Under sugar-sufficient conditions, phosphorylation levels of calcium-dependent protein kinase 5 (CPK5) are elevated by G6P-mediated suppression of protein phosphatases, enhancing defense responses before pathogen invasion. Subsequently, recognition of bacterial flagellin activates sugar transporters, leading to increased cellular G6P, which elicits CPK5-independent signaling promoting synthesis of the phytohormone salicylic acid (SA) for antibacterial defense. In contrast, while perception of fungal chitin does not promote sugar influx or SA accumulation, chitin-induced synthesis of the antifungal compound camalexin requires basal sugar influx activity. By monitoring sugar levels, plants determine defense levels and execute appropriate outputs against bacterial and fungal pathogens. Together, our findings provide a comprehensive view of the roles of sugar in defense.
Yoshihiro Inoue, Thi Vy Trinh Phuong, Suthitar Singkaravanit-Ogawa, Ru Zhang, Kohji Yamada, Taiki Ogawa, Junya Ishizuka, Yoshihiro Narusaka and Yoshitaka Takano : Selective deployment of virulence effectors correlates with host specificity in a fungal plant pathogen., The New Phytologist, 2023.
(Summary)
The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C. orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C. orbiculare 'core' effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C. orbiculare to cucurbit hosts, but not to the Solanaceae host N. benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C. orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts.
Kohji Yamada, Toya Yamamoto, Kanon Uwasa, Keishi Osakabe and Yoshitaka Takano : The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems., Fungal genetics and biology : FG & B, 2023.
(Summary)
Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.
Hiroki Irieda, Yoshihiro Inoue, Masashi Mori, Kohji Yamada, Yuu Oshikawa, Hiromasa Saitoh, Aiko Uemura, Ryohei Terauchi, Saeko Kitakura, Ayumi Kosaka, Suthitar Singkaravanit-Ogawa and Yoshitaka Takano : Conserved fungal effector suppresses PAMP-triggered immunity by targeting plant immune kinases., Proceedings of the National Academy of Sciences of the United States of America, Vol.116, No.2, 496-505, 2018.
(Summary)
displayed significantly reduced virulence on rice and barley, its hosts. Our study therefore reveals that a broad range of filamentous fungi maintain and utilize the core effector NIS1 to establish infection in their host plants and perhaps also beneficial interactions, by targeting conserved and central PRR-associated kinases that are also known to be targeted by bacterial effectors.
Kohji Yamada, Yuriko Osakabe and Kazuko Yamaguchi-Shinozaki : A C-terminal motif contributes to the plasma membrane localization of Arabidopsis STP transporters., PLoS ONE, Vol.12, No.10, e0186326, 2017.
(Summary)
Membrane trafficking is highly organized to maintain cellular homeostasis in any organisms. Membrane-embedded transporters are targeted to various organelles to execute appropriate partition and allocation of their substrates, such as ions or sugars. To ensure the fidelity of targeting and sorting, membrane proteins including transporters have sorting signals that specify the subcellular destination and the trafficking pathway by which the destination is to be reached. Here, we have identified a novel sorting signal (called the tri-aromatic motif) which contains three aromatic residues, two tryptophans and one histidine, for the plasma membrane localization of sugar transporters in the STP family in Arabidopsis. We firstly found that a C-terminal deletion disrupted the sugar uptake activity of STP1 in yeast cells. Additional deletion and mutation analyses demonstrated that the three aromatic residues in the C-terminus, conserved among all Arabidopsis STP transporters, were critical for sugar uptake by not only STP1 but also another STP transporter STP13. We observed that, when the tri-aromatic motif was mutated, STP1 was largely localized at the endomembrane compartments in yeast cells, indicating that this improper subcellular localization led to the loss of sugar absorption. Importantly, our further analyses uncovered that mutations of the tri-aromatic motif resulted in the endoplasmic reticulum (ER) retention of STP1 and STP13 in plant cells, suggesting that this motif is involved at the step of ER exit of STP transporters to facilitate their plasma membrane localization. Together, we here identified a novel ER export signal, and showed that appropriate sorting via the tri-aromatic motif is important for sugar absorption by STP transporters.
