The hypersensitive response (HR) is a common feature of plant immune responses and a type of programmed cell death. However, little is known about the induction mechanism of HR cell death. We report that overexpression of OsNAC4, which encodes a plant-specific transcription factor, leads to HR cell death accompanied by the loss of plasma membrane integrity, nuclear DNA fragmentation and typical morphological changes. In OsNAC4 knock-down lines, HR cell death is markedly decreased in response to avirulent bacterial strains. After induction by an avirulent pathogen recognition signal, OsNAC4 is translocated into the nucleus in a phosphorylation-dependent manner. A microarray analysis showed that the expression of 139 genes including OsHSP90 and IREN, encoding a Ca 2 þ -dependent nuclease, were different between the OsNAC4 knock-down line and control line during HR cell death. During the induction of HR cell death, OsHSP90 is involved in the loss of plasma membrane integrity, whereas IREN causes nuclear DNA fragmentation. Overall, our results indicate that two important events occurring during HR cell death are regulated by independent pathways.

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Plants have sensitive perception systems that recognize various pathogen-derived molecules. We previously reported that rice detects flagellin from a rice-incompatible strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, which induces subsequent immune responses involving cell death. The mechanism of flagellin perception in rice, however, has remained obscure. In this study, we found that flg22, a peptide derived from the flagellin N-terminus, induced weak immune responses without cell death in cultured rice cells. To elucidate the mechanism by which flg22 induced signaling in rice, we characterized OsFLS2, the rice ortholog of AtFLS2, which mediates flg22 perception. Heterologous expression of OsFLS2 functions in Arabidopsis, showing the conservation of the flg22 signaling pathway across divergent plant taxa. OsFLS2-overexpressing rice cultured cells generated stronger immune responses with the induction of cell death following stimulation with flg22 and flagellin. However, examination of the growth rate of the compatible strain in inoculated OsFLS2-overexpressing rice could not confirm bacterial growth suppression compared with wild-type rice. These results suggest that rice possesses a conserved flagellin perception system utilizing the FLS2 receptor which, when upregulated, hardly affects resistance against compatible A. avenae.

SummaryEL5, a rice gene responsive to N-acetylchitooligosaccharide elicitor, encodes a RING-H2 ®nger protein with structural features common to the plant-speci®c ATL family. We show that the fusion protein of EL5 with maltose binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitinactivating enzyme (E1), and the Ubc4/5 subfamily of the ubiquitin-conjugating enzyme (E2). EL5 possesses the activity to catalyse the transfer of ubiquitin to the MBP moiety, and the RING-H2 ®nger motif of EL5 is necessary for this activity. Thus, we concluded that EL5 represents a ubiquitin ligase (E3). We also show that two rice E2s (OsUBC5a, OsUBC5b) of the Ubc4/5 subfamily function as E2 which catalyses EL5-mediated ubiquitination, and OsUBC5b was induced by elicitor, as well as EL5. These results strongly suggest that EL5 and OsUBC5b have roles in plant defense response through the turnover of protein(s) via the ubiquitin/proteasome system.

EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of twostranded ␤-sheets (␤1, Ala 147 -Phe 149 ; ␤2, Gly 156 -His 158 ), one ␣-helix (Cys 161 -Leu 166 ), and two large N-and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N-and C-terminal loops and the ␣-helix. We demonstrated that the fusion protein with EL5-(96 -181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.Upon sensing the invasion of microorganisms, plants evoke a variety of defense reactions, including the synthesis of antimicrobial compounds (phytoalexins) and proteins. Many of these biochemical reactions are based on the activation of defenserelated genes. In some cases, the level of protein accumulation and the rapidity of gene induction in the host plant are correlated to the degree of its disease resistance. Therefore, it might be possible to control disease resistance by modifying the regulatory factors for the expression of defense-related genes.Such regulatory factors could be elements of signal transduction pathways leading from the recognition of invading pathogens to the activation of defense-related genes. Most of the defense responses are reproducible in suspension-cultured cells treated with specific substances called elicitor (1). Chitin fragments (N-acetylchitooligosaccharides) can act as elicitors (2), which induce the transient expression of several "early responsive" genes, such as EL5 (3). EL5 is a RING finger protein, which is structurally related to proteins of the Arabidopsis ATL family. These proteins are characterized by a transmembrane domain (domain I), basic domain (domain II), conserved domain (domain III), and RING-H2 finger domain (domain IV) followed by the C-terminal region with highly diverse amino acid sequences (4). Although some ATL family genes resemble EL5 in being induced in early stages of the defense responses (5), their biochemical function is obscure. Recently, it was shown that the fusion protein of EL5 with maltose-binding protein (MBP) 1 was polyubiquitinated by incubation with ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2). ...

Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI). We previously reported that rice recognizes flagellin from the rice-incompatible N1141 strain of Acidovorax avenae and subsequently induces immune responses. Cell extracts isolated from flagellin-deficient N1141 (Δfla1141) still induced PTI responses, suggesting that Δfla1141 possesses an additional PAMP distinct from flagellin. Here, we show that elongation factor Tu (EF-Tu), one of the most abundant bacterial proteins, acts as a PAMP in rice and causes several PTI responses. In Brassicaceae species, EF-Tu and an N-acetylated peptide comprising the first 18 amino acids of the N-terminus, termed elf18, are fully active as inducers of PTI responses. By contrast, elf18 did not cause any immune responses in rice, whereas an EF-Tu middle region comprising Lys176 to Gly225, termed EFa50, is fully active as a PAMP in rice. In the leaves of rice plants, EF-Tu induced H2O2 generation and callose deposition, and also triggered resistance to coinfection with pathogenic bacteria. Taken together, these data demonstrate that rice recognizes EFa50, which is distinct from elf18, and that this epitope induces PTI responses.

Reactive oxygen species (ROS) are proposed to function as diffusible signaling molecules in plant immune response. Rice respiratory burst oxidase homologs (Osrboh genes) are proposed to play a role in ROS generation. We examined a role in rice immune responses of four Osrboh homologs, OsrbohA, OsrbohB, OsrbohD, and OsrbohE. OsrbohA and OsrbohD transcripts were induced after inoculation with an incompatible N1141 strain of Acidovorax avenae, whereas OsrbohaB and OsrbohE mRNA levels did not obviously change even after inoculation with the incompatible strain. We examined the function of the Osrboh genes in ROS generation and in the plant immune response using RNAi-based knockdown in rice cells. OsrbohA and OsrbohE knockdown lines showed that rapid H 2 O 2 generation is caused by OsrbohA, whereas OsrbohE is involved in late H 2 O 2 production during the immune response. Hypersensitive cell death was decreased only in the OsrbohA knockdown line. We further demonstrated that among immune related genes, the induction of EL2 and LOX genes is controlled by ROS generated by OsrbohE, whereas expression of Cht-1 gene is regulated by both OsrbohA and OsrbohE. These results indicate that the ROS molecules generated by OsrbohA and OsrbohE regulate different signaling pathways in the plant immune response. the Arabidopsis genome (Foreman et al. 2003). Of these rboh in Arabidopsis, AtrbohD and AtrbohF were shown to be necessary for ROS generation in the plant immune responses (Torres et al. 2002). Furthermore, NtbohD from Nicotiana tabacum and NbrbohA and NbrbohB from N. benthamiana were required for ROS accumulation and resistance (Simon-Plas et al. 2002;Yoshioka et al. 2003), suggesting that individual isoforms have different functions and participate in multiple distinct signaling pathways.Acidovorax avenae is a Gram-negative bacterium that causes a seedling disease characterized by the formation of brown stripes on the sheaths of infected plants (Kadota et al. 1991). A. avenae can infect a wide range of monocotyledonous plants, including rice, oats, Italian millet, and maize. However, individual strains of the pathogen can infect only one or a few host species (Nishiyama et al. 1979;Kadota et al. 1991;Kadota et al. 1996). We recently reported that several immune responses, such as H 2 O 2 generation, hypersensitive cell death accompanied by clear 180-bp nucleosomal DNA laddering, and immune-related genes were induced when cultured rice cells were inoculated with a riceincompatible strain of A. avenae. These responses were completely abolished when a rice-compatible strain of A. avenae was used as the inoculating pathogen (Che et al. 1999;Tanaka et al. 2001;Iwano et al. 2002;Tanaka et al. 2003;Fujiwara et al. 2004). To identify the specific elicitor related to the induction of these immune responses in cultured rice cells, a strain-specific antibody was raised against the incompatible strain (N1141) and then absorbed by the compatible strain (H8301). The specific antibody detected flagellin protein, a component of t...

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