Transcriptional Characteristics of Xa21-mediated Defense Responses in Rice.
Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most destructive bacterial disease of rice. The cloned rice gene Xa21 confers resistance to a broad spectrum of Xoo races. To identify genes involved in Xa21‐mediated immunity, a whole‐genome oligonucleotide microarray of rice was used to profile the expression of rice genes between incompatible interactions and mock treatments at 0, 4, 8, 24, 72 and 120 h post inoculation (hpi) or between incompatible and compatible interactions at 4 hpi, respectively. A total of 441 differentially expressed genes, designated as XDGs (Xa21 mediated differentially expressed genes), were identified. Based on their functional annotations, the XDGs were assigned to 14 categories, including defense‐related, signaling, transcriptional regulators. Most of the defense‐related genes belonged to the pathogenesis‐related gene family, which was induced dramatically at 72 and 120 hpi. Interestingly, most signaling and transcriptional regulator genes were downregulated at 4 and 8 hpi, suggesting that negative regulation of cellular signaling may play a role in the Xa21‐mediated defense response. Comparison of expression profiles between Xa21‐ and other R gene‐mediated defense systems revealed interesting common responses. Representative XDGs with supporting evidences were also discussed.
To guard themselves against pathogen infection, plants have developed sophisticated recognition and defense systems through evolution, including basal defense and resistance (R) gene mediated defense. Basal defense occurs at the early stage of plant‐pathogen interactions and is often mediated by the recognition of pathogen‐associated molecular patterns (PAMPs) through plant pattern recognition receptors, while R gene mediated resistance is typically detectable later and controlled by the specific recognition between the R protein and the corresponding pathogen effectors ([1]).
Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important diseases of rice. So far, nearly 30 major R genes for resistance to Xoo have been identified and six of them have been cloned: Xa1, Xa3/Xa26, xa5, xa13, Xa21, and Xa27 ([37]; [52]; [14]; [40]; [13]; [6]; [15]). Of the six cloned disease resistance genes (R), Xa21 and Xa26 encode receptor‐like kinases (RLKs) belonging to the non‐RD kinase family ([7]). Xa3/Xa26 family members were constitutively expressed in leaf tissue, while XA3/XA26 protein might function as a monomer or form a hetero‐dimer with non‐family member protein in disease resistance ([50]). The kinase domain of XA21 is capable of autophosphorylation specifically at the serine and threonine residues ([23]) and the three phosphorylated residues Ser686, Thr688 and Ser689 in the juxtamembrane domain have been implicated in stability of the XA21 protein ([51]). Moreover, the accumulation of the XA21 protein and the full Xa21‐mediated resistance depends on the levels of the ubiquitin ligase XB3 that interacts with XA21 ([47]). It has also been reported that XB15, a protein phosphatase 2C, negatively regulates Xa21‐mediated innate immunity ([30]). Transgenic plants overexpressing OsWRKY62.1, a XA21 binding WRKY transcription factor (XB10), are compromised in basal defense and Xa21‐mediated resistance to Xoo ([32]). Recently, another XA21 binding protein (XB24), an ATPase, was reported to enhance XA21 autophosphorylation and inhibit XA21‐mediated resistance ([3]). Different from many bacterial effectors, which are secreted by a type III secretion system, AvrXa21 activity requires the function of a type I secretion system that represents a previously undescribed family of signaling molecules ([20]). Recently, by analyzing biologically active fractions from Xoo supernatants identified a 194‐amino acid protein designated Ax21 as the activator of XA21‐mediated immunity and confirmed that Ax21 is a pathogen‐associated molecular pattern. Thus, XA21 is a disease resistance gene because it is the single polymorphic determinant in rice that confers resistance to strains of bacteria expressing sulfated Ax21, and also encodes a pattern recognition receptor because it is required for recognition of a particular modified peptide epitope that is conserved across a microbial genus ([21]). This is certainly a great step forward towards fully understanding the nature of Xa21‐mediated immunity. In future, to reveal a comprehensive picture of the Xa21‐mediated resistance, an overall profiling of genes involved in Xa21‐mediated immunity by a high‐throughput approach may provide more candidates than a yeast two‐hybrid system, by which the above‐mentioned XBs were identified ([47]; [30]; [32]).
