Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Barley (Hordeum vulgare) mildew locus o (mlo) mutants exhibit strong resistance to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei. MLO, a seven-transmembrane protein localized at the plasma membrane is thought to be involved in intracellular calcium signaling. However, its molecular function and the mechanism by which mlo mutations confer resistance to penetration by the fungus remain poorly understood. A large number of mlo alleles with different amino acid substitutions at each intracellular loop have been found in various cultivars. However, it is difficult to analyze how each amino acid is involved in penetration resistance by comparing these cultivars because they differ substantially in their genetic background and in the presence or absence of resistance genes recognizing avirulence factors from the pathogen. In this study, we used a CRISPR/Cas9-mediated genetic modification system to generate mlo mutants in the transformable cultivar Golden Promise to enable complementation experiments with the aim of elucidating the molecular function of MLO. An mlo mutant with a thymine insertion in the second exon and penetration resistance to B. graminis f. sp. hordei was obtained. Susceptibility was restored in cells in which the MLO-mCherry gene was introduced using particle bombardment, indicating that this mlo mutant could be a useful genetic tool for complementation experiments using transgenes expressing a variety of mlo alleles.

DOI： 10.1007/s10327-023-01120-w

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To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the ArabidopsisMulti-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacumMAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs-knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants' sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.

DOI： 10.3390/ijms23042006

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1094/MPMI-05-21-0120-A

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Authorship：Lead author,　Last author,　Corresponding author  

DOI： 10.2520/myco.72-1-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Inflammation is the cause and/or result of many diseases in peripheral tissues and the central nervous system. Recent findings suggested that inflammation in peripheral tissue induces an inflammatory response in the brain that activates glial cells, which, in turn, induce neuronal cell dysfunction. Therefore, anti-inflammatory compounds are important for the suppression of chronic inflammation and prevention of disease. The present study revealed microglial activation in the hippocampus of the brain two days after the peripheral administration of lipopolysaccharide (LPS). Furthermore, the expression of the synaptic vesicle membrane protein, synaptophysin, in the CA3 stratum lucidum of the hippocampus was down-regulated 7 days after the LPS injection. The administration of tocotrienols, a type of vitamin E, significantly attenuated these changes in the hippocampus. Collectively, the present results demonstrated the spread of peripheral inflammatory responses to the brain, in which glial activation and neuronal dysfunction were induced, while tocotrienols exerted anti-inflammatory effects and protected neurons from damage.

DOI： 10.3390/neuroglia2010009

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Loss-of-function mutation of the MILDEW RESISTANCE LOCUS O (Mlo) gene confers durable and broad-spectrum resistance to powdery mildew fungi in various plants, including barley. In combination with the intracellular nucleotide-binding domain and leucine-rich repeat receptor (NLR) genes, which confer the race-specific resistance, the mlo alleles have long been used in barley breeding as genetic resources that confer robust non-race-specific resistance. However, a Japanese Blumeria graminis f. sp. hordei isolate, RACE1, has been reported to have the potential to overcome partially the mlo-mediated penetration resistance, although this is yet uncertain because the putative effects of NLR genes in the tested accessions have not been ruled out. In this study, we examined the reproducibility of the earlier report and found that the infectious ability of RACE1, which partially overcomes the mlo-mediated resistance, is only exerted in the absence of NLR genes recognizing RACE1. Furthermore, using the transient-induced gene silencing technique, we demonstrated that RACE1 can partially overcome the resistance in the host cells with suppressed MLO expression but not in plants possessing the null mutant allele mlo-5.

DOI： 10.1371/journal.pone.0256574

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© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Pyridine nucleotides such as a nicotinamide adenine dinucleotide (NAD) are known as plant defense activators. We previously reported that nicotinamide mononucleotide (NMN) enhanced disease resistance against fungal pathogen Fusarium graminearum in barley and Arabidopsis. In this study, we reveal that the pretreatment of nicotinamide (NIM), which does not contain nucle-otides, effectively suppresses disease development of Fusarium Head Blight (FHB) in wheat plants. Correspondingly, deoxynivalenol (DON) mycotoxin accumulation was also significantly decreased by NIM pretreatment. A metabolome analysis showed that several antioxidant and antifungal compounds such as trigonelline were significantly accumulated in the NIM-pretreated spikes after in-oculation of F. graminearum. In addition, some metabolites involved in the DNA hypomethylation were accumulated in the NIM-pretreated spikes. On the other hand, fungal metabolites DON and ergosterol peroxide were significantly reduced by the NIM pretreatment. Since NIM is relative sta-ble and inexpensive compared with NMN and NAD, it may be more useful for the control of symptoms of FHB and DON accumulation in wheat and other crops.

DOI： 10.3390/ijms22062968

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)  

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), induces disease resistance to the Fusarium head blight fungus Fusarium graminearum in Arabidopsis and barley, but it is unknown at which stage of the infection it acts. Since the rate of haustorial formation of an obligate biotrophic barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was significantly reduced in NMN-treated coleoptile epidermal cells, the possibility that NMN induces resistance to the biotrophic stage of F. graminearum was investigated. The results show that NMN treatment caused the wandering of hyphal growth and suppressed the formation of appressoria-like structures. Furthermore, we developed an experimental system to monitor the early stage of infection in real-time and analyzed the infection behavior. We observed that the hyphae elongated windingly by NMN treatment. These results suggest that NMN potentiates resistance to the biotrophic invasion of F. graminearum as well as Bgh.

DOI： 10.3390/ijms22052696

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Language：English   Publisher：Japan International Research Center for Agricultural Sciences  

<p><i>Rice necrosis mosaic virus</i> (RNMV) is a fungus-transmitted bymovirus that leads to losses in rice yield. This research tested ten rice cultivars (cvs) with different levels of resistance to RNMV. The lowest levels of RNMV RNA were found in two high-resistance cvs; the highest levels were found in the two low-resistance cvs. The <i>RNA-dependent RNA polymerase 6 </i>gene in rice (<i>OsRDR6</i>) was found to be the most highly expressed in two high-resistance cvs and the least expressed in two low-resistance cvs although its basal level of constitutive expression was similar among cvs. Plant growth and yields were also tested. The extent of RNMV RNA accumulation affected plant height, panicle/tiller numbers, and seed weight. The RNMV induced <i>OsRDR6 </i>expression level in the rest of cvs was more or less inversely correlated to RNMV RNA accumulation. The observed results suggest a close relationship between RNMV resistance and RNMV induced <i>OsRDR6 </i>expression level in these cvs.</p>

DOI： 10.6090/jarq.55.127

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In the present study, we have shown the transcriptional changes in a chlorosis model transgenic tobacco plant, i-amiCHLI, in which an artificial micro RNA is expressed in a chemically inducible manner to silence the expression of CHLI genes encoding a subunit of a chlorophyll biosynthetic enzyme. Comparison to the inducer-treated and untreated control non-transformants and untreated i-amiCHLI revealed that 3568 and 3582 genes were up- and down-regulated, respectively, in the inducer-treated i-amiCHLI plants. Gene Ontology enrichment analysis of these differentially expressed genes indicated the upregulation of the genes related to innate immune responses, and cell death pathways, and the downregulation of genes for photosynthesis, plastid organization, and primary and secondary metabolic pathways in the inducer-treated i-amiCHLI plants. The cell death in the chlorotic tissues with a preceding H2O2 production was observed in the inducer-treated i-amiCHLI plants, confirming the activation of the immune response. The involvement of activated innate immune response in the chlorosis development was supported by the comparative expression analysis between the two transgenic chlorosis model systems, i-amiCHLI and i-hpHSP90C, in which nuclear genes encoding different chloroplast proteins were similarly silenced.

