Hyperphosphorylation of a mitochondrial protein,prohibitin, is induced by calyculin A in a rice lesion-mimic mutant cdr1

The rice (Oryza sativa) lesion-mimic mutants, cell death and resistance (cdr), show spontaneous cell death on the entire leaf and exhibited significant resistance to the rice blast fungus. Our previous studies showed that CDR1 and CDR2 genes negatively regulated the phosphorylation steps leading to the activation of NADPH oxidase, which is associated with oxidative burst. To identify novel factors involved in the phosphorylation steps, the phosphorylation level of total proteins was compared between cdr mutants and wild type using two-dimensional gel electrophoresis. Here, we show that the phosphorylation level of four proteins in cdr1 was increased as compared with the wild type after calyculin A treatment. Partial amino acid sequences revealed that one of the four proteins is homologous to prohibitin (PHB), which has been shown to be associated with senescence and cell death and to function as a chaperone in the assembly of mitochondrial respiratory chain complex in yeast and mammals. Analysis of green fluorescent protein fusions indicated that rice PHB (OsPHB1) was targeted to mitochondria as found in yeast and mammals, suggesting a possibility that PHB is involved in defense response and/or programmed cell death through the mitochondrial function.     

Probenazole-induced accumulation of salicylic acid confers resistance to Magnaporthe grisea in adult rice plants

Probenazole (PBZ) is the active ingredient of Oryzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Oryzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pre-treatment of inoculated fourth leaves of young wild-type rice plants at the 4-leaf stage with PBZ did not influence the development of whitish expanding lesions (ELs) in the susceptible interaction without the accumulation of SA and pathogenesis-related (PR) proteins. However, PBZ pre-treatment increased accumulation of SA and PR proteins in the eighth leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction (HR) lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.     

Proteome analysis of programmed cell death and defense signaling using the rice lesion mimic mutant cdr2

We have previously identified three lesion-mimic mutants, cell death and resistance (cdr), in rice. These mutants induce a series of defense responses, including expression of defense-related genes and high accumulation of phytoalexins, indicating that the cdr mutants are useful materials to study programmed cell death and defense signaling in rice. Here, we carried out a proteome analysis of the cdr2 mutant. Total proteins prepared from the wild type and the cdr2 mutant at three different stages of lesion formation were compared using two-dimensional electrophoresis. We found a total of 37 proteins that were differentially expressed between cdr2 and wild type. Among them, 28 spots were up-regulated and nine were down-regulated in the cdr2 mutant. All the protein spots were identified by mass spectrometric analysis. These differentially regulated proteins included defense-related proteins. In addition, 27 proteins were classified as metabolic enzymes, suggesting that the programmed cell death that occurs in the cdr2 mutant is associated with active metabolic changes. Our study shows that proteome analysis is a useful approach to study programmed cell death and defense signaling in plants.     

Characterization of PBZ1, a probenazole-inducible gene, in suspension-cultured rice cells

Probenazole (PBZ) induces non-race specific resistance in rice plants against rice blast fungus and PBZ1 was identified as a PBZ-inducible gene from rice. The induction of PBZ1 expression in suspension-cultured rice cells was investigated. Northern blot analysis indicated that PBZ1 was induced by PBZ in a dose-dependent manner. Enzyme-linked immunosorbent assay (ELISA) showed a dose and time-dependent accumulation of PBZ1 protein. Both mRNA and protein analysis showed that PBZ1 was not induced by salicylic acid or an active metabolite, 1,2-benzisothiazole-1,1-dioxide.     

Cloning and Characterization of a Probenazole-Inducible Gene for an Intracellular Pathogenesis-Related Protein in Rice

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) inducesdisease resistance in rice against rice blast fungus. To investigatethe molecular mechanism of probenazole-induced resistance, weisolated and characterized a cDNA clone of a probenazole-induciblegene in rice, which encoded a protein designated PBZ1. Sequenceanalysis revealed that significant homology at the amino acidlevel exists between the predicted PBZ1 protein and intracellularpathogenesis-related (IPR) proteins. Accumulation of PBZ1 mRNAwas not induced by wounding, but markedly induced by inoculationwith rice blast fungus. In addition, it was induced sooner byinoculation with rice blast fungus. In addition, it was inducedsooner by inoculation with an incompatible race than that witha compatible race. On the other hand, when the accumulationof the PBZ1 mRNA was examined after treatment with probenazole-relatedcompounds, it was not fully correlated with anti-rice blastactivity. However, it was induced after treatement with N-cyanomethyl-2-chloro-isonicotinamide(NCI), which belongs to another group of compounds known toinduce disease resistance. Thus, although the accumulation ofthe PBZ1 mRNA was not fully correlated with anti-rice blastactivity, our findings suggest that the PBZ1 gene has an importantfunction during the disease resistance response in rice. (Received June 19, 1995; Accepted October 13, 1995)     

Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus, Magnaporthe grisea

Proteomic approaches using two-dimensional gel electrophoresis (2-DE) were adopted to identify proteins from rice leaf that are differentially expressed in response to the rice blast fungus, Magnaporthe grisea. Microscopic observation of inoculated leaf with M. grisea revealed that callose deposition and hypersensitive response was clearly visible in incompatible interactions but excessive invading hypha with branches were evident in compatible interactions. Proteins were extracted from leaves 24, 48, and 72 hours after rice blast fungus inoculation. Eight proteins resolved on the 2-DE gels were induced or increased in the inoculated leaf. Matrix-assisted laser desorption/ionization-time of flight analysis of these differentially displayed proteins showed them to be two receptor-like protein kinases (RLK), two beta-1.3-glucanases (Glu1, Glu2), thaumatin-like protein (TLP), peroxidase (POX 22.3), probenazole-inducible protein (PBZ1), and rice pathogenesis-related 10 (OsPR-10). Of these proteins, RLK, TLP, PBZ, and OsPR-10 proteins were induced more in the incompatible interactions than in compatible ones. A phytohormone, jasmonic acid also induced all eight proteins in leaves. To confirm whether the expression profile is equal to the 2-DE data, seven cDNA clones were used as probes in Northern hybridization experiments using total RNA from leaf tissues inoculated with incompatible and compatible rice blast fungal races. The genes encoding POX22.3, Glu1, Glu2, TLP, OsRLK, PBZ1, and OsPR-10 were activated in inoculated leaves, with TLP, OsRLK, PBZ1, and OsPR-10 being expressed earlier and more in incompatible than in compatible interactions. These results suggest that early and high induction of these genes may provide host plants with leading edges to defend themselves. The localization of two rice PR-10 proteins, PBZ1 and OsPR-10, was further examined by immunohistochemical analysis. PBZ1 accumulated highly in mesophyll cells under the attachment site of the appressorium. In contrast, OsPR-10 expression was mainly localized to vascular tissue.     

Proteomics analysis of rice lesion mimic mutant (spl1) reveals tightly localized probenazole-induced protein (PBZ1) in cells undergoing programmed cell death

Numerous reports have predicted/hypothesized a role for probenazole-induced protein (PBZ1) as a molecular marker in rice self-defense mechanism. However, the precise function of PBZ1 remains unknown. In the present study, we examined PBZ1 as a putative cell death marker in rice. For this, we focused our attention on a rice lesion mimic mutant (LMM), spotted leaf 1 ( spl1), which has been used to study the programmed cell death (PCD) phenomenon during lesion development in leaf. Using two-dimensional gel electrophoresis (2-DGE), 18 colloidal Coomassie brilliant blue stained protein spots were found to be differentially expressed in the leaves of spl1 mutant. After analysis of these spots by MALDI-TOF-MS, we identified the PBZ1 protein to be highly inducible in spl1. On the basis of these results, we proceeded to verify whether PBZ1 is highly expressed in the tissues undergoing PCD in rice. To do so, we performed immunoblot analysis and immunolocalization and used transgenic lines carrying the PBZ1 promoter fused with GFP. Results demonstrated that the expression levels and localizations of PBZ1 dramatically coincided with tissues undergoing PCD, namely, during leaf senescence, root aerenchyma formation, coleoptiles senescence, root cap, and seed aleurone layer. Furthermore, localization of the PBZ1 protein was also tightly correlated with TUNEL signal in the seed aleurone layer. As DNA fragmentation is a hallmark of PCD, this result clearly indicates a role for PBZ1 in rice tissues undergoing PCD. In conclusion, our results provide strong support for the hypothesis that PBZ1 is a molecular marker in rice defense response, and can serve as a novel potential marker for cell death/PCD in rice.     

