxidase 1 (ACC oxidase 1)

BAD38208 (1e-171)

Cell structure

 Os.49281.1.S1_s_at

AK062457

4.40

Putative glycine rich protein

BAD61563 (1e-11)

 Os.38152.1.S1_at

AK065615

3.41

LrgB-like family protein expressed

AAP55171 (1e-73)

 OsAffx.11838.1.S1_at

9629.m07254

2.53

Putative bundle sheath cell specific protein 1

BAB63632 (8e-60)

 Os.55444.1.S1_at

AK108103

2.34

Glycine-rich protein

NP_193893 (4e-08)

 Os.10370.1.S1_at

AK062370

2.32

Putative glycine rich protein

BAD61558 (1e-14)

 Os.10370.1.S1_x_at

AK062370

2.27

Putative glycine rich protein

BAD61558 (1e-14)

Disease/defense

 Os.10401.1.S1_s_at

AK070510

2.65

Putative MtN3 (disease resistant allele XA13)

BAD13168 (1e-141)

 Os.51641.1.S1_at

AK063248

2.55

Putative pathogenesis-related protein/PR1 like

BAC84818 (2e-67)

 Os.37893.1.S1_at

AK100346

2.46

Phenylalanine ammonia-lyase

AAO72666 (0.0)

 Os.37890.1.S1_s_at

AK061288

2.09

Protease inhibitor/seed storage/Lipid Transfer Protein family putative

AAX96637 (4e-39)

 Os.9679.1.S1_at

AK060077

2.03

Disease resistance-responsive family protein putative expressed

ABA91787 (1e-90)

 Os.46305.1.S1_at

AK122171

2.01

Leucine Rich Repeat family protein expressed

ABA96188 (0.0)

 Os.51641.1.S1_x_at

AK063248

2.86

Putative pathogenesis-related protein/ PR1 like

BAC84818 (5e-68)

Energy

 Os.12181.1.S1_s_at

AK098872

3.26

Chlorophyll a/b-binding protein CP26 precursor - maize

AAX95978 (7e-146)

 Os.37713.1.S1_at

AK070051

3.23

Putative photosystem I antenna protein

BAC83072 (5e-55)

 Os.28216.2.S1_a_at

AK119545

2.59

Putative chlorophyll A-B binding protein of LHCII type III chloroplast precursor (CAB)

BAC83393 (2e-153)

 OsAffx.18836.1.S1_at

9639.m01283

2.58

Chlorophyll a/b-binding protein CP26 precursor - maize

AAX95980 (9e-113)

 Os.5869.3.S1_x_at

CB667246

2.02

Chlorophyll a/b-binding protein CP24 precursor

AAD27882 (8e-78)

Intracellular traffic

 Os.2759.1.S1_s_at

AK099409

2.46

Mitochondrial carnitine/acylcarnitine carrier putative expressed

AAP55124 (3e-116)

 Os.11654.1.S1_at

AK065544

2.26

SOUL heme-binding protein-like

BAD32927 (8e-174)

Metabolism

 Os.11789.1.S1_at

AK099538

3.18

Putative lysine decarboxylase-like protein

AAN61486 (1e-111)

 Os.26441.2.S1_at

AK119461

2.84

Putative beta-glycosidase

CAD36515 (0.0)

 Os.7669.1.S1_at

CB643976

2.69

Putative fatty acid elongase

AAR96244 (5e-57)

 Os.6595.1.S1_a_at

AK062262

2.65

Magnesium-chelatase subunit H family protein expressed

ABF95686 (0.0)

 Os.14145.1.A1_at

AK070312

2.56

Ferric reductase

BAD18962 (0.0)

 Os.27713.1.A1_at

AK120567

2.54

Beta-ketoacyl-coA synthase putative expressed

ABA94525 (0.0)

 Os.12025.1.S1_a_at

AK099966

2.43

Hydrolase/alpha/beta fold family protein

NP_175660 (3e-153)

 Os.15570.1.S1_at

AK068586

2.40

Putative glucosyltransferase-3 UDP-glucoronosyl and UDP-glucosyl transferase

BAC83960 (0.0)

 Os.25109.1.S1_at

AK105536

2.35

Mannan endo-1 4-beta-mannosidase 7 (OsMAN7)

Q2RBB1 (0.0)

 Os.17814.2.S1_x_at

AK068064

2.33

Putative malate dehydrogenase

BAC20686 (0.0)

 Os.12839.1.S1_at

AK060512

2.26

Putative beta-keto acyl reductase

BAD22122 (1e-103)

 Os.26486.1.S1_at

AK120227

2.15

Putative glossy1 protein/Sterol desaturase

BAD33619 (0.0)

 OsAffx.27508.100.S1_s_at

AK108972

2.15

Putative methionyl aminopeptidase

BAD08973 (3e-18)

 Os.9494.1.S1_s_at

CF292249

2.14

Short-chain dehydrogenase/reductase SDR family protein

NP_001053404 (6e-18)

 Os.9724.1.S1_at

AK104714

2.14

Very-long-chain fatty acid condensing enzyme putative expressed

ABF94686 (0.0)

 Os.22241.1.S1_s_at

AK099444

2.13

Nitrilase-associated protein putative expressed

ABA98636 (2e-13)

 Os.14118.1.S1_at

AK099064

2.09

Putative lipase

AAP33477 (3e-100)

 Os.36960.1.S1_at

AK062283

2.08

Glucosyltransferase NTGT2-like

BAD82532 (0.0)

 Os.11403.1.S1_at

AK073262

2.07

Putative N-carbamyl-L-amino acid amidohydrolase

BAD45389 (0.0)

 Os.8569.1.S1_at

AK060559

2.01

Beta-carotene hydroxylase putative expressed

ABF93742 (9e-98)

 Os.20755.1.S1_at

AK068040

2.13

UDP-glucoronosyl/UDP-glucosyl transferase family protein

NP_196793 (2e-87)

Otherf

 Os.51579.1.S1_at

AK063100

5.03

No significant similarity found

 

 Os.37849.1.A1_at

AK068289

3.73

Unnamed protein product

BAA90498 (2e-07)

 Os.17536.1.S1_at

AK063180

2.90

Unknown protein

BAD36443 (3e-63)

 Os.34400.1.S1_at

AK071545

2.61

Protein domain with at least 5 transmembrane alpha-helices

NP_001042652 (2e-102)

 OsAffx.24703.1.S1_s_at

9630.m04191

2.60

No significant similarity found

 

 Os.11944.1.S1_at

AK070758

2.53

No significant similarity found

 

 Os.55256.1.S1_at

AK107743

2.51

Expressed protein

AAP54198 (8e-09)

 Os.24338.1.A1_at

AK111667

2.47

Os01g0908300 .