High‐throughput approaches, such as serial analysis of gene expression, cDNA‐amplified fragment length polymorphism, suppression subtractive hybridization, microarray, proteomic analysis and yeast two‐hybrid, have been used to investigate molecular mechanisms of disease resistance in a variety of plant species. Of those mentioned, microarray analysis allows simultaneous measurement of the transcriptional levels of thousands of genes, making it possible to globally identify defense‐related genes in plant‐pathogen interactions ([39]; [12]). To our knowledge, this approach has been applied to a number of R gene‐mediated resistance systems, such as Arabidopsis‐RPM1, ‐RPP4, ‐RPP7, ‐RPP8, ‐RCY1, ‐Rpt2 and ‐RPS2 ([41]), tomato‐Pto and ‐Prf ([27], [28]) and soybean‐RPG1 ([58]). The resulting expression profiles provide a more comprehensive view of plant defense mechanisms ([49]), such as the quantitative differences between the responses in incompatible and compatible interactions ([41]). Microarrays were also used to characterize the rice‐pathogen interactions, such as the interactions of rice‐Xoo ([22]; [19]), ‐flagellin ([10]), ‐lipopolysaccharides ([8]), ‐fungal elicitor ([17]), ‐rice dwarf virus ([35]) and ‐planthopper ([4]); however, in most of these studies, the number of probes on the arrays were rather limited.
In the current research, comprehensive screening of genes involved in the Xa21‐mediated Xoo resistance was carried out by using the whole‐genome oligonucleotide microarray of rice. A total of 441 rice genes were identified to be differentially expressed between the incompatible, mock, and compatible Xoo‐inoculations. These genes were assigned to 14 categories, including defense‐related, signaling and transcriptional regulators.
Results
Rational and experimental design
The japonica rice cultivar TP309 is susceptible to both Xoo PR6 and PR10. The transgenic line 4021‐3 expressing a c‐Myc tagged Xa21 is resistant to Xoo PR6, but susceptible to Xoo PR10 ([46]). To identify genes related to Xa21‐mediated resistance, a whole rice genome oligonucleotide microarray was used to compare the gene expression profiles between 4021‐3 plants inoculated with Xoo PR6 (R) and those mock‐inoculated (M) as reported by van Poecke ([32]) at 0, 4, 8, 24, 72 and 120 hpi. In addition, expression profiles of the incompatible and two compatible interactions, lacking either an avirulence gene (SAvr‐) or a resistance gene (SR‐), were also compared at 4 hpi to identify differentially expressed genes at the early stage.
Validation of the microarray data
The whole genome oligo microarray system had been evaluated by our previous transcriptomic analysis of a superhybrid rice in its reliability ([48]). To validate our microarray data in this experiment, 13 XDGs (five of them showed differential expression at two time points) were validated by three independent replications of reverse transcription‐polymerase chain reaction (RT‐PCR) reactions. These XDGs, listed in Table S1, include five PR genes, four signaling‐related genes, one hormone and stress‐related gene, and three energy‐related genes. The rice actin gene Os03g50890 was used as the control as it is constitutively expressed in the microarray experiments. RT‐PCR results showed that 15 RT‐PCR reactions had consistent results with those in the microarray analysis, including 11 upregulating and four downregulating genes. However, RT‐PCR failed to detect significant difference in the expression of Os07g03730 (4 hpi), Os08g01310 and Os12g43130 (Figure S1). In summary, the RT‐PCR results showed 83% consistency with the microarray data, close to the result obtained earlier ([48]).
Summary of the array data
Five hundred and six differentially expressed oligos were identified in the R‐M comparisons at 4, 8, 24, 72 and 120 hpi, and in the R‐ SAvr‐ and R‐SR‐ comparisons at 4 hpi. These oligos and their corresponding genes were analyzed by BLASTN, of which 470 oligos represented 441 known or putative genes (Table S2). These genes were designated as XDGs (Xa21‐mediated differentially expressed genes). The remaining oligos were not evaluated since no significant hits were found in the sequence databases. Within 441 XDGs, 211 showed upregulation, 207 were downregulated, and 23 were up/downregulated at different time points. Notably, 126 XDGs (29%) showed a fold change greater than 4, and 303 XDGs (69%) showed a fold change of greater than 2.
Among the 441 XDGs in the R‐M comparisons, the differentially expressed genes at each time point were as follows: 47 XDGs (4 hpi), 75 XDGs (8 hpi), 88 XDGs (24 hpi), 75 XDGs (72 hpi) and 169 XDGs (120 hpi) (Tables S3–S7). And the unique XDG numbers at each time point were as follows: 62 (8 hpi), 42 (24 hpi), 40 (72 hpi) and 105 (120 hpi). To identify the genes that are differentially expressed in the early stage of Xa21‐mediated response, the XDGs between the R‐SAvr‐ and between R‐SR‐ were surveyed at 4 hpi. Fifty‐six XDGs, including 35 upregulated and 21 downregulated genes, were identified in the R‐SAvr‐ comparison, and 40 XDGs, including 32 upregulated and eight downregulated genes, were identified in the R‐SR‐ comparison (Tables S8–S9). In total, 143 XDGs were identified in the three interactions (R‐M, R‐SAvr‐and R‐SR‐) of profiles at 4 hpi; however, only two overlapping XDGs with annotation [ATP synthase alpha (Os09g08910) and 16 kDa membrane protein (Os04g33830)] were found (Table S2).
