Early and specific gene expression triggered by rice resistance gene Pi33 in response to infection by ACE1 avirulent blast fungus

Authors


Author for correspondence: J.-B. Morel Tel: +33 499624 837 Fax: +33 499624 822 Email: jbmorel@cirad.fr

Summary

  • • Our view of genes involved in rice disease resistance is far from complete. Here we used a gene-for-gene relationship corresponding to the interaction between atypical avirulence gene ACE1 from Magnaporthe grisea and rice resistance gene Pi33 to better characterize early rice defence responses induced during such interaction.
  • • Rice genes differentially expressed during early stages of Pi33/ACE1 interaction were identified using DNA chip-based differential hybridization and QRT-PCR survey of the expression of known and putative regulators of disease resistance.
  • • One hundred genes were identified as induced or repressed during rice defence response, 80% of which are novel, including resistance gene analogues. Pi33/ACE1 interaction also triggered the up-regulation of classical PR defence genes and a massive down-regulation of chlorophyll a/b binding genes. Most of these differentially expressed genes were induced or repressed earlier in Pi33/ACE1 interaction than in the gene–for–gene interaction involving Nipponbare resistant cultivar.
  • • Besides demonstrating that an ACE1/Pi33 interaction induced classical and specific expression patterns, this work provides a list of new genes likely to be involved in rice disease resistance.

Introduction

The hypersensitive response (HR) in plants is often controlled by gene-for-gene systems where an avirulence gene in the pathogen matches the allelic form of a particular resistance gene in the plant. Downstream of this recognition event, signalling transduction pathways controlled by regulator genes are activated and lead to differential expression of defence-related genes (Dangl & Jones, 2001; Van Loon et al., 2006). Thus three types of genes are involved in the HR response: resistance genes, regulator genes and defence genes. Pathogenesis-related (PR) proteins fall into the latter class of defence genes (Van Loon et al., 2006). Our current picture of which genes are involved in disease resistance in monocotyledons is rather poor. By contrast, extensive studies in the plant model Arabidopsis have demonstrated that between 11% (Schenk et al., 2000) and 22% (Tao et al., 2003) of the genes are differentially regulated in a single situation (for review, see Eulgem, 2005). Most importantly, using Pseudomonas syringae pv. tomato, Tao et al. (2003) demonstrated that late compatible and early incompatible interactions show similar responses. Considering regulator genes that were identified in Arabidopsis by a forward genetic approach, most of the time they were shown to be differentially expressed upon infection. For example, the EDS5 gene was shown to be induced by virulent and avirulent strains of Pseudomonas syringae pv. tomato in Arabidopsis (Nawrath et al., 2002).

With the completion of rice and Magnaporthe grisea genomes, rice blast disease has become a model of choice to study host reactions in monocotyledons (Dean et al., 2005). Several large-scale approaches have been used to identify genes and proteins that are differentially expressed upon infection by M. grisea, other pathogens and related stresses. This led to the identification of genes that are potentially differentially regulated upon infection by M. grisea (Kim et al., 2001; Rauyaree et al., 2001; Jantasuriyarat et al., 2005) or related conditions (Xanthomonas oryzae pv. oryzae: Chu et al., 2004; Rice Yellow Mottle Virus: Ventelon-Debout et al., 2003). Differential cDNA libraries (SSH: Xiong et al., 2001; Lu et al., 2004; Shim et al., 2004; differential display: Kim et al., 2000) combined with reverse northern were also used. Although it is always difficult to compare such different studies, some common elements emerge:

  • • As in other systems, incompatible and compatible interactions seem to differ mainly quantitatively (Wen et al., 2003; Lu et al., 2004).
  • • Except for classical defence genes such as PR1, PR3 (chitinases), PBZ1 and metallothionein, there are few overlaps between the differentially expressed genes isolated in these studies. From reports on genes differentially expressed upon infection, it appears that only 18% were found in at least two independent studies (Droc et al., 2006; this study). This suggests that our knowledge of defence response in rice is far from complete.
  • • Among the common patterns of gene expression emerging from those studies, several found repression of genes involved in photosynthesis (Zhou et al., 2002; Lu et al., 2004; Shim et al., 2004).
  • • Many of the genes identified appear to belong to multigene families, and appropriate tools, such as quantitative RT-PCR, are needed to study their specific expression.
  • • Besides transcriptional factors, such as WRKYs (Zhou et al., 2002) and MYBs (Xiong et al., 2001), and with the exception of the Mlo gene (Zhou et al., 2002), no major regulator gene known to be required for disease resistance in Arabidopsis or monocotyledons was found in the thousands of genes differentially expressed upon infection.

