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Identification of novel genes differentially expressed in compatible and incompatible interactions between rice and Pseudomonas avenae
Plant Science 162 (2002) 449 /458 www.elsevier.com/locate/plantsci
Identification of novel genes differentially expressed in compatible and incompatible interactions between rice and Pseudomonas avenae Fang-Sik Che *, Tetsuyuki Entani, Taizou Marumoto, Masatoshi Taniguchi, Seiji Takayama, Akira Isogai Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5, Takayama Ikoma, Nara 630-0101, Japan Received 16 July 2001; received in revised form 23 October 2001; accepted 22 November 2001
Abstract Establishment of a plant /pathogen interaction involves differential gene expression in both organisms. To isolate rice genes induced during either compatible or incompatible interactions, we performed a comparative analysis of expression patterns in cultured rice cells, inoculated with either compatible or incompatible strains of Pseudomonas avenae, by fluorescence differential display (FDD). Using 52 sets of arbitrary primer combinations, two cDNAs (IAI1 and IAI2 ) expressed in incompatible straininoculated rice cells and one cDNA (CAI1 ) expressed in compatible strain inoculated cells, were identified. A hydropathy profile revealed that the IAI1 protein spans the membrane at a putative transmembrane domain. The deduced amino acid sequence of IAI2 shares considerable homology with Bowman /Birk proteinase inhibitors over the majority of the sequence. The deduced CAI1 protein is rich in glycine, containing a putative signal peptide with a potential cleavage site, suggesting its membership in the glycinerich protein (GRP) family. GRPs are thought to be structural cell wall proteins. When a GFP fusion with the CAI1 gene was introduced into onion cells by bombardment, green fluorescence of the cell wall was observed. These data suggest that CAI1 is involved in the repair of cell wall injuries by the compatible strain of P. avenae . # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bowman /Birk proteinase inhibitors; Differential display; Glycine-rich protein; Rice; Pseudomonas avenae
1. Introduction The host range of bacteria pathogenic to plants is generally limited to certain species of plants. ‘Compatible’ relationships lead to intercellular bacterial growth and symptom development within the host and no disease symptoms are observable in ‘incompatible’ cases. In addition to preformed physical and chemical barriers, such as thickened cuticles or preformed antimicrobial
Abbreviations: CAI, compatible strain of P. avenae -induced gene; FDD, fluorescence differential display; GFP, green fluorescent protein; GRP, glycine-rich protein; IAI, incompatible strain of P. avenae -induced gene; PCR, polymerase chain reaction The nucleotide sequence data reported will appear in the DDBJ/ EMBL/GenBank Nucleotide Sequence Database under the accession numbers AB059237 (IAI1 cDNA), AB059238 (IAI2 cDNA), AB059239 (CAI1 cDNA). * Corresponding author. Tel.: 81-743-72-5452; fax: 81-743-725459. E-mail address: [email protected] (F.-S. Che).
compounds, pathogenic attack induces a wide variety of defense responses [1,2]. These defenses are triggered rapidly and coordinately, allowing the plant to become resistant to pathogen invasion. The defense mechanisms of susceptible plants respond more slowly after infection. Thus, both the timely recognition of an invading microorganism and the rapid and effective induction of the defense responses differentiate resistant plants from susceptible ones [3,4]. Inducible defense follows a basic pathway of recognition and signal transduction to activate defensive responses, such as the generation of reactive oxygen species (the oxidative burst) [5,6], hypersensitive cell death [7], the deposition of both hydroxyproline-rich glycoproteins [8] and cell wall lignins [9], the accumulation of phytoalexins [10] and the synthesis of antimicrobial PR proteins [5]. The longterm systemic induction of additional or overlapping sets of genes follows generating proteinase inhibitors protecting the gene products associated with induced
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 5 8 5 - 4
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systemic resistance. The response of the host plant thus requires complex changes in gene expression patterns. Pseudomonas avenae (Acidovorax avenae), a Gramnegative bacterium, causes a seedling disease, characterized by the formation of brown stripes on the sheaths of infected plants. The wide host range of P. avenae throughout the monocotyledonous plants includes rice, oats, Italian millet and maize, individual strains of the pathogen, however, infect only one or a few host species. Strains isolated from rice, such as H8201 or H8301, can only infect rice plants, while the N1141 strain, isolated from finger millet, cannot infect rice even after inoculation into rice tissues. The rice-incompatible strain, N1141, causes hypersensitive cell death accompanied by DNA cleavage and typical apoptotic morphological changes; the rice-compatible strain, H8301, does not induce any of these changes [11]. In addition to the difference in the hypersensitive cell death induction, several different phenomena were observed in cultured rice cells inoculated with the incompatible and the compatible strains. The EL2 gene is known to be expressed with N -acetylchitoheptaose within 15 min to induce a set of defense reactions in cultured rice cells [12,13]. The accumulation of EL2 mRNA is detectable within 3 h after inoculation with the incompatible strain, N1141, whereas in H8301-inoculated cultured cells, it is not detectable. These results indicate that the N1141 strain causes a defense response in cultured rice cells, but the compatible strain H8301 does not [11]. Furthermore, we have demonstrated that the P. avenae flagellin is involved in the induction of the defense responses in incompatible hosts using the purified flagellin and flagellin-deficient mutants [14]. However, our understanding of the molecular interaction between P. avenae and the rice plant is limited and the genetic determinants of P. avenae host species specificity remain elusive. Determination of genes differentially expressed in the compatible and incompatible interactions would allow a greater understanding of the molecular mechanism involved in the interaction between P. avenae and the rice plant. mRNA differential display is commonly employed to identify genes differentially expressed between a particular tissue in the control state and an altered state. To identify genes differentially expressed in rice plants in response to compatible and incompatible strains, we applied mRNA fluorescent differential display to the compatible and incompatible interaction between P. avenae and rice. Using 52 sets of arbitrary primer combinations, we identified two cDNAs (IAI1 and 2) induced in the cells inoculated with the incompatible strain and one cDNA (CAI1 ) induced in the cells inoculated with the compatible strain. We also report the characterization and localization of CAI1 gene, induced by the compatible strain, and predict the possible role of this gene during the interaction between P. avenae and rice.