Functional categories of XDGs
Based on their functional annotations, the 441 XDGs were assigned to 14 categories: cell cytoskeleton and development, defense related, energy, fatty acid, heat shock, hormone and stress, membrane, oxidation, primary metabolism, protein modification and degradation, signaling and R gene‐like, transcriptional regulators, miscellaneous and unknown (Table 1). The differential expression pattern and the numbers of XDGs at each time point are shown in Figures 1 and 2.
1 Functional categories of XDGs (Xa21 mediated differentially expressed genes) based on their annotations and potential cellular functions
| Categories | Selection criteria | Examples | # XDGs | % |
|---|
| Cell cytoskeleton and development | Associated with actin, cell cycle and embryogenesis | Beta‐expansin, esterase, DMC1 homolog | 11 | 2.5 |
| Defense related | Pathogenesis‐related (PR) proteins, protease inhibitors, elicitor induced and HR related | PR‐1, PR‐10, metallothionein‐like protein, PAL, thaumatin‐like protein, chitinase, bowman‐birk serine protease inhibitor | 32 | 7.3 |
| Energy | Energy acquisition or production, photosynthesis light reactions, ATP‐related | Chlorophyll a‐b binding protein, photosystem, early light‐induced protein, ATP synthases, cytochromes, thioredoxin | 58 | 13.2 |
| Fatty acid | Fatty acid metabolism | Acyl‐CoA‐binding protein, GDSL‐like Lipase/Acylhydrolase, LTP 2 | 8 | 1.8 |
| Heat shock | Heat‐shock proteins | HSP70, HSP82, HSP90, ClpX, DNAJ | 14 | 3.2 |
| Hormone and stress | Hormone and stress regulated | SAUR, ARP1, cytokinin‐repressed protein CR9, drought induced protein, wound‐responsive protein | 7 | 1.6 |
| Membrane | Membrane associated, transporters | ABC transporters, Aquaporin, ChaC‐like protein, MIP, sugar transporter | 19 | 4.3 |
| Oxidation | Related to oxidative stress | Alternative oxidase, catalase, GST, lipoxygenase, cytochrome P450 | 18 | 4.1 |
| Primary metabolism | Metabolism of sugars, amino acids and nucleotides | Alpha‐glucosidase, amino acid synthesis, ribulose, glycosyl hydrolase, thiamine biosynthesis | 43 | 9.8 |
| Protein modification and degradation | Protein modification and degradation | Clp protease proteolytic subunit, GrpE, F‐box, ubiquitin, U‐box | 16 | 3.6 |
| Signaling | Signal transduction related and R gene | Protein kinase, calcium associated, MAP3K‐like, Phosphatase, NB‐ARC, LRR | 36 | 8.2 |
| Transcriptional regulators | Transcription factor, DNA or RNA binding | Myb‐like, AP2, WRKY, GRAS, zinc finger, NAC transcription factor | 37 | 8.4 |
| Miscellaneous | Does not fit into any of the above categories | Polygalacturonase, terpene synthase, barwin, nodulin, elongation factor | 50 | 11.3 |
| Unknown | Matched a hypothetical or uncharacterized gene | Expressed protein, hypothetical protein | 93 | 21.1 |
| Total | | | 441 | 100.0 |
1 GDSL, GDSL esterases and lipases with consensus amino acid sequence of Gly, Asp, Ser, and Leu around the active site Ser; GRAS, GRAS (GAI, RGA, SCR) gene family, GAI, RGA and SCR were derived from gibberellin‐acid insensitive (GAI) and the repressor of gibberelllin‐acid 1 (RGA) and scarecrow (SCR), respectively; GST, glutathione S‐transferase; LRR, leucine‐rich repeat; MAP3K, mitogen‐activated protein kinase kinase kinase; MIP, major intrinsic protein; NAC, NAC domain was defined from the petunia NAM and arabidopsis ATAF1 and CUC2 genes; NB‐ARC, nucleotide‐binding adaptor shared by APAF‐1, R proteins, and CED‐4 domain; PAL, phenylalanine ammonia‐lyase; WRKY, defined by the conserved amino acid sequence WRKYGQK.
Graph: 1 Classification and expression patterns XDGs (Xa21 mediated differentially expressed genes) identified in R‐M interactions.
XDGs identified in R‐M interactions at four time points. The Xa21 line 4021‐3 was inoculated with Xoo PR6 containing avrXa21. XDGs were identified at the indicated time points as compared with zero time. X‐axis represents the number of XDGs; dark and white bars denote up‐ and downregulated XDGs, respectively. Y‐axis indicates the functional classification of XDGs.
Graph: 2 Classification and expression patterns XDGs (Xa21 mediated differentially expressed genes) identified in R‐S interactions.
In the R‐Savr‐ interaction, XDGs were identified by a comparison of gene expression profiles of 4021‐3 inoculated with Xoo PR6 and Xoo PR10 (lacking avrXa21), respectively. In the R‐SR‐ interaction, XDGs were based on the susceptible control TP309 (lacking Xa21) and 4021‐3 inoculated with Xoo PR6, respectively. X‐axis represents the number of XDGs; dark and white bars denote up‐ and downregulated XDGs, respectively. Y‐axis indicates the functional classification of XDGs.