Kohji Yamada, Yusuke Saijo, Hirofumi Nakagami and Yoshitaka Takano : Regulation of sugar transporter activity for antibacterial defense in Arabidopsis., Science, Vol.354, No.6318, 1427-1430, 2016.
(Summary)
Microbial pathogens strategically acquire metabolites from their hosts during infection. Here we show that the host can intervene to prevent such metabolite loss to pathogens. Phosphorylation-dependent regulation of sugar transport protein 13 (STP13) is required for antibacterial defense in the plant Arabidopsis thaliana STP13 physically associates with the flagellin receptor flagellin-sensitive 2 (FLS2) and its co-receptor BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1). BAK1 phosphorylates STP13 at threonine 485, which enhances its monosaccharide uptake activity to compete with bacteria for extracellular sugars. Limiting the availability of extracellular sugar deprives bacteria of an energy source and restricts virulence factor delivery. Our results reveal that control of sugar uptake, managed by regulation of a host sugar transporter, is a defense strategy deployed against microbial infection. Competition for sugar thus shapes host-pathogen interactions.
Kohji Yamada, Misuzu Yamashita-Yamada, Taishi Hirase, Tadashi Fujiwara, Kenichi Tsuda, Kei Hiruma and Yusuke Saijo : Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1., The EMBO Journal, Vol.35, No.1, 46-61, 2015.
(Summary)
Pathogens infect a host by suppressing defense responses induced upon recognition of microbe-associated molecular patterns (MAMPs). Despite this suppression, MAMP receptors mediate basal resistance to limit host susceptibility, via a process that is poorly understood. The Arabidopsis leucine-rich repeat (LRR) receptor kinase BAK1 associates and functions with different cell surface LRR receptors for a wide range of ligands, including MAMPs. We report that BAK1 depletion is linked to defense activation through the endogenous PROPEP peptides (Pep epitopes) and their LRR receptor kinases PEPR1/PEPR2, despite critical defects in MAMP signaling. In bak1-knockout plants, PEPR elicitation results in extensive cell death and the prioritization of salicylate-based defenses over jasmonate-based defenses, in addition to elevated proligand and receptor accumulation. BAK1 disruption stimulates the release of PROPEP3, produced in response to Pep application and during pathogen challenge, and renders PEPRs necessary for basal resistance. These findings are biologically relevant, since specific BAK1 depletion coincides with PEPR-dependent resistance to the fungal pathogen Colletotrichum higginsianum. Thus, the PEPR pathway ensures basal resistance when MAMP-triggered defenses are compromised by BAK1 depletion.
Annegret Ross, Kohji Yamada, Kei Hiruma, Misuzu Yamashita-Yamada, Xunli Lu, Yoshitaka Takano, Kenichi Tsuda and Yusuke Saijo : The Arabidopsis PEPR pathway couples local and systemic plant immunity., The EMBO Journal, Vol.33, No.1, 62-75, 2013.
(Summary)
Recognition of microbial challenges leads to enhanced immunity at both the local and systemic levels. In Arabidopsis, EFR and PEPR1/PEPR2 act as the receptor for the bacterial elongation factor EF-Tu (elf18 epitope) and for the endogenous PROPEP-derived Pep epitopes, respectively. The PEPR pathway has been described to mediate defence signalling following microbial recognition. Here we show that PROPEP2/PROPEP3 induction upon pathogen challenges is robust against jasmonate, salicylate, or ethylene dysfunction. Comparative transcriptome profiling between Pep2- and elf18-treated plants points to co-activation of otherwise antagonistic jasmonate- and salicylate-mediated immune branches as a key output of PEPR signalling. Accordingly, as well as basal defences against hemibiotrophic pathogens, systemic immunity is reduced in pepr1 pepr2 plants. Remarkably, PROPEP2/PROPEP3 induction is essentially restricted to the pathogen challenge sites during pathogen-induced systemic immunity. Localized Pep application activates genetically separable jasmonate and salicylate branches in systemic leaves without significant PROPEP2/PROPEP3 induction. Our results suggest that local PEPR activation provides a critical step in connecting local to systemic immunity by reinforcing separate defence signalling pathways.