NP_001045142 (8e-51)

 Os.5066.1.S1_at

AK073193

2.45

Os01g0219300

NP_001042419 (4e-44)

 Os.8395.1.S1_at

AK069514

2.31

Expressed protein

ABA98963 (3e-70)

 OsAffx.23005.1.S1_x_at

AK108349

2.31

Unknown protein

BAD67880 (2e-17)

 OsAffx.23005.1.S1_at

AK108349

2.30

Unknown protein

BAD67880 (2e-17)

 Os.10615.1.S1_x_at

AK058921

2.22

No significant similarity found

 

 Os.10524.2.S1_at

AK060098

2.21

FHA domain containing protein expressed

ABA95708 (4e-83)

 Os.5648.1.S1_at

AU172533

2.21

No significant similarity found

 

 OsAffx.14410.1.S1_s_at

9632.m05269

2.13

Os04g0635000 .

NP_001054004 (6e-11)

 Os.25052.1.S1_at

CA763439

2.10

Hypothetical protein OsJ_000675

EAZ10850 (2e-23)

 Os.49304.1.A1_at

AK063613

2.08

Hypothetical protein OsI_013772

EAY92539 (1e-34)

 Os.27659.1.S1_x_at

AK069734

2.03

TMS membrane protein/tumour differentially expressed protein

ABE91594 (4e-133)

 Os.11190.1.S1_at

AK058995

2.01

Os04g0402700/Hypothetical protein

NP_001052695 (7e-28)

Protein synthesis/turnover

 Os.11707.1.A1_at

CB656443

4.46

Putative cysteine proteinase

AAM34401 (7e-37)

Secondary metabolism

 Os.9685.1.S1_a_at

AK067949

2.51

Putative cinnamoyl CoA reductase

BAD28656 (2e-171)

 Os.46551.1.S1_at

AK064736

2.31

Putative chalcone flavonoid 3′ - hydroxylase

AAN04937 (0.0)

Signal transduction

 OsAffx.16737.1.S1_at

AK067579

3.35

Phototropic-responsive protein putative/a light-activated serine-threonine protein kinase

NP_187478 (3e-31)

 Os.12110.1.S1_at

AK064985

2.88

RPT2 expressed /nonphototropic hypocotyl 1 (NPH1)/a serine-threonine protein kinase

ABA91174 (0.0)

 Os.55822.1.A1_at

AK108850

2.39

Putative serine/threonine protein phosphatase 2A (PP2A) regulatory subunit B’

BAD67828 (1e-95)

 Os.7676.1.S1_at

AY476807

2.30

Extracellular calcium sensing receptor

AAS00828 (1e-146)

 Os.7140.1.S1_at

AK061014

2.28

GUN4-like family protein expressed/regulator of chlorophyll synthesis and intracellular signaling

ABA92587 (4e-110)

 Os.45531.1.S1_at

CB621884

2.19

Receptor-like kinase

AAF78017 (1e-48)

 Os.7304.1.S1_at

AK069537

2.16

Putative protein kinase

BAB92217 (0.0)

 Os.28255.1.S1_at

AK070519

2.07

ABC1 family protein /Mn2+-dependent serine/threonine protein kinase

NP_565025 (0.0)

Stress

 Os.53572.1.S1_at

AK073356

3.66

Universal stress protein USP1-like protein

BAC16006 (2e-84)

 Os.37549.1.S1_at

AK102998

3.35

Auxin/aluminum-responsive protein putative

NP_197415 (3e-10)

 Os.15692.1.S1_at

AK069017

2.68

Cytochrome P450 family protein expressed

ABF97430 (0.0)

 Os.5377.1.S1_at

AK105891

2.52

Selenium-binding protein-like (pentatricopeptide repeat-containing protein)

BAD07861 (1e-152)

 Os.34316.1.S1_at

AK071882

2.37

Hypothetical protein OsJ_021319/DnaJ domains

EAZ37836 (9e-21)

 Os.17037.1.S2_a_at

CB671899

2.20

Putative Altered Response to Gravity/DnaJ domain

BAD73072 (7e-21)

 Os.35448.1.S1_at

AK067480

2.20

Thioredoxin h-like protein

AAN63618 (4e-17)

Transcription

 Os.46600.1.S1_at

AK058809

3.77

Helix-loop-helix DNA-binding domain containing protein

AAP53429 (7e-113)

 Os.56880.1.S1_at

AK110526

3.66

Basic-leucine zipper (bZIP) transcription factor domain containing protein

NP_001043273 (5e-39)

 Os.31716.2.A1_at

BI305433

3.03

Scl1 protein/GRAS family transcription factor

AAF00139 (2e-24)

 Os.49848.1.S1_at

AK111960

2.71

Putative MYB-related protein 1

BAD07916 (4e-71)

 Os.25588.2.S1_x_at

AK101674

2.67

Putative MYB protein

BAC79723 (5e-88)

 Os.3386.1.S1_x_at

AK062487

2.63

MYB protein

BAD36195 (7e-26)

 Os.27085.1.A1_at

CB656188

2.34

Putative typical P-type R2R3 MYB protein/SANT domain containing protein

BAD10148 (2e-12)

 Os.46081.1.S1_at

AK060177

2.18

Hypothetical protein OsJ_029817/transcription factor activity

EAZ15608 (2e-96)

 Os.16025.1.S1_s_at

CA764046

2.14

Putative bZIP protein HY5

BAD32844 (1e-07)

 Os.19539.1.S1_at

AK058932

2.04

Putative DNA helicase

BAD08079 (0.0)

 Os.11409.1.S1_at

AK063523

2.04

Basic helix-loop-helix putative expressed

ABF93851 (6e-36)

 Os.17902.1.S1_at

AK111634

2.03

MYB17 protein

CAD44611 (7e-34)

 Os.26437.1.A1_s_at

CB673064

2.02

bZIP transcription factor family protein expressed

ABA99796 (6e-51)

 Os.23030.1.S1_at

AK060509

2.05

Arabidopsis NAC domain containing protein 83; transcription factor

NP_196822 (4e-24)

Transporter

 Os.11804.1.S1_at

CB624264

3.27

Tryptophan/tyrosine permease family protein expressed .