There were 36 signaling and R gene‐like XDGs. Nineteen of them encode protein kinases, RLKs or phosphatases (Table S10), supporting the notion that protein phosphorylation and dephosphorylation may play roles in Xa21‐mediated immunity. Notably, most of these kinases and RLKs were downregulated, suggesting a negative regulatory role in this defense system.
Almost all of the XDGs occurring at the late stage (72 and 120 hpi) belong to the category of defense‐related genes, of which 29 PR genes, including the genes coding for PR‐1, PR‐10, PRB1‐3, PRMS, PR Bet v I, endo‐1,3‐beta‐glucosidase, endochitinase, phenylalanine ammonia‐lyase, metallothionein‐like protein, thaumatin‐like protein and thionin‐like peptide, were upregulated dramatically at late stages (Figure 3, Table S11). In addition to PR genes, Xylanase inhibitor protein (Os05g15770), Bowman‐Birk serine protease inhibitor family protein (Os01g03680, Os01g03340), and pectinesterase inhibitor domain containing protein (Os08g01670) were downregulated significantly at early stage (8 hpi) (Table S2).
Graph: 3 Time course induction of nine representative pathogenesis‐related (PR) genes in the R‐M interaction.
Each data point represents an induction level of indicated gene by Xoo PR6 after normalized with that of mock inoculation.
Thirty‐seven XDGs encode transcription regulators, of which 10 were upregulated and 27 were downregulated. Among the upregulated genes, three XDGs encode myb‐like DNA‐binding domain proteins, two encode AP2 domain containing proteins, and five encode for ZIM motif family proteins and NAC‐domain containing proteins (Table 2). Five downregulated transcription factors belong to the zinc finger family, and this observation is consistent with the fact that most differentially expressed zinc finger‐containing genes were downregulated in the arabidopsis‐Pseudomonas syringae pv. tomato (Pst) DC3000 interaction ([42]). Interestingly, in the R‐M comparisons, all of the transcription regulators were downregulated at 4, 8 and 24 hpi, suggesting a negative role in the Xa21‐mediated defense response. In support of this hypothesis, the transcription factor OsWRKY62 has been shown to function as a negative regulator in Xa21‐mediated resistance ([32]).
2 Thirty‐seven transcriptional factors and transcription factor‐related XDGs (Xa21 mediated differentially expressed genes)
| Oligo ID | XDGs | Brief annotation | log2(R/M) | R/S1# 4 h | R/S2## 4 h |
|---|
| 4 h | 8 h | 24 h | 72 h | 120 h |
|---|
| Os010958_01 | 001‐122‐G01 | AP2 domain containing protein | | | −1.21 | | | | 2.09 |
| Os018037_01 | Os02g43790 | AP2 domain containing protein | | −1.21 | | | | | |
| Os023819_01 | Os08g41030 | AP2 domain containing protein | | | | 0.86 | | | |
| Os053208_01 | Os02g07930 | B‐box zinc finger family protein | | | | | | 0.93 | |
| Os053203_01 | Os11g32880 | CarD‐like transcriptional regulator family protein | | | | −1.38 | | | |
| Os002428_01 | Os01g57944 | DNA‐directed RNA polymerase alpha chain | −0.81 | | | | | | |
| Os056698_01 | Os03g22510 | Flowering promoting factor‐like 1, putative | | | | | −0.82 | | |
| Os000976_01 | Os02g16000 | GAMYB‐binding protein, putative | | | | | −0.70 | | |
| Os057385_02 | Os12g43600 | Glycine‐rich RNA‐binding protein GRP1A | −0.83 | −0.89 | | | | | |
| Os009699_01 | Os07g38030 | GRAS family transcription factor containing protein | −1.03 | | | | | | |
| Os020246_01 | Os11g47870 | GRAS family transcription factor containing protein | | | | | 2.10 | | |
| Os002967_01 | Os03g25120 | Heat shock transcription factor family protein | −1.54 | | | | | | |
| Os014311_01 | Os01g11910 | Helix‐loop‐helix DNA‐binding domain containing | | | | | 1.95 | | |
| Os005941_01 | Os09g31300 | Helix‐loop‐helix DNA‐binding domain containing | | | | | −1.04 | | |
| Os057927_01 | CK068197 | HMG box family protein | | | −2.19 | | −1.66 | | |
| Os000416_01 | Os08g39450 | Multiple stress‐responsive zinc‐finger protein ISAP1 | | −0.95 | | | | | |
| Os000997_01 | Os02g09480 | Myb‐like DNA‐binding domain containing protein | | −1.15 | | | | | |
| Os045752_01 | Os06g46560 | Myb‐like DNA‐binding domain containing protein | | | | | 2.33 | | |
| Os004771_01 | Os05g34110 | Myb‐like DNA‐binding domain | −0.73 | | | | | | |