2. Materials and methods 2.1. Bacterial strains and cultures P. avenae H8201 (MAFF 301502), isolated from rice, and strain N1141 (MAFF 301141), isolated from finger millet, were utilized as previously described [15]. Each P. avenae strain was maintained at 30 8C on Pseudomonas F agar plates (Difco) as described [11]. 2.2. Fluorescent differential display Fluorescent differential display (FDD) was performed as previously described [16,17]. Suspension cultures of the Oc line of rice cells [18] were grown at 30 8C under light irradiation. Cells were diluted in fresh medium every 7 days. Experiments were performed 4 days after transfer. The cultured cells were incubated at 30 8C with bacteria (108 cfu ml 1) for variable time lengths after inoculation. mRNAs were prepared from cultured rice cells 5, 15, 30 and 60 min after inoculation of either the compatible or the incompatible strain using ISOGEN (Nippon gene). First-strand cDNAs were synthesized by reverse transcriptase (Super Script II, Gibco BRL) using 3?-anchored oligo-dT primers (5?-GT15VN-3?). Second strand synthesis and polymerase chain reaction (PCR) were performed using rhodamine-labeled 3?-anchored primers (XRITC-GT15MA, XRITC-GT15MT, XRITC-GT15MG, XRITC-GT15MC) and arbitrary 10-mer primers (Operon Technologies, Inc.). We utilized Taq-DNA polymerase and reaction buffer (TOYOBO, Osaka, Japan) for PCR analysis (94 8C 3 min, 40 8C 2 min, 72 8C 5 min, followed by 25 cycles of 95 8C 15 s, 40 8C 2 min, 72 8C 1 min, and 72 8C 5 min). The PCR products were separated by electrophoresis on 5% polyacrylamide gels containing 7 M urea and fluorescent images were analyzed with an FM-BIO (TaKaRa). Differentially expressed bands were removed from the gels, eluted by boiling, reamplified using the same primer pairs, cloned into the PCR 2.1 vector (Invitrogen) and sequenced. To eliminate false positive clones, PCR products, separated by the polyacrylamide gel, were blotted onto a Hybond-N membrane (Amersham Pharmacia Biotech) and analyzed by hybridization to each cDNA clone. The missing 5? regions of the cDNA fragments were obtained using a Marathon cDNA Amplification kit (Clontech), according to the manufacturer’s instructions. Total RNA was isolated from cultured rice cells 1 h after inoculation with either the N1141 or the H8201 strain. Poly(A)/ RNA was isolated using a Micro fast track kit (Invitrogen) and single-strand cDNA was synthesized using a polyT-NM primer. After doublestrand cDNA synthesis and adapter ligation, PCR was performed using both the adapter primer and each genespecific primer. The amplified fragments were cloned
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with a TA cloning kit (Invitrogen). Five clones from each PCR reaction were sequenced with a DNA sequencer (model 377; PE Applied Biosystems). PCR and cloning procedures were independently repeated twice to confirm DNA sequences. 2.3. RNA isolation and Northern blot analysis Following incubation with each bacterial strain (or without bacteria as a control), the cultured rice cells were ground in liquid nitrogen. Total RNA was extracted with ATA (aurintricarbosylic acid) [19] and RNAs (15 mg) were electrophoresed on a formaldehyde denaturing 1% (w/v) agarose gel in 1 / MOPS buffer (20 mM MOPS /KOH, pH 7.0, 5 mM sodium acetate, and 1 mM EDTA). RNA was then transferred to a Hybond-N membrane (Amersham Pharmacia Biotech), according to standard protocols. Each cDNA fragment was then labeled with [32P]dCTP to be utilized as a hybridization probe. 2.4. Southern blot analysis Genomic DNA was isolated from cultured rice cells with a Nucleon plant-DNA extraction kit (Amersham Pharmacia Biotech) according to the manufacturer’s protocol. Following digestion with Hin d III, Eco RI and Bgl II, DNA (10 mg) was electrophoresed on a 0.7% (w/v) agarose gel. Fractionated DNA was then transferred to a Hybond N membrane (Amersham Pharmacia Biotech). Two CAI1 cDNAs (corresponding to the ORF and 3?-UTR sequences) were labeled with an AlkPhos Direct labeling kit (Amersham Pharmacia Biotech), according to the manufacturer’s instructions. Both the hybridization and washing were performed at 65 8C. The signals were detected by a CDP-Star chemiluminescent detection reagent system (Amersham Pharmacia Biotech). 2.5. Localization of GFP-Fused CAI1 Protein To construct the GFP expression vector, we PCRamplified the GFP coding sequence from the pGFP2 vector, as described [20]. The targeting sequences, corresponding to the translated region of CAI1 cDNA, were PCR-amplified from the full length CAI1 cDNA using the primer pair, 5?-ACTGCCATGGATGAGCGCTATGGCGTTATCG-3? and 5?-CAGTACTAGTGCAGCGGATAGAGCTTCCTC-3?. The amplified fragment was cloned into the pCR 2.1 vector and digested with Nco I and Spe I. This fragment was cloned, in-frame, between the Nco I and Spe I sites of the pCR 2.1 vector, containing the GFP2 gene. The plasmid was further digested with Nco I and Sac I; the resulting fragment was cloned between the Nco I and Sac I sites of the expression vector, pBI221 (Novagen,
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Madison, USA). Gold particles (2.5 mg of 1-mm gold particles), coated with 2.5 mg of the plasmid DNA construct by CaCl2/spermidine precipitation as described [21], bombarded onion epidermal cells through the use of a particle gun (Bio-Rad PDS-100/He). Following an overnight incubation at 25 8C, we observed transient expression of the GFP-fused protein using a fluorescence microscope (Leica) with Micro Mover-W camera (Photometrics), fitted with a tripleband filter (#81 series PINKEL #1 Filter SET, Chroma Technology). GFP fluorescence was observed at a 495 nm excitation wavelength and an emission wavelength of 530 nm.
3. Results 3.1. Isolation of differentially expressed cDNAs between compatible strain and incompatible strain-inoculated rice cells mRNA samples were prepared from cultured rice cells 5, 15, 30 and 60 min after the inoculation of either compatible or incompatible strains and cDNAs were then synthesized from these samples. Using four rhodamine-labeled, 3?-anchored primers in combination with 13 arbitrary primers, we performed 52 independent FDD-PCRs. From the differences in displayed patterns, 10 cDNA fragments were initially found to change their levels between the incompatible and the compatible strain-inoculated cells within 60 min after inoculation (data not shown). The cDNAs amplified from the cultured rice cells were excised from the gel, re-amplified, and cloned. We could observe two cDNA fragments (A1 and A2) that are induced by the incompatible strain, five cDNA fragments (B1, B2, B3, B4, and B5) are induced by both strains, two cDNA fragments (C1 and C2) are suppressed by the incompatible strain and one cDNA fragment (E1) is induced by the compatible strain. The sequence analysis of the cDNA fragments revealed that B1 and B2 were the same clone. B3 and C2 were determined to be ribosomal RNA. Therefore, B2, B3 and C2 were omitted from further analysis. To confirm the differential expression of the other identified genes, preliminary Northern blot analysis was performed utilizing mRNA isolated from cultured rice cells taken 0, 15 and 30 min after the inoculations. The expression patterns of A1, A2 and E1 were similar to the patterns obtained by FDD. The expression patterns of the other clones (B1, B4, B5 and C1), however, were the same in cultured rice cells inoculated with either the incompatible or the compatible strains. To isolate fulllength cDNA clones of A1, A2 and E1, we performed 5?and 3?-RACE methods using mRNAs extracted from either incompatible strain- (for A1 and A2) or compatible strain- (for E1) inoculated cultured rice cells. The
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Fig. 1. Deduced amino acid sequence and the structural features of the IAI1 gene (accession number: AB059237) product. (A) The derived one-letter
Fig. 2. Alignment of the deduced amino acid sequences of both IAI2 (accession number: AB059238) and the maize WIP1 (accession number: X71396). Amino acids that are shaded black are identical to the consensus sequence. The predicted N-terminal signal sequence of IAI2 is underlined with a thick line.
Fig. 3. Deduced amino acid sequence of the CAI1 gene (DDBJ accession number AB059239 for the gene). The box indicates the putative N-terminal signal sequence. The multiple glycine residues are underlined.
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Fig. 5. Southern blot analysis of CAI1 in genomic DNA isolated from rice leaf. Genomic DNA (20 mg) was digested with restriction enzymes (Hin d III, Eco RI and Bgl II) and subjected to Southern blot analysis. Hybridizations were performed using probes corresponding to the ORF (left gel image) and 3?-UTR (right gel image) sequences of the CAI1 cDNA, labeled by random priming. The sizes of standard DNA fragments (in kb) are shown on the left.