Nico Tintor, Annegret Ross, Kazue Kanehara, Kohji Yamada, Li Fan, Birgit Kemmerling, Thorsten Nürnberger, Kenichi Tsuda and Yusuke Saijo : Layered pattern receptor signaling via ethylene and endogenous elicitor peptides during Arabidopsis immunity to bacterial infection., Proceedings of the National Academy of Sciences of the United States of America, Vol.110, No.15, 6211-6216, 2013.
(Summary)
Recognition of molecular patterns characteristic of microbes or altered-self leads to immune activation in multicellular eukaryotes. In Arabidopsis thaliana, the leucine-rich-repeat receptor kinases FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR (EFR) recognize bacterial flagellin and elongation factor EF-Tu (and their elicitor-active epitopes flg22 and elf18), respectively. Likewise, PEP1 RECEPTOR1 (PEPR1) and PEPR2 recognize the elicitor-active Pep epitopes conserved in Arabidopsis ELICITOR PEPTIDE PRECURSORs (PROPEPs). Here we reveal that loss of ETHYLENE-INSENSITIVE2 (EIN2), a master signaling regulator of the phytohormone ethylene (ET), lowers sensitivity to both elf18 and flg22 in different defense-related outputs. Remarkably, in contrast to a large decrease in FLS2 expression, EFR expression and receptor accumulation remain unaffected in ein2 plants. Genome-wide transcriptome profiling has uncovered an inventory of EIN2-dependent and EFR-regulated genes. This dataset highlights important aspects of how ET modulates EFR-triggered immunity: the potentiation of salicylate-based immunity and the repression of a jasmonate-related branch. EFR requires ET signaling components for PROPEP2 activation but not for PROPEP3 activation, pointing to both ET-dependent and -independent engagement of the PEPR pathway during EFR-triggered immunity. Moreover, PEPR activation compensates the ein2 defects for a subset of EFR-regulated genes. Accordingly, ein2 pepr1 pepr2 plants exhibit additive defects in EFR-triggered antibacterial immunity, compared with ein2 or pepr1 pepr2 plants. Our findings suggest that the PEPR pathway not only mediates ET signaling but also compensates for its absence in enhancing plant immunity.
Yuriko Osakabe, Naoko Arinaga, Taishi Umezawa, Shogo Katsura, Keita Nagamachi, Hidenori Tanaka, Haruka Ohiraki, Kohji Yamada, So-Uk Seo, Mitsuru Abo, Etsuro Yoshimura, Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki : Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis., The Plant Cell, Vol.25, No.2, 609-624, 2013.
(Summary)
Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K(+) uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K(+) channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using (86)radioactive rubidium ion ((86)Rb(+)) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.
Miki Fujita, Yasunari Fujita, Satoshi Iuchi, Kohji Yamada, Yuriko Kobayashi, Kaoru Urano, Masatomo Kobayashi, Kazuko Yamaguchi-Shinozaki and Kazuo Shinozaki : Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis., Proceedings of the National Academy of Sciences of the United States of America, Vol.109, No.16, 6343-6347, 2012.