| Os057372_02 | Os06g24070 | Myb‐like DNA‐binding domain | | | | | | 1.19 | |
| Os053441_01 | Os08g06110 | Myb‐like DNA‐binding domain | | | | | 6.17 | 1.92 | |
| Os054996_01 | Os04g43680 | Myb‐related protein Myb4, putative | | −0.81 | | | | | |
| Os017943_01 | Os07g12340 | NAC‐domain containing protein 2, putative | | −1.39 | | | | | |
| Os057929_01 | Os11g08210 | NAC‐domain containing protein 2, putative | | | | | | | 1.21 |
| Os021895_01 | Os08g28180 | Pentatricopeptide, putative | | | −0.88 | | | | |
| Os014166_01 | Os08g02160 | Putative NAC transcription factor | | | | −0.74 | | | |
| Os055303_01 | Os02g39060 | RNA recognition motif family protein | −0.91 | | | | | | |
| Os014369_01 | Os05g51150 | Sigma‐70, region 4 family protein | | | | | | −1.97 | |
| Os007237_01 | Os03g14850 | SRF‐type transcription factor family protein | | | −2.13 | | −1.69 | | |
| Os009048_01 | Os02g45120 | Transcription factor S‐II family protein | | | −1.15 | | | | |
| Os055619_01 | Os05g46020 | WRKY DNA binding domain containing protein | | −1.05 | | | | | |
| Os022161_01 | 001‐208‐D06 | ZIM motif family protein | −0.66 | | | | | | 1.27 |
| Os014440_01 | Os02g53530 | Zinc finger protein, putative | | −0.90 | | | | | |
| Os002415_01 | Os03g43840 | Zinc finger protein, putative | | | −1.05 | | | | |
| Os040711_01 | Os09g03500 | Zinc finger, C2H2 type family protein | −1.22 | | | | | | |
| Os051257_01 | Os02g35329 | Zinc finger, C3HC4 type OR E3 ubiquitin ligase | | −1.52 | | | | | |
| Os052123_01 | Os06g04920 | Zn‐finger in Ran binding protein | | | | −0.69 | | | |
- 2 R/S1# indicates log2[R/S(Avr−)] and R/S2## indicates log2[R/S(R−)].
- 3 GAMYB: Gibberellins acid (GA) inducible MYB (v‐myb avian myeloblastosis viral oncogene homolog) type transcription factor; GRAS: GRAS (GAI, RGA, SCR) gene family, GAI, RGA and SCR were derived from Gibberellin‐acid Insensitive (GAI) and the Repressor of Gibberelllin‐acid 1 (RGA) and Scarecrow (SCR), respectively; HMG: High mobility group (HMG) proteins are a family of relatively low molecular weight non‐histone components in chromatin; NAC: NAC domain was defined from the petunia NAM and arabidopsis ATAF1 and CUC2 genes; SRF: The name SRF derives from STRUBBELIG RECEPTOR FAMILY; WRKY: The WRKY domain is defined by the conserved amino acid sequence WRKYGQK; ZIM: The gene was named ZIM for Zinc‐finger protein expressed in Inflorescence Meristem.
Most of the energy‐related XDGs belong to the photosynthesis‐related gene family. These XDGs were downregulated at 24, 72 and 120 hpi, which is consistent with observations from other studies on plant defense responses to bacterial and fungal pathogens, and to phloem‐feeding insects ([28]; [58]; [16]). It is possible that the downregulation of photosynthesis‐related genes induces leaf senescence in R gene‐mediated disease resistance ([34]).
Among the 14 categories, the largest group was that of unknown genes accounting for 21% of the 441 XDGs. These data therefore provide evidence to link to the Xa21‐mediated defense response.
Discussion
Comparison of XDGs with genes involved in other defense systems
To illustrate common components of plant defense pathways, XDGs were compared with the gene expression datasets obtained in other R gene‐mediated defense systems. A total of 95 XDGs and their homologs were identified (Table S12). These include 51, 26, 22 and five XDGs found in RPG1‐ ([58]), xa13‐ ([5]), Pto‐ ([28]) and Xa1/Xa3‐ ([25]) mediated defense responses, respectively. For example, the genes encoding PR Bet v I (Os12g36880) and thaumatin‐like protein (Os12g43380) were upregulated at 72 hpi in the resistance response mediated by Xa21 and Xa1/Xa3, whereas, the barwin coding gene (Os11g37950) was upregulated in the Xa21‐, RPG1‐ and Pto‐mediated defense processes. Five unknown genes (Os01g14690, Os01g10400, Os04g11400, Os10g42040 and Os12g09720) were differentially expressed in both Xa21‐ and xa13‐mediated disease resistances. The homologs of the unknown gene Os01g14690 in arabidopsis and potato were also upregulated in the arabidopsis‐Pst DC3000 and the tobacco‐virus interaction, respectively. Interestingly, the expression patterns of these upregulated genes were comparable, suggesting similar kinetics of defense activation and response in the R gene‐mediated systems.