We are currently studying the interaction between rice resistance gene Pi33 (Berruyer et al., 2003) and M. grisea avirulence gene ACE1 (Böhnert et al., 2004). In rice cultivar IR64, the Pi33 gene belongs to a 5 Mb introgression from O. rufipogon located on the small arm of O. sativa chromosome 8 (E. Ballini et al., unpublished). Pi33 is also found in some wild rice species, suggesting an ancient origin (E. Ballini et al., unpublished). However, the Pi33 resistance gene is probably a classical resistance gene as it maps to a small interval from the rice chromosome 8 containing several classical resistance gene analogues (Berruyer et al., 2003; E. Ballini et al., unpublished). Avirulence gene ACE1 encodes a putative hybrid protein between a polyketide synthase and a nonribosomal peptide synthase. Mutagenesis studies strongly support the hypothesis that the metabolite produced by this enzyme, rather than the enzyme itself, is the avirulence molecule (Böhnert et al., 2004). Such an interaction between a fungal metabolite and a disease resistance gene is unique among known avirulence/resistance interactions in fungi (Böhnert et al., 2004). As this interaction is original, this avirulence gene may induce plant defence mechanisms that differ from other avirulence/resistance interactions, at least during the early phases of recognition. Therefore we decided to explore rice defence reactions in this particular avirulence/resistance interaction. Nontargeted differential gene expression was performed using DNA chips to identify genes up- or down-regulated during early stages in this interaction. Additionally, the expression of genes known to be required for disease resistance in rice or other plants was monitored during ACE1/Pi33 interaction. Whereas more than 50 regulator genes required for disease resistance are known in dicots (Hammond-Kosack & Parker, 2003), only 14 have been described in cereals, mostly in barley (Ayliffe & Lagudah, 2004), and rice, including rice genes SPL11 (Zeng et al., 2004), OsMT2b (Wong et al., 2004), OsRac1 (Ono et al., 2001), NRR (Chern et al., 2005), SPL7 (Yamanouchi et al., 2002), OsEDR1 (Kim et al., 2003), OsEREBP (Kim et al., 2000), OsLSD1 (Wang et al., 2005) and OsMAPK6 (Lieberherr et al., 2005). Some of these disease resistance regulator genes are differentially expressed upon infection. The corresponding rice genes are probably involved in the control of disease resistance and as such have been used as putative markers of disease resistance induction in rice. Finally, there is a need for the identification of novel genes in rice that are differentially expressed during infection. Our strategy towards this goal involves a combination of DNA chip-based differential screening and an expression survey of selected disease resistance regulator genes. This first screening was followed by the monitoring of the expression of a selected number of differentially expressed genes during different compatible/incompatible interactions to identify patterns specific of Pi33/ACE1 interaction.

Materials and Methods

Plants and fungus

Oryza sativa L. (rice) plants and Magnaporthe grisea (Hebert) Barr were grown and inoculated as described in Berruyer et al. (2003). The IR64 rice cultivar contains the Pi33 resistance gene matching the ACE1 avirulence gene. The M. grisea PH14 isolate does not contain the ACE1 avirulence gene unless transformed here with the 31C12 cosmid (Böhnert et al., 2004). The Nipponbare/CL26 interaction is incompatible (Fig. 1c).

Figure 1.

Oxidative burst in the Pi33/ACE1 interaction. IR64 plants carrying the Pi33 resistance gene were inoculated with an avirulent strain (PH14:ACE1), virulent strain (PH14) or gelatin control. (a) The oxidative burst (H2O2) was measured using DAB staining. Spots where DAB staining was visible were counted under the microscope and normalized by measuring the length of leaf observed. The mean (+ SD) of two independent experiments is shown. (b) Examples of DAB staining in the incompatible interaction (1 day postinoculation (dpi)) and in the compatible interaction (5 dpi). Bar, 1 mm (c) Symptoms 5 dpi in resistant IR64 (inoculated with PH14:ACE1 isolate), susceptible IR64 (inoculated with PH14 isolate) and resistant Nipponbare inoculated with CL26.

DAB staining

The protocol for DAB staining was adapted from Thordal-Christensen et al. (1997). Diaminobenzidine (Sigma D-8001) was solubilized to 1 mg ml−1 of water, excised leaves were dipped overnight (in the dark) and tissues were cleared with ethanol/chloroform (4 : 1) overnight at room temperature.m

RNA extraction

For RT-QPCR applications, frozen tissue was ground in liquid nitrogen. Approximately 500 µl of powder was then treated with 500 µl TLES buffer (Tris PH8100 mm, LiCl 100 mm, EDTA PH8 10 mm and SDS 1%), 500 µl warmed water saturated phenol (Acros Organics, Halluin, France), and vortexed for 30 s. A volume of 500 µl of CHCl3 : isoamylalcohol (24 : 1) was then added and the samples were vortexed for 30 s. The samples were then centrifuged (30 min, 13 000 rpm) and the RNA was precipitated from the supernatant overnight using one volume LiCl 4 m. RNA pellets were then washed with 70% ethanol and resuspended in distilled water.

Primer design

Primers were designed using the Beacon designer v4 software (Stratagene, La Jolla, CA, USA). When it was available, the sequence used was the full-length cDNA (Kikuchi et al., 2003). In order to reduce specificity issues, available 3′ UTR sequences were chosen for primer design. Primer pairs for which efficiency was between 90 and 110%, as determined by standard amplification curves, were used for expression studies. Specific amplification was checked using denaturation curves of QPCR products. All primer sequences are available in the Supplementary Material (Table S1). The rice genome sequence (TIGR pseudomolecules v3) was used for annotation and BLAST searches using the OryGenesDB database and tool box (http://orygenesdb.cirad.fr/; Droc et al., 2006).

RT and QPCR

RNA samples (5 µg) were denaturated for 5 min at 65°C with oligo dT (3.5 µm) and dNTP (1.5 µm). They were then subjected to reverse transcription for 60 min at 37°C with 200 U of reverse transcriptase M-MLV (Promega, Madison, WI, USA), and DTT (5 mm) in the appropriate buffer. Two microlitres of cDNA (dilution 1/100) were then used for quantitative RT-PCR. Quantitative RT-PCR mixtures contained PCR buffer, dNTP (0.25 mm), MgCl2 (2.5 mm), forward and reverse primers (300 µm), 1 U of HotGoldStar polymerase and SYBR Green PCR mix as per the manufacturer's recommendations (Eurogentec, Seraing, Belgium). Amplification was performed as follows: 95°C for 10 min; 40 cycles of 95°C for 15 s, 62°C for 1 min and 72°C for 30 s; then 95°C for 1 min and 55°C for 30 s. The quantitative RT-PCR (QRT-PCR) reactions were performed using a MX3000P machine (Stratagene) and data were extracted using the MX3000P software. The amount of plant RNA in each sample was normalized using actin (Os03g50890; Table S1) as internal control. For Table 1 and Fig. 3, the calculation of expression ratios (infected vs gelatin treated plants) was done according to Pfaffl (2001). For Fig. 1, the calculation of gene expression was done using the measured efficiency for each gene (data not shown).