(Summary)
Polyamines (PAs) are ubiquitous, polycationic compounds that are essential for the growth and survival of all organisms. Although the PA-uptake system plays a key role in mammalian cancer and in plant survival, the underlying molecular mechanisms are not well understood. Here, we identified an Arabidopsis L-type amino acid transporter (LAT) family transporter, named RMV1 (resistant to methyl viologen 1), responsible for uptake of PA and its analog paraquat (PQ). The natural variation in PQ tolerance was determined in 22 Arabidopsis thaliana accessions based on the polymorphic variation of RMV1. An RMV1-GFP fusion protein localized to the plasma membrane in transformed cells. The Arabidopsis rmv1 mutant was highly resistant to PQ because of the reduction of PQ uptake activity. Uptake studies indicated that RMV1 mediates proton gradient-driven PQ transport. RMV1 overexpressing plants were hypersensitive to PA and PQ and showed elevated PA/PQ uptake activity, supporting the notion that PQ enters plant cells via a carrier system that inherently functions in PA transport. Furthermore, we demonstrated that polymorphic variation in RMV1 controls PA/PQ uptake activity. Our identification of a molecular entity for PA/PQ uptake and sensitivity provides an important clue for our understanding of the mechanism and biological significance of PA uptake.
Mario Serrano, Kazue Kanehara, Martha Torres, Kohji Yamada, Nico Tintor, Erich Kombrink, Paul Schulze-Lefert and Yusuke Saijo : Repression of sucrose/ultraviolet B light-induced flavonoid accumulation in microbe-associated molecular pattern-triggered immunity in Arabidopsis., Plant Physiology, Vol.158, No.1, 408-422, 2011.
(Summary)
Recognition of microbe-associated molecular patterns (MAMPs) leads to the generation of MAMP-triggered immunity (MTI), which restricts the invasion and propagation of potentially infectious microbes. It has been described that the perception of different bacterial and fungal MAMPs causes the repression of flavonoid induction upon light stress or sucrose application. However, the functional significance of this MTI-associated signaling output remains unknown. In Arabidopsis (Arabidopsis thaliana), FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR act as the pattern recognition receptors for the bacterial MAMP epitopes flg22 (of flagellin) and elf18 (of elongation factor [EF]-Tu), respectively. Here, we reveal that reactive oxygen species spiking and callose deposition are dispensable for the repression of flavonoid accumulation by both pattern recognition receptors. Importantly, FLS2-triggered activation of PATHOGENESIS-RELATED (PR) genes and bacterial basal defenses are enhanced in transparent testa4 plants that are devoid of flavonoids, providing evidence for a functional contribution of flavonoid repression to MTI. Moreover, we identify nine small molecules, of which eight are structurally unrelated, that derepress flavonoid accumulation in the presence of flg22. These compounds allowed us to dissect the FLS2 pathway. Remarkably, one of the identified compounds uncouples flavonoid repression and PR gene activation from the activation of reactive oxygen species, mitogen-activated protein kinases, and callose deposition, corroborating a close link between the former two outputs. Together, our data imply a model in which MAMP-induced repression of flavonoid accumulation serves a role in removing the inherent inhibitory action of flavonoids on an MTI signaling branch.
Kohji Yamada, Motoki Kanai, Yuriko Osakabe, Haruka Ohiraki, Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki : Monosaccharide absorption activity of Arabidopsis roots depends on expression profiles of transporter genes under high salinity conditions., The Journal of Biological Chemistry, Vol.286, No.50, 43577-43586, 2011.
(Summary)
Plant roots are able to absorb sugars from the rhizosphere but also release sugars and other metabolites that are critical for growth and environmental signaling. Reabsorption of released sugar molecules could help reduce the loss of photosynthetically fixed carbon through the roots. Although biochemical analyses have revealed monosaccharide uptake mechanisms in roots, the transporters that are involved in this process have not yet been fully characterized. In the present study we demonstrate that Arabidopsis STP1 and STP13 play important roles in roots during the absorption of monosaccharides from the rhizosphere. Among 14 STP transporter genes, we found that STP1 had the highest transcript level and that STP1 was a major contributor for monosaccharide uptake under normal conditions. In contrast, STP13 was found to be induced by abiotic stress, with low expression under normal conditions. We analyzed the role of STP13 in roots under high salinity conditions where membranes of the epidermal cells were damaged, and we detected an increase in the amount of STP13-dependent glucose uptake. Furthermore, the amount of glucose efflux from stp13 mutants was higher than that from wild type plants under high salinity conditions. These results indicate that STP13 can reabsorb the monosaccharides that are released by damaged cells under high salinity conditions. Overall, our data indicate that sugar uptake capacity in Arabidopsis roots changes in response to environmental stresses and that this activity is dependent on the expression pattern of sugar transporters.