XDGs were also compared with the genes involved in other defense systems including basal defense ([54]; [57], [56]; [8]), hypersensitive response (HR) ([11]; [43]; [18]), systemic acquired resistance (SAR) ([26]; [45]), induced systemic resistance (ISR) ([44]) and Benzothiadiazole (BTH)‐inducible response ([36]). A total of 106 XDGs could find their homologs in the disease resistance of other plant species, which includes categories in basal defense, HR, SAR/ISR or BTH inducible response (Table S13). For example, the XDG Os11g47760, encoding a heat shock cognate 70 kDa protein, had a homolog upregulated in SAR in arabidopsis at 120 hpi; the arabidopsis homolog of Os01g7134 for glucan endo‐1,3‐beta‐glucosidase was upregulated in SAR in arabidopsis at 72 hpi and 120 hpi. These coincidences may suggest a conserved nature of plant defense pathways.
Representative XDGs with supporting information
Based on our microarray data and published reports, a list of representative XDGs with supporting information were proposed as follows.
Os03g14860, encodes a G‐patch domain‐containing protein, and increased tenfold at 120 hpi in the Xa21‐mediated incompatible interaction. G‐patch is a conserved domain found in the splicing factor 45, SON DNA binding protein, and D‐type Retrovirus‐polyproteins, suggesting that proteins with this domain may be involved in RNA‐protein interactions. An arabidopsis homolog of Os03g14860 is required for basal resistance against the virulent bacterial pathogen Pseudomonas syringae maculicola ES4326 AvrB at 3 d post inoculation and for the R gene mediated resistance specified by RPM1, RPS4 and RPP4 ([55]). Therefore, the induction of Os03g14860 might suggest it plays a role in Xa21‐mediated basal resistance, which might be different from arabidopsis flagellin‐induced early basal defense.
Os11g37950 encodes a putative barwin protein and was upregulated 1.55 and 3.68‐fold at 72 and 120 hpi, respectively. One of its homologs was reported to be upregulated in the wheat‐Fusarium graminearum resistance response at 12 hpi, and the accumulation of its transcript increased in a time dependent manner ([33]). In addition, its arabidopsis homolog (U01880) interacts in vivo with Pti4, a transcription factor associated with the tomato R protein Pto ([2]), Furthermore, the expression of U01880 was shown to be upregulated in the Pti4‐expressing transgenic arabidopsis plants. Therefore, it will be interesting to test the function of Os11g37950 in XA21‐mediated pathway.
Os06g46950 is the rice EF‐hand Ca2+‐binding protein CCD1. We found that the gene coding for Os06g46950 was downregulated for 1.92‐fold at 8 hpi in the Xa21‐mediated incompatible interaction. In line with this observation, its arabidopsis homolog (At4g27280) is downregulated at 1 hpi with the treatment of the bacterial flagellin peptide flg22, but upregulated in response to the bacterial elicitors elf26 and elf18 ([29]; [56]). These results suggested a possible role of Os06g46950 involved in both R gene and PAMP mediated defense responses.
Os03g43840, encoding a zinc finger protein, was downregulated at 24 hpi for 2.07‐fold in the resistance response. The arabidopsis zinc finger protein LSD1 has been proposed to negatively regulate a plant cell death pathway by monitoring a superoxide‐dependent signal ([9]). In the arabidopsis‐Pst DC3000 compatible interaction, the LSD1 gene was upregulated at 7 hpi ([42]).
Os01g03340 and Os01g03680 encode bowman‐birk type bran trypsin inhibitor; they were downregulated 2.06‐ and 2.48‐fold at 8 hpi, respectively. Yeast‐two hybrid experiment has shown that their gene products interact with the kinase domain of rice blast resistance gene Pi‐d2 (WY Song et al. unpubl. data, 2009).
Based on the published reports ([47]; [30]; [31]; [3]), the transcription of XA21‐binding proteins (XB3, XB10, XB15 and XB24) in the interaction between rice and Xoo were not revealed. We checked the transcription of XBs in our experiments and found that they have low expression and none of them are differentially expressed at the time points we studied. The possible reason is that their regulation in Xa21‐mediated disease resistance responses might occur at translational or post‐translational level, which cannot be detected by microarray. It will be interesting to carry out experiments to investigate the expression of XB proteins by means of immunoassay.