Table 1.  Expression ratios of genes selected from targeted approach and microarray. Two to three experiments were used to establish mean ratios between infected and control plants (gelatin). Ratios above 1.5 and below −1.5 are highlighted in bold and italic, respectively. Ratios between 1 and −1 were adjusted to 1. R, resistant; S, susceptible
GenePutative functionR-IR64 (Pi33) PH14:ACE1/gelatin R-Nipponbare CL26/gelatinS-IR64 (Pi33) PH14/gelatin
1 dpi2 dpi1 dpi2 dpi1 dpi2 dpi
Genes from targeted
approach
Os08g06280 OsLSD1 −3.8 −1.9 1.6 −2.1 1.7 −1.6
Os07g18750 DIR1 −2.7 2.6 1.8 −3.2 2.7 −1.4
Os01g69080 SSI2 −3.1 1.9 1.0 −1.9 1.41.0
Os05g02070 OsMT2b −2.0 1.6 1.0 −1.6 −1.21.0
Os05g49140 OsMAPK6 −3.5 2.2 −1.1−1.4 2.8 1.5
Os01g43540 OsSGT1 1.0 1.5 1.2 −1.9 1.01.0
Os12g38210 OsSPL11 1.0 1.6 1.0 −1.7 1.0−1.1
Os03g06410 OsEDR1 1.0 2.7 2.1 −1.6 2.6 1.0
Os01g57770 SABP2 1.0 1.9 1.6 1.5 2.5 1.0
Os05g45410 OsSPL7 2.5 −3.2 1.3 2.4 1.21.0
Os01g03940 OsNRR 2.0 1.7 1.0 1.8 −3.9 2.5
Os02g02980 EDS5 1.6 1.5 1.01.01.01.0
Os01g63420 COI1 6.3 1.5 1.4 −1.7 1.0 −2.8
Os10g42430 JIN 1.9 1.21.0−1.1 3.8 −2.3
Genes from microarray
Os07g48020Peroxidase 4.3 10.2 −2.0 5.6 1.9 2.5
Os04g24220Wall-associated kinase 2.0 4.9 −2.5 1.6 16.2 1.0
Os10g40930Oxidoreductase 2.4 3.1 1.0 2.0 2.6 1.3
Os03g1695033 kDa secretory protein 2.3 2.9 1.0 5.0 4.8 −1.6
Os07g35560Chitinase 5.1 24.8 1.0 4.8 16.2 8.1
Os01g58290Subtilase family protein 2.6 7.7 1.0 2.7 2.5 3.0
Os01g64110Chitinase III 2.2 2.9 1.0 2.0 3.2 1.0
Os06g03580Zinc finger, C3HC4 type 2.1 3.9 1.0 1.5 7.0 1.7
Os11g39370LRR kinase 1.7 2.3 −1.1 1.5 15.6 1.5
Os09g09830Heavy metal-associated domain 2.2 2.5 1.6 1.0 6.8 −1.3
Os06g50300Hsp90 1.9 2.2 1.01.0 2.8 1.0
Os02g08440WRKY09 2.5 5.9 1.01.0 1.8 1.0
Os08g10310LRR kinase 7.0 4.1 1.0−1.3 3.1 1.0
Figure 3.

Repression of proteases (a) and chlorophyll a/b binding genes (b) during Magnaporthe grisea infection. Expression ratios between infected and control plants (gelatin). R, resistant IR64 (Pi33)/PH14:ACE1 vs gelatin control; S, susceptible IR64 (Pi33)/PH14 vs gelatin control; R-NB, resistant Nipponbare/CL26 vs gelatin. QRT-PCR measurements were made on two to three independent experiments.

Microarray hybridization

RNA labelling and hybridization of the Agilent oligo chips (70-mers; Agilent G4137A, http://www.chem.agilent.com) were carried out according to the manufacturer's protocol. One dye swap and two technical repeats were done for each comparison.

Rice defence gene list

Genes for which differential expression is available in publications were from Cooper et al. (2003), Fujiwara et al. (2006), Han et al. (2004), Kim et al. (2001), Lu et al. (2004), Ramalingam et al. (2003), Shim et al. (2004), Shimono et al. (2003), Wen et al. (2003), Xiong et al. (2001), Zhou et al. (2002), Tsunezuka et al. (2005), Güimil et al. (2005) and Jantasuriyarat et al. (2005). This represents a total of 923 nonredundant rice genes for 1131 accession numbers (Table S2). For Jantasuriyarat et al., 2005), it is important to mention that only a subset of the genes from this publication was included in our database. We focused on ESTs that showed important induction or repression ratios between infected and control library. This represents 201 induced and 100 repressed EST in infected or SPL11 lesion mimic mutant tissues.

Origin of putative and known rice disease regulators

This corresponds to five genes originally described in Arabidopsis (COI1, DIR1, EDS5, JIN and SSI2), one gene from barley (SGT1; Schulze-Lefert & Vogel, 2000) and one from tobacco (SA binding protein SABP2; Slaymaker et al., 2002; Kumar & Klessig, 2003). The original sequences were blasted on the rice genome to identify the best blast mutual hit. Seven known rice disease regulators were also included (OsLSD1, OsSPL7, OsEDR1, OsMAPK6, OsMT2b, OsNRR and OsSPL11). Details can be found in Table S3.