Yasunari Fujita, Kazuo Nakashima, Takuya Yoshida, Takeshi Katagiri, Satoshi Kidokoro, Norihito Kanamori, Taishi Umezawa, Miki Fujita, Kyonoshin Maruyama, Kanako Ishiyama, Masatomo Kobayashi, Shoko Nakasone, Kohji Yamada, Takuya Ito, Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki : Three SnRK2 protein kinases are the main positive regulators of abscisic acid signaling in response to water stress in Arabidopsis., Plant & Cell Physiology, Vol.50, No.12, 2123-2132, 2009.
(Summary)
Responses to water stress are thought to be mediated by transcriptional regulation of gene expression via reversible protein phosphorylation events. Previously, we reported that bZIP (basic-domain leucine zipper)-type AREB/ABF (ABA-responsive element-binding protein/factor) transcription factors are involved in ABA signaling under water stress conditions in Arabidopsis. The AREB1 protein is phosphorylated in vitro by ABA-activated SNF1-related protein kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). Consistent with this, we now show that SRK2D/E/I and AREB1 co-localize and interact in nuclei in planta. Our results show that unlike srk2d, srk2e and srk2i single and double mutants, srk2d srk2e srk2i (srk2d/e/i) triple mutants exhibit greatly reduced tolerance to drought stress and highly enhanced insensitivity to ABA. Under water stress conditions, ABA- and water stress-dependent gene expression, including that of transcription factors, is globally and drastically impaired, and jasmonic acid (JA)-responsive and flowering genes are up-regulated in srk2d/e/i triple mutants, but not in other single and double mutants. The down-regulated genes in srk2d/e/i and areb/abf triple mutants largely overlap in ABA-dependent expression, supporting the view that SRK2D/E/I regulate AREB/ABFs in ABA signaling in response to water stress. Almost all dehydration-responsive LEA (late embryogenesis abundant) protein genes and group-A PP2C (protein phosphatase 2C) genes are strongly down-regulated in the srk2d/e/i triple mutants. Further, our data show that these group-A PP2Cs, such as HAI1 and ABI1, interact with SRK2D. Together, our results indicate that SRK2D/E/I function as main positive regulators, and suggest that ABA signaling is controlled by the dual modulation of SRK2D/E/I and group-A PP2Cs.
Kohji Yamada, Yuichiro Nakatsu, Akio Onogi, Junji Ueda and Tomomasa Watanabe : Specific intracellular localization and antiviral property of genetic and splicing variants in bovine Mx1., Viral Immunology, Vol.22, No.6, 389-395, 2009.
