Construction and commissioning of mid-infrared self-amplified spontaneous emission free-electron laser at compact energy recovery linac.

The mid-infrared range is an important spectrum range where materials exhibit a characteristic response corresponding to their molecular structure. A free-electron laser (FEL) is a promising candidate for a high-power light source with wavelength tunability to investigate the nonlinear response of materials. Although the self-amplification spontaneous emission (SASE) scheme is not usually adopted in the mid-infrared wavelength range, it may have advantages such as layout simplicity, the possibility of producing a single pulse, and scalability to a short-wavelength facility. To demonstrate the operation of a mid-infrared SASE FEL system in an energy recovery linac (ERL) layout, we constructed an SASE FEL setup in cERL, a test facility of the superconducting linac with the ERL configuration. Despite the adverse circumstance of space charge effects due to the given boundary condition of the facility, we successfully established the beam condition at the undulators and observed FEL emission at a wavelength of 20 µm. The results show that the layout of cERL has the potential for serving as a mid-infrared light source.

Stimulated Excitation of an Optical Cavity by a Multibunch Electron Beam via Coherent-Diffraction-Radiation Process.

With a low emittance and short-bunch electron beam at a high repetition rate realized by a superconducting linac, stimulated excitation of an optical cavity at the terahertz spectrum range is shown. The electron beam passes through small holes in the cavity mirrors without being destroyed. A sharp resonance structure which indicates wideband stimulated emission via coherent diffraction radiation is observed while scanning the round-trip length of the cavity.

CD2-1, the C-Terminal Region of Flagellin, Modulates the Induction of Immune Responses in Rice.

Flagellin from the rice avirulent N1141 strain of Acidovorax avenae functions as a pathogen-associated molecular pattern (PAMP) and induces PAMP-triggered immunity (PTI) in rice. To study the recognition mechanism of flagellin in rice, we attempted to define one or more regions of the flagellin protein required to activate the PTI response. Based on domain classification, we produced four fragments of N1141 flagellin: N-terminal D0, D1. and D2 domains (ND0-2), N-terminal D2, D3, and C-terminal D2 domains (ND2-CD2), C-terminal D2, D1, and D0 domains (CD2-0), and C-terminal D2 and D1 domains (CD2-1). The C-terminal CD2-1 and CD2-0 fragments induced PTI responses in cultured rice cells. Synthetic flg22, which is sufficient to produce the flagellin response in Arabidopsis, and the N-terminal flagellin fragments containing flg22 region elicited very weak immune responses in rice. OsFLS2, the rice ortholog of AtFLS2, which mediates flg22 recognition, was not involved in CD2-0 or CD2-1 recognition in rice. In addition, CD2-0 triggered resistance to coinfection with pathogenic bacteria. Taken together, these data suggest that rice mainly recognizes flagellin CD2-1 by a receptor distinct from OsFLS2 and that this epitope recognition leads to PTI responses.

Glycan moiety of flagellin in Acidovorax avenae K1 prevents the recognition by rice that causes the induction of immune responses.

Abstract Recognition of pathogen-associated molecular patterns (PAMPs) such as flagellin, a main component of the bacterial flagellum, constitutes the first layer of plant immunity and is referred to as PAMP-triggered immunity (PTI). The rice avirulent N1141 strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, induces PTI including H2O2 generation, while flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. Mass spectrometry analyses revealed that total 1,600-Da and 2,150-Da of glycan residues were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin, suggesting that the glycan in K1 flagellin prevent epitope recognition in rice. We identified three genes in K1 flagella operon, which regulate structural modification of glycan in K1 flagellin. The immature glycan-attached flagellin from three genes deletion mutant, KΔ3FG, induced H2O2 generation in cultured rice cells, whereas the K1 mature-type flagellin did not cause a detectable increase in H2O2. The data indicate that the immature glycan of flagellin from KΔ3FG cannot prevent the epitope recognition in rice.

Two distinct EF-Tu epitopes induce immune responses in rice and Arabidopsis.

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.

Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin.

Plants have a sensitive system that detects various pathogen-derived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H2O generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H2O2 generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferase-deficient mutants showed that 1,600- and 2,150-Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (Ser¹78, Ser¹8³, Ser²¹², and Thr³5¹) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, S178A and S183A, K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at Ser¹78 and Ser¹8³ in K1 flagellin prevent epitope recognition in rice.

Characterization of receptor proteins using affinity cross-linking with biotinylated ligands.