DOI： 10.3390/ijms21197044

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

To further develop barley breeding and genetics, more information on gene functions based on the analysis of the mutants of each gene is needed. However, the mutant resources are not as well developed as the model plants, such as Arabidopsis and rice. Although genome editing techniques have been able to generate mutants, it is not yet an effective method as it can only be used to transform a limited number of cultivars. Here, we developed a mutant population using 'Mannenboshi', which produces good quality grains with high yields but is susceptible to disease, to establish a Targeting Induced Local Lesions IN Genomes (TILLING) system that can isolate mutants in a high-throughput manner. To evaluate the availability of the prepared 8043 M3 lines, we investigated the frequency of mutant occurrence using a rapid, visually detectable waxy phenotype as an indicator. Four mutants were isolated and single nucleotide polymorphisms (SNPs) were identified in the Waxy gene as novel alleles. It was confirmed that the mutations could be easily detected using the mismatch endonuclease CELI, revealing that a sufficient number of mutants could be rapidly isolated from our TILLING population.

DOI： 10.3390/plants9091153

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RNA-seq analysis of a transgenic tobacco plant, i-hpHSP90C, in which chloroplast HSP90C genes can be silenced in an artificially inducible manner resulting in the development of chlorosis, revealed the up- and downregulation of 2746 and 3490 genes, respectively. Gene ontology analysis of these differentially expressed genes indicated the upregulation of ROS-responsive genes; the activation of the innate immunity and cell death pathways; and the downregulation of genes involved in photosynthesis, plastid organization, and cell cycle. Cell death was confirmed by trypan blue staining and electrolyte leakage assay, and the H2O2 production was confirmed by diaminobenzidine staining. The results collectively suggest that the reduced levels of HSP90C chaperone lead the plant to develop chlorosis primarily through the global downregulation of chloroplast- and photosynthesis-related genes and additionally through the light-dependent production of ROS, followed by the activation of immune responses, including cell death.

DOI： 10.3390/ijms21124202

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

High humidity decreases the penetration rate of barley powdery mildew Blumeria graminis f. sp. hordei. However, the mechanism is not well understood. In this study, the morphological and cytochemical analyses revealed that substances containing proteins leaked from the tip of the appressorial germ tube of conidia without the formation of appressorium under a high humidity condition. In addition, exposure to high humidity prior to the formation of appressorium caused the aberrant formation of the appressorial germ tube without appressorium formation, resulting in failure to penetrate the host cell. These findings suggest that the formation and maturation of the appressorium requires a low humidity condition, and will be clues to improve the disease management by humidity control.

DOI： 10.3390/pathogens9010045

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© 2020 The American Phytopathological Society Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a major threat to economically important cucurbit crops worldwide. An attenuated strain (SH33b) derived from a severe strain (SH) of CGMMV caused a reduction in the viral RNA accumulation and the attenuation of symptoms, and it has been successfully used to protect muskmelon plants against severe strains in Japan. In this study, we compared GFP-induced silencing suppression by the 129K protein and the methyltransferase domain plus intervening region (MTIR) of the 129K protein between the SH and SH33b strains, respectively. As a result, silencing suppression activity (SSA) in the GFP-silenced plants was inhibited efficiently by the MTIR and 129K protein of SH strain, and it coincided with drastically reduced accumulation of GFP-specific small interfering RNAs (siRNAs) but not by that of SH33b strain. Furthermore, analyses of siRNA binding capability (SBC) by the MTIR of 129K protein and 129K protein using electrophoretic mobility shift assay revealed that SBC was found with the MTIR and 129K protein of SH but not with that of SH33b, suggesting that a single amino acid mutation (E to G) in the MTIR is responsible for impaired SSA and SBC of SH33b. These data suggest that a single amino acid substitution in the intervening region of 129K protein of CGMMV resulted in attenuated symptoms by affecting RNA silencing suppression.

DOI： 10.1094/PHYTO-12-18-0478-FI

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Authorship：Last author   Language：English   Publisher：Mycological Society of Japan ; 1994-  

© 2019 The Mycological Society of Japan We aimed to develop an artificial membrane system to observe the infection process of the obligate biotrophic powdery mildew fungi without the use of living plant cells. The conidia of Blumeria graminis and Erysiphe pisi conidia were inoculated on a formvar membrane laid on an artificial medium. Germinated conidia frequently formed appressoria and then penetrated the membrane to form haustorium-like structures in the artificial medium. Secondary hyphae elongation was also observed after the formation of haustorium-like structures. These results suggested that the formvar membrane laid on artificial medium induced the formation of haustorium-like structures that have roles in the formation of secondary hyphae.

DOI： 10.1016/j.myc.2019.06.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer-Verlag Wien  

The TOM1/TOM3 genes from Arabidopsis are involved in the replication of tobamoviruses. Tomato homologs of these genes, LeTH1, LeTH2 and LeTH3, are known. In this study, we examined transgenic tomato lines where inverted repeats of either LeTH1, LeTH2 or LeTH3 were introduced by Agrobacterium. Endogenous mRNA expression for each gene was detected in non-transgenic control plants, whereas a very low level of each of the three genes was found in the corresponding line. Small interfering RNA was detected in the transgenic lines. Each silenced line showed similar levels of tobamovirus resistance, indicating that each gene is similarly involved in virus replication.

DOI： 10.1007/s00705-018-3747-4

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Although the use of stable transformants is indispensable to elucidate mechanisms underlying molecular plant-pathogen interactions, this approach remains difficult to apply to crops. Alternatively, biolistic transformation has often been used as a transient expression method in various plants. In this study, we developed a method for in situ biolistic transformation without separating leaves from barley seedlings by using a hand-held particle bombardment system because unwounded leaves are preferable for analyzing interactions between barley and Blumeria graminis f. sp. hordei which requires healthy living cells. As a result, we found that the infection rate in intact leaves was higher than in separated leaves and that the transformation efficiencies in leaves were higher when plants were grown in vermiculite rather than in culture soil. Furthermore, we determined the appropriate inoculation time after bombardment to analyze the incompatible interaction and successfully monitored the gradual occurrence of cell death over time. Our system was suitable for relatively long-term follow-up analysis of the fate of each single cell during plant-pathogen interactions.

DOI： 10.1007/s10327-017-0712-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer India  

Recent studies with Y satellite RNA (Y-sat) of cucumber mosaic virus have demonstrated that Y-sat modifies the disease symptoms in specific host plants through the silencing of the magnesium protoporphyrin chelatase I subunit (CHLI), which is directed by the Y-sat derived siRNA. Along with the development of peculiar yellow phenotypes, a drastic decrease in CHLI-transcripts and a higher accumulation of Y-sat derived siRNA were observed. To investigate the molecular mechanisms underlying the Y-sat—induced chlorosis, especially whether or not the reduced expression of CHLI causes the chlorosis simply through the reduced production of chlorophyll or it triggers some other mechanisms leading to the chlorosis, we have established a new experimental system with an inducible silencing mechanism. This system involves the expression of artificial microRNAs targeting of Nicotiana tabacum CHLI gene under the control of chemically inducible promoter. The CHLI mRNA levels and total chlorophyll content decreased significantly in 2 days, enabling us to analyze early events in induced chlorosis and temporary changes therein. This study revealed that the silencing of CHLI did not only result in the decreased chlorophyll content but also lead to the downregulation of chloroplast and photosynthesis-related genes expression and the upregulation of defense-related genes. Based on these results, we propose that the reduced expression of CHLI could activate unidentified signaling pathways that lead plants to chlorosis.