(Summary)
In bovine Mx1, only an amino acid substitution between Ile and Met at position 120 was detected by the nucleotide sequence and mismatched PCR-RFLP technique. The Ile variant was assumed to distribute mainly in the bovine population since the gene frequency was 79.3%. Furthermore, we cloned water buffalo Mx1 cDNA, which showed 51 nucleotide and 20 amino acid substitutions in comparison with that of the cow. Another kind of Mx1 cDNA, bovine Mx1B cDNA, was found and it was deduced to cause 27 amino acid substitutions at the N-terminus compared to the original Mx1 by alternative splicing. However, no variation was detected in 27 amino acids specific for Mx1B among 29 cows and a water buffalo. We established four kinds of mRNA-expressing 3T3 cells and Vero cells. When infection experiments were performed using recombinant vesicular stomatitis virus (VSVDeltaG*-G), bovine Ile and Met types and water buffalo Mx1 mRNA-expressing cell lines showed equally positive antiviral activities (p < 0.05). On the other hand, bovine Mx1B mRNA-expressing cell lines did not have activity against VSVDeltaG*-G. Intracellular localization of bovine Mx1 and Mx1B proteins was examined by a transiently GFP-fused expression system in 3T3 cells. Bovine Mx1 was localized in the cytoplasm, while bovine Mx1B was mainly localized in the nucleus. An arginine-rich nuclear localization signal was found in 27 amino acids specific for Mx1B. N-terminus-deleted Mx1B was only localized in the cytoplasm, and the deleted Mx1B-expressing cell lines showed significantly positive antiviral activities (p < 0.05) against VSVDeltaG*-G.
Kohji Yamada, Yuriko Osakabe, Junya Mizoi, Kazuo Nakashima, Yasunari Fujita, Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki : Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides., The Journal of Biological Chemistry, Vol.285, No.2, 1138-1146, 2009.
(Summary)
Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using beta-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K(m) for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.
Kyonoshin Maruyama, Migiwa Takeda, Satoshi Kidokoro, Kohji Yamada, Yoh Sakuma, Kaoru Urano, Miki Fujita, Kyouko Yoshiwara, Satoko Matsukura, Yoshihiko Morishita, Ryosuke Sasaki, Hideyuki Suzuki, Kazuki Saito, Daisuke Shibata, Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki : Metabolic pathways involved in cold acclimation identified by integrated analysis of metabolites and transcripts regulated by DREB1A and DREB2A., Plant Physiology, Vol.150, No.4, 1972-1980, 2009.
(Summary)
DREB1A/CBF3 and DREB2A are transcription factors that specifically interact with a cis-acting dehydration-responsive element (DRE), which is involved in cold- and dehydration-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Overexpression of DREB1A improves stress tolerance to both freezing and dehydration in transgenic plants. In contrast, overexpression of an active form of DREB2A results in significant stress tolerance to dehydration but only slight tolerance to freezing in transgenic plants. The downstream gene products for DREB1A and DREB2A are reported to have similar putative functions, but downstream genes encoding enzymes for carbohydrate metabolism are very different between DREB1A and DREB2A. We demonstrate that under cold and dehydration conditions, the expression of many genes encoding starch-degrading enzymes, sucrose metabolism enzymes, and sugar alcohol synthases changes dynamically; consequently, many kinds of monosaccharides, disaccharides, trisaccharides, and sugar alcohols accumulate in Arabidopsis. We also show that DREB1A overexpression can cause almost the same changes in these metabolic processes and that these changes seem to improve freezing and dehydration stress tolerance in transgenic plants. In contrast, DREB2A overexpression did not increase the level of any of these metabolites in transgenic plants. Strong freezing stress tolerance of the transgenic plants overexpressing DREB1A may depend on accumulation of these metabolites.
Kohji Yamada, Yuichiro Nakatsu, Akio Onogi, Akiko Takasuga, Yoshikazu Sugimoto, Junji Ueda and Tomomasa Watanabe : Structural and functional analysis of the bovine Mx1 promoter., Journal of Interferon and Cytokine Research, Vol.29, No.4, 217-226, 2009.