(compatible strain of P. a venae -ınduced gene), respec¯ ¯ ¯ tively. 3.2. Sequence analyses and characterization of IAI1, IAI2 and CAI1 cDNAs
Fig. 4. Accumulation of IAI1 (A), IAI2 (B) and CAI1 (C) mRNAs in cultured rice cells, inoculated with either the compatible (H8201) or incompatible (N1141) strains of P. avenae . Total RNA was isolated from infected cells after incubation with the bacterium. Fifteen micrograms of RNA were analyzed in each lane, utilizing each cDNA as a probe. The relative intensity of the bands is quantitated in the lower bar graph. Ethidium bromide-stained gels (below blots) demonstrate equal RNA loading.
full-length cDNAs corresponding to A1, A2 and E1 cDNA fragments were designated IAI1 (ıncompatible strain of P. a venae -ınduced gene), IAI2¯ and CAI1 ¯ ¯
Two of the three identified cDNAs (IAI1 and IAI2) were induced by inoculation with incompatible strain. The sequence analysis demonstrated that the IAI1 cDNA is composed of 2467 bp, containing a 2124-bp open reading frame, a 165-bp 5? untranslated region (5?UTR) and a 178-bp 3?-UTR sequence. The IAI1 predicted protein is 708 amino acids in length, possessing a calculated molecular mass of 78.2 kDa and an isoelectric point of 8.8 (Fig. 1A). The amino acid sequence deduced from the IAI1 cDNA did not demonstrate similarity to any known gene products and IAI1 is, therefore, a novel gene. The deduced amino acid sequence also revealed that a putative ATP binding domain similar to that of the GHMP kinase family [22] existed within the N-terminal domain of IAI1 (Ile-355 to Ser-365). In addition, a short repeat motif (PXYVPT; position 113/138 and 138 /193) and several putative phosphorylation sites including a five serine stretch (position 361/365) were observed within the protein. A hydropathy profile of the protein, previously described by Kyte and Doolittle [23], revealed that the IAI1 protein is extremely hydrophilic with a hydrophobic domain in the central region of the protein (Fig. 1B).
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TMHMM software, a commercially available transmembrane predictor program, indicated that this hydrophobic domain, between Cys-388 and Val-412, is the putative transmembrane domain. The software also predicted that the N-terminus resides inside the plant cell with the C-terminus located on the outside, suggesting that the protein may penetrate the membrane at this transmembrane domain. The IAI2 cDNA sequence revealed the presence of an ORF of 300 nucleotides, coding for a predicted polypeptide of 100 amino acids with calculated molecular mass of 10.7 kDa and the sequence also reveals a 86-bp 5?-UTR and a 2867-bp 3?-UTR. The deduced amino acid sequence of IAI2 revealed a typical amino-terminal signal sequence (Met-1 to Ala-22, underlined sequence in Fig. 2), an N-glycosylation consensus sequence (Asn50, Phe-51, Ser-52) and a high composition of cysteine residues. A BLAST homology search demonstrated that IAI2 is a novel gene of rice and it shares considerable total sequence homology with the wound induced protein (WIP1) of maize (73% identity), belonging to the family of Bowman /Birk proteinase inhibitors [24]. High conservation of spaced cysteine residues is common for these proteins. Alignment of the IAI2 and WIP1 sequences revealed that all the conserved cysteine residues are present in IAI2. The 947-bp cDNA of CAI1 exhibits an open reading frame coding for a 185 amino acid protein with a calculated molecular mass of 15.5 kDa. The CAI1 cDNA contained both a 146-bp 5?-UTR and a 246-bp 3?-UTR sequence. Comparison of the deduced protein sequence with known proteins demonstrated that CAI1 is a novel gene. The deduced protein is rich in glycine, constituting approximately 54% of the total amino acids (underlined amino acid residues in Fig. 3). The sequence also contains a putative signal peptide (circled sequence in Fig. 3) with a potential cleavage site between amino acid residues 20 and 21 (predicted with PSORT), typical of the glycine-rich protein (GRP) family [25]. To examine whether these three genes are unique or belong to multigene families, sequence alignments between the full length cDNA and known rice EST were performed. Several ESTs possessing the sequence similarity with IAI1 or IAI2 were obtained. The sequences of all obtained ESTs were identical to those of IAI1 or IAI2, suggesting that IAI1 and IAI2 are unique genes. Several ESTs were also identified as gene homologues of CAI1 . The sequence identities of these ESTs to CAI1 are widely distributed (85 /100%), indicating that CAI1 belongs to mutignen families and some homologues of CAI1 exist in rice. The presence of CAI homologue genes in rice was further confirmed by genomic Southern blot hybridization analysis utilizing two probes, corresponding to the open reading frame and 3?-UTR of CAI1 cDNA, respectively (Fig. 5). Genomic DNA, isolated from
young leaves of rice, was digested with Hin d III, Eco RI and Bgl II. Hybridization to each probe was performed under high-stringency conditions. Two to three strong bands and several weak bands were observed using the cDNA ORF region as a probe. Only a single band was observed in each lane when utilizing the 3?-UTR of the cDNA as the hybridization probe. Therefore, CAI1 gene is a single gene and several genes homologous to CAI1 are likely to exist in rice. 3.3. Induction patterns of mRNA in cultured rice cells by inoculations of either compatible or incompatible strains We examined the changes in IAI1, IAI2 and CAI1 mRNA levels in cultured rice cells, following inoculation with either N1141 (incompatible) or H8201 (compatible) strains of P. avenae in a time course experiment. Inoculation with the incompatible N1141 strain resulted in the rapid accumulation (1 h) of IAI1 mRNA in cultured rice cells, peaking 3 h after inoculation. No significant increase in IAI1 mRNA was observed until 6 h after inoculation with the compatible strain (Fig. 4A). Utilizing the IAI2 cDNA as a probe, we observed a somewhat different pattern from that seen for the IAI1 mRNA. IAI2 mRNA levels, low in uninoculated cultured rice cells, were induced 0.5 h after inoculation with the incompatible N1141 strain and gradually increased to 6 h after inoculation. IAI2 mRNA was also induced at 0.5 h after inoculation with the compatible strain. Accumulation, however, peaked 1 h after incubation, decreasing slowly to become undetectable at 6 h after inoculation (Fig. 4B). We also examined the induction pattern of CAI1 mRNA utilizing the full length CAI1 cDNA as a probe. The transcriptional activation time course of this gene demonstrated that no significant accumulation of CAI1 mRNA was seen in cultured rice cells incubated with water alone (Fig. 4C). The mRNA rapidly accumulated when the cells were inoculated with the compatible strain, declining to become undetectable 3 h after inoculation. In contrast, no mRNA accumulation was observed at any time point examined after inoculation with the incompatible strain. Since CAI1 belongs to multigene family, Northern blot analysis was also performed using 3?-UTR of CAI1 cDNA as probe. The same induction patterns with that seen for the full length cDNA as probe were obtained (data not shown), suggesting that expression pattern in Fig. 4C shows that of CAI1 cDNA but not CAI1 homologues. 3.4. Localization of CAI1 gene products We further characterized the CAI1 gene in the present study because the discovery of mRNA species induced during compatible interaction is rare. The sequence analysis of CAI1 demonstrated that the gene product
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was rich in glycine residues, likely belonging to the family of GRP thought to be structural cell wall components [26]. The presence of a possible signal peptide within the N-terminal region of CAI1 indicates that CAI1 may exist within the plant cell wall. To determine the localization of the CAI1 protein, we created a green fluorescent protein (GFP)-conjugate to act as a reporter. A cDNA, encoding the full length ORF of CAI1, was fused to the GFP2 gene. The fusion was placed under the control of the cauliflower mosaic virus 35S promoter (CAI /GFP ). This construct was introduced into onion epidermal cell by bombardment and transient expression was observed by fluorescence microscopy. Strong green fluorescence was observed on the cell walls of cells bombarded with the CAI /GFP construct (Fig. 6A). In onion epidermal cell bombarded with a control GFP construct, green fluorescence, originating from the GFP, did not display this cell wall-specific pattern (Fig. 6B), indicating the CAI1 protein is transported out of the plasma membrane to be found within the cell wall.