In summary, the whole genome rice oligo microarray was used to perform expression profiling of Xa21‐mediated immunity and a total of 441 genes were identified to be differentially expressed. One interesting observation is that transcription factors and other signal transduction‐related genes tend to be downregulated at 4 and 8 hpi, and PR genes tend to be upregulated at 72 and 120 hpi. These results suggest that negative regulation of cell signaling may play an important role in the early stage, while the induction of well‐characterized PR genes occurs in the late stage of this pathosystem. Moreover, profile comparisons between Xa21‐ and other R gene‐ mediated defense systems in rice or other plants suggest that XDGs may be linked to multiple defense pathways, including basal defense, HR, SAR and BTH responding. A number of representative XDGs with supporting evidence in response to disease resistance were discussed. However, the present gene expression profiling in Xa21‐mediated resistance is preliminary; to better understand the mechanism of Xa21‐mediated immunity, it is important to compare the expression level among R, S and mock treatment systematically and also to further investigate the biological functions of some XDGs relative to the nature of the Xa21 immunity. Our continuing work to verify the involvement of these XDGs through reverse‐genetic approaches may facilitate identification of the critical components in Xa21‐mediated resistance networks.
Materials and Methods
Bacterial pathogens
Xoo Philippine race 6 (PR6) and race 10 (PR10) were obtained from Professor Zhang Qi's laboratory (Chinese Academy of Agricultural Sciences) and used for the pathogen inoculations. Bacteria were subcultured on PSA (0.5% bacto peptone, 2% sucrose, 0.05% L‐glutamic acid) agar media (1.5%, w/v) at 30 °C for 72 h prior to inoculation.
Plant materials and pathogen inoculation
Rice seeds were imbibed in water overnight at 30 °C and placed on moist filter paper. After germination, the seeds were grown in soil in a greenhouse until the seedling stage. The seedlings were transferred to experimental field plots located at the Institute of Genetics and Developmental Biology in Beijing. Plants were grown in the field for approximately 9 weeks in preparation for inoculation.
Bacterial cells of Xoo were suspended in sterile distilled water and the inoculation concentration was adjusted to approximately 109 cells per milliliter. Rice leaves were inoculated with the bacterial suspension using the leaf clipping method. Mock inoculated plants were treated in a similar fashion except sterile distilled water was used in place of Xoo.
RNA preparation
Inoculated rice leaves were collected approximately 1 cm from the inoculation site at 0, 4, 8, 24, 72 and 120 h post inoculation. Three independent replicate samples were collected at each time point for microarray and RT‐PCR analysis. Total RNA was isolated using a QIAGEN Co., Ltd. (Shanghai, China) RNeasy Plant Mini Kit following the manufacturer's protocols. The quality and quantity of RNA samples were determined by gel electrophoresis using a NanoDrop ND‐1000 spectrophotometer (NanoDrop Technology, Wilmington, DE, USA).
Rice whole‐genome oligonucleotide array
The whole‐genome array was developed based on annotated and predicted genes from the genome assembly of indica rice 93‐11 ([53]). Using a SpotArray72 microarray printer (PerkinElmer, Waltham, MA, USA) in the microarray laboratory at Beijing Genomic Institute, 70‐mer oligonucleotides were arrayed onto a set of two poly‐L‐lysine coated microscope slides and the detailed description of whole genome rice microarray was reported recently ([48]).
Homology analysis
BLASTN was used to align 70‐mer oligos and their corresponding genes collected in the following rice genome databases: The Institute of Genomic Research (TIGR) rice pseudomolecule release 4.0, KOME (version from 24 January 2006), UniGene (build #60), and the rice expressed sequence tag (EST) database downloaded from the National Center for Biotechnology Information (1 May 2006). The cutoff criteria include overlapping base pairs greater than 50 and identity greater than 95%. BLASTP or BLASTX was used to analyze the homolog between the identified rice genes and their homologs in other plant species. The BLASTP cutoff criteria was set to the E value of 1E‐10, the identity greater than 20%, and the blast hit length no less than 100 amino acids or 50% of the full‐length protein. The BLASTX cutoffs were set to the E value of 1E‐10, the identity greater than 50%, and the blast hit length no less than 60 amino acids or 50% of the full‐length protein.
Fluorescent labeling and hybridization
Probe labeling and hybridization were performed as described by Ma et al. ([24]). Briefly, RNA was labeled by indirect incorporation of amino‐allyl‐dUTP (aa‐dUTP; Sigma, St. Louis, MO, USA) during reverse transcription followed by coupling with either Cy3 or Cy5 monofunctional dye (Amersham Pharmacia Biotech., GE Healthcare Bio‐Sciences AB, Uppsala, Sweden). After purification, the labeled probe was added to the hybridization solution and then hybridized for 12 to 18 h in a 42 °C water bath. After washing, the chip was dried for 5 min by spinning at 1000 rpm and then for scanning. Microarray hybridization at each time point was carried out in three replications.
Scan and data analysis
The detailed protocol for scan and data analysis was reported ([48]). The microarray was scanned at Cy3 and Cy5 wavelengths using the ScanArray Lite (Perkin‐Elmer) at 5 nm resolution. Axon Genepix Pro 5.1 was used for the raw data extractions. Contaminated spots identified manually were eliminated. The pre‐processed data were normalized using the LOWESS (Locally Weighted Linear Regression) scale implemented in R package limma 2.0 (http://bioinf.wehi.edu.au). All spots from each microarray were included in the analysis, but zero weight was given to all the spots whose Genepix flags were less than 0.