ABF95016 (3e-111)

 Os.52259.1.S1_at

AK065945

2.55

Major Facilitator Superfamily protein expressed

ABA91097 (0.0)

 Os.5210.1.S1_at

AK072183

2.49

Putative sodium-dicarboxylate cotransporter

BAD09278 (0.0)

 Os.19896.2.S1_x_at

CB623166

2.47

Putative sodium transporter

CAD37186 (1e-64)

 Os.41637.1.S1_at

CA766063

2.40

MDR-like ABC transporter

CAD59585 (8e-14)

 Os.27393.1.S1_s_at

AK061521

2.39

MDR-like ABC transporter

CAD59590 (0.0)

 Os.57530.1.S1_x_at

AJ491855

2.27

Putative sodium transporter

CAD37198 (0.0)

 Os.25449.1.S1_at

AK108531

2.21

Integral membrane transporter family protein

NP_180886 (1e-62)

 Os.17299.1.S1_at

CB655994

2.18

Putative glycerol 3-phosphate permease

AAS82603 (7e-112)

 Os.52960.1.S1_x_at

AK072316

2.17

Putative amino acid permease

AAP46198 (3e-146)

 Os.17681.1.S1_at

AK072059

2.14

Monosaccharide transporter 1

BAB19862 (4e-158)

 Os.21635.1.S1_at

AF543419

2.11

Major facilitator superfamily antiporter

AAN33182 (0.0)

 Os.52621.1.S1_x_at

AK068351

2.04

Nitrate transporter putative

AAT85061 (8e-162)

Transposons

 Os.16593.1.S1_at

BI803439

2.14

Retrotransposon protein putative Ty3-gypsy subclass expressed

ABA92095 (1e-34)

 Os.24273.1.A1_at

AK102986

2.07

Hypothetical protein osi_009106/transposase activity

EAY87873 (5e-30)

aAffymetrix Probe set ID of downregulated gene

bThe public representative ID of these genes obtained from Affymetrix NetAffx analysis center

cPLIER normalized absolute fold change value for downregulation

dThe putative function of the gene based on BLASTX analysis

eAccession ID of the homologous protein based upon which the function has been assigned. The corresponding BLASTX E- score is shown in brackets

fGenes encoding hypothetical proteins or those with no significant matches in the databases are kept in this category

Increased expression of functions involved in the JA-mediated defense pathway during ClsA-induced HR

A rice transcription factor (OsRERJ1) gene that had been previously found to be induced by methyl JA treatment (Kiribuchi et al. 2004) was upregulated (~5.3-fold) following ClsA treatment. The gene for a putative ethylene binding protein/APETALA2 type of transcription factor (OsAP2/ERF) was upregulated (4- to 7-fold) following ClsA treatment. Two different probe sets for this gene are present on this chip: one probe set (Os.15849.1.S1_s_at) exhibited a 4.3-fold upregulation while the other (OsAffx.17366.1.S1_at) exhibited a 6.5-fold upregulation. This gene is homologous to subfamily 3 of AP2/ERF transcription factors in Arabidopsis; one member of this subfamily (AtERF1) is involved in integration of the JA and ET pathways (Lorenzo et al. 2003) whereas another member (AtERF2) functions as an activator in JA signaling (McGrath et al. 2005). The genes for five ZIM motif family proteins are upregulated following ClsA treatment (Table 3). The Arabidopsis homologs of four of these proteins have been shown to be upregulated by JA (Chini et al. 2007; Thines et al. 2007). These proteins function as negative regulators of the JA signaling pathway, and their upregulation has been attributed to serve in dampening the JA pathway after its initial induction. Furthermore, the ZIM family proteins repress a MYC transcription factor, which is the key activator of the JA signaling pathway (Lorenzo et al. 2004). A gene for the rice ortholog (Os.46443.1.s1_at) of this MYC factor (OsMYC) is upregulated 1.49-fold by ClsA treatment.
Table 3

List of Cellulase Responsive Rice Genes Associated with Jasmonic-Acid-Mediated Defense Pathway

Probe set IDa

Public IDb

FCAc

Gene descriptiond

Homolog (E score)e

OsAffx.17366.1.S1_at f

9636.m03709

6.54

Ethylene-binding protein-AP2 domain containing

BAD38371 (4e-41)

Os.6043.1.S1_at

AB040744.1

5.30

RERJ1(Helix-loop-helix domain containing transcriptin factor)

AB040744 (8e-111)

Os.15849.1.S1_s_at f

AK062882.1

4.34

AP2 domain-containing transcription factor-like

BAD17116 (4e-15)

Os.55259.1.S1_at

AK107750.1

3.77

ZIM motif family protein, expressed

ABF94311 (1e-07)

Os.4671.1.S1_a_at

AF435970.1

3.47

Jacalin lectin family protein, putative

P24120 (2e-78)

Os.46849.1.S1_at

AK107854

3.46

ZIM motif family protein, expressed

AAP53563 (7e-23)

Os.9923.1.S1_s_at

AK120087.1

2.58

ZIM motif family protein, expressed

ABF94311 (9e-57)

Os.8741.1.S1_at

AK102303.1

2.57

Poly polymerase catalytic domain containing protein/RADICAL-INDUCED CELL DEATH1

ABF94777 (0.0)

Os.35343.1.A1_at

AK067922.1

2.51

No apical meristem protein (NAC domain containing)

ABA95706 (2e-116)

Os.10356.1.S1_at

AK073589.1

2.09

ZIM motif family protein, expressed

ABF94310 (2e-82)

Os.50513.1.S1_at

AK121554.1

2.05

Putative 12-oxophytodienoate reductase (OPR2)

BAD61319 (0.0)

Os.12012.1.S1_at

AK061602.1

1.97

ZIM motif family protein, expressed

AAP53568 (5e-83)

Os.22000.1.S1_at

AK061042.1

1.95

Endochitinase/ putative PR3 like

BAA03749 (9e-166)

Os.8778.1.S1_a_at

AB040743.1

1.63

Cis-12-oxo-phytodienoic acid-reductase 1 (OPR1)

BAD26703 (0.0)

Os.3415.1.S1_s_at

AB016497.1

1.51

Chitinase/ putative PR3 like

BAA31997 (2e-135)