Results

Induction of defence mechanisms during ACE1/Pi33 interaction

Since ACE1/Pi33 interaction differs from other gene-for-gene relationships, we first examined whether classical defence mechanisms were triggered in this interaction. Plants of rice indica cultivar IR64 carrying Pi33 were inoculated with M. grisea isogenic strains containing ACE1 (PH14-ACE1) or not (PH14). A typical HR was observed as soon as 2–3 dpi (days postinoculation) in the incompatible interaction (IR64/PH14-ACE1). Disease symptoms were visible after 5 dpi only in the compatible interaction (IR64/PH14, Fig. 1c). Using DAB staining, we observed that an oxidative burst was triggered as early as 1 dpi in the incompatible interaction (Fig. 1a,b). This burst was also detected at 2 dpi in the compatible interaction. Thus, as in other gene-for-gene interactions (Wojtaszek, 1997), the Pi33/ACE1 interaction is associated with an oxidative burst which is a hallmark of plant defence response. We monitored the expression of PBZ1 (Midoh & Iwata, 1996; Kim et al., 2004) and PR1b (Agrawal et al., 2000), which are classical pathogen-induced rice defence genes. In the incompatible Pi33/ACE1 interaction, PBZ1 and PR1b were induced at a high level at 2 dpi, whereas they were induced to a lower degree in the compatible interaction (Fig. 2). According to these criteria, the ACE1/Pi33 interaction is similar to other known gene-for-gene interactions, as there is an earlier and stronger induction of defence mechanisms (oxidative burst and induction of PR genes) in the incompatible interaction than in the compatible one.

Figure 2.

Examples of gene induction in the Pi33/ACE1 interaction. Gene expression was measured (0, 1 and 2 dpi) by QRT-PCR. Open bars, gelatin control; grey bars, susceptible IR64 (Pi33)/PH14; closed bars, resistant IR64 (Pi33)/PH14:ACE1. Gene expression level (arbitrary units) was normalized using actin (Os03g50890) as an internal reference (see Materials and Methods section). The mean (+ SD) of three experiments is shown. PBZ1: Os12g36880, similar to D38170; PR1b: Os01g28450, similar to U89895.

Identification of differentially expressed genes during Pi33/ACE1 interaction using a targeted approach

Among the large number of Arabidopsis and rice genes involved in disease resistance (Hammond-Kosack & Parker, 2003; see Introduction), we picked 14 (Table 1) to examine whether these genes would be differentially regulated in the Pi33/ACE1 interaction. These genes were selected on the basis that they showed, to some extent, differential expression upon microbial infection (Table S3). The corresponding homologous genes were identified in the rice genome and their expression was monitored by quantitative RT-PCR during the incompatible Pi33/ACE1 interaction as well as other resistant or susceptible situations. Data presented in Table 1 demonstrate that our strategy to select known regulator genes was fruitful to identify genes that are differentially regulated in the Pi33/ACE1 interaction. At 1 dpi, five genes (OsSPL7, EDS5, JIN, OsNRR, COI1) were up-regulated upon infection in a Pi33/ACE1 incompatible interaction, five (OsLSD1, OsMAPK6, SSI2, OsMT2b and DIR1) were down-regulated, while four (OsSGT1, OsSPL11, OsEDR1 and SABP2) were not differentially expressed (Table 1). The majority of these genes were also up-regulated at 2 dpi, except for OsLSD1 and OsSPL7, which were both repressed. Most of these genes were also differentially expressed during the other incompatible interaction tested between cv. Nipponbare and M. grisea isolate CL26. However, striking differences were observed. First, differential expression occurred earlier in the incompatible Pi33/ACE1 interaction than in the other incompatible interaction. Second, one gene (EDS5) was only regulated in the incompatible Pi33/ACE1 interaction and not in the other interactions tested. Finally, OsSPL11 and OsSGT1 showed opposite differential expression between Nipponbare/CL26 and Pi33/ACE1 incompatible interactions. These results suggest that the types of genes and their expression pattern strongly differ between Pi33/ACE1- and Nipponbare/CL26 incompatible interactions.

Identification of genes differentially expressed during Pi33/ACE1 interaction using microarrays

We have used microarrays to systematically identify the rice genes differentially expressed during ACE1/Pi33 interaction. To focus on the initial phases of infection, three differential hybridizations were performed: incompatible (IR64/PH14-ACE1 vs IR64/gelatin-treated) at two time points (1 and 2 dpi), and compatible (IR64/PH14 vs IR64/gelatin-treated) at 2 dpi. Four technical replicates were performed allowing a statistical comparison based on a Student t-test on absolute values from hybridization signals. With a P-value of 0.05, we identified 79 and 22 oligo probes up-regulated, respectively, more than 1.5-fold and twofold compared with noninoculated leaves. Down-regulated oligo probes were found in large numbers with 876 and 51 genes being down-regulated 1.5-fold and twofold, respectively, compared with noninoculated leaves. With regard to these values, to select genes for further studies, we applied 1.5-fold and twofold ratios as cut-offs for up- and down-regulated genes, respectively. Based on these criteria, 57 nonredundant genes are up-regulated (ratio > 1.5; Table 2) and 42 nonredundant genes are down-regulated genes (ratio < −2; Table 3). Among up-regulated genes, 18 (67%) were common to incompatible and compatible interactions (Table 2), whereas only one (1/42) down-regulated gene was common to incompatible and compatible interactions (Table 3).