DOI： 10.1007/s13337-017-0360-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer India  

Chlorosis is one of the most common symptoms of plant diseases, including those caused by viruses and viroids. Recently, a study has shown that Peach latent mosaic viroid (PLMVd) exploits host RNA silencing machinery to modulate the virus disease symptoms through the silencing of chloroplast-targeted heat shock protein 90 (Hsp90C). To understand the molecular mechanisms of chlorosis in this viroid disease, we established an experimental system suitable for studying the mechanism underlying the chlorosis induced by the RNA silencing of Hsp90C in transgenic tobacco. Hairpin RNA of the Hsp90C-specific region was expressed under the control of a dexamethasone-inducible promoter, resulted in the silencing of Hsp90C gene in 2 days and the chlorosis along with growth suppression phenotypes. Time course study suggests that a sign of chlorosis can be monitored as early as 2 days, suggesting that this experimental model is suitable for studying the molecular events taken place before and after the onset of chlorosis. During the early phase of chlorosis development, the chloroplast- and photosynthesis-related genes were downregulated. It should be noted that some pathogenesis related genes were upregulated during the early phase of chlorosis in spite of the absence of any pathogen-derived molecules in this system.

DOI： 10.1007/s13337-017-0361-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The complete nucleotide sequences of Beet pseudoyellows virus (BPYV)-MI (cucumber isolate; Matsuyama, Idai) genomic RNAs 1 and 2 were determined and compared with the previously sequenced Japanese cucumber strain (BPYV-JC) and a strawberry strain (BPYV-S). The RNA 2 of BPYV-MI showed 99 % nucleotide sequence identity with both BPYV-JC and -S having highly conserved eight ORFs. In contrast, the RNA1 of BPYV-MI showed sequence identities of 98 and 86 % with BPYV-JC and -S, respectively. Phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) coding sequences from three fully sequenced BPYV strains and five partially sequenced cucurbit-infecting BPYV strains from Japan and South Africa has shown that cucurbit-infecting strains are closer to each other than to BPYV-S. In addition, the strawberry strain BPYV-S has an ORF2 in the downstream of RdRp gene in RNA1, but all the cucumber strains, BPYV-JC, -MI, and those from South Africa, lacked the ORF2 of RNA1, highlighting the difference between common BPYV cucumber strains and a unique strawberry strain.

DOI： 10.1007/s11262-016-1376-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVR(a) gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVR(a) genes and identified AVR(a1) and AVR(a13), encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVR(a1) and AVR(a13) are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVR(A1) directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVR(A1). Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

DOI： 10.1073/pnas.1612947113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Here we report a novel method to extract nucleic acids for virus detection. Plant tissue in a filter paper sandwich was hit with a hammer, and the crude sap was adsorbed by the filter paper, as in hammer blotting for tissue-print immunodetection of plant viruses. Nucleic acids were extracted from the paper with a guanidine-containing buffer and purified through standard extraction and precipitation. Both viral and cellular RNAs in the extracted RNA were detectable by RT-PCR. The blots can be stored at 4 A degrees C for more than a few days to detect viral RNA, but not cellular RNA.

DOI： 10.1007/s10327-016-0673-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER INDIA  

We examined the transmission of RNA silencing signal in non-transgenic tomato and tobacco scions grafted onto the tobacco Sd1 rootstocks, which is silenced in both NtTOM1 and NtTOM3 required for tobamovirus multiplication. When the non-transgenic tomato scions were grafted onto the Sd1 rootstocks, RT-PCR analysis of the scions showed the reduced level of mRNA compared with that before grafting in both LeTH3 and LeTH1, tomato homologs of NtTOM1 and NtTOM3, respectively. siRNAs from both genes were detected in the scions after grafting but not before grafting. Further tomato scions were inoculated with Tomato mosaic virus (ToMV) and used for virus infection. They showed very low level of virus accumulation. Necrotic responding tobacco to tobamovirus was grafted onto the rootstock of Sdl. RT-PCR analysis showed low level expression of both NtTOM1 and NtTOM3 in the scions but siRNA was detected after grafting. When the leaves of scions were inoculated with ToMV or Tobacco mosaic virus, they produced very few local necrotic lesions (LNLs) while the control scions did many LNLs. These results suggest that RNA silencing was transmitted to non-transgenic tomato and tobacco scions after grafting onto the Sd1 rootstocks and that virus resistance was induced in the scions.

DOI： 10.1007/s13562-015-0334-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

We investigated graft transmission of high-temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA-silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high-temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high-temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks.

DOI： 10.1111/pbi.12429

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

The complete nucleotide sequence of Rice necrosis mosaic virus (RNMV) RNA1 was determined to be 7178 nt long with one large open reading frame, potentially encoding a polyprotein of 258 kDa with the features of a typical bymovirus. The nucleotide sequence showed 56 % identity with that of Barley mild mosaic virus (BaMMV) and 46 % with those of Oat mosaic virus, Barley yellow mosaic virus and Wheat yellow mosaic virus. RNA2 was 3579 nt long, encoding a 110-kDa polyprotein, and showed 42 and 35/26 % identity with BaMMV and others, respectively. Phylogenetic analysis showed that bymoviruses were classified into two subgroups.

DOI： 10.1007/s10327-015-0618-7

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RNA-dependent RNA polymerases (RDRs) play key roles in gene silencing. The rice RDR6 gene was analyzed in response to viral, bacterial and fungal pathogens, after inoculation of a rice mutant line of OsRDR6, shl2-rol, with Cucumber mosaic virus, Rice necrosis mosaic virus, Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae. Compared with the wild type, the mutant line accumulated more viral RNA after inoculation with the viruses and developed more severe symptoms after inoculation with the bacterium or fungus. Thus, the OsRDR6-mediated RNA silencing pathway seems to participate in defense against not only viruses, but also bacterial and fungal pathogens.

DOI： 10.1007/s10327-015-0630-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB.

DOI： 10.1105/tpc.15.00213

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To study the precise mechanisms underlying the chlorosis caused by plant viruses, we previously established a synchronous experimental system using transgenic plants expressing Cauliflower mosaic virus multifunctional protein, Tav (transactivator/viroplasmin), under the control of an artificially inducible promoter. Shortly after the induction of Tav expression, pathogenesis-related protein (PR) 1a gene expression is upregulated in the transgenic tobacco lines, which show visible chlorosis within a week. The present study showed that the expression of Tav also induces some salicylic acid (SA)- and ethylene-responsive PR genes. In contrast to transiently expressed Tav, which suppressed Agrobacterium-induced and SA-induced PR1a expression, the artificial induction of Tav from the transgene did not affect SA-induced PR1a expression, rather it alone induced PR1a expression. In a deletion analysis, chlorosis and PR1a induction function in transgenic tobacco were mapped to a region in Tav that had been shown to have a role in pathogenesis in a susceptible host, elicitation of the hypersensitive response in a resistant host, suppression of RNA silencing, and the suppression of Tomato bushy stunt virus P19-mediated cell death in tobacco. The results suggest that Tav-induced chlorosis results from a host response, which accompanies PR1a induction, to pathogenic function of Tav.