Os.46443.1.S1_at

AY536428.1

1.46

MYC protein/ATMYC2 (JASMONATE INSENSITIVE 1) ortholog

AAS66204 (0.0)

aAffymetrix Probe set ID

bThe public representative ID obtained from Affymetrix NetAffx analysis center

cPLIER normalized absolute fold change value

dThe putative function of the gene based on BLASTX analysis

eAccession ID of the homologous protein based upon which the function has been assigned. The corresponding BLASTX E score is shown in brackets

fThese are two probe set IDs for the same gene

Allene oxide synthase is a key enzyme in the JA biosynthetic pathway (Turner et al. 2002). The Affymetrix rice array contains two paralogous genes that encode allene oxide synthase. One of them (Affymetrix probe set ID, Os.8266.1.A1_at) was found to be upregulated ~2.5-fold by the PLIER algorithm and ~1.95-fold by RMA but was not identified at p < 0.05. The other gene (Affymetrix probe set ID, Os.7678.1.S1_at) has been earlier shown to be involved in JA biosynthesis in rice (Mei et al. 2006). It was identified as being upregulated by the PLIER algorithm (~1.7-fold at p < 0.05) but was not selected as being upregulated using RMA. We assessed the expression of this gene (Os.7678.1.S1_at) by quantitative real-time PCR and found that it was upregulated ~2.0-fold at p < 0.05 following ClsA treatment (Fig. 3; Supplementary Table S2, for the list of primers). Furthermore, as described above, pathway analysis had revealed the upregulation of two 12-oxophytodienoate reductase genes (OsOPR2, Os.50513.1.S1_at; OsOPR1, Os.8778.1.S1_a_at) that are predicted to be involved in JA biosynthesis (Table 3). ClsA treatment also induced the expression of two paralogous rice chitinase (PR-3) genes whose Arabidopsis ortholog is involved in a JA-mediated defense pathway (Thomma et al. 1998). ClsA treatment led to increased expression of a rice ortholog of the Arabidopsis RADICAL-INDUCED CELL DEATH-1 gene which has been postulated to modulate methyl JA responses (Ahlfors et al. 2004). A gene encoding a jacalin-related lectin like protein which is homologous to a JA-induced protein of wheat and barley (Lee et al. 1996; Wang and Ma 2005) was ~3.5-fold induced by ClsA treatment.

The gene for the rice ortholog of Arabidopsis COPINE1 (a negative regulator of SA-associated systemic-acquired resistance; Jambunathan et al. 2001) was ~3-fold upregulated during ClsA-induced HR. Interestingly, genes that are putatively involved in SA modification were upregulated. Two different probe sets (Os.10546.1.S1_s_at and OsAffx.30138.1.S1_at) for a gene encoding a putative UDP-glucose/salicylic acid glucosyltransferase (OsSGT; predicted to be involved in glucosylation and inactivation of SA; Chen et al. 1995) were upregulated ~2.9-fold. The gene for a putative benzothiadiazole-induced S-adenosyl-l-methionine:salicylic acid carboxyl methyltransferase1 (OsBSMT1; which is predicted to be involved in volatilizing SA; Koo et al. 2007) was ~2.0-fold upregulated. Glycosylated SA is an inactive storage form of SA (Chen et al. 1995) whereas methylated SA is volatile (Koo et al. 2007); thus, both modifications might lead to overall reduction of the cellular pool of SA. Increased expression of the JA pathway has been suggested to lead to a downregulation of the SA pathway (Takahashi et al. 2004; Kunkel and Brooks 2002; Spoel et al. 2003).

Validation of differentially expressed biologically significant genes by real-time PCR analysis

The expression profile of a few selected biologically significant genes was also examined by SYBR green based real-time PCR. Primers were designed to amplify 130–170 bp gene fragments of eight upregulated genes (OsAP2/ERF, OsRERJ1, OsMYC, OsOPR2, OsAOS, OsCOPINE1, OsSGT, and OsBSMT1) and one downregulated gene (OsMtN3; a host susceptibility factor; see Supplementary Table S2; for list of primers). The results indicate that OsAP2/ERF, OsRERJ1, OsMYC, OsOPR2, OsAOS, OsCOPINE1, OsSGT, and OsBSMT1 are upregulated while the OsMtN3 gene is down regulated following ClsA treatment (Fig. 2).
Fig. 2

Real-time PCR analysis of some differentially expressed genes. Total RNA was isolated from either ClsA- or buffer-treated rice leaves; first-strand cDNA was synthesized, and gene expression was assayed by SYBR green-based real-time PCR. The genes that were assayed are the following: OsAP2 (AP2/ERF transcription factor), OsRERJ1 (methyl JA induced helix loop helix rice transcription factor), OsOPR2 (a 12-oxophytodienoate reductase), OsMYC (a key transcription factor in the JA-mediated defense pathway), OsAOS (Allene Oxide Synthase), OsCOPINE1, OsSGT (salicylic acid glucosyltransferase), OsBSMT1 (benzothiadiazole-induced S-adenosyl-L methionine:salicylic acid carboxyl methyltransferase1), and OsMtN3 (Os8N3; a host susceptibility factor). The relative fold changes were calculated by using 2-ddCT method using the expression of buffer treated leaves as a comparison. The OsGAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene was used as an endogenous control. Mean and standard deviation from at least four biological replicates for each gene are presented.

Wild-type X. oryzae pv. oryzae suppressed ClsA-induced expression of OsAP2/ERF and OsRERJ1

Rice seedlings were infiltrated with either ClsA or ClsA along with wild-type X. oryzae pv. oryzae in order to determine if the ClsA-induced expression of OsAP2/ERF, and OsRERJ1 is suppressed by co-treatment with the bacterium. Expression of these genes was analyzed by SYBR green-based quantitative real-time PCR in three independent biological replicates. The relative fold change was calculated in comparison to their expression in buffer-treated leaves and the OsGAPDH gene as an endogenous control. Although there was variability between individual experiments, overall, the results indicated that co-treatment with wild-type X. oryzae pv. oryzae suppressed the induction of expression of OsAP2/ERF and OsRERJ1 by ClsA at significance level p < 0.05 (Table 4).
Table 4

Suppression of Cellulase-induced Expression of OsRERJ1 and OsAP2/ERF Transcription Factor Genes by Wild-type X. oryzae pv. oryzae

Genesa

Experiment-1b

Experiment-2b

Experiment-3b

ClsAc

ClsA + Xooc

ClsAc

ClsA + Xooc

ClsAc

ClsA + Xooc

OsAP2/ERF

5.56

1.33

5.11

1.43

2.64

0.66

OsRERJ1

8.26

1.48

9.45

1.7

5.86

1.36

aRice genes upregulated following cellulase treatment

bFifteen days old rice seedlings were infiltrated with either cellulase (ClsA) or cellulase along with wild type X. oryzae pv. oryzae (ClsA + Xoo). Buffer-treated leaves were used as control. Total RNA was isolated from each of the samples, converted into cDNA and subjected to real-time PCR analysis

cThe relative fold changes were calculated by 2–ddCT method in comparison to expression in buffer treated leaves. The OsGAPDH (Glyceraldehyde-3-phosphate dehydro-genase) gene was used as an endogenous control

Sequence features of OsAP2/ERF

The OsAP2 (APETALA2)/ERF gene that is upregulated following ClsA treatment is an 849 bp intron less gene encoding a 282 amino acid protein. Sequence analysis of the OsAP2/ERF using prosite (http://www.expasy.ch/prosite) has revealed that it contains a single AP2/ERF domain which spans from 138–195 amino acid residues (Supplementary Fig. S2). The presence of A and D amino acid at position 151 and 156 is a characteristic of AP2/ERF proteins which distinguishes them from related DREB proteins wherein the V and E amino acids are present at the corresponding positions (Sakuma et al. 2002). AP2/ERF domain has two sub-domains: YRG, involved in DNA binding and RAYD, which is probably involved in DNA binding through the interaction of its hydrophobic phase with the major groove of DNA (Okamuro et al. 1997). The protein also contains nuclear localization signal sequences. The protein is predicted to contain two α helices and four β sheets. Amino acid residues in the pink box are predicted to be in direct contact with DNA (Allen et al. 1998; Nakano et al. 2006). Besides this, presence of a putative zinc finger motif (C×2C×4C×2~4C) on the N-terminal region of the protein suggests its involvement in DNA binding or protein–protein interactions. Due to presence of this motif, the protein has been previously classified to be a member of Xb subgroup of AP2/ERF proteins of rice (Nakano et al. 2006).