Table 2.  Expression ratios of 57 genes up-regulated in IR64 (Pi33)/PH14–ACE1 interaction. Genes in italic and bold are those chosen for QRT-PCR confirmation of data. All except Os12g30820 were confirmed. The genes for which accession numbers are indicated in bold are those presented in more detail in Table 1. Expression ratios in bold are those for which ratios are > 1.5 with an associated P-value < 0.05
Agilent IDTIGR IDPutative functionIR64 (Pi33) PH14:ACE1/gelatin PH14/gelatinExisting published expression
1 dpi2 dpi2 dpi
AZR05361Os10g36840Putative ferulate-5-hydroxylase (Oryza sativa) 1.60 1.191.18 
AZR06995 Os04g40990 oj991113_30.4 (Oryza sativa (japonica cultivar-group)) 1.59 −1.57−1.9 
AZR06501 Os02g08440 WRKY transcription factor, putative, expressed 1.52 1.311.36 
AZR06000 Os07g34520 Isocitrate lyase, putative, expressed 1.81 1.51 −1.42Yes
AZR06114 Os07g26110 EST 1.61 1.58 1.47 
AZR00863 Os01g71350 Glucan endo-1,3-beta-glucosidase GII precursor 1.53 1.86 2.27  
AZR07054Os03g58790ATPase 2, putative, expressed 1.50 1.78 2.52  
AZR03696EST1.85 1.66 1.52 
AZR03556Os01g70490Potassium transporter 5, putative, expressed1.32 1.84 1.43 
AZR00830Os02g40240Leucine Rich Repeat family protein, expressed−1.13 1.71 1.46Yes
AZR02616Os04g56430Receptor-like protein kinase 6, putative, expressed−1.99 2.10 −1.09Yes
AZR04751 Os07g35560 Chitinase1.31 3.16 4.08 Yes
AZR02475 Os04g10160 Cytochrome P450 CYP99A1, putative, expressed1.16 3.08 2.12  
AZR05828Os11g37950Barwin, putative, expressed−1.09 2.97 4.55  
AZR01867 Os01g42410 PDR5-like ABC transporter, putative, expressed1.21 2.35 1.90  
AZR04963 Os04g24220 Wall-associated kinase−1.15 2.29 2.19  
AZR01852 Os12g36110 Calmodulin-binding protein, putative, expressed1.02 2.09 2.05  
AZR06906Os12g36850Pathogenesis-related protein PR-10b (Oryza sativa subsp. indica)−1.29 1.98 2.10 Yes
AZR03112Os10g28080Class III chitinase1.21 1.95 3.11  
AZR00482 Os06g03580 Zinc finger, C3HC4 type family protein1.26 1.95 2.17  
AZR06915Os01g73200Peroxidase 12 precursor, putative, expressed1.24 1.93 1.63  
AZR02876 Os10g40930 Putative dioxygenase1.14 1.78 1.94  
AZR01190Os03g03440Putative phosphoribosylanthranilate transferase1.06 1.76 1.57 Yes
AZR06021 Os12g30820 EST−1.29 1.73 1.73  
AZR05864Os01g58290Subtilase family protein−1.19 1.73 2.13  
AZR06128Os10g39140Oxidoreductase, 2OG-Fe oxygenase family protein, expressed1.16 1.67 1.72  
AZR05868Os05g43170Calreticulin-3 precursor, putative, expressed1.05 1.60 2.03  
AZR06450 Os08g10310 LRR kinase1.231.60 2.67  
AZR02673 Os11g39370 LRR kinase1.101.54 1.75  
AZR01833 Os07g48020 Peroxidase 2 precursor, putative, expressed1.451.46 2.65 Yes
AZR06271Os01g41820Cytochrome P450 family protein, expressed−1.201.46 1.63  
AZR01976 Os09g09830 Heavy metal-associated domain containing protein, expressed1.011.40 1.51  
AZR04574Os06g40650Copine family protein, expressed−1.071.38 1.53  
AZR06379Os03g16390Putative Avr9/Cf-9 rapidly elicited protein1.031.37 1.75  
AZR02349 Os02g57280 EST−1.031.37 2.51  
AZR00712Os03g24930Protein kinase APK1A, chloroplast precursor, putative, expressed1.471.36 1.97  
AZR02733Os01g55810P0403C05.2 (Oryza sativa (japonica cultivar-group))−1.261.35 1.52  
AZR05843Os04g30330Protein kinase domain containing protein, expressed−1.151.31 1.51  
AZR04276Os01g38250Putative peptidylprolyl isomerase (Oryza sativa (japonica cultivar-group))−1.421.30 1.73  
AZR05576Os01g57070Putative zinc protease (Oryza sativa (japonica cultivar-group))1.091.27 1.66  
AZR01394Os11g44560Protein kinase domain containing protein, expressed−1.021.26 1.52  
AZR00912Os08g17680Stromal cell-derived factor 2-like protein precursor, putative, expressed1.051.26 1.61  
AZR04350Os03g05620Phosphate: H+ symporter family protein, expressed1.171.25 1.63  
AZR00322Os01g38240Putative peptidylprolyl isomerase (Oryza sativa (japonica cultivar-group))−1.351.21 1.59  
AZR06659Os01g48610B1144G04.12 (Oryza sativa (japonica cultivar-group))1.101.21 1.84  
AZR02294 Os06g50300 Hsp90−1.041.19 1.55  
AZR03995 Os03g16950 33 kDa secretory protein, putative, expressed1.461.19 1.73  
AZR03912Os04g15690’Unknown protein; protein id: At1g76020.1 (Arabidopsis thaliana)‘−1.031.15 1.79  
AZR01286Os03g55240Cytochrome P450 family protein, expressed−1.291.12 1.53  
AZR01338 Os01g64110 Chitinase III1.391.11 1.87  
AZR06427Os05g16740SHR5-receptor-like kinase, putative, expressed−1.341.07 1.53  
AZR05757Os04g52640SHR5-receptor-like kinase, putative, expressed−1.16−1.06 1.64  
AZR03333 Os10g10130 Calcium binding EGF domain containing protein, expressed−1.12−1.09 1.53 Yes
AZR01858 Os11g25260 GDA1/CD39 family protein, expressed−1.65−1.10 1.96  
AZR00341Os01g68740Expressed protein−1.20−1.13 1.54  
AZR04075Os11g25330GDA1/CD39 family protein, expressed−1.70−1.19 1.73  
AZR06380Os01g64120Ferredoxin-2, chloroplast precursor, putative, expressed−1.14−1.22 1.52  
Table 3.  Expression ratios of 42 genes down-regulated in the IR64 (Pi33)/PH14–ACE1 interaction. Expression ratios in bold are those for which ratios are less than −2 with associated P-value < 0.05
Agilent IDTIGR IDPutative functionIR64 (Pi33) PH14: ACE1/gelatin PH14/gelatinExisting published expression
1 dpi2 dpi2 dpi
AZR05140Os01g52240Chlorophyll a/b binding protein 2, chloroplast precursor, putative, expressed −3.01 −1.41−1.15 
AZR02014Starch branching enzyme rbe4 (Oryza sativa) −2.70 −1.24−1.22 
AZR00828Os04g38410Chlorophyll a/b binding protein CP24 10B, chloroplast precursor, putative −2.63 −1.59−1.14Yes
AZR06965Os01g41710Chlorophyll a/b binding protein 2, chloroplast precursor, putative, expressed −2.58 −1.46−1.01 
AZR03292Os11g32650Chalcone synthase, putative, expressed −2.38 −1.99−1.24Yes
AZR05281Os03g20700Magnesium-chelatase subunit H family protein, expressed −2.26 1.041.29 
AZR03049Os05g50750AAA-type ATPase family protein, putative, expressed −2.22 −1.25−1.16 
AZR01577Os05g44210Alpha,alpha-trehalose-phosphate synthase, putative, expressed −2.15 −1.05−1.00 
AZR06951Os06g06290GDSL-like Lipase/Acylhydrolase family protein, expressed −2.12 −1.821.03 
AZR06598Os03g52630Endo-1,4-beta-glucanase Cel1, putative, expressed −2.09 −1.31−1.30 
AZR06715Os04g36720Ferric reductase-like transmembrane component family protein, putative, expressed −2.05 −1.81−1.33 
AZR05987Os02g18450GTP-binding protein TypA/BipA homolog, putative, expressed −2.00 −1.101.09Yes
AZR00656Os03g14650EST −2.62 −2.14 −1.25 
AZR00910Os10g21280Ribulose bisphosphate carboxylase large chain precursor, putative, expressed−1.66 −2.34 −1.04Yes
AZR05721Os09g38090Expressed protein−1.56 −2.09 −1.10 
AZR06984Os07g10840Seed imbibition protein, putative, expressed−1.56 −2.58 −1.27 
AZR04269Os02g13060EST−1.55 −2.06 −1.28 
AZR05446Os07g15460Metal transporter Nramp1, putative, expressed−1.55 −2.55 −1.72 
AZR02650Os01g46720Protein kinase domain containing protein, expressed−1.53 −2.11 −1.35 
AZR00021Os10g36000Remorin, C-terminal region family protein, expressed−1.52 −2.14 −1.05 
AZR05968Os08g04430EST−1.48 −2.13 −1.16 
AZR05929Os12g02340Nonspecific lipid-transfer protein 3 precursor, putative, expressed−1.47 −2.34 −1.09 
AZR04727Os08g02630Expressed protein−1.43 −2.09 1.00 
AZR05211Os10g18340Unknown protein (Oryza sativa) >gi|22655738|gb|AAN04155.1−1.36 −2.31 −1.40Yes
AZR05823Os04g47220Aquaporin RWC3, putative, expressed−1.36 −2.08 −1.24 
AZR06883EST−1.34 −2.59 −1.10 
AZR04953Os02g33070Hypothetical protein−1.30 −2.01 1.08 
AZR03973Os01g52750Subtilisin N-terminal Region family protein, expressed−1.29 −2.04 −1.02 
AZR05034Os12g02570EST−1.25 −2.02 −1.11 
AZR02228Os06g22960Aquaporin TIP-type RB7-18C, putative, expressed−1.25 −2.04 −1.33Yes
AZR00145Os01g14950Importin alpha-1b subunit, putative, expressed−1.22 −2.59 −1.10Yes
AZR04300Os01g74410Myb-like DNA-binding domain containing protein, expressed−1.21 −2.27 −1.12Yes
AZR00994Os07g05360Photosystem II 10 kDa polypeptide, chloroplast precursor, putative, expressed−1.20 −2.13 −1.71Yes
AZR04934EST−1.18 −2.25 −1.20 
AZR03740Os06g48500EST−1.17 −2.02 −1.01 
AZR05305Os06g21210Glycine rich protein family protein, expressed−1.17 −3.73 −1.33Yes
AZR02117Os12g25200Chloride channel protein CLC-a, putative, expressed−1.16 −2.04 −1.15 
AZR00543Os01g63190Multicopper oxidase family protein, expressed−1.02 −2.45 1.08 
AZR03226Hypothetical protein (Oryza sativa (japonica cultivar-group))1.20 −2.27 −1.22 
AZR05231Os08g35760Auxin-binding protein ABP20 precursor, putative, expressed1.31 −2.99 −1.84 
AZR03028Os04g40630TAZ zinc finger family protein, expressed−1.71 −2.55 −2.18 Yes
AZR00960Os02g52390Peptidase M16 inactive domain containing protein, expressed−1.601.44 −3.08 Yes