DOI： 10.1007/s10327-015-0600-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding -glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H2O2), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H2O2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.

DOI： 10.1111/pbi.12239

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HAP (CBF/NF-Y) transcription factors have important functions in regulating plant growth, development, and stress responses. In this study, we examined whether the endogenous gene OsHAP2E and the GUS transgene driven by the promoter of OsHAP2E respond to virus infection. RT-PCR analyses showed OsHAP2E expression was induced after inoculation with Cucumber mosaic virus (CMV) or Rice necrosis mosaic virus (RNMV). After inoculating OsHAP2E::GUS-transgenic plants with either virus, the levels of GUS expression increased significantly. The expression levels of GUS or OsHAP2E reached a plateau 5 days after inoculation of rice with CMV, which paralleled the accumulation of CMV RNA level. Furthermore, transgenically over-expressed lines of OsHAP2E (OsHAP2E-OX) had lower levels of CMV and RNMV RNAs than in in nontransgenic control plants. The OsHAP2E-OX lines developed no significant symptoms from RNMV while control plants had yellowing and stunting. These results suggested that OsHAP2E is induced by virus infection and contributes to resistance against viral pathogens.

DOI： 10.1007/s10327-014-0564-9

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The underlying molecular mechanism of chlorosis, a typical symptom of plant viral diseases, remains poorly understood. To establish an experimental system to determine the molecular changes during chlorosis, especially in the early phase, we generated transgenic tobacco plants expressing Cauliflower mosaic virus Transactivator/viroplasmin (Tav) under the control of a chemically inducible promoter. Induction of Tav resulted in visible chlorosis in ten days, a statistically significant decrease in chlorophyll content in two days, decreased expression of chloroplast protein genes, and abnormal thylakoid stacks, indicating that this system reproduces the common features of chlorosis in virus-infected plants. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.pmpp.2014.08.005

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Plants control CO2 uptake and water loss by modulating the aperture of stomata located in the epidermis. Stomatal opening is initiated by the activation of H+-ATPases in the guard-cell plasma membrane. In contrast to regulation of H+-ATPase activity, little is known about the translocation of the guard cell H+-ATPase to the plasma membrane. Here we describe the isolation of an Arabidopsis gene, PATROL1, that controls the translocation of a major H+-ATPase, AHA1, to the plasma membrane. PATROL1 encodes a protein with a MUN domain, known to mediate synaptic priming in neuronal exocytosis in animals. Environmental stimuli change the localization of plasma membrane-associated PATROL1 to an intracellular compartment. Plasma membrane localization of AHA1 and stomatal opening require the association of PATROL1 with AHA1. Increased stomatal opening responses in plants overexpressing PATROL1 enhance the CO2 assimilation rate, promoting plant growth.

DOI： 10.1038/ncomms3215

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The translocation of effector proteins into the host plant cells is essential for pathogens to suppress plant immune responses. The oomycete pathogen Phytophthora infestans secretes AVR3a, a crucial virulence effector protein with an N-terminal RXLR motif that is required for this translocation. It has been reported that the RXLR motif of P. sojae Avr1b, which is a close homolog of AVR3a, is required for binding to phosphatidylinositol monophosphates (PIPs). However, in our previous report, AVR3a as well as Avr1b bind to PIPs not via RXLR but via lysine residues forming a positively-charged area in the effector domain. In this report, we examined whether other RXLR effectors whose structures have been determined bind to PIPs. Both P. capsici AVR3a11 and Hyaloperonospora arabidopsidis ATR 1 have an RXLR motif in their N-terminal regions but did not bind to any PIPs. These results suggest that the RXLR motif is not sufficient for PIP binding. © 2013 Landes Bioscience.

DOI： 10.4161/psb.23865

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The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography-electrospray ionization-tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis.

DOI： 10.1073/pnas.1108434108

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The oomycete pathogen Phytophthora infestans causes potato late blight, one of the most economically damaging plant diseases worldwide. P. infestans produces AVR3a, an essential modular virulence effector with an N-terminal RXLR domain that is required for host-cell entry. In host cells, AVR3a stabilizes and inhibits the function of the E3 ubiquitin ligase CMPG1, a key factor in host immune responses including cell death triggered by the pathogen-derived elicitor protein INF1 elicitin. To elucidate the molecular basis of AVR3a effector function, we determined the structure of Phytophthora capsici AVR3a4, a close homolog of P. infestans AVR3a. Our structural and functional analyses reveal that the effector domain of AVR3a contains a conserved, positively charged patch and that this region, rather than the RXLR domain, is required for binding to phosphatidylinositol monophosphates (PIPs) in vitro. Mutations affecting PIP binding do not abolish AVR3a recognition by the resistance protein R3a but reduce its ability to suppress INF1-triggered cell death in planta. Similarly, stabilization of CMPG1 in planta is diminished by these mutations. The steady-state levels of non-PIP-binding mutant proteins in planta are reduced greatly, although these proteins are stable in vitro. Furthermore, overexpression of a phosphatidylinositol phosphate 5-kinase results in reduction of AVR3a levels in planta. Our results suggest that the PIP-binding ability of the AVR3a effector domain is essential for its accumulation inside host cells to suppress CMPG1dependent immunity.

DOI： 10.1073/pnas.1106002108

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Linolenic acid (18:3) and its derivative jasmonic acid (]A) are important molecules in disease resistance in many dicotyledonous plants. We have previously used 18:3- and JA-deficient rice (F78Ri) to investigate the roles of fatty acids and their derivatives in resistance to the blast fungus Magnaporthe grisea JA. Yara, T. Yaeno, J.-L. Montillet, M. Hasegawa, S. Seo, K. Kusumi, K. Iba, Enhancement of disease resistance to Magnaporthe grisea in rice by accumulation of hydroxy linoleic acid, Biochem. Biophys. Res. Commun. 370 (2008) 344-347; A. Yara, T. Yaeno, M. Hasegawa, H. Seto, J.-L. Montillet, K. Kusumi, S. Seo, K. Iba, Disease resistance against Magnaporthe grisea is enhanced in transgenic rice With Suppression of omega-3 fatty acid desaturases, Plant Cell Physiol. 48 (2007) 1263-1274]. However, because F78Ri plants are suppressed in the first step of the JA biosynthetic pathway, we could not confirm the specific contribution of JA to disease resistance. In this paper, we generated two JA-deficient rice lines (AOCRi and OPRRi) with suppressed expression of the genes encoding allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR), which catalyze late steps in the JA biosynthetic pathway. The levels of disease resistance in the AOCRi and OPRRi lines were equal to that in wild-type plants. Our data suggest that resistance to M. grisea is not dependent on JA synthesis. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.08.157