(Summary)
The bovine Mx1 promoter region was found to contain 4 IFN-stimulated response elements (ISREs), 7 GC boxes, 2 IL-6 responsive elements, 2 NFB-binding sites and 2 AP-1-binding sites. Among Holstein, Charolai, and Brahman breeds, 5 nucleotide substitutions were detected in the promoter region. After the Mx1 promoter region from Holstein had been constructed with pGL-basic expression vector, the transfected cells showed promoter activity after IFN induction. Several artificial deletion mutants were prepared to determine the important regulatory elements responsible for the promoter activity, and it was found that ISRE has a key function in IFN response. The proximal ISRE1 showed potential induction by IFN. Furthermore, the proximal GC boxes were found to be essential for IFN response in the bovine Mx1 promoter with the deletion mutants. In this case, the 2 GC boxes exhibited a synergistic activation in the IFN response. Mithramycin A, an agent that inhibits gene expression selectively by coating GC boxes, was used, and Mx mRNA expression in MDBK cells was suppressed by this chemical. Therefore, GC boxes were also shown to be essential for IFN response in the bovine Mx1 gene.
(Keyword)
Animals / Cattle / GTP-Binding Proteins / Gene Expression Regulation / Interferon Type I / Myxovirus Resistance Proteins / Promoter Regions, Genetic / Protein Conformation
E H A Babiker, Y Nakatsu, Kohji Yamada, A Yoneda, A Takada, J Ueda, H Hata and T Watanabe : Bovine and water buffalo Mx2 genes: polymorphism and antiviral activity., Immunogenetics, Vol.59, No.1, 59-67, 2006.
(Summary)
Millennia-long selective pressure of single-strand RNA viruses on the bovine Mx locus has increased the advantages of using the bovine Mx protein to evaluate the ultimate significance of the antiviral role of Mx proteins. The conclusions of research based only on the bovine Mx1 protein showed the need for comprehensive studies that demonstrate the role of all isoforms, individually or together, especially in the presence of a second isoform, the bovine Mx2 gene. This study provides information about bovine and water buffalo Mx2 genes, as well as their allelic polymorphism and basic antiviral potential. Observation of an Mx2 cDNA sequence (2,381 bp) obtained from 15 animals from 11 breeds using primers based on a previous sequence (NCBI accession no. AF335147) revealed several nucleotide substitutions, with eight different alleles and two amino acid exchanges: Gly to Ser at position 302 and Ile to Val at position 354, though the latter was found only in the NCBI database. A water buffalo Mx2 cDNA sequence was identified for the first time, revealing 46 nucleotide substitutions with 12 amino acid variations, in addition to a 9-bp insertion in the 5' untranslated region UTR, compared with the bovine Mx2 cDNA. Transfected 3T3 cells expressing bovine Mx2 mRNAs coding Gly or Ser at position 302, water buffalo Mx2 mRNA, positive control bovine Mx1 mRNA-expressing cells, and negative control parental 3T3 were subjected to infection with recombinant vesicular stomatitis virus (VSVDeltaG*-G), as were empty pCI-neo vector-transfected cells. The positive control and all cells expressing Mx2 mRNAs displayed significantly higher levels of antiviral activity against VSVDeltaG*-G (P < 0.01) than did the negative controls.
Y Nakatsu, Kohji Yamada, J Ueda, A Onogi, P G Ables, M Nishibori, H Hata, A Takada, K Sawai, Y Tanabe, M Morita, M Daikohara and T Watanabe : Genetic polymorphisms and antiviral activity in the bovine MX1 gene., Animal Genetics, Vol.35, No.3, 182-187, 2004.
(Summary)
Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.
Kohji Yamada and Yuriko Osakabe : Sugar compartmentation as an environmental stress adaptation strategy in plants., Seminars in Cell & Developmental Biology, Vol.S1084-9521, No.16, 30399-8, Dec. 2017.
(Summary)
The sessile nature of plants has driven their evolution to cope flexibly with ever-changing surrounding environments. The development of stress tolerance traits is complex, and a broad range of cellular processes are involved. Recent studies have revealed that sugar transporters contribute to environmental stress tolerance in plants, suggesting that sugar flow is dynamically fluctuated towards optimization of cellular conditions in adverse environments. Here, we highlight sugar compartmentation mediated by sugar transporters as an adaptation strategy against biotic and abiotic stresses. Competition for sugars between host plants and pathogens shapes their evolutionary arms race. Pathogens, which rely on host-derived carbon, manipulate plant sugar transporters to access sugars easily, while plants sequester sugars from pathogens by enhancing sugar uptake activity. Furthermore, we discuss pathogen tactics to circumvent sugar competition with host plants. Sugar transporters also play a role in abiotic stress tolerance. Exposure to abiotic stresses such as cold or drought stress induces sugar accumulation in various plants. We also discuss how plants allocate sugars under such conditions. Collectively, these findings are relevant to basic plant biology as well as potential applications in agriculture, and provide opportunities to improve crop yield for a growing population.