We selected 24 genes from the 57 up-regulated genes (Table 2) for further studies of their expression using QRT-PCR. With the exception of Os12g30820, the expression of 23/24 genes (96%) was confirmed with RNA from the original microarray hybridization, suggesting that the quality of the microarray hybridization is correct. However, only 13 of these 23 genes (Table 1) were also up-regulated in the three independent biological replicates. Genes encoding leucin-rich repeat (LRR) proteins, protein kinases and receptor-like kinases are a major class of genes up-regulated during Pi33/ACE1 interaction (11% of the 57 up-regulated and none of the 42 down-regulated genes; Tables 2, 3). These results strongly suggest that genes involved in signal perception and transduction are well represented among up-regulated genes. The putative functions of down-regulated genes are strikingly different as they are related to photosynthesis (chlorophyll a/b binding proteins; discussed later), protein degradation (proteases, 5%) and water transport (aquaporins, 5%). The survey of published data on induction of rice genes upon infection identifies a set of 923 genes differentially expressed upon infection by M. grisea or X. oryzae oryzae (Droc et al., 2006; also see http://orygenesdb.cirad.fr). Using this ‘rice defence gene list’ as a reference (Table S2), we show that 86% of the induced genes (Table 2) and 75% of the repressed genes (Table 3) in Pi33/ACE1 interaction were not described as such before.