Agrobacterium-mediated transient transfer of OsAP2/ERF induces defense responses such as a PCD reaction in rice roots and callose deposition in leaves

PCD is a form of plant defense response (Pennell and Lamb 1997). We treated rice roots with Agrobacterium strain LBA4404/pOsAP2/ERF and stained with propidium iodide (PI) followed by confocal microscopy in order to determine if transient transfer of OsAP2/ERF induces PCD. Cell-wall-associated fluorescence was observed in roots treated with Agrobacterium strain LBA4404/pSB11; very few cells took up PI stain, and the distribution of the stain indicated that the nucleus remained compact (Fig. 3). In contrast, the PI stain was taken up by all the cells in rice roots treated with LBA4404/pOsAP2/ERF indicating that extensive cell death has been induced (Fig. 3). As expected, cell death was not observed in roots treated with water, and extensive cell death was observed in roots treated with ClsA. Syringe infiltration of rice leaves with LBA4404/pOsAP2/ERF strain but not LBA4404/pSB11 strain induced callose deposition in rice leaves. As expected, infiltration with ClsA induced callose deposition while only background levels of callose deposition were observed in leaves infiltrated with water (Fig. 3, Table 5).
Fig. 3

Transient transfer of OsAP2/ERF induces callose deposition in rice leaves and cell death in rice roots. Rice leaves were syringe infiltrated with one of the following: water, ClsA, LBA4404/ pSB11, and LBA4404/pOsAP2/ERF. After 15 h, they were stained with aniline blue and examined under epifluorescence microscope. White spots in these pictures are indicative of callose deposition. b cellulase and d LBA4404/pOsAP2/ERF induce increased callose deposition compared with either a water or c LBA4404/pSB11. Rice roots were also treated either with water, cellulase, LBA4404/pSB11, LBA4404/pOsAP2/ERF and stained with propidium iodide (PI), and examined under a confocal microscope. Treatment with either water (e) or LBA4404/pSB11 (g) did not induce cell death (intake of PI), whereas treatment with cellulase (f) and LBA4404/pOsAP2/ERF (h) induced cell death and extensive nuclear fragmentation. Similar results were obtained in at least three independent experiments.

Table 5

Transient Transfer of OsAP2/ERF Induces Callose Deposition in Rice Leaves

Mean number of callose deposits/0.6 mm2 area ± SDa

Expb

Water

Cellulase

LBA4404/pSB11

LBA4404/pOsAP2/ERF

1

13.30 ± 4.08

141.20 ± 37.43

23.10 ± 10.07

87.00 ± 15.29

2

11.70 ± 3.09

154.00 ± 37.21

29.00 ± 15.81

97.50 ± 35.73

3

14.40 ± 3.50

126.10 ± 29.17

25.50 ± 6.43

80.00 ± 16.47

aRice leaves were syringe infiltrated with one of the following: water, cellulase (100 µg ml−1), LBA4404/pSB11, and LBA4404/pOsAP2/ERF, stained with aniline blue, and examined under epifluorescence microscope. Mean and standard deviation were calculated for number of callose deposits from a leaf area of 0.60 mm2

bData were collected from at least ten leaves in each experiment. ANOVA was performed, and the values obtained after infiltration with LBA4404/pOsAP2/ERF were found to be significantly different (p < 0.05) as compared to all other treatments

Prior treatment with LBA4404/pOsAP2/ERF strain induces resistance to subsequent X. oryzae pv. oryzae infection in rice

The midveins of leaves from 40-day-old rice plants were preinjected with one of the following: water and saturated culture of LBA4404/pSB11 and LBA4404/pOsAP2/ERF. All leaves were subsequently infected with X. oryzae pv. oryzae. Across four independent experiments, all the leaves (100%) that had been preinjected with water exhibited lesions induced by X. oryzae pv. oryzae. About 72–77% of the leaves which were preinjected with LBA4404/pSB11 exhibited X. oryzae pv. oryzae-induced lesions. Interestingly, only 30–50% of leaves that were preinjected with LBA4404/pOsAP2/ERF exhibited disease lesions (Table 6). Among the leaves that exhibited disease lesions, the lesion lengths were 3.56 ± 1.24 to 5.58 ± 1.73 cm, 7.71 ± 5.30 to 10.8 ± 7.36 cm, and 10.18 ± 3.66 to 11.86 ± 4.82 cm long following preinjection with LBA4404/pOsAP2/ERF, LBA4404/pSB11, and water, respectively.
Table 6

Transient Transfer of OsAP2/ERF Induces Resistance Against Xanthomonas oryzae pv. oryzae Infection

Treatmentsa

% of infected leaves (no. of infected leaves/total no. of leaves)b

Mean lesion length of leaves showing disease symptoms (cm) ± SDc

Exp 1

Exp 2

Exp 3

Exp 4

Exp 1

Exp 2

Exp 3

Exp 4

Water

100 (19/19)

100 (28/28)

100 (21/21)

100 (25/25)

11.2 ± 7.01

10.50 ± 6.42

11.86 ± 4.82

10.18 ± 3.66

LBA4404/ pSB11

72 (18/25)

75 (21/28)

77 (14/18)

76 (19/25)

10.8 ± 7.36

9.38 ± 5.93

7.71 ± 5.30

8.68 ± 5.75

LBA4404/ pOsAP2/ERF

30 (7/23)

50 (10/20)

50 (9/18)

48 (12/25)