4. Discussion In order to isolate rice genes differentially expressed after inoculation of either the compatible or the incompatible strains of P. avenae , we performed a comparative gene expression analysis using FDD. Using 52 sets of arbitrary primer combination, we identified IAI1 and IAI2 as genes induced by the incompatible strain and CAI1 as a compatible strain-induced gene. Assuming the presence of 15 000 individual transcripts in cultured rice cells, it would be necessary to use over 450 random primer sets to screen all the available transcripts by FDD-PCR with 95% probability, approximately 100 bands are visualized per lane [16,27]. Therefore, our FDD analysis observed greater than 10% of all transcripts (more than 1500 individual transcripts) in cultured rice cells. We identified three positive clones by FDD analysis, suggesting that nearly 30 genes are differentially expressed in the cultured rice cells within 60 min after inoculation with either the compatible or incompatible strains. Hypersensitive cell death, oxidative burst, Ca2 influx and cytoplasmic pH change are characteristic early events observed following introduction of an incompatible pathogen. Our FDD analysis suggests that several genes are differentially expressed between the compatible and the incompatible straininfected rice cells, but the number of the genes are comparatively small. The induction of such drastic early events during the incompatible interaction, therefore, may result from the expression of relatively few genes. This result, obtained by FDD analysis, is supported by previously obtained data detailing that several early
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events are cyclohexamide-insensitive, not requiring de novo protein synthesis [28]. The deduced amino acid sequence of IAI2 is a cysteine-rich polypeptide possessing a 22 amino acid N-terminal signal sequence. Both the structural features and amino acid similarity indicate that IAI2 can be classified as Bowman /Birk type proteinase inhibitors (BBI). BBIs possess two different specificities within a single molecule; one is specific for trypsin and the other inhibits the action of chymotrypsin. IAI2 has two possible interaction sites for chymotrypsin (Phe51Ser and Thr78Tyr), but none for trypsin [24,29], suggesting IAI2 is a double-headed chymotrypsin inhibitor. BBIs possess a putative N-terminal secretory signal sequence, suggesting these proteins may be transported to accumulate within the cell wall [24,30,31]. BBIs also inhibit the activities of trypsin and chymotrypsin derived from microbes both in vitro and in vivo [32]. Many bacterial plant pathogens secrete proteases including trypsin or chymotrypsin type proteinases, which contribute to their aggressiveness when colonizing plant tissues but are not required for growth on synthetic media [33,34]. This idea of an extracellular protease has been evaluated by transposon mutagenesis in Xanthomonas campestris pv. oryzae , the agent of rice leaf blight. Populations of protease-defective mutants in rice plants were 10- to 100-fold smaller than those observed for the wild-type strain, suggesting an important role for the protease in growth [35]. Similarly, protease-deficient mutant of Xanthomonas campestris pv. campestris , the black rot pathogen, lacking both serine- and metalloproteinase activity, demonstrated considerable loss of virulence in pathogenicity tests when introduced into mature turnip leaves [33]. These findings, in conjunction with the fact that the IAI2 is induced only during incompatible interactions, suggests that IAI2 may prevent pathogen infection by proteinase inhibition. The deduced IAI1 amino acid sequence demonstrates that IAI1 possesses a putative single transmembrane domain. The several receptor kinases have been known to be transmembrane proteins in plant and play an important role in perception and transduction of signals. Xa21 of rice is one of the most widely studied. This gene, conferring resistance to Xanthomonas oryzae pv. oryzae race 6, is composed of several discrete domains, functionally identified by homologies to previously studied genes [36]. An N-terminal domain, containing 23 imperfect copies of a 24 amino acid leucine-rich repeat (LRR), is implicated in protein/protein interactions [37]. After the LRR, the protein contains a putative transmembrane domain. The C-terminal domain is indicative of a protein kinase, containing conserved serine /threonine specificity. Although IAI1 has a putative ATP binding domain similar to that of the GHMP kinase family [22], effective LRR motif and kinase motif could not be found in either the N-terminal
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Fig. 6. Localization of expression from green fluorescent protein (GFP) fused to CAI1. Cells were bombarded with constructs coding for either GFP fused to CAI1 (A) or control GFP (B). The fluorescence of GFP was observed at excitation wavelength 495 nm and emission wavelength 530 nm (A and B). Bar represents 10 mm.
domain or the C-terminal domain of IAI1. The accumulation of IAI1 mRNA in the early stages of infection with incompatible strains (Fig. 4A) suggests that IAI1 plays an important role in defense signal transduction during the early stages of incompatible interactions. Further study is needed to clarify the function of IAI1 in the rice /P. avenae interaction. The CAI1 sequence, excluding the putative signal peptide, was rich in Gly, Ser and Tyr (Fig. 3), a feature consistent with glycine-rich cell wall proteins. Glycinerich proteins (GRP), classified into different families [38], have been found in different tissues from several plant species [25,39,40]. The most typical GRP proteins (CL-GRP family) have a cytokeratin-like domain, consisting of glycine stretches with interspersed tyrosine residues, preceded by a charged N-terminal domain and putative signal peptide. This GRP type has been identified from Arabidopsis thaliana [41], Petunia hybrida [39], and bean [42]. A second GRP family, capable of binding RNA (RNA-GRP family), is characterized by a domain containing two typical RNA-binding motifs [43,44]. CAI1 possesses a putative signal peptide consisting of the 20 N-terminal amino acids, followed by a short charged domain (Fig. 3), suggesting that CAI1 belongs in CL-GRP family. The expression of CL-GRP genes is highly influenced by external stimuli, such as wounding [45], abscisic acid, pathogen infection [46 /48], water stress [41], salicylic acid [49] and light [50]. GRP1.8 of French bean, a member of the CL-GRP family, is localized specifically to the modified primary cell walls of protoxylem elements and continuously deposited into the cell walls during elongation growth.
This property of GRP1.8 suggested that GRP1.8 functions as part of a repair mechanism to strengthen the protoxylem [26,51]. We previously reported that the morphological changes in cultured rice cells, inoculated with the incompatible strain of P. avenae [11], included cytoplasmic condensation and shrinkage, essential morphological characteristics of animal apoptosis. These features were observed only in cultured rice cells inoculated with incompatible N1141 strain and inoculation of rice cells with the H8301 compatible strain weakened the cell wall surrounding the bacterial contact area, but did not induce the characteristic morphological changes of apoptosis. Cell wall weakening occurred not only in the middle lamella, the intercellular cement, but also in the secondary cell wall, proximal to the cytosol [11]. Degradation of the cell wall is caused by many types of plant pathogenic bacteria, resulting in the liquefaction of the pectic substances holding plant cells together. CAI1 may be involved in the repair of cell wall, damaged by the lytic enzymes of the P. avenaecompatible strain because cell wall repair is an important characteristic of the CL-GRP family of proteins.
Acknowledgements FSC and TE contributed equally to this paper. We are grateful to Tokiko Nakanishi and Hiroko Sato for their excellent technical support. This work was supported by a Grant-in-aid for Scientific Research on Priority Area (A) of Ministry of Education, Culture, Sports, Science and Technology (Molecular Mechanisms of Plant /
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Pathogenic Microbe Interaction) and ‘Research for the Future’ Program of the Japan Society for the Promotion of Science (JSPS-RFTF96R16001).