Genes up-regulated during Pi33/ACE1 interaction

Several contrasting biological situations were used to survey the expression patterns of 13 up-regulated genes from the microarray experiment validated by QRT-PCR (Table 1). We used the original IR64 (Pi33) and isolate PH14:ACE1 interaction as well as another incompatible interaction between Nipponbare japonica cultivar and avirulent M. grisea isolate CL26 (Fig. 1c). As a compatible interaction, we used the infection of IR64 rice cultivar by virulent M. grisea isolate PH14. Most of these 13 genes had induction ratios close to two, although two transcripts had higher ratios (10 and 25 for peroxidase Os07g48020 and chitinase Os07g35560, respectively). Results obtained from a representative experiment are displayed for two representative patterns in Fig. 2. The Os01g58290 pattern is representative of five genes (Os07g48020, Os07g35560, Os06g03580, Os11g39370 and Os01g58290) up-regulated in both incompatible Pi33/ACE1 and compatible IR64/PH14 interactions. Os09g09830 is representative of eight genes (Os04g24220, Os10g40930, Os03g16950, Os01g64110, Os08g10310, Os06g50300, Os02g08440 and Os09g09830) still up-regulated in incompatible Pi33/ACE1 at 2 dpi while their up-regulation decreases in the corresponding compatible interaction. The incompatible interaction between rice cv. Nipponbare and M. grisea isolate CL26 was used to survey the expression of these genes in another gene-for-gene interaction besides Pi33/ACE1. Overall, the expression patterns of these genes are quite different in incompatible Pi33/ACE1 (IR64) and Nipponbare interactions (Table 1), as only a single gene Os09g09830 displayed induction in both incompatible interactions. Other genes up-regulated in Pi33/ACE1 interaction were either not differentially expressed (Os06g50300, Os02g08440 and Os08g10310) or up-regulated only later (2 dpi) in a Nipponbare/CL26 interaction.

Genes down-regulated in Pi33/ACE1 interaction

Among the 42 genes down-regulated during Pi33/ACE1 interaction, we identified two proteases (Table 3) and four more in an extended data set (data not shown). The expression patterns of three of these proteases (Os01g37910, Os11g13670 and Os09g27030) were surveyed using QRT-PCR. These three proteases were shown to be down-regulated in the three biological replicates corresponding to a Pi33/ACE1 interaction at 2 dpi (Fig. 3a) and in a Nipponbare/CL26 incompatible interaction (data not shown). However, they were also down-regulated in the compatible interaction at 2 dpi (Fig. 3a), suggesting that this differential expression is related to M. grisea infection rather than to the induction of specific plant defence mechanisms. Three chlorophyll a/b binding proteins were also repressed during Pi33/ACE1 interaction at 1 dpi (Table 3). In addition, the microarray experiment indicated that 11 chlorophyll a/b binding proteins were also repressed at 1 dpi in the Pi33/ACE1 interaction (data not shown). We monitored the expression of five chlorophyll a/b binding genes by QRT-PCR (Fig. 3b) and observed an early repression of these genes in both Pi33/ACE1 and Nipponbare/CL26 incompatible interactions. By contrast, these chlorophyll a/b binding genes were up-regulated at 2 dpi in the Pi33/ACE1 incompatible (Fig. 3b) and IR64/PH14 compatible interactions (data not shown). This suggests that down-regulation of chlorophyll a/b binding genes is associated with the onset of plant defence mechanisms.