Proteomic analysis of jasmonic acid-regulated proteins in rice leaf blades

Jasmonates play a critical role in plant defense against pathogens through regulation of the expression of defense-related genes. To study the role of jasmonic acid (JA) in the rice self-defense mechanism, a proteomic approach was applied. When 3-week-old rice cv. Java 14 was treated with 100 microM JA for 3 days, numerous necrotic brown spots were observed on the leaf blade. Three-week-old rice was treated with JA and proteins from cytosolic and membrane fractions of leaf blade were separated by two-dimensional polyacrylamide gel electrophoresis. A total of 305 proteins were detected in both cytosolic and membrane fractions. When rice plant was treated with 100 microM JA for 2 days, 12 proteins were up-regulated and 2 proteins were down-regulated. Out of them, 8 proteins were changed in dose dependence manner, while 4 proteins were changed in a time course manner. Among them, pathogenesis-related protein 5 (PR5) and probenazole inducible protein 1 (PBZ1) were significantly induced by 100 microM JA for 2 days. These results suggest that PR5 and PBZ1 are important proteins expressed down-stream of JA signals in rice cv. Java 14.     

How phenology influences physiology in deciduous forest spring ephemerals

The protein phosphatase inhibitor cantharidin activates defense responses in rice leaves when applied exogenously at concentrations ranging from 100 to 500 μ M . Responses include the accumulation of the major rice phenolic phytoalexin sakuranetin and the lactone phytoalexin momilactone A. Accumulation of sakuranetin was preceded by an induction of phenylalanine ammonia lyase (PAL) activity and an increase in the activity of naringenin 7- O -methyltransferase (NOMT), the key enzyme in sakuranetin biosynthesis. Cantharidin also strongly induced accumulation of the probenazole (PBZ)-inducible protein (PBZ1) and two novel, related proteins named PBZ2 and PBZ3. Endothall, a herbicide and potent protein phosphatase inhibitor, but not its inactive analog (1,4-dimethylendothall) also induced sakuranetin accumulation, increased activity of NOMT and accumulation of the 3 PBZ proteins. In contrast, two other protein phosphatase inhibitors, calyculin A and microcystin LR, did not activate these defense responses. Induction of NOMT and PAL activity, and sakuranetin accumulation, was completely blocked by cycloheximide. Leaf segments treated with cantharidin and endothall showed brownish and orange colored lesions, respectively, similar to the lesion mimic mutants of rice. These results indicate a direct role for protein phosphorylation/dephosphorylation events in the activation of defense responses in rice, in particular on the accumulation of antifungal phytoalexins and the PBZ proteins.     

Regulatory mechanisms of ROI generation are affected by rice spl mutations

Reactive oxygen intermediates (ROIs) play a pivotal role in the hypersensitive response (HR) in disease resistance. NADPH oxidase is a major source of ROI; however, the mechanisms of its regulation are unclear. Rice spl mutants spontaneously form lesions which resemble those occurring during the HR, suggesting that the mutations affect regulation of the HR. We found that spl2, spl7 and spl11 mutant cells accumulated increased amounts of H(2)O(2) in response to rice blast fungal elicitor. Increased accumulation of ROIs was suppressed by inhibition of NADPH oxidase in the spl cells, and was also observed in the ozone-exposed spl plants. These mutants have sufficient activities of ROI-scavenging enzymes compared with the wild type. In addition, spl7 mutant cells accumulated higher amounts of H(2)O(2) when treated with calyculin A (CA), an inhibitor of protein phosphatase. Furthermore, spl2 mutant plants exhibited accelerated accumulation of H(2)O(2) and increased rates of cell death in response to wounding. These results suggest that the spl2, spl7 and spl11 mutants are defective in the regulation of NADPH oxidase, and the spl7 mutation may give rise to enhancement of the signaling pathway which protein dephosphorylation controls, while the spl2 mutation affects both the pathogen-induced and wound-induced signaling pathways.     

gid1, a gibberellin-insensitive dwarf mutant, shows altered regulation of probenazole-inducible protein (PBZ1) in response to cold stress and pathogen attack