[18]
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Identification of novel genes differentially expressed in compatible and incompatible interactions between rice and Pseudomonas avenae Fang-Sik Che *, Tetsuyuki Entani, Taizou Marumoto, Masatoshi Taniguchi, Seiji Takayama, Akira Isogai Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5, Takayama Ikoma, Nara 630-0101, Japan Received 16 July 2001; received in revised form 23 October 2001; accepted 22 November 2001
Abstract Establishment of a plant /pathogen interaction involves differential gene expression in both organisms. To isolate rice genes induced during either compatible or incompatible interactions, we performed a comparative analysis of expression patterns in cultured rice cells, inoculated with either compatible or incompatible strains of Pseudomonas avenae, by fluorescence differential display (FDD). Using 52 sets of arbitrary primer combinations, two cDNAs (IAI1 and IAI2 ) expressed in incompatible straininoculated rice cells and one cDNA (CAI1 ) expressed in compatible strain inoculated cells, were identified. A hydropathy profile revealed that the IAI1 protein spans the membrane at a putative transmembrane domain. The deduced amino acid sequence of IAI2 shares considerable homology with Bowman /Birk proteinase inhibitors over the majority of the sequence. The deduced CAI1 protein is rich in glycine, containing a putative signal peptide with a potential cleavage site, suggesting its membership in the glycinerich protein (GRP) family. GRPs are thought to be structural cell wall proteins. When a GFP fusion with the CAI1 gene was introduced into onion cells by bombardment, green fluorescence of the cell wall was observed. These data suggest that CAI1 is involved in the repair of cell wall injuries by the compatible strain of P. avenae . # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bowman /Birk proteinase inhibitors; Differential display; Glycine-rich protein; Rice; Pseudomonas avenae
1. Introduction The host range of bacteria pathogenic to plants is generally limited to certain species of plants. ‘Compatible’ relationships lead to intercellular bacterial growth and symptom development within the host and no disease symptoms are observable in ‘incompatible’ cases. In addition to preformed physical and chemical barriers, such as thickened cuticles or preformed antimicrobial
Abbreviations: CAI, compatible strain of P. avenae -induced gene; FDD, fluorescence differential display; GFP, green fluorescent protein; GRP, glycine-rich protein; IAI, incompatible strain of P. avenae -induced gene; PCR, polymerase chain reaction The nucleotide sequence data reported will appear in the DDBJ/ EMBL/GenBank Nucleotide Sequence Database under the accession numbers AB059237 (IAI1 cDNA), AB059238 (IAI2 cDNA), AB059239 (CAI1 cDNA). * Corresponding author. Tel.: 81-743-72-5452; fax: 81-743-725459. E-mail address: [email protected] (F.-S. Che).
compounds, pathogenic attack induces a wide variety of defense responses [1,2]. These defenses are triggered rapidly and coordinately, allowing the plant to become resistant to pathogen invasion. The defense mechanisms of susceptible plants respond more slowly after infection. Thus, both the timely recognition of an invading microorganism and the rapid and effective induction of the defense responses differentiate resistant plants from susceptible ones [3,4]. Inducible defense follows a basic pathway of recognition and signal transduction to activate defensive responses, such as the generation of reactive oxygen species (the oxidative burst) [5,6], hypersensitive cell death [7], the deposition of both hydroxyproline-rich glycoproteins [8] and cell wall lignins [9], the accumulation of phytoalexins [10] and the synthesis of antimicrobial PR proteins [5]. The longterm systemic induction of additional or overlapping sets of genes follows generating proteinase inhibitors protecting the gene products associated with induced
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 5 8 5 - 4
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systemic resistance. The response of the host plant thus requires complex changes in gene expression patterns. Pseudomonas avenae (Acidovorax avenae), a Gramnegative bacterium, causes a seedling disease, characterized by the formation of brown stripes on the sheaths of infected plants. The wide host range of P. avenae throughout the monocotyledonous plants includes rice, oats, Italian millet and maize, individual strains of the pathogen, however, infect only one or a few host species. Strains isolated from rice, such as H8201 or H8301, can only infect rice plants, while the N1141 strain, isolated from finger millet, cannot infect rice even after inoculation into rice tissues. The rice-incompatible strain, N1141, causes hypersensitive cell death accompanied by DNA cleavage and typical apoptotic morphological changes; the rice-compatible strain, H8301, does not induce any of these changes [11]. In addition to the difference in the hypersensitive cell death induction, several different phenomena were observed in cultured rice cells inoculated with the incompatible and the compatible strains. The EL2 gene is known to be expressed with N -acetylchitoheptaose within 15 min to induce a set of defense reactions in cultured rice cells [12,13]. The accumulation of EL2 mRNA is detectable within 3 h after inoculation with the incompatible strain, N1141, whereas in H8301-inoculated cultured cells, it is not detectable. These results indicate that the N1141 strain causes a defense response in cultured rice cells, but the compatible strain H8301 does not [11]. Furthermore, we have demonstrated that the P. avenae flagellin is involved in the induction of the defense responses in incompatible hosts using the purified flagellin and flagellin-deficient mutants [14]. However, our understanding of the molecular interaction between P. avenae and the rice plant is limited and the genetic determinants of P. avenae host species specificity remain elusive. Determination of genes differentially expressed in the compatible and incompatible interactions would allow a greater understanding of the molecular mechanism involved in the interaction between P. avenae and the rice plant. mRNA differential display is commonly employed to identify genes differentially expressed between a particular tissue in the control state and an altered state. To identify genes differentially expressed in rice plants in response to compatible and incompatible strains, we applied mRNA fluorescent differential display to the compatible and incompatible interaction between P. avenae and rice. Using 52 sets of arbitrary primer combinations, we identified two cDNAs (IAI1 and 2) induced in the cells inoculated with the incompatible strain and one cDNA (CAI1 ) induced in the cells inoculated with the compatible strain. We also report the characterization and localization of CAI1 gene, induced by the compatible strain, and predict the possible role of this gene during the interaction between P. avenae and rice.