Kohji Yamada : 安定的な食糧供給に向けて –病害抵抗性作物作製への指針-, 生物工学学会誌, Vol.93, 490, Aug. 2015.
Proceeding of International Conference:
1.
Yuriko Osakabe, Kira Nozomu, Ueta Risa, Sakamoto Hideki, Takahito Watanabe, Takayanagi Eiko, Hara Chihiro, Hashimoto Ryosuke, Kohji Yamada and Keishi Osakabe : Development of in planta-regeneration system for plant genome editing, Frontiers in Genome Engineering 2019, Kobe Convention Center, Nov. 25-27, 2019, Nov. 2019.
2.
Shimada Kanari, Iuchi Satoshi, Iuchi Atsuko, Kohji Yamada, Keishi Osakabe and Yuriko Osakabe : IDENTIFICATION OF AN ARABIDOPSIS MUTANT WITH ALTERED ROOT HAIR FORMATION, International Conference on Arabidopsis Research 2018 (ICAR2018), Turku Finland, Jul. 2018.
Ryosuke Hashimoto, Kohji Yamada, Keishi Osakabe and Yuriko Osakabe : CRISPR/Cas9によるトマトNADキナーゼ2遺伝子の変異体作製と機能解析, 第61回日本植物生理学会年会, 大阪大学吹田キャンパス, 2020年3月19日-21日, Mar. 2020.
5.
Kanari Shimada, 井内 聖, 井内 敦子, Kohji Yamada, Keishi Osakabe and Yuriko Osakabe : Identification of an Arabidopsis mutant with altered root hair formation, 第60回日本植物生理学会年会, 名古屋大学, 2019年3月13日-15日, Mar. 2019.
6.
Ryosuke Hashimoto, 宮城 敦子, 澤田 有司, 佐藤 心郎, Kohji Yamada, 平井 優美, 川合 真紀, Keishi Osakabe and Yuriko Osakabe : Functional analysis of chloroplast-localized NAD kinase in plant abiotic stress responses, 第60回日本植物生理学会年会, 名古屋大学, 2019年3月13日-15日, Mar. 2019.
7.
Kanari Shimada, 井内 聖, 井内 敦子, Kohji Yamada, Keishi Osakabe and Yuriko Osakabe : 根毛形成に異常を示すシロイヌナズナ変異体の解析, 第36回日本植物分子生物学会(金沢)大会, 金沢商工会議所会館, 2018年8月26∼28日, Aug. 2018.
8.
島田 佳南里, 井内 聖, 井内 敦子, Kohji Yamada, Keishi Osakabe and Yuriko Osakabe : Identification of an Arabidopsis mutant with altered root hair formation, 第59回日本植物生理学会年会, Mar. 2018.
9.
阿部 千尋, 上田 梨紗, 橋本 諒典, Kohji Yamada, Yuriko Osakabe and Keishi Osakabe : New plant breeding technique of commercial tomato cultivars by using CRISPR/Cas9, 第59回日本植物生理学会年会, Mar. 2018.
10.
Chihiro Abe, Risa Ueta, 橋本 諒典, Kohji Yamada, Yuriko Osakabe and Keishi Osakabe : 栽培品種トマトにおけるCRISPR/Cas9システムを用いた育種技術基盤の構築, 第35回日本植物細胞分子生物学会(さいたま)大会, Aug. 2017.