A recessive gibberellin (GA)-insensitive dwarf mutant of rice, gibberellin-insensitive dwarf1 (gid1), has been identified, which shows a severe dwarf phenotype and contains high concentrations of endogenous GA. To elucidate the function of gid1, proteins regulated downstream of gid1 were analysed using a proteomic approach. Proteins extracted from suspension-cultured cells of gid1 and its wild type were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of a total of 962 proteins identified from the suspension-cultured cells, 16 were increased and 14 were decreased in gid1 compared with its wild type. Among the proteins hyper-accumulated in gid1 were osmotin, triosephosphate isomerase, probenazole inducible protein (PBZ1) and pathogenesis-related protein 10. Of these four genes, only the expression of PBZ1 was increased by exogenous GA3 application. Expression of this gene was also enhanced in shoots of the wild type by cold stress or by rice blast fungus infection. Under normal growth conditions, there was more PBZ1 protein in gid1 than in the wild type. In addition, gid1 showed increased tolerance to cold stress and resistance to blast fungus infection. The entcopalyl diphosphate synthase (OsCPS) genes, which encode enzymes at the branch point between GA and phytoalexin biosynthesis, were expressed differentially in gid1 relative to the wild type. Specifically, OsCPS1, which encodes an enzyme in the GA biosynthesis pathway, was down-regulated and OsCPS2 and OsCPS4, which encode enzymes in phytoalexin biosynthesis, were up-regulated in gid1. These results suggest that the expression of PBZ1 is regulated by GA signalling and stress stimuli, and that gid1 is involved in tolerance to cold stress and resistance to blast fungus.     

Antagonism of Chk1 signaling in the G2 DNA damage checkpoint by dominant alleles of Cdr1

Activation of the Chk1 protein kinase by DNA damage enforces a checkpoint that maintains Cdc2 in its inactive, tyrosine-15 (Y15) phosphorylated state. Chk1 downregulates the Cdc25 phosphatases and concomitantly upregulates the Wee1 kinases that control the phosphorylation of Cdc2. Overproduction of Chk1 causes G(2) arrest/delay independently of DNA damage and upstream checkpoint genes. We utilized this to screen fission yeast for mutations that alter sensitivity to Chk1 signaling. We describe three dominant-negative alleles of cdr1, which render cells supersensitive to Chk1 levels, and suppress the checkpoint defects of chk1Delta cells. Cdr1 encodes a protein kinase previously identified as a negative regulator of Wee1 activity in response to limited nutrition, but Cdr1 has not previously been linked to checkpoint signaling. Overproduction of Cdr1 promotes checkpoint defects and exacerbates the defective response to DNA damage of cells lacking Chk1. We conclude that regulation of Wee1 by Cdr1 and possibly by related kinases is an important antagonist of Chk1 signaling and represents a novel negative regulation of cell cycle arrest promoted by this checkpoint.     

The Protein Kinase Cdr2, Related to Nim1/Cdr1 Mitotic Inducer, Regulates the Onset of Mitosis in Fission Yeast

Cdc2–Cyclin B, the protein kinase that catalyzes the onset of mitosis, is subject to multiple forms of regulation. In the fission yeast Schizosaccharomyces pombe and most other species, a key mode of Cdc2–Cyclin B regulation is the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the protein kinases Wee1 and Mik1 and removed by the phosphatase Cdc25. These proteins are also regulated, a notable example being the inhibition of Wee1 by the protein kinase Nim1/Cdr1. The temperature-sensitive mutation cdc25–22 is synthetic lethal with nim1/cdr1 mutations, suggesting that a synthetic lethal genetic screen could be used to identify novel mitotic regulators. Here we describe that such a screen has identified cdr2⁺, a gene that has an important role in the mitotic control. Cdr2 is a 775 amino acid protein kinase that is closely related to Nim1 and mitotic control proteins in budding yeast. Deletion of cdr2 causes a G2-M delay that is more severe than that caused by nim1/cdr1 mutations. Genetic studies are consistent with a model in which Cdr2 negatively regulates Wee1. This model is supported by experiments showing that Cdr2 associates with the N-terminal regulatory domain of Wee1 in cell lysates and phosphorylates Wee1 in vitro. Thus, Cdr2 is a novel mitotic control protein that appears to regulate Wee1.     

Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis-related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1-1 and coi1-1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR-1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1-1 and coi 1-1 mutant plants. Thus SA and NPR1 appear to be required for PBZ- and BIT-mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1-mediated defense signaling pathway upstream of SA.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

The cdr2+ Gene Encodes a Regulator of G2/M Progression and Cytokinesis in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells respond to nutrient deprivation by altering G₂/M cell size control. The G₂/M transition is controlled by activation of the cyclin-dependent kinase Cdc2p. Cdc2p activation is regulated both positively and negatively. cdr2⁺ was identified in a screen for regulators of mitotic control during nutrient deprivation. We have cloned cdr2⁺ and have found that it encodes a putative serine-threonine protein kinase that is related to Saccharomyces cerevisiae Gin4p and S. pombe Cdr1p/Nim1p. cdr2⁺ is not essential for viability, but cells lacking cdr2⁺ are elongated relative to wild-type cells, spending a longer period of time in G₂. Because of this property, upon nitrogen deprivation cdr2⁺ mutants do not arrest in G₁, but rather undergo another round of S phase and arrest in G₂ from which they are able to enter a state of quiescence. Genetic evidence suggests that cdr2⁺ acts as a mitotic inducer, functioning through wee1⁺, and is also important for the completion of cytokinesis at 36°C. Defects in cytokinesis are also generated by the overproduction of Cdr2p, but these defects are independent of wee1⁺, suggesting that cdr2⁺ encodes a second activity involved in cytokinesis.     

A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway

Plant roots have important roles not only in absorption of water and nutrients, but also in stress tolerance such as desiccation, salt, and low temperature. We have investigated stress-response proteins from rice roots using 2-dimensional polyacrylamide-gel electrophoresis and found a rice protein, RO-292, which was induced specifically in roots when 2-week-old rice seedlings were subjected to salt and drought stress. The full-length RO-292 cDNA was cloned, and was determined to encode a protein of 160 amino acid residues (16.9 kDa, pI 4.74). The deduced amino acid sequence showed high similarity to known rice PR10 proteins, OsPR10a/PBZ1 and OsPR10b. RO-292 mRNA accumulated rapidly upon drought, NaCl, jasmonic acid and probenazole, but not by exposure to low temperature or by abscisic acid and salicylic acid. The RO-292 gene was also up-regulated by infection with rice blast fungus. Interestingly, induction was observed almost exclusively in roots, thus we named the gene RSOsPR10 (root specific rice PR10). The present results indicate that RSOsPR10 is a novel rice PR10 protein, which is rapidly induced in roots by salt, drought stresses and blast fungus infection possibly through activation of the jasmonic acid signaling pathway, but not the abscisic acid and salicylic acid signaling pathway.     

Rice lesion mimic mutants with enhanced resistance to diseases

Lesion mimic mutants are characterized by the formation of necrotic lesions in the absence of pathogens. Such genetic defects often result in enhanced resistance to pathogen infection and constitutive expression of defense response genes. To understand the genetic mechanisms leading to these mutations, we characterized 21 lesion mimic mutants isolated from IR64 rice mutant populations produced by mutagenesis with diepoxybutane (D), gamma rays (G), and fast neutrons (F). Four mutations are controlled by single dominant genes, one of which is inherited maternally. Five lesion mimics are allelic to known spotted leaf (spl) mutants spl1, spl2, spl3, or spl6. In total, 11 new lesion mimic mutations, named spl16, spl17, and spl19 through Spl27, were established based on allelism tests. Two lesion mimics, spl17 and Spl26 showed enhanced resistance to multiple strains of Magnaporthe oryzae, the rice blast pathogen, and Xanthomonas oryzae pv. oryzae, the bacterial blight (BB) pathogen. Co-segregation analyses of blast and BB resistance and lesion mimic phenotypes in segregating populations of spl17 and Spl26 indicate that enhanced resistance to the two diseases is conferred by mutations in the lesion mimic genes. A double mutant produced from two independent lesion mimics showed more severe lesions and higher level of resistance to X. o. pv. oryzae than their single mutant parents indicating a synergistic effect of the two mutations. In mutants that exhibit enhanced disease resistance to both pathogens, increases in expression of defense response genes PR-10a, POX22.3, and PO-C1 were correlated with lesion mimic development and enhancement of resistance. These lesion mimic mutants may provide essential materials for a comprehensive dissection of the disease resistance pathways in rice.