2. Materials and methods 2.1. Bacterial strains and cultures P. avenae H8201 (MAFF 301502), isolated from rice, and strain N1141 (MAFF 301141), isolated from finger millet, were utilized as previously described [15]. Each P. avenae strain was maintained at 30 8C on Pseudomonas F agar plates (Difco) as described [11]. 2.2. Fluorescent differential display Fluorescent differential display (FDD) was performed as previously described [16,17]. Suspension cultures of the Oc line of rice cells [18] were grown at 30 8C under light irradiation. Cells were diluted in fresh medium every 7 days. Experiments were performed 4 days after transfer. The cultured cells were incubated at 30 8C with bacteria (108 cfu ml 1) for variable time lengths after inoculation. mRNAs were prepared from cultured rice cells 5, 15, 30 and 60 min after inoculation of either the compatible or the incompatible strain using ISOGEN (Nippon gene). First-strand cDNAs were synthesized by reverse transcriptase (Super Script II, Gibco BRL) using 3?-anchored oligo-dT primers (5?-GT15VN-3?). Second strand synthesis and polymerase chain reaction (PCR) were performed using rhodamine-labeled 3?-anchored primers (XRITC-GT15MA, XRITC-GT15MT, XRITC-GT15MG, XRITC-GT15MC) and arbitrary 10-mer primers (Operon Technologies, Inc.). We utilized Taq-DNA polymerase and reaction buffer (TOYOBO, Osaka, Japan) for PCR analysis (94 8C 3 min, 40 8C 2 min, 72 8C 5 min, followed by 25 cycles of 95 8C 15 s, 40 8C 2 min, 72 8C 1 min, and 72 8C 5 min). The PCR products were separated by electrophoresis on 5% polyacrylamide gels containing 7 M urea and fluorescent images were analyzed with an FM-BIO (TaKaRa). Differentially expressed bands were removed from the gels, eluted by boiling, reamplified using the same primer pairs, cloned into the PCR 2.1 vector (Invitrogen) and sequenced. To eliminate false positive clones, PCR products, separated by the polyacrylamide gel, were blotted onto a Hybond-N membrane (Amersham Pharmacia Biotech) and analyzed by hybridization to each cDNA clone. The missing 5? regions of the cDNA fragments were obtained using a Marathon cDNA Amplification kit (Clontech), according to the manufacturer’s instructions. Total RNA was isolated from cultured rice cells 1 h after inoculation with either the N1141 or the H8201 strain. Poly(A)/ RNA was isolated using a Micro fast track kit (Invitrogen) and single-strand cDNA was synthesized using a polyT-NM primer. After doublestrand cDNA synthesis and adapter ligation, PCR was performed using both the adapter primer and each genespecific primer. The amplified fragments were cloned
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with a TA cloning kit (Invitrogen). Five clones from each PCR reaction were sequenced with a DNA sequencer (model 377; PE Applied Biosystems). PCR and cloning procedures were independently repeated twice to confirm DNA sequences. 2.3. RNA isolation and Northern blot analysis Following incubation with each bacterial strain (or without bacteria as a control), the cultured rice cells were ground in liquid nitrogen. Total RNA was extracted with ATA (aurintricarbosylic acid) [19] and RNAs (15 mg) were electrophoresed on a formaldehyde denaturing 1% (w/v) agarose gel in 1 / MOPS buffer (20 mM MOPS /KOH, pH 7.0, 5 mM sodium acetate, and 1 mM EDTA). RNA was then transferred to a Hybond-N membrane (Amersham Pharmacia Biotech), according to standard protocols. Each cDNA fragment was then labeled with [32P]dCTP to be utilized as a hybridization probe. 2.4. Southern blot analysis Genomic DNA was isolated from cultured rice cells with a Nucleon plant-DNA extraction kit (Amersham Pharmacia Biotech) according to the manufacturer’s protocol. Following digestion with Hin d III, Eco RI and Bgl II, DNA (10 mg) was electrophoresed on a 0.7% (w/v) agarose gel. Fractionated DNA was then transferred to a Hybond N membrane (Amersham Pharmacia Biotech). Two CAI1 cDNAs (corresponding to the ORF and 3?-UTR sequences) were labeled with an AlkPhos Direct labeling kit (Amersham Pharmacia Biotech), according to the manufacturer’s instructions. Both the hybridization and washing were performed at 65 8C. The signals were detected by a CDP-Star chemiluminescent detection reagent system (Amersham Pharmacia Biotech). 2.5. Localization of GFP-Fused CAI1 Protein To construct the GFP expression vector, we PCRamplified the GFP coding sequence from the pGFP2 vector, as described [20]. The targeting sequences, corresponding to the translated region of CAI1 cDNA, were PCR-amplified from the full length CAI1 cDNA using the primer pair, 5?-ACTGCCATGGATGAGCGCTATGGCGTTATCG-3? and 5?-CAGTACTAGTGCAGCGGATAGAGCTTCCTC-3?. The amplified fragment was cloned into the pCR 2.1 vector and digested with Nco I and Spe I. This fragment was cloned, in-frame, between the Nco I and Spe I sites of the pCR 2.1 vector, containing the GFP2 gene. The plasmid was further digested with Nco I and Sac I; the resulting fragment was cloned between the Nco I and Sac I sites of the expression vector, pBI221 (Novagen,
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Madison, USA). Gold particles (2.5 mg of 1-mm gold particles), coated with 2.5 mg of the plasmid DNA construct by CaCl2/spermidine precipitation as described [21], bombarded onion epidermal cells through the use of a particle gun (Bio-Rad PDS-100/He). Following an overnight incubation at 25 8C, we observed transient expression of the GFP-fused protein using a fluorescence microscope (Leica) with Micro Mover-W camera (Photometrics), fitted with a tripleband filter (#81 series PINKEL #1 Filter SET, Chroma Technology). GFP fluorescence was observed at a 495 nm excitation wavelength and an emission wavelength of 530 nm.
3. Results 3.1. Isolation of differentially expressed cDNAs between compatible strain and incompatible strain-inoculated rice cells mRNA samples were prepared from cultured rice cells 5, 15, 30 and 60 min after the inoculation of either compatible or incompatible strains and cDNAs were then synthesized from these samples. Using four rhodamine-labeled, 3?-anchored primers in combination with 13 arbitrary primers, we performed 52 independent FDD-PCRs. From the differences in displayed patterns, 10 cDNA fragments were initially found to change their levels between the incompatible and the compatible strain-inoculated cells within 60 min after inoculation (data not shown). The cDNAs amplified from the cultured rice cells were excised from the gel, re-amplified, and cloned. We could observe two cDNA fragments (A1 and A2) that are induced by the incompatible strain, five cDNA fragments (B1, B2, B3, B4, and B5) are induced by both strains, two cDNA fragments (C1 and C2) are suppressed by the incompatible strain and one cDNA fragment (E1) is induced by the compatible strain. The sequence analysis of the cDNA fragments revealed that B1 and B2 were the same clone. B3 and C2 were determined to be ribosomal RNA. Therefore, B2, B3 and C2 were omitted from further analysis. To confirm the differential expression of the other identified genes, preliminary Northern blot analysis was performed utilizing mRNA isolated from cultured rice cells taken 0, 15 and 30 min after the inoculations. The expression patterns of A1, A2 and E1 were similar to the patterns obtained by FDD. The expression patterns of the other clones (B1, B4, B5 and C1), however, were the same in cultured rice cells inoculated with either the incompatible or the compatible strains. To isolate fulllength cDNA clones of A1, A2 and E1, we performed 5?and 3?-RACE methods using mRNAs extracted from either incompatible strain- (for A1 and A2) or compatible strain- (for E1) inoculated cultured rice cells. The
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Fig. 1. Deduced amino acid sequence and the structural features of the IAI1 gene (accession number: AB059237) product. (A) The derived one-letter
Fig. 2. Alignment of the deduced amino acid sequences of both IAI2 (accession number: AB059238) and the maize WIP1 (accession number: X71396). Amino acids that are shaded black are identical to the consensus sequence. The predicted N-terminal signal sequence of IAI2 is underlined with a thick line.
Fig. 3. Deduced amino acid sequence of the CAI1 gene (DDBJ accession number AB059239 for the gene). The box indicates the putative N-terminal signal sequence. The multiple glycine residues are underlined.
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Fig. 5. Southern blot analysis of CAI1 in genomic DNA isolated from rice leaf. Genomic DNA (20 mg) was digested with restriction enzymes (Hin d III, Eco RI and Bgl II) and subjected to Southern blot analysis. Hybridizations were performed using probes corresponding to the ORF (left gel image) and 3?-UTR (right gel image) sequences of the CAI1 cDNA, labeled by random priming. The sizes of standard DNA fragments (in kb) are shown on the left.