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Salicylic acid (SA) is a primary factor responsible for exerting diverse immune responses in plants and is synthesized in response to attack by a wide range of pathogens. The Arabidopsis (Arabidopsis thaliana) sid2 mutant is defective in a SA biosynthetic pathway involving ISOCHORISMATE SYNTHASE1 (ICS1) and consequently contains reduced levels of SA. However, the sid2 mutant as well as ICS-suppressed tobacco (Nicotiana benthamiana) still accumulate a small but significant level of SA. These observations along with previous studies suggest that SA might also be synthesized by another pathway involving benzoic acid (BA). Here we isolated a benzoic acid hypersensitive1-Dominant (bah1-D) mutant that excessively accumulated SA after application of BA from activation-tagged lines. This mutant also accumulated higher levels of SA after inoculation with Pseudomonas syringae pv tomato DC3000. Analysis of the bah1-D sid2 double mutant suggested that the bah1-D mutation caused both ICS1-dependent and-independent accumulation. In addition, the bah1-D mutant showed SA-dependent localized cell death in response to P. syringae pv tomato DC3000. The T-DNA insertional mutation that caused the bah1-D phenotypes resulted in the suppression of expression of the NLA gene, which encodes a RING-type ubiquitin E3 ligase. These results suggest that BAH1/NLA plays crucial roles in the ubiquitination-mediated regulation of immune responses, including BA-and pathogen-induced SA accumulation, and control of cell death.

DOI： 10.1104/pp.108.124529

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Linoleic acid (18:2) and linolenic acid (18:3) are sources for various oxidized metabolites called oxylipins, some of which inhibit growth of fungal pathogens. In a previous study, we found disease resistance to rice blast fungus Magnaporthe grisea enhanced in 18:2-accumulating transgenic rice (F78Ri) in which the conversion from 18:2 to 18:3 was suppressed. Here, we demonstrate that 18:2-derived hydroperoxides and hydroxides (HPODEs and HODEs, respectively) inhibit growth of M. grisea more strongly than their 18:3-derived counterparts. Furthermore, in F78Ri plants, the endogenous levels of HPODEs and HODEs increased significantly, compared with wild-type plants. These results suggest that the increased accumulation of antifungal oxylipins, such as HPODEs and HODEs, causes the enhancement of disease resistance against M. grisea. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.03.083

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Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91(phox) and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern-induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac-Rboh interaction was supported by further studies using in vitro pull-down assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca2+ concentration may regulate Rac-Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac-Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca2+ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.

DOI： 10.1105/tpc.107.055624

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Linolenic acid (18:3) is the most abundant fatty acid in plant membrane lipids and is a source for various oxidized metabolites, called oxylipins. 18:3 and oxylipins play important roles in the induction of defense responses to pathogen infection and wound stress in Arabidopsis. However, in rice, endogenous roles for 18:3 and oxylipins in disease resistance have not been confirmed. We generated 18:3-deficient transgenic rice plants (F78Ri) with co-suppression of two omega-3 fatty acid desaturases, OsFAD7 and OsFAD8. that synthesize 18:3. The F78Ri plants showed enhanced resistance to the phytopathogenic fungus Magnaporthe grisea. A typical 18:3-derived oxylipin, jasmonic acid (JA), acts as a signaling molecule in defense responses to fungal infection in Arabidopsis. However, in F78Ri plants, the expression of JA-responsive pathogenesis-related genes, PBZI and PR1b, was induced after inoculation with M. grisea, although the JA-mediated wound response was suppressed. Furthermore, the application of JA methyl ester had no significant effect on the enhanced resistance in F78Ri plants. Taken together, our results indicate that, although suppression of fatty acid desaturases involves the concerted action of varied oxylipins via diverse metabolic pathways, 18:3 or 18:3-derived oxylipins, except for JA, may contribute to signaling on defense responses of rice to M. grisea infection.

DOI： 10.1093/pcp/pcm107

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DOI： 10.1007/978-1-4020-6635-1_32

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Salicylic acid (SA) is known to be an essential component responsible for disease resistance in dicotyledonous plants. In rice, however, tissue contains extremely high endogenous levels of SA that do not increase after pathogen infection, suggesting that the SA has other major functions in healthy leaves. Although involvement of SA in oxidative-stress response is known in some dicotyledonous plants, antioxidative role of SA in rice is obscure. In this study, we examined the involvement of SA in the protection against oxidative stress in rice, using transgenic plants expressing the bacterial nahG gene that encodes salicylate hydroxylase, an SA-degrading enzyme. In SA-deficient NahG rice, the glutathione pool size was constitutively diminished as compared with control plants. NahG seedlings showed a delayed development phenotype, an increased susceptibility to oxidative stress and they developed light-induced lesions in their leaves without pathogen infection. Conversely, treatment with an activator of the SA-mediated defense-signaling pathway, probenazole, increased the glutathione pool size and suppressed lesion formation. These results suggest that in rice, SA has an important role in the response to highlight-induced oxidative stress, through its regulatory effects on glutathione homeostasis.

DOI： 10.1111/j.1399-3054.2006.00786.x

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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 H2O2 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 H2O2 when treated with calyculin A (CA), an inhibitor of protein phosphatase. Furthermore, spl2 mutant plants exhibited accelerated accumulation of H2O2 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.

DOI： 10.1093/pcp/pcj074

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The Arabidopsis FAD7 gene encodes a plastid omega-3 fatty acid desaturase that catalyzes the desaturation of dienoic fatty acids to trienoic fatty acids in chloroplast membrane lipids. The expression of FAD7 was rapidly and locally induced by ozone exposure, which causes oxidative responses equivalent to pathogen-induced hypersensitive responses and subsequently activates various defense-related genes. This induction was reduced in salicylic acid (SA)-deficient NahG plants expressing SA hydroxylase, but was unaffected in etr1 and jar1 mutants, which are insensitive to ethylene and jasmonic acid (JA), respectively. The SA dependence of the FAD7 induction was confirmed by the exogenous application of SA. SA-induced expression of FAD7 in the npr1 mutant which is defective in an SA signaling pathway occurred to the same extent as in the wild type. Furthermore, in the sid2 mutant which lacks an enzyme required for SA biosynthesis, the expression of FAD7 was induced by ozone exposure. These results suggest that the ozone-induced expression of FAD7 gene requires SA, but not ethylene, JA, NPR1 and SID2.

DOI： 10.1093/pcp/pci253

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Trienoic fatty acids (TAs) are the major polyunsaturated fatty acid species in the membrane lipids in plant cells. TAs are crucial for the adaptation to abiotic stresses, especially low- or high-temperature stress. We show that TAs in chloroplast membrane lipids are involved in defense responses against avirulent bacterial pathogens. Avirulent pathogen invasion of plants induces a transient production of reactive oxygen intermediates (ROI), programmed cell death and subsequent disease resistance. The Arabidopsis fad7fad8 mutation, which prevents the synthesis of TAs in chloroplast lipids, caused the reduction in ROI accumulation in leaves inoculated with Pseudomonas syringae pv. tomato DC3000 (avrRpm1). Linolenic acid, the most abundant TA, activated the NADPH oxidase that is responsible for ROI generation. TAs were transferred from chloroplast lipids to extrachloroplast lipids coincident with ROI accumulation after inoculation with Pst DC3000 (avrRpm1). Furthermore, the fad7fad8 mutant exhibited reduced cell death and was compromised in its resistance to several avirulent P. syringae strains. These results suggest that TAs derived from chloroplast lipids play an important role in the regulation of plant defense responses.