The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.

Role of OsHSP90 and IREN, Ca2+ dependent nuclease, in plant hypersensitive cell death induced by transcription factor OsNAC4.

The hypersensitive response (HR) is a form of programmed cell death (PCD) commonly associated with the immune response in plants. HR cell death is often characterized by DNA fragmentation, loss of plasma membrane integrity, protein degradation and typical morphological changes such as plasma membrane shrinkage and nuclear condensation. Initiation of HR cell death requires de novo protein synthesis, suggesting that HR cell death induction involves a transcriptional network regulated by a key factor. We recently identified the OsNAC4 gene, which encodes a plant-specific transcription factor that exhibited rapid but transient transcriptional activation during the early stages of HR cell death. Overexpression of OsNAC4 in rice plants induced cell death accompanied by all characteristics of HR cell death: DNA fragmentation, loss of plasma membrane integrity, and protein degradation. In OsNAC4 RNAi knock-down lines exposed to an avirulent bacterial strain, the cellular response was characterized by a marked decrease in HR cell death compared to wild-type rice cells. Gene expression profiling, which compared rice cells and OsNAC4 knock-down transformants using a rice cDNA microarray, demonstrated that OsNAC4 controls the transcription of at least 139 genes including OsHSP90, involved in loss of plasma membrane integrity, and IREN, which encodes novel plant nuclease involved in cleavage of nuclear DNA. Here we report that although OsNAC4 overexpression caused rapid protein degradation during HR cell death, neither IREN nor OsHSP90 were involved. Thus, three important processes that accompany HR cell death are regulated by independent signaling pathways that are collectively induced by OsNAC4.

The transcription factor OsNAC4 is a key positive regulator of plant hypersensitive cell death.

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+)-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.

Analysis of flagellin perception mediated by flg22 receptor OsFLS2 in rice.

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.

Characterization of an elicitor-induced rice WRKY gene, OsWRKY71.

Expression of OsWRKY71, a rice WRKY gene, was induced by biotic elicitors and pathogen infection. It was also found that OsWRKY71 has features characteristic of a transcriptional repressor. Microarray analysis revealed that several elicitor-induced defense-related genes were upregulated in rice cells overexpressing OsWRKY71. These results indicate that the activation of defense-related genes by OsWRKY71 was probably indirect.

Involvement of the elicitor-induced gene OsWRKY53 in the expression of defense-related genes in rice.

We present a detailed characterization of the chitin oligosaccharide elicitor-induced gene OsWRKY53. OsWRKY53 was also induced in suspension-cultured rice cells by a fungal cerebroside elicitor and in rice plants by infection with the blast fungus Magnaporthe grisea. A fusion of OsWRKY53 with green fluorescent protein was detected exclusively in the nuclei of onion epidermal cells, and OsWRKY53 protein specifically bound to W-box elements. A transient assay using the particle bombardment method showed that OsWRKY53 is a transcriptional activator. A microarray analysis revealed that several defense-related genes, including pathogenesis-related protein genes such as PBZ1, were upregulated in rice cells overexpressing OsWRKY53. Finally, overexpression of OsWRKY53 in rice plants resulted in enhanced resistance to M. grisea. These results strongly suggest that OsWRKY53 is a transcription factor that plays important roles in elicitor-induced defense signaling pathways in rice.

A new method of defense response analysis using a transient expression system in rice protoplasts.

We established a new plant defense response assay using a transient expression system in rice protoplasts. The assay system sensitively detected defense induction by flagellin, which had previously been assigned to a specific elicitor. Our assay system provides a rapid and efficient way to dissect rice defense mechanisms.

High precision NMR structure and function of the RING-H2 finger domain of EL5, a rice protein whose expression is increased upon exposure to pathogen-derived oligosaccharides.

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 two-stranded beta-sheets (beta1, Ala(147)-Phe(149); beta2, Gly(156)-His(158)), one alpha-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 alpha-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.

EL5, a rice N-acetylchitooligosaccharide elicitor-responsive RING-H2 finger protein, is a ubiquitin ligase which functions in vitro in co-operation with an elicitor-responsive ubiquitin-conjugating enzyme, OsUBC5b.

EL5, a rice gene responsive to N-acetylchitooligosaccharide elicitor, encodes a RING-H2 finger protein with structural features common to the plant-specific ATL family. We show that the fusion protein of EL5 with maltose binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating 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 finger 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.