3.57 ± 1.51

5.0 ± 1.56

3.56 ± 1.24

5.58 ± 1.73

aMidveins of leaves of 40 days old rice plants (TN-1) were preinjected with either water alone, or water containing ~109 cells of LBA4404/pSB11 or LBA4404/pOsAP2/ERF. After 6 h, Xanthomonas oryzae pv. oryzae was inoculated onto the midvein, 2 to 3 cm below the point of initial injection, by pricking with a needle that had been used to touch a freshly grown colony of Xanthomonas oryzae pv. oryzae. Lesion lengths were measured 10 days after inoculation

b18–28 leaves were used in each experiment and four independent replications were performed

cLesion lengths were measured 10 days after inoculation. Mean and standard deviation were calculated for each of the treatments. ANOVA was performed and the values obtained after treatment with LBA4404/pOsAP2/ERF were found to be significantly different (p < 0.05) as compared to all other treatments

Discussion

X. oryzae pv. oryzae secretes a cocktail of at least five cell-wall-degrading enzymes, including ClsA, to damage the plant cell wall during infection. Besides these five enzymes, the X. oryzae pv. oryzae genome additionally encodes five cellulases, three xylanases, one pectinase as well as several other categories of cell-wall-degrading enzymes (Lee et al. 2005). It is possible that many of these enzymes are expressed only during in planta growth, and it is likely that the action of each one of these enzymes will induce rice innate immune responses. In spite of the apparent importance of cell-wall-degrading enzymes for induction of host innate immunity (Jha et al. 2007), very little information is available about plant functions that participate in the elaboration of innate responses that are induced following their action.

To address this lacuna, we have performed transcriptional profiling of rice gene expression changes that occur following cell-wall damage initiated by treatment with X. oryzae pv. oryzae ClsA. In our experiments, we have infiltrated 5 μg of ClsA protein into approximately 1 cm2 of leaf area although we find that infiltration of even 4 μg of enzyme is sufficient to induce a HR like response. In the laboratory, approximately 4 μg of ClsA is secreted per milliliter of a saturated culture (approx. 1 × 109 cell) of X. oryzae pv. oryzae. During infection, 1 cm2 of leaf area supports 1 × 109 cells of X. oryzae pv. oryzae, and we expect that these cells would secrete about 4 μg of ClsA. As a number of other cell-wall-degrading enzymes, besides ClsA, are secreted by X. oryzae pv. oryzae, we expect that the total amounts of cell-wall-degrading enzymes secreted by X. oryzae pv. oryzae is going to be much higher than that used in this experiment. Besides the above, there is evidence that secretion by T2S of V. cholerae and P. aeruginosa occurs in a polar manner because the T2S apparatus is localized to one pole of the cell, possibly at the point of attachment to host surface (Scott et al. 2001; Senf et al. 2008). If the T2S of X. oryzae pv. oryzae is similarly localized, the local concentrations of cell-wall-degrading enzymes are likely to be quite high during infection.

We have performed homology searches using BLASTX for each of the 267 genes that were found to be differentially expressed (≥2-fold at p < 0.05) following ClsA treatment. A striking observation is that a large number of putative transcription factors (26 upregulated and 15 downregulated) are differentially expressed following ClsA treatment. This is suggestive of global changes in rice gene expression during the HR like reaction. The microarray analysis has revealed the downregulation of genes for several chlorophyll a/b binding proteins and suppression of the rice ortholog of GUN4, a critical activator of chlorophyll synthesis in Arabidopsis (Larkin et al. 2003). The gene ontology enrichment analysis also indicated that functions involved in photosynthesis are downregulated. This suggests an overall downregulation of synthesis of chlorophyll and chlorophyll-binding proteins during ClsA-induced cell death and a possible reduction in photosynthetic efficiency during this process. It was also interesting to note that the gene which exhibits maximum upregulation in our microarray is homologous to a gene encoding a senescence associated protein of Arabidopsis (AT1G74940.1) suggesting a possible correlation between some events associated with senescence and HR. Three condensing enzymes and a ß-ketoacyl reductase that are homologous to Arabidopsis functions that participate in cuticle development were also downregulated following ClsA treatment. Defects in cuticle development have been shown to cause enhanced resistance to Botrytis cinareae and enhanced disease symptoms in response to an avirulent strain of Pseudomonas syringae pv. tomato in Arabidopsis thaliana (Chassot et al. 2007; Bessire et al. 2007; Tang et al. 2007).

We had earlier demonstrated that co-treatment with wild-type X. oryzae pv. oryzae results in suppression of ClsA-induced rice defense responses. We demonstrate here, using real-time PCR, that co-treatment with wild-type X. oryzae pv. oryzae also results in suppression of ClsA-induced expression of the rice transcription factor genes OsAP2/ERF and OsRERJ1. AP2/ERF members are downstream components of both the ethylene and jasmonatic acid (JA) pathways and are key to the integration of both signals (Lorenzo et al. 2003; Zhang et al. 2004a, b). The OsRERJ1 gene is upregulated by JA (Kiribuchi et al. 2004). Chini et al. (2007) and Thines et al. (2007) have demonstrated that several Arabidopsis ZIM family transcription factors serve as negative regulators of the JA pathway through repression of a MYC transcription factor (JASMONATE INSENSITIVE1, JIN1; Lorenzo et al. 2004), which is the key activator of JA responsive genes. JA promotes ubiquitination and degradation of these ZIM transcription factors, resulting in expression of MYC and activation of the JA responsive genes. Transcription of the genes for these ZIM transcription factors is upregulated by JA, and this has been attributed to serve in damping the JA pathway (Chini et al. 2007; Thines et al. 2007). The microarray data presented here indicates that rice homologs of these ZIM transcription factors and OsMYC (or OsJIN1) are upregulated by ClsA treatment. Besides the above, we have found that several genes that encode functions predicted to be involved in JA biosynthesis are upregulated following ClsA treatment. These results, taken together, suggest that the JA-mediated pathway might be associated with ClsA-induced HR. This needs to be confirmed using rice lines that are either mutated or knocked down for the JA pathway.

Nakano et al. (2006) indicate that the genome sequences of Arabidopsis and rice contain 122 and 139 ERF proteins, respectively, which are subdivided into I to X subgroups. Our sequence analysis indicates that the OsAP2/ERF studied here belongs to Xb subgroup of the ERF subfamily which is characterized by the presence of Cys repeats at its N-terminal region. We have treated rice leaves and roots with LBA4404/pOsAP2/ERF in order to assess the effect of transient transfer of OsAP2/ERF on induction of innate immune responses. Rice leaves infiltrated with LBA4404/pOsAP2/ERF showed a significant increase in callose deposition as compared to leaves treated with LBA4404/pSB11 (control). Similarly, rice roots treated with LBA4404/pOsAP2/ERF exhibit programmed cell death, which is absent in roots treated with LBA4404/pSB11. Prior injection of LBA4404/pOsAP2/ERF in rice mid-veins induces resistance against subsequent infection with X. oryzae pv. oryzae. Treatment with LBA4404/pSB11 also induces a certain degree of resistance against subsequent bacterial infection as evident from fewer leaves showing X. oryzae pv. oryzae induced disease lesions. Although this resistance is significantly lesser than that induced by LBA4404/pOsAP2/ERF, it suggests that Agrobacterium can also induce a certain amount of resistance. Overall, these results suggest that transient overexpression of OsAP2/ERF induces rice resistance responses. However, these results need to be confirmed by making stably transgenic rice lines that overexpress OsAP2/ERF, preferably under the control of an inducible promoter.