DOI： 10.1111/j.1365-313X.2004.02260.x

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Other Link： https://opac1.lib.ehime-u.ac.jp/iyokan/TD00003399

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Pathogens deliver a number of effector proteins into plant cells to suppress PAMP (pathogen-associated molecular pattern)-triggered immunity (PTI). Resistant plants are able to recognize the effectors by the resistance (R) proteins and induce strong immune responses. AVR3a, an effector protein secreted from potato blight pathogen <i>Phytophthora infestans</i>, is translocated into plant cells and suppresses PTI induced by the recognition of INF1. However, its underlying mechanism is still unclear. Because the molecular function of AVR3a is not predictable from the amino acid sequences, we performed protein structural analysis. The NMR analysis revealed that AVR3a protein has a positively charged surface area, which is important for binding a phosphatidylinositol phosphate. AVR3a with a point mutation in the area was not able to suppress INF1-induced PTI, although it was still recognized by R3a, a potato R protein. These data suggest that the binding of a phosphatidylinositol phosphate plays an important role for the virulence functions of AVR3a. We will discuss the molecular relationship between the lipid-binding area and the virulence function.

DOI： 10.14841/jspp.2011.0.0294.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Pathogens deliver a number of effector proteins into plant cells to suppress immune responses. Resistant plants are able to recognize the effectors and induce strong immune responses including the oxidative burst and programmed cell death. Although many genes involved in plant-pathogen interactions have been identified, little is known about the mechanisms of effector recognition and subsequent signal transduction. Because the function of effectors is often not predictable from the amino acid sequences, it is difficult to study the mechanism of interaction between effectors and plant proteins and how effectors suppress immune responses. To elucidate the molecular basis of the interactions, we screened 261 proteins for their solubility and performed protein structural analyses. We will report the structural features of <i>Phytophthora</i>-derived effector proteins and their putative function.

DOI： 10.14841/jspp.2010.0.0081.0

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Pathogens dispatch numerous proteins called effector to plant cells to disturb plant defense responses. On the other hand, plants recognize the effectors and induce strong defense responses including the oxidative burst and programmed cell death. Although the genes involved in the plant-pathogen interactions have been identified, little is known about the mechanisms of the effector recognition and of the subsequent signal transduction. It is especially difficult to study the function of effectors because the amino acid sequences are not highly conserved. To elucidate the molecular basis for the mechanism of interaction between effectors and plant defense-related proteins, we study the three-dimensional structure of proteins by X-ray crystallography and NMR spectroscopy. We screened for proteins which were highly expressed and soluble by using the high-throughput cell-free protein expression system.

DOI： 10.14841/jspp.2009.0.0969.0

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Linolenic acid (18:3) is a source for various oxidized metabolites, such as jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (OPDA), which are involved in defense responses to pathogen infection as signaling molecules. OPDA is synthesized by allene oxide cyclase (AOC), and then is converted to JA by OPDA reductase (OPR). To study roles of JA and OPDA in rice disease resistance to a blast fungus <I>Magnaporthe</I> <I>grisea</I>, we generated an OPDA/JA-deficient line (AOCRi) and a JA-deficient line (OPRWRi) with suppression of the <I>AOC</I> or <I>OPR</I> genes. The levels of the disease resistance in the AOCRi and OPRWRi lines were equal to that in wild-type plants. Furthermore, in the two lines, two pathogenesis-related genes (<I>PBZ1</I> and <I>PR1b</I>) were normally induced after inoculation with <I>M</I>. <I>grisea</I>. These results suggest that the disease resistance to <I>M</I>. <I>grisea</I> in rice is induced without any relation to JA and OPDA synthesis.

DOI： 10.14841/jspp.2008.0.0941.0

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Salicylic acid (SA) plays important roles in plant immune responses. In <i>Arabidopsis</i>, two pathways are proposed for the biosynthesis of SA: the isochorismate (IC) pathway and the benzoate (BA) pathway. Concerning the IC pathway, the <i>sid2</i> mutant which is defective in IC synthase gene (<i>ICS1</i>) has been isolated. However, mutants related to the BA pathway have not been identified. To study the BA pathway in plant immune responses, we isolated the <i>bah1-D</i> mutant (<i><u>b</u>enzoic <u>a</u>cid <u>h</u>ypersensitive<u>1</u>-<u>D</u>ominant</i>) which accumulates higher amounts of SA in response to the application of BA. The <i>bah1-D</i> mutant is resistant to <i>Pseudomonas syringae</i> DC3000 and accumulates higher amount of SA. Furthermore, HR-like cell death is observed in the <i>bah1-D</i> mutant after inoculation with <i>Pst</i> DC3000. We will also report other characteristics of the <i>bah1-D</i> mutant.

DOI： 10.14841/jspp.2008.0.0966.0

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Linoleic acid (18:2) and linolenic acid (18:3) are the sources for various oxidized metabolites, oxylipins, which are crucial for the induction of pathogen defense responses in plants. We show that 18:2-derived oxylipins play an important role in resistance to a phytopathogenic fungus, <I>Magnaporthe</I> <I>grisea</I>, in rice. We generated 18:2-accumulating transgenic rice plants (F78Ri) using RNA-interference of two &omega;-3 fatty acid desaturases, OsFAD7 and OsFAD8, which desaturate 18:2 to 18:3. The F78Ri plants exhibited enhanced resistance to <I>M</I>. <I>grisea</I> in a non-race-specific manner. 18:2-derived hydroperoxides and hydroxides (HPODEs and HODEs, respectively) inhibited <I>in</I> <I>vitro</I> growth of <I>M</I>. <I>grisea</I> more strongly than their respective 18:3-derived counterparts. In F78Ri plants, the endogenous levels of HPODEs and HODEs increased significantly, compared with wild-type plants. These results indicate that, in F78Ri plants, the increased accumulation of HPODEs and HODEs results in elevated antifungal activity against <I>M</I>. <I>grisea</I>, and therefore enhanced disease resistance.

DOI： 10.14841/jspp.2007.0.593.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Salicylic acid (SA) plays important roles both in the hypersensitive response and in the systemic acquired response (SAR). Two pathways are proposed for the biosynthesis of SA in Arabidopsis: the benzoic acid (BA) pathway and the isochorismate pathway. In the latter, a mutant defective in isochorismate synthase has been isolated and this mutant is known to be involved in SAR. However, mutants related to the former pathway have not been identified. To study the BA pathway in plant immune responses, we isolated the BA-sensitive mutant <i>bah1</i> (<i><u>b</u>enzoic <u>a</u>cid <u>h</u>ypersensitive <u>1</u></i>). The <i>bah1</i> mutant accumulated high levels of SA and expressed the <i>PR1</i> gene after the application of BA, suggesting that the BA pathway for SA biosynthesis is activated in the <i>bah1</i> mutant. Moreover, the <i>bah1</i> mutant was resistant to <i>Pseudomonas syringae</i>.