(compatible strain of P. a venae -ınduced gene), respec¯ ¯ ¯ tively. 3.2. Sequence analyses and characterization of IAI1, IAI2 and CAI1 cDNAs
Fig. 4. Accumulation of IAI1 (A), IAI2 (B) and CAI1 (C) mRNAs in cultured rice cells, inoculated with either the compatible (H8201) or incompatible (N1141) strains of P. avenae . Total RNA was isolated from infected cells after incubation with the bacterium. Fifteen micrograms of RNA were analyzed in each lane, utilizing each cDNA as a probe. The relative intensity of the bands is quantitated in the lower bar graph. Ethidium bromide-stained gels (below blots) demonstrate equal RNA loading.
full-length cDNAs corresponding to A1, A2 and E1 cDNA fragments were designated IAI1 (ıncompatible strain of P. a venae -ınduced gene), IAI2¯ and CAI1 ¯ ¯
Two of the three identified cDNAs (IAI1 and IAI2) were induced by inoculation with incompatible strain. The sequence analysis demonstrated that the IAI1 cDNA is composed of 2467 bp, containing a 2124-bp open reading frame, a 165-bp 5? untranslated region (5?UTR) and a 178-bp 3?-UTR sequence. The IAI1 predicted protein is 708 amino acids in length, possessing a calculated molecular mass of 78.2 kDa and an isoelectric point of 8.8 (Fig. 1A). The amino acid sequence deduced from the IAI1 cDNA did not demonstrate similarity to any known gene products and IAI1 is, therefore, a novel gene. The deduced amino acid sequence also revealed that a putative ATP binding domain similar to that of the GHMP kinase family [22] existed within the N-terminal domain of IAI1 (Ile-355 to Ser-365). In addition, a short repeat motif (PXYVPT; position 113/138 and 138 /193) and several putative phosphorylation sites including a five serine stretch (position 361/365) were observed within the protein. A hydropathy profile of the protein, previously described by Kyte and Doolittle [23], revealed that the IAI1 protein is extremely hydrophilic with a hydrophobic domain in the central region of the protein (Fig. 1B).
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TMHMM software, a commercially available transmembrane predictor program, indicated that this hydrophobic domain, between Cys-388 and Val-412, is the putative transmembrane domain. The software also predicted that the N-terminus resides inside the plant cell with the C-terminus located on the outside, suggesting that the protein may penetrate the membrane at this transmembrane domain. The IAI2 cDNA sequence revealed the presence of an ORF of 300 nucleotides, coding for a predicted polypeptide of 100 amino acids with calculated molecular mass of 10.7 kDa and the sequence also reveals a 86-bp 5?-UTR and a 2867-bp 3?-UTR. The deduced amino acid sequence of IAI2 revealed a typical amino-terminal signal sequence (Met-1 to Ala-22, underlined sequence in Fig. 2), an N-glycosylation consensus sequence (Asn50, Phe-51, Ser-52) and a high composition of cysteine residues. A BLAST homology search demonstrated that IAI2 is a novel gene of rice and it shares considerable total sequence homology with the wound induced protein (WIP1) of maize (73% identity), belonging to the family of Bowman /Birk proteinase inhibitors [24]. High conservation of spaced cysteine residues is common for these proteins. Alignment of the IAI2 and WIP1 sequences revealed that all the conserved cysteine residues are present in IAI2. The 947-bp cDNA of CAI1 exhibits an open reading frame coding for a 185 amino acid protein with a calculated molecular mass of 15.5 kDa. The CAI1 cDNA contained both a 146-bp 5?-UTR and a 246-bp 3?-UTR sequence. Comparison of the deduced protein sequence with known proteins demonstrated that CAI1 is a novel gene. The deduced protein is rich in glycine, constituting approximately 54% of the total amino acids (underlined amino acid residues in Fig. 3). The sequence also contains a putative signal peptide (circled sequence in Fig. 3) with a potential cleavage site between amino acid residues 20 and 21 (predicted with PSORT), typical of the glycine-rich protein (GRP) family [25]. To examine whether these three genes are unique or belong to multigene families, sequence alignments between the full length cDNA and known rice EST were performed. Several ESTs possessing the sequence similarity with IAI1 or IAI2 were obtained. The sequences of all obtained ESTs were identical to those of IAI1 or IAI2, suggesting that IAI1 and IAI2 are unique genes. Several ESTs were also identified as gene homologues of CAI1 . The sequence identities of these ESTs to CAI1 are widely distributed (85 /100%), indicating that CAI1 belongs to mutignen families and some homologues of CAI1 exist in rice. The presence of CAI homologue genes in rice was further confirmed by genomic Southern blot hybridization analysis utilizing two probes, corresponding to the open reading frame and 3?-UTR of CAI1 cDNA, respectively (Fig. 5). Genomic DNA, isolated from
young leaves of rice, was digested with Hin d III, Eco RI and Bgl II. Hybridization to each probe was performed under high-stringency conditions. Two to three strong bands and several weak bands were observed using the cDNA ORF region as a probe. Only a single band was observed in each lane when utilizing the 3?-UTR of the cDNA as the hybridization probe. Therefore, CAI1 gene is a single gene and several genes homologous to CAI1 are likely to exist in rice. 3.3. Induction patterns of mRNA in cultured rice cells by inoculations of either compatible or incompatible strains We examined the changes in IAI1, IAI2 and CAI1 mRNA levels in cultured rice cells, following inoculation with either N1141 (incompatible) or H8201 (compatible) strains of P. avenae in a time course experiment. Inoculation with the incompatible N1141 strain resulted in the rapid accumulation (1 h) of IAI1 mRNA in cultured rice cells, peaking 3 h after inoculation. No significant increase in IAI1 mRNA was observed until 6 h after inoculation with the compatible strain (Fig. 4A). Utilizing the IAI2 cDNA as a probe, we observed a somewhat different pattern from that seen for the IAI1 mRNA. IAI2 mRNA levels, low in uninoculated cultured rice cells, were induced 0.5 h after inoculation with the incompatible N1141 strain and gradually increased to 6 h after inoculation. IAI2 mRNA was also induced at 0.5 h after inoculation with the compatible strain. Accumulation, however, peaked 1 h after incubation, decreasing slowly to become undetectable at 6 h after inoculation (Fig. 4B). We also examined the induction pattern of CAI1 mRNA utilizing the full length CAI1 cDNA as a probe. The transcriptional activation time course of this gene demonstrated that no significant accumulation of CAI1 mRNA was seen in cultured rice cells incubated with water alone (Fig. 4C). The mRNA rapidly accumulated when the cells were inoculated with the compatible strain, declining to become undetectable 3 h after inoculation. In contrast, no mRNA accumulation was observed at any time point examined after inoculation with the incompatible strain. Since CAI1 belongs to multigene family, Northern blot analysis was also performed using 3?-UTR of CAI1 cDNA as probe. The same induction patterns with that seen for the full length cDNA as probe were obtained (data not shown), suggesting that expression pattern in Fig. 4C shows that of CAI1 cDNA but not CAI1 homologues. 3.4. Localization of CAI1 gene products We further characterized the CAI1 gene in the present study because the discovery of mRNA species induced during compatible interaction is rare. The sequence analysis of CAI1 demonstrated that the gene product
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was rich in glycine residues, likely belonging to the family of GRP thought to be structural cell wall components [26]. The presence of a possible signal peptide within the N-terminal region of CAI1 indicates that CAI1 may exist within the plant cell wall. To determine the localization of the CAI1 protein, we created a green fluorescent protein (GFP)-conjugate to act as a reporter. A cDNA, encoding the full length ORF of CAI1, was fused to the GFP2 gene. The fusion was placed under the control of the cauliflower mosaic virus 35S promoter (CAI /GFP ). This construct was introduced into onion epidermal cell by bombardment and transient expression was observed by fluorescence microscopy. Strong green fluorescence was observed on the cell walls of cells bombarded with the CAI /GFP construct (Fig. 6A). In onion epidermal cell bombarded with a control GFP construct, green fluorescence, originating from the GFP, did not display this cell wall-specific pattern (Fig. 6B), indicating the CAI1 protein is transported out of the plasma membrane to be found within the cell wall.