Discussion

The Pi33/ACE1 interaction triggers classical defence reactions

Given the unusual nature of the ACE1 avirulence gene (Böhnert et al., 2004), we wondered whether rice defence mechanisms triggered by this avirulence gene would be novel. As shown in Fig. 1(a,b), an oxidative burst was triggered by the ACE1/Pi33 incompatible interaction as in other plant disease defence reactions (Wojtaszek, 1997) and classical plant defence genes including known PR proteins were induced (Fig. 2). Transcriptome analysis of early stages of plant fungal interactions (1–2 dpi) further supported the assertion that this gene-for-gene interaction is similar to others, as most genes differentially expressed during the Pi33/ACE1 incompatible interaction were also differentially expressed in susceptible plants (Fig. 2, Tables 1, 2), although later, as frequently observed for plant defence genes (Tao et al., 2003). An additional characteristic of expression patterns observed during the Pi33/ACE1 incompatible interaction is the high frequency of down-regulated genes (Table 3). For example, two putative cysteine proteases (Os01g37910 and Os09g27030) and one putative serine protease (Os11g13670) were down-regulated in both incompatible Pi33/ACE1 and compatible IR64/PH14 interactions (Fig. 3a). Repression of cysteine proteases in rice leaves infected by M. grisea has already been observed in rice. Fujiwara et al. (2006) showed that another rice cysteine protease, Os01g67980, is repressed by a constitutively active form of OsRac1 mimicking disease resistance. Cooper et al. (2003) also showed that jasmonic acid, a key signalling molecule during rice disease resistance, repressed the expression of the same cysteine protease Os09g27030 we identified as differentially expressed during infection. In susceptible plants, down-regulation of cysteine proteases may be triggered by the pathogen in order to suppress basal rice defence. Triggering of resistance by the Pi33/ACE1 interaction is possibly not sufficient to counteract this defence suppression induced at early stages of the infection process. Finally, chlorophyll a/b binding genes were early and strongly down-regulated in the Pi33/ACE1 interaction (Fig. 3b), a phenomenon already observed in other gene-for-gene interactions between rice and M. grisea or X. oryzae (Zhou et al., 2002; Lu et al., 2004; Shim et al., 2004).

Microarray analysis of the Pi33/ACE1 interaction identifies novel putative defence genes

A transcriptomics analysis of the ACE1/Pi33 interaction was performed using Agilent DNA chips, allowing the identification of rice genes that are differentially expressed upon infection (Tables 2, 3). About 80% (79 genes) of the regulated genes we identified were not previously reported, even though similar analyses of rice leaves infected with M. grisea or X. oryzae were performed with a comparable number of genes being analysed (Chu et al., 2004; Lu et al., 2004; Jantasuriyarat et al., 2005). This is consistent with the observation that, among the 923 rice genes putatively involved in disease resistance (Droc et al., 2006; see Table S2), only 18% were reported as being differentially expressed during disease resistance in at least two studies. Indeed, whereas resistance gene analogues (LRR proteins and receptor-like kinases) represented 11% of the 57 up-regulated genes we identified (Table 2), none were reported in Lu et al. (2004) or Jantasuriyarat et al. (2005). Eleven of the novel rice genes we identified (Table 1) encode protein functions known to be up-regulated in rice or other plants upon infection (Van Loon et al., 2006). For example, 15 WRKY transcription factors were recently reported to be up-regulated upon M. grisea infection (Ryu et al., 2006). However, the WRKY09 gene we identified was not reported in this study, probably as a result of the lower sensitivity of the technique used. Similarly, an Arabidopsis wall-associated kinase (Wak1) was shown to be involved in plant defence (He et al., 1998) and a wall-associated like kinase (WAKL22) was shown to be required for disease resistance in Arabidopsis infected with Fusarium (Diener & Ausubel, 2005), and hsp90 proteins (for review, see Sangster & Queitsch, 2005), subtilase and proteases (Xia, 2004) have frequently been found to be involved in or associated with induction of plant disease resistance.

The Pi33/ACE1 interaction regulation pathway

Global comparison between Pi33/ACE1 and Nipponbare/CL26 incompatible interactions provided some information on the Pi33 regulation pathway. Most regulated genes were differentially expressed in both Pi33/ACE1 and Nipponbare/CL26 incompatible interactions (Table 1), although differential expression was observed earlier in the Pi33/ACE1 interaction than in the Nipponbare/CL26 interaction for 15 out of 17 genes (Table 1). This observation suggests that the Pi33 resistance gene is more efficient at triggering a defence response than the resistance gene from Nipponbare involved in the recognition of CL26. This could be related to the fact that the ACE1 gene is only expressed at an early stage of infection (1 dpi) during penetration of the fungus into the first host epidermal cell (Böhnert et al., 2004). However, we cannot rule out the possibility that background differences for both host and pathogen may lead to these results.

Five genes differentially expressed in the two incompatible interactions tested were not expressed in the compatible interaction IR64/PH14 (SSI2, OsMT2b, OsSGT1, OsSPL11 and OsSPL7; Table 1). These genes encode known or potential regulators of disease resistance (Table S3) involved in signalling disease resistance: the SGT1 gene is involved in the initial recognition between the avirulence product and the resistance gene (Azevedo et al., 2006), the OsMT2b gene is a negative regulator of the oxidative burst (Wong et al., 2004), and the three genes, SSI2, OsSPL7 and OsSPL11, are negative regulators of cell death (Kachroo et al., 2001; Yamanouchi et al., 2002; Zeng et al., 2004). Their expression during additional compatible interactions needs to be tested to establish if they are good markers of rice resistance.

Genes up- or down-regulated specifically during a particular gene-for-gene incompatible interaction have been only rarely described, despite numerous attempts. As such, only one gene (a proteasome component, BF108345) was identified as being up-regulated specifically during resistance triggered by an avirulent M. grisea isolate recognized by rice resistance gene Pi1(t) (Wen et al., 2003). In our experiments, we could only identify one gene, EDS5, as specifically up-regulated during the Pi33/ACE1 incompatible interaction (Table 1). Interestingly, the EDS5 gene codes a MATE (multidrug and toxin extrusion) transporter protein (Nawrath et al., 2002). How this relates to the secondary metabolite produced by the ACE1 gene will need further investigation. More gene-for–gene interactions need to be tested to establish if EDS5 is a specific marker of rice Pi33-triggered resistance by ACE1.

Acknowledgements

EV is funded by a joint grant from INRA and the Languedoc-Rousillon region. EB is funded by the French Ministry of Research. Part of this work was supported by the French programme Génoplante (projects OsCrR1 and B8). We thank V. Chalvon, C. Michel and L. Fontaine for technical assistance. We thank Christophe Périn for critical reading of the manuscript.

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