DOI： 10.14841/jspp.2007.0.935.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Jasmonic acid (JA) is known to be an important signaling molecule in defense responses against pathogen attack. JA is synthesized in the octadecanoid pathway, which employs several enzymes including allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid (OPDA) reductase (OPR). Recent studies in <I>Arabidopsis</I> suggest that OPDA, an intermediate in the biosynthesis of JA, is also involved in defense responses. However, little is known about physiological roles of OPDA in defense responses in rice. We generated transgenic rice plants in which the expression of the genes encoding AOC and OPR (<I>OsAOC</I>, <I>OsOPR1</I> and <I>OsOPR3</I>) is suppressed by RNAi method. JA content was undetectable in leaves of the <I>OsAOC</I>-suppressed transformant, and was reduced by approximately 3% in leaves of the <I>OsOPR1</I>-<I>OsOPR3</I>-co-suppressed transformant. We will also report the OPDA content in the <I>OsAOC</I>-suppressed rice.

DOI： 10.14841/jspp.2006.0.766.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Jasmonic acid (JA) plays an important role as a signaling molecule in plant defense responses to wound stress and pathogen infection. Although there are various JA-inducible pathogenesis-related (PR) genes in rice, JA does not enhance the disease resistance against rice blast fungus <I>Magnaporthe</I> <I>grisea</I>. To resolve this discrepancy, we generated and analyzed JA-deficient rice plants (F7Ri) with co-suppression of both two intrinsic plastidial &omega;-3 fatty acid desaturases, OsFAD7-1 and OsFAD7-2, that supply the JA precursor linolenic acid. Unexpectedly, in F7Ri plants, the disease resistance against <I>M</I>. <I>grisea</I> was enhanced in a non-race-specific manner. Furthermore, the enhanced resistance in F7Ri was not restored by the methyl-jasmonate treatment. These results suggest that JA is not an effective regulator of the fungal resistance. Moreover, this study provides a novel insight that the oxidized derivatives of polyunsatulated fatty acids may be involved in rice-specific fungal resistance.

DOI： 10.14841/jspp.2006.0.763.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Rapid accumulation of reactive oxygen intermediates (e.g., H<SUB>2</SUB>O<SUB>2</SUB>) is a striking early event in the hypersensitive response including programmed cell death. Signaling pathways leading to these responses, however, remain to be dissolved. To elucidate the signaling pathways, eleven rice lesion mimic mutants <I>spl</I> (<I>spl1</I>-<I>11</I>) with spontaneous cell death on their leaves, were investigated using suspension-cultured cell system. Three of the mutants (<I>spl2</I>, <I>spl7</I>, <I>spl11</I>) were found to accumulate higher amount of H<SUB>2</SUB>O<SUB>2</SUB> than the wild type, when treated with elicitor, indicating that these mutations are involved in accumulation of H<SUB>2</SUB>O<SUB>2</SUB>. Among three mutants, only <I>spl7</I> mutant accumulated higher amounts of H<SUB>2</SUB>O<SUB>2</SUB> than the wild type, when treated with calyculin A (CA), an inhibitor of protein phosphatase. Furthermore, <I>spl2</I> mutant plant exhibited accelerated accumulation of H<SUB>2</SUB>O<SUB>2</SUB> in response to wounding. We discuss the possible roles of these <I>spl</I> mutations in H<SUB>2</SUB>O<SUB>2</SUB> generation.

DOI： 10.14841/jspp.2006.0.769.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Salicylic acid (SA) is an important signal molecule in plant disease resistance. The benzoic acid (BA) pathway for the biosynthesis of SA has been proposed. However, genes involved in the BA pathway are not known. We reported that <I>bah1</I> (<I>benzoic acid hypersensitive 1</I>) mutant involved in the BA pathway was isolated (Yaeno et al., PCP 46: Suppl. S125, 2005). <I>bah1</I> mutant showed an inhibited growth and enhanced expression of SA-inducible <I>PR1</I> gene in the presence of BA. Furthermore, the accumulation of SA occurred in <I>bah1</I> mutant by application of BA. cDNA microarray analysis showed enhanced expression of defense-related genes and several P450 genes, candidates for the gene encoding an enzyme converting BA to SA.

DOI： 10.14841/jspp.2006.0.224.0

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Authorship：Lead author   Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

DOI： 10.18978/jscrp.41.2_150

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Jasmonic acid (JA) is known to be an important signaling molecule in defense response against wounding. JA is synthesized in the octadecanoid pathway, which employs several enzymes including allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-oxo-phytodienoic acid reductase (OPR). We generated transgenic rice plants in which the expression of the genes encoding AOS, AOC, and OPR (<I>OsAOS</I>, <I>OsAOC</I>, <I>OsOPR1</I> and <I>OsOPR3</I> ) is suppressed by RNAi method. JA content in leaves of the <I>OsAOS</I>-suppressed transformant was reduced approximately 62% of the wild-type level. JA content was undetectable in leaves of the <I>OsAOC</I>-suppressed transformant. JA content in leaves of the <I>OsOPR1</I>-and <I>OsOPR3</I>-co-suppressed transformant was reduced approximately 3% of the wild-type level. These materials will provide a useful tool to elucidate the functions of the intermediates (e.g., 12-oxo-phytodienoic acid) of the octadecanoid pathway in rice defense responses.

DOI： 10.14841/jspp.2005.0.388.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Salicylic acid (SA) is an important signal molecule in plant disease resistance. Two pathways for the biosynthesis of SA are proposed: the isochorismate pathway and the benzoic acid (BA) pathway. In the former, <I>ICS1</I> gene encoding isochorismate synthase has been identified. However, genes involved in the latter pathway are not known. To study the BA pathway for SA biosynthesis, mutant screening was performed. The growth of wild-type is inhibited in the presence of SA. On the other hand, BA has no such inhibitory effect. Based on this property, we isolated <I><U>b</U>enzoic <U>a</U>cid <U>h</U>ypersensitive</I> (<I>bah</I>) <I>1</I> mutant that exhibit an inhibited growth in the presence of BA. <I>bah1</I> grew normally in the absence of BA. The transcript of SA-inducible <I>PR1</I> gene accumulated in <I>bah1</I> in the presence of BA. These results raise a possibility that the accumulation of SA occurs in <I>bah1</I> by application of BA.

DOI： 10.14841/jspp.2005.0.406.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Plants respond to insect attack and pathogen by activating the expression of genes involved in disease resistance. In rice, jasmonic acid (JA) is assumed to play an important role as a signaling molecule in defense responses. The synthesis of linolenic acid (LA), the precursor of JA, is catalyzed by two plastidial &omega;-3 fatty acid desaturases which are encoded by two genes, <I>FAD7-1</I> and <I>FAD7-2</I>. To determine the role of JA, we generated transgenic rice in which the expression of both <I>FAD7-1</I> and <I>FAD7-2</I> is suppressed by RNAi method. LA content in leaves of the <I>FAD</I>-suppressed transformant (F7Ri) was reduced to approximately 13% of the wild-type level. Wound-induced JA accumulation of the F7Ri was reduced to approximately 10% of the wild-type level. These results indicate that F7Ri line is a useful tool for studying the role of JA in rice.