4. Discussion In order to isolate rice genes differentially expressed after inoculation of either the compatible or the incompatible strains of P. avenae , we performed a comparative gene expression analysis using FDD. Using 52 sets of arbitrary primer combination, we identified IAI1 and IAI2 as genes induced by the incompatible strain and CAI1 as a compatible strain-induced gene. Assuming the presence of 15 000 individual transcripts in cultured rice cells, it would be necessary to use over 450 random primer sets to screen all the available transcripts by FDD-PCR with 95% probability, approximately 100 bands are visualized per lane [16,27]. Therefore, our FDD analysis observed greater than 10% of all transcripts (more than 1500 individual transcripts) in cultured rice cells. We identified three positive clones by FDD analysis, suggesting that nearly 30 genes are differentially expressed in the cultured rice cells within 60 min after inoculation with either the compatible or incompatible strains. Hypersensitive cell death, oxidative burst, Ca2 influx and cytoplasmic pH change are characteristic early events observed following introduction of an incompatible pathogen. Our FDD analysis suggests that several genes are differentially expressed between the compatible and the incompatible straininfected rice cells, but the number of the genes are comparatively small. The induction of such drastic early events during the incompatible interaction, therefore, may result from the expression of relatively few genes. This result, obtained by FDD analysis, is supported by previously obtained data detailing that several early
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events are cyclohexamide-insensitive, not requiring de novo protein synthesis [28]. The deduced amino acid sequence of IAI2 is a cysteine-rich polypeptide possessing a 22 amino acid N-terminal signal sequence. Both the structural features and amino acid similarity indicate that IAI2 can be classified as Bowman /Birk type proteinase inhibitors (BBI). BBIs possess two different specificities within a single molecule; one is specific for trypsin and the other inhibits the action of chymotrypsin. IAI2 has two possible interaction sites for chymotrypsin (Phe51Ser and Thr78Tyr), but none for trypsin [24,29], suggesting IAI2 is a double-headed chymotrypsin inhibitor. BBIs possess a putative N-terminal secretory signal sequence, suggesting these proteins may be transported to accumulate within the cell wall [24,30,31]. BBIs also inhibit the activities of trypsin and chymotrypsin derived from microbes both in vitro and in vivo [32]. Many bacterial plant pathogens secrete proteases including trypsin or chymotrypsin type proteinases, which contribute to their aggressiveness when colonizing plant tissues but are not required for growth on synthetic media [33,34]. This idea of an extracellular protease has been evaluated by transposon mutagenesis in Xanthomonas campestris pv. oryzae , the agent of rice leaf blight. Populations of protease-defective mutants in rice plants were 10- to 100-fold smaller than those observed for the wild-type strain, suggesting an important role for the protease in growth [35]. Similarly, protease-deficient mutant of Xanthomonas campestris pv. campestris , the black rot pathogen, lacking both serine- and metalloproteinase activity, demonstrated considerable loss of virulence in pathogenicity tests when introduced into mature turnip leaves [33]. These findings, in conjunction with the fact that the IAI2 is induced only during incompatible interactions, suggests that IAI2 may prevent pathogen infection by proteinase inhibition. The deduced IAI1 amino acid sequence demonstrates that IAI1 possesses a putative single transmembrane domain. The several receptor kinases have been known to be transmembrane proteins in plant and play an important role in perception and transduction of signals. Xa21 of rice is one of the most widely studied. This gene, conferring resistance to Xanthomonas oryzae pv. oryzae race 6, is composed of several discrete domains, functionally identified by homologies to previously studied genes [36]. An N-terminal domain, containing 23 imperfect copies of a 24 amino acid leucine-rich repeat (LRR), is implicated in protein/protein interactions [37]. After the LRR, the protein contains a putative transmembrane domain. The C-terminal domain is indicative of a protein kinase, containing conserved serine /threonine specificity. Although IAI1 has a putative ATP binding domain similar to that of the GHMP kinase family [22], effective LRR motif and kinase motif could not be found in either the N-terminal
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Fig. 6. Localization of expression from green fluorescent protein (GFP) fused to CAI1. Cells were bombarded with constructs coding for either GFP fused to CAI1 (A) or control GFP (B). The fluorescence of GFP was observed at excitation wavelength 495 nm and emission wavelength 530 nm (A and B). Bar represents 10 mm.
domain or the C-terminal domain of IAI1. The accumulation of IAI1 mRNA in the early stages of infection with incompatible strains (Fig. 4A) suggests that IAI1 plays an important role in defense signal transduction during the early stages of incompatible interactions. Further study is needed to clarify the function of IAI1 in the rice /P. avenae interaction. The CAI1 sequence, excluding the putative signal peptide, was rich in Gly, Ser and Tyr (Fig. 3), a feature consistent with glycine-rich cell wall proteins. Glycinerich proteins (GRP), classified into different families [38], have been found in different tissues from several plant species [25,39,40]. The most typical GRP proteins (CL-GRP family) have a cytokeratin-like domain, consisting of glycine stretches with interspersed tyrosine residues, preceded by a charged N-terminal domain and putative signal peptide. This GRP type has been identified from Arabidopsis thaliana [41], Petunia hybrida [39], and bean [42]. A second GRP family, capable of binding RNA (RNA-GRP family), is characterized by a domain containing two typical RNA-binding motifs [43,44]. CAI1 possesses a putative signal peptide consisting of the 20 N-terminal amino acids, followed by a short charged domain (Fig. 3), suggesting that CAI1 belongs in CL-GRP family. The expression of CL-GRP genes is highly influenced by external stimuli, such as wounding [45], abscisic acid, pathogen infection [46 /48], water stress [41], salicylic acid [49] and light [50]. GRP1.8 of French bean, a member of the CL-GRP family, is localized specifically to the modified primary cell walls of protoxylem elements and continuously deposited into the cell walls during elongation growth.
This property of GRP1.8 suggested that GRP1.8 functions as part of a repair mechanism to strengthen the protoxylem [26,51]. We previously reported that the morphological changes in cultured rice cells, inoculated with the incompatible strain of P. avenae [11], included cytoplasmic condensation and shrinkage, essential morphological characteristics of animal apoptosis. These features were observed only in cultured rice cells inoculated with incompatible N1141 strain and inoculation of rice cells with the H8301 compatible strain weakened the cell wall surrounding the bacterial contact area, but did not induce the characteristic morphological changes of apoptosis. Cell wall weakening occurred not only in the middle lamella, the intercellular cement, but also in the secondary cell wall, proximal to the cytosol [11]. Degradation of the cell wall is caused by many types of plant pathogenic bacteria, resulting in the liquefaction of the pectic substances holding plant cells together. CAI1 may be involved in the repair of cell wall, damaged by the lytic enzymes of the P. avenaecompatible strain because cell wall repair is an important characteristic of the CL-GRP family of proteins.
Acknowledgements FSC and TE contributed equally to this paper. We are grateful to Tokiko Nakanishi and Hiroko Sato for their excellent technical support. This work was supported by a Grant-in-aid for Scientific Research on Priority Area (A) of Ministry of Education, Culture, Sports, Science and Technology (Molecular Mechanisms of Plant /
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Pathogenic Microbe Interaction) and ‘Research for the Future’ Program of the Japan Society for the Promotion of Science (JSPS-RFTF96R16001).
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