Intensity‐dependent Z‐scores (sliding window = 50, threshold = 1.96) ([38]) were used in combination with a fold change of 1.5 to identify differentially expressed genes for each pair of hybridized samples. To detect the genes that are significantly regulated and to eliminate those that have inconsistent expression data among three replicated microarray experiments, significant genes were selected if they were differentially expressed (if the Z‐score was greater than 1.96 and the fold change was greater than 1.5) in at least two experimental samples. The Z‐score and fold change of each XDG was calculated as the average of replication experiments at each time point.
RT‐PCR assays
Total RNA was extracted from the plants samples that were used to isolate RNA for the microarray experiments. To prevent genomic DNA contamination, the RNA samples were treated with RNase‐free DNase (QIAGEN). First‐strand cDNA was synthesized from the total RNA using M‐MLV reverse transcriptase (Promega, Madison, WI, USA) following the manufacturer's protocol in a 30 μL reaction mix. PCR was carried out using 10 μL diluted cDNA as a template to the final volume of 30 μL in 1× buffer, 1.56 mM MgCl2, 0.2 mM dNTP, 10 pM of each specific primer and 2 U of Taq DNA polymerase (TaKaRa Biotechnology Co., Ltd., Dalian China). Actin was used as a control. Primers and their PCR programs are listed in Table S1.
(Co‐Editor: Daoxin Xie)
Acknowledgements
This work was funded by grants from the National Natural Science Foundation of China to GL (30670175), WS (30328019), and LZ (3073007), from the State Key Basic Research and Development Plan of China to LZ (2006CB101904), and from the Chinese Academy of Sciences to LZ (KSCX2‐YW‐N‐005). The authors are grateful to Terry Davoli for critical reading of the manuscript.
Figure S1. Validation of XDGs (Xa21 mediated differentially expressed genes) identified by the microarray assays. Expression of 13 XDGs was validated by reverse transcription‐polymerase chain reaction (RT‐PCR) reactions (five XDGs were tested at two time points). Log2 expression ratio of each XDG from the microarray assays is shown. R, the transgenic line 4021‐3 (containing Xa21) inoculated with Xoo PR6 (containing avrXa21); M, 4021‐3 inoculated with sterile distilled water; SAvr‐: 4021‐3 inoculated with Xoo PR10 (lacking avrXa21). RT‐PCR assays of the actin gene Os03g50890 were used as control.
Table S1. Primers and programs used for reverse transcription‐polymerase chain reaction (RT‐PCR) verification.
Table S2. The detailed information of 506 differentially expressed oligos.
Table S3. Forty‐seven XDGs (Xa21 mediated differentially expressed genes) between R‐M interactions at 4 h post inoculation (hpi).
Table S4. Seventy‐five XDGs (Xa21 mediated differentially expressed genes) between R‐M interactions at 8 h post inoculation (hpi).
Table S5. Eighty‐eight XDGs (Xa21 mediated differentially expressed genes) between R‐M interactions at 24 h post inoculation (hpi).
Table S6. Seventy‐five XDGs (Xa21 mediated differentially expressed genes) between R‐M interactions at 72 h post inoculation (hpi).
Table S7. One hundred and sixty‐nine XDGs (Xa21 mediated differentially expressed genes) between R‐M interactions at 120 h post inoculation (hpi).
Table S8. Fifty‐six XDGs (Xa21 mediated differentially expressed genes) between R‐S (Avr‐) interactions at 4 h post inoculation (hpi).
Table S9. Forty XDGs (Xa21 mediated differentially expressed genes) between R‐S (R‐) interactions at 4 h post inoculation (hpi).
Table S10. Thirty‐six signaling‐related XDGs (Xa21 mediated differentially expressed genes).
Table S11. Twenty‐nine pathogenesis‐related XDGs (Xa21 mediated differentially expressed genes).
Table S12. Comparison among 95 XDGs (Xa21 mediated differentially expressed genes) with the genes regulated in other R gene‐mediated defense responses.
Table S13. Comparison among 106 XDGs (Xa21 mediated differentially expressed genes) with the genes involved in basal defense, hypersensitive response (HR), systemic acquired resistance (SAR) and benzothiadiazole (BTH)‐response.
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Footnotes
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Available online on 17 February 2011 at http://www.jipb.net and http://www.wileyonlinelibrary.com/journal/jipb
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By Qiang Gan; Hui Bai; Xianfeng Zhao; Yong Tao; Haipan Zeng; Yuning Han; Wenyuan Song; Lihuang Zhu and Guozhen Liu
Reported by Author; Author; Author; Author; Author; Author; Author; Author; Author