DOI： 10.14841/jspp.2005.0.390.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Rapid accumulation of reactive oxygen intermediates (e.g., H<SUB>2</SUB>O<SUB>2</SUB>) is a striking early event in the hypersensitive response including programmed cell death. Signalling pathways leading to these responses, however, remaine to be dissolved. To elucudate the signaling pathways, eleven rice lesion mimic mutants <I>spl</I> (<I>spl1~11</I>) with spontaneous cell death on their leaves, were investigated using suspension-cultured cell. Three of the mutants (<I>spl2</I>, <I>spl7</I>, <I>spl11</I>) were found to accumulate higher amount of H<SUB>2</SUB>O<SUB>2</SUB> than the wild type, when treated with elicitor, indicating that these mutations are involved in accumulation of H<SUB>2</SUB>O<SUB>2</SUB>. Calyculin A (CA), an inhibitor of protein phosphatase, enhanced production of H<SUB>2</SUB>O<SUB>2</SUB> in <I>spl7</I> but not in the wild type, <I>spl2</I> and <I>spl11</I>. Moreover, CA-enhanced H<SUB>2</SUB>O<SUB>2</SUB> production in <I>spl7</I> was reduced by addition of a Ca<SUP>2+</SUP> chelator EGTA. These results imply that <I>spl7</I> participates in regulation of H<SUB>2</SUB>O<SUB>2</SUB> production at a protein dephosphorylate- and Ca<SUP>2+</SUP> - dependent step.

DOI： 10.14841/jspp.2005.0.407.0

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Language：Japanese  

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

In rice, the physiological role of jasmonic acid (JA), which is assumed to play an important role as a signal molecule in defense responses to insect herbivory in dicotyledonous plants, has remained unclear. The synthesis of linolenic acid (LA), the precursor of JA, in rice is catalyzed by two plastidial &omega;-3 fatty acid desaturases encoded by two genes, <I>FAD7-1</I> and <I>FAD7-2</I>. To determine the role of JA, we generated transgenic rice in which the expression of both <I>FAD7-1</I> and <I>FAD7-2</I> is suppressed by RNA interference method. LA content in leaves of the <I>FAD</I>-suppressed transformant (F7Ri) was reduced to approximately 13% of the wild-type level. The wound induction of a wound- and JA-responsive gene, <I>JAmyb</I>, was strongly suppressed in F7Ri. From these results, it was inferred that the reduction of LA content in F7Ri affected the wound-induced JA biosynthesis and hence the wound induction of <I>JAmyb</I>.

DOI： 10.14841/jspp.2004.0.563.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Salicylic acid (SA) is an essential component responsible for disease resistance in some dicotyledonous plants. Increase in SA level is required for induction of systemic acquired resistance in these plants. Rice has the extremely higher levels of SA relative to the dicotyledonous plants, and SA levels does not increase after pathogen infection, suggesting that the role of SA in rice differ from that in dicotyledonous plants. In this study, we characterized transgenic rice plants that express the bacterial <I>nahG</I> gene encoding salicylate hidroxylase, the enzyme that degrades SA. <I>nahG</I>-expressed rice seedlings showed delayed development phenotype, and developed lesions in their leaves without pathogen infection under high-light conditions. In such <I>nahG</I> plants, glutathione pool size decreased and the redox state of glutathione were reduced. These results suggest that the SA in rice is required for maintaining defense mechanism against oxidative damages caused by high-light stress.

DOI： 10.14841/jspp.2004.0.564.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Cell death is the most striking event during the hypersensitive responses (HR), that are characterized by recognition of elicitor and transient burst of reactive oxygen species (ROS, e.g., H2O2). To investigate the mechanisms leading to cell death, rice lesion mimic mutants <I>spl</I> (<U>sp</U>otted <U>l</U>eaf), that cause aberrant regulation of cell death, were analyzed. H2O2 accumulation and degree of cell death were compared between the <I>spl</I> lines and wild type (TC65) using suspension-cultured cells treated with elicitor of rice blast fungus, <I>Magnaporthe grisea</I>. Cell death was enhanced in <I>spl3</I> but not in <I>spl7</I> and <I>spl11</I>. However, increased accumulation of H2O2 occurred in <I>spl7</I> and <I>spl11</I> but not in <I>spl3</I>. These results suggest that the mutated gene of <I>spl3</I> is responsible for execution of cell death, and that the genes of <I>spl7</I> and <I>spl11</I> are involved in accumulation of H2O2.

DOI： 10.14841/jspp.2004.0.565.0

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Language：Japanese   Publisher：The Japanese Society of Plant Physiologists  

Pathogen invasion of plants induces a transient production of reactive oxygen species (e.g., O₂^-, H₂O₂). O₂^- generation is catalyzed by NADPH oxidase. Molecular mechanism underlying activation of NADPH oxidase remains unclear. As reported previously (Yaeno <i>et al</i>. (2003) <i>Plant Cell Physiol</i>. 44: suppl, s149), &alpha;-linolenic acid (LA), the most abundant trienoic fatty acids (TAs) in the chloroplast membrane lipids, is involved in the activation of NADPH oxidase. In <i>Arabidopsis</i> wild-type leaves inoculated with <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 (<i>avrRpm1</i>), the levels of free LA increased coincident with H₂O₂ accumulation. Further, the LA levels of extrachloroplast membrane phospholipids increased substantially. This increase was accompanied by the transfer of hexadecatrienoic acid, a TA species usually acylated in chloroplast galactolipids, to extrachloroplast phospholipids. These results suggest that LA liberated from chloroplast membrane lipids is utilized for the activation of NADPH oxidase which is localized in the plasma membrane.

DOI： 10.14841/jspp.2004.0.631.0

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Publisher：The Japanese Society of Plant Physiologists  

&omega;-3 fatty acid desaturase, which catalyzes the synthesis of trienoic fatty acids, is assumed to play an important role in plant adaptation to environmental stresses. In this study, two novel rice &omega;-3 desaturase genes (<I>OsFAD7-1</I> and <I>OsFAD7-2</I>), with an extensive sequence similarity to the chloroplast-localized <I>Arabidopsis</I> &omega;-3 desaturase (<I>AtFAD7</I>), were characterized. The deduced amino acid sequences of OsFAD7-1 and OsFAD7-2 were 73% and 78% identical to that of AtFAD7, respectively. The N-termini of these genes contained a putative chloroplast transit peptide. RT-PCR analysis showed that their mRNAs highly accumulated in the leaf tissues. These observations suggest that these two genes encode the chloroplast-localized &omega;-3 desaturase. To clarify the physiological role of OsFAD7-1 and OsFAD7-2, transgenic rice plants, in which the corresponding intrinsic &omega;-3 desaturases are silenced by RNAi technique, were created. We have obtained several transgenic lines with reduced levels of TAs.

DOI： 10.14841/jspp.2003.0.495.0

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Publisher：The Japanese Society of Plant Physiologists  

Pathogen invasion of plants induces a transient production of reactive oxygen species (e.g., superoxide) in the oxidative burst. Superoxide generation is probably catalyzed by NADPH oxidase. Animal NADPH oxidase is well characterized, but the molecular mechanism underlying activation of NADPH oxidase in plants remains unclear. Trienoic fatty acids (TAs) are the major polyunsaturated fatty acid in the membrane lipids in plant cells. TAs are crucial for adaptation to abiotic stresses, especially to low- or high-temperature stress. We report that &alpha;-linolenic acid (LA), the most abundant TA species in the chloroplast membrane lipids, is critical for generation of superoxide. Impairment of LA synthesis in the chloroplast membranes impaired superoxide accumulation in the oxidative burst, leading to reduced resistance to avirulent bacterial pathogens. LA was the most effective fatty acid for stimulating NADPH oxidase activity <i>in vitro</i>.

DOI： 10.14841/jspp.2003.0.498.0

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publisher：Japanese Society of Plant Physiologists  

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