Besides the cell-wall-degrading enzymes secreted by X. oryzae pv. oryzae, several other molecules are known to be elicitors of innate immune responses in rice. The OsFLS2 protein, a receptor kinase, has been shown to be the receptor for the flg22 peptide (Takai et al. 2008); Xa21, a rice resistance protein and receptor kinase, is the receptor for AvrXa21 peptide (Lee et al. 2009), and the rice chitin-binding protein CeBiP, a transmembrane protein with particular extracellular motifs, is the receptor for chitin (Kaku et al. 2006). The small GTPase OsRAC1 is involved in the immune responses induced by N-acetylchitooligosaccharide and spingolipids (Nakashima et al. 2008). None of these above mentioned proteins were differentially expressed (2-fold level) in the microarray experiments described here. However, several putative receptor-like protein kinases are differentially expressed following ClsA treatment, and it remains to be determined whether these are involved either in elicitor perception or in further signal processing. X. oryzae pv. oryzae lipopolysaccharide is also an inducer of rice defense responses, but the receptor has not yet been characterized (Desaki et al. 2006).

In summary, we have performed microarray analysis of gene expression changes that are induced in rice by ClsA treatment. A number of rice functions, particularly transcription factors, are differentially expressed under these conditions. Initial studies using Agrobacterium-mediated transient gene transfers suggest that overexpression of one of the highly upregulated transcription factor genes, OsAP2/ERF, results in induction of callose deposition, cell death, and enhanced resistance against subsequent infection by Xanthomonas oryzae pv. oryzae. Future studies will be aimed at understanding the role of OsAP2/ERF and other differentially expressed genes in ClsA-induced innate immunity in rice.

Methods

RNA isolation

Fifteen-day-old seedlings of the susceptible rice cultivar TN-1 were grown in a greenhouse in the months of December/January and shifted to a growth chamber (28°C; 80% relative humidity; light intensity of 440 μmole/m2/s; 12/12 h light/dark cycle) 48 h before the treatment. The adaxial surfaces of the leaves were syringe infiltrated, with approximately 10 μl of ClsA (500 µg/ml) purified from the culture supernatant of wild type X. oryzae pv. oryzae (strain BXO43; our laboratory wild type) or with buffer (10 mM potassium phosphate buffer pH 6.0) alone (as described in Jha et al. (2007)). In this procedure, approximately, 30–40 μl of either ClsA or buffer are taken into a needle less 1 ml syringe for infiltration, and we are able to infiltrate ~10 μl of the solution into the leaf while the rest of the solution either falls off the adaxial surface or passes right through to the abaxial surface. Twenty to 30 leaf pieces covering the infiltrated zone from each of the treatments were harvested 12 h after infiltration. At this time point, HR like symptoms are not observed in ClsA-treated leaves, and it would take a further 24 h for these symptoms to be visible (Supplementary Fig. S1). The leaf tissues were ground to a fine powder (in liquid nitrogen using a mortar and pestle) and subjected to total RNA isolation by using Trizol (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. The quality of the isolated RNA was assessed by agarose gel electrophoresis and quantitated using a spectrophotometer.

Microarray analysis

Eight micrograms of total RNA isolated from ClsA- and buffer-treated leaves were converted to double-strand cDNA using an Affymetrix (Santa Clara, CA) one cycle labeling kit following the manufacturer’s instructions. The synthesized double-stranded cDNA was column purified and was further subjected to RNA amplification using an Affymetrix IVT labeling kit. The amplified RNA (cRNA) generated in this manner was quantitated by using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE). The normalized cRNA was fragmented (into 50–200 bp fragments), hybridized to rice gene chips, washed, stained, and scanned as per Affymetrix protocols. The experiment was repeated with three different biological replicates using RNA isolated from three batches of rice leaves treated with either X. oryzae pv. oryzae ClsA or buffer.

Data analysis

The data were analyzed by using the GCOS of Affymetrix. The present calls ranged between 40% and 42% in ClsA-treated leaves and between 38% and 40% in the buffer-treated leaves. All the CEL (cell intensity) files generated by GCOS were uploaded to Avadis™ software version 4.3 (an Affymetrix approved software manufactured by Strand Life Sciences, Bangalore, India) for further analysis. The CEL files of the buffer-treated samples were grouped as control whereas the ClsA-treated sample files were grouped as treatment. The data were normalized using RMA (Irizarry et al. 2003a, b) and PLIER (Affymetrix Inc 2005) algorithms available in Avadis and subjected to differential expression analysis as per the manufacturer’s instructions. The genes identified by both the algorithms as being either ≥2.0- or ≥1.5-fold differentially expressed with p < 0.05 were selected. Q-value-based significance analysis was performed for rice genes that were commonly differentially expressed at fold change 1.5 and p < 0.05. Q values were generated from p values for all 862 genes using the online tool (http://genomics.princeton.edu/storeylab/qvalue/index.html) with settings of interval 0.01 and smoother method with FDR 0.02 (Storey and Tibshirani 2003). GO enrichment analysis was performed to identify overrepresented GO terms amongst 1.5-fold change differentially expressed rice genes following ClsA treatment. The Affymetrix probe set IDs of the ≥1.5-fold upregulated and downregulated rice genes were subjected to Gene Ontology Enrichment Analysis Software Toolkit analysis using an online tool accessible at http://omicslab.genetics.ac.cn/GOEAST following default standard setting (Zheng and Wang 2008). The ≥2.0-fold differentially expressed genes were subjected to NCBI BLASTX analysis to manually curate their functions. A gene was assigned the function of its orthologs if it showed significant homology with expect value ≥1 × 10−7. Based upon the assigned functions, the differentially expressed genes were further categorized into 14 different functional categories as per Bevan et al. (1998) with some minor changes. We did not have any differentially expressed genes that were predicted to be involved in protein destination and storage. Therefore, we dropped this category. Some of the differentially expressed genes were predicted to function in protein synthesis, while others appeared to encode functions involved in protein degradation. We formed a new category called protein synthesis/turnover into which we clubbed genes involved in protein synthesis as well as protein degradation. Genes related to abiotic stress were put into a new category called “stress” that is distinct from genes related to biotic stress which were placed under the category of “disease/defense.” We did this because we wanted to separate genes related to biotic stress from those related to abiotic stress. The genes having unclear, unclassified or no significant similarity in the available databases were grouped together in a category called “others”. In order to identify Arabidopsis orthologs of rice genes, BLASTX searches were performed through TAIR (The Arabidopsis Information Resource; www.arabidopsis.org).

For the ≥1.5-fold differentially expressed genes, the locus information corresponding to the Affymetrix probe set IDs was obtained from the National Science Foundation rice oligonucleotide array project (http://www.ricearray.org/matrix.search. shtml) through the rice genome server of The Institute for Genome Research, Rockville, MA (Yuan et al. 2005; Ouyang et al. 2007). These were subjected to pathway analysis using RiceCyc 1.2 software (Jaiswal et al. 2006) available at http://dev.gramene.org/pathway.

Data submission

All the six CEL files (generated by GCOS) and the Avadis™ processed (PLIER and RMA normalized) files are deposited at the NCBI microarray repository, Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE8216 with details provided about the experimental procedures that are minimum information about a microarray experiment compliant.

Real-time PCR

Two micrograms of freshly isolated RNA were converted into first-strand cDNA as per the recommended protocol by using Superscript™ II reverse transcriptase (Invitrogen) and oligo-dT primer. One microliter of the 1:100-fold diluted cDNAs were subjected to real-time PCR analysis using SYBR green PCR master mix (Applied Biosystem, Barrington, UK) following the manufacturer’s instructions using gene specific primers designed to amplify 130–170 bp fragments of each gene of interest. After 10 min of initial denaturation at 95°C, the samples were subjected to the cycling parameters of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s (for 40 cycles) using a 7900 HT sequence detection system (Applied Biosystems, Foster City, CA). The relative expression of the gene between ClsA and buffer-treated samples was calculated using the 2−ddCT method with OsGAPDH as an endogenous control (Livak and Schmittgen 2001). The real-time PCR assays were performed at least three times with the same cDNA sample and repeated at least three times with independently isolated RNA samples.

Co-infiltration of ClsA and wild-type X. oryzae pv. oryzae into rice leaves

Three milliliters of wild-type X. oryzae pv. oryzae cultures were grown to saturation in Peptone Sucrose broth (pH 7.2) (Ray et al. 2000). The cells were pelleted and washed in double-distilled water following centrifugation. The pelleted cells were resuspended (at a concentration of approximately 3 × 109 cells/ml) in 1 ml of purified X. oryzae pv. oryzae ClsA (final conc. 500 μg/ml) and syringe infiltrated into the leaves as described above. The total RNA isolated from the zone of infiltration in the leaves was subjected to quantitative real-time PCR analysis (as described above). A Student’s t test was performed to check the level of significance for this suppression.

Sequence analysis of OsAP2/ERF

Gene sequence of OsAP2/ERF (LOC_Os08g36920) was downloaded from TIGR (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/), and BLASTX analysis was performed in Rice Genome Annotation Project BLAST Search (http://rice.Plantbiology.msu.edu/blast.shtml). Amino acid sequence of the gene was used to do a domain search (http://www.expasy.ch/prosite). Secondary structure prediction was performed with Jpred web server (http://www.compbio.dundee.ac.uk/www-jpred).

Cloning OsAP2/ERF in Agrobacterial T-DNA vector and transfer to Agrobacterium strain LBA4404

OsAP2/ERF full-length gene was PCR amplified by using a pair of primers, each containing a BamHI site and cloned into the BamHI site between CaMV35S promoter and PolyA on pSB11 binary vector (Ramesh et al. 2004) (Supplementary Fig. S3). This construct was transformed into Escherichia coli DH5α cells. Positive clones were screened using gene and vector-specific primers and confirmed by sequencing. Plasmids from positive clones were further electroporated into electro-competent cells of Agrobacterium LBA4404.

Callose deposition assay

Rice leaves were syringe infiltrated with one of the following: water, ClsA (~100 μg/ml), LBA4404/pSB11, and LBA4404/pOsAP2/ERF strains (approximately 109 CFU/ml resuspended in water). After 12 to 15 h, the leaves were heated with lactophenol at 65°C to remove chlorophyll, stained with aniline blue for 2 to 3 h, washed with water, mounted on a slide in 50% glycerol, and analyzed by an Axioplan2 epifluorescence microscope, using a blue filter (excitation wavelength 365 nm and emission wavelength above 420 nm long pass [LP]) and ×10 objective (Hauck et al. 2003).

Cell death assay

Rice seeds were surface sterilized with sodium hypochlorite and germinated under sterile conditions on a filter paper placed on 0.5% agar in Petri dishes for 3 to 4 days. Root tips, 1 to 2 cm long, were treated with one of the following: water, ClsA (~500 μg/ml), LBA4404/pSB11, and LBA4404/pOsAP2/ERF. Rice roots were treated with LBA4404/pSB11 and LBA4404/pOsAP2/ERF for 2 h, and roots were subsequently transferred to water for another 14 h. After incubation for a total of 16 h at 28°C, roots were washed in 1X PBS and stained with PI by vacuum infiltration for 10 to 15 min. The roots were mounted on a microscopic slide in 50% glycerol in 1X PBS; 0.3-μm thick longitudinal optical sections were acquired on a Zeiss LSM-510 Meta confocal microscope using 63X oil immersion (NA 1.4) and were further projected to obtain the image of 2 to 3 μm total thickness. HeNe laser at 543 nm excitation and emission above 560 nm (LP) was used to detect PI internalization. All images were analyzed using LSM software and further edited using Photoshop (Adobe, San Jose, CA, USA).

Rice resistance assay

The midveins of leaves of approximately 40 days old TN-1 rice plants were injected with 40 to 50 μl of water alone, LBA4404/pSB11 or LBA4404/pOsAP2/ERF cells (resuspended in water at ~109 cells/ml) using a 1-ml hypodermic syringe and needle. Six hours later, the midveins of the leaves were inoculated with X. oryzae pv. oryzae, 2 to 3 cm below the point of initial injection, by pricking with a needle that had been used to touch a fresh bacterial colony. After 10 days, the leaves were observed for the appearance of visible disease lesions (discoloration of midvein and surrounding regions).

Notes

Declarations

Acknowledgements

GJ was supported by a fellowship from the Council of Scientific and Industrial Research. MD was supported by a research associateship from the Department of Biotechnology, Government of India. This work was supported, in part, by a grant to RVS from the Department of Biotechnology. We thank Dr. K. V. Rao for providing us the pSB11 vector.

Authors’ Affiliations

(1)
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research
(2)
Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research

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