The rice OsERF101 transcription factor regulates the NLR Xa1-mediated immunity induced by perception of TAL effectors.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) initiate immune responses by recognizing pathogen effectors. The rice gene Xa1 encodes an NLR with an N-terminal BED domain, and recognizes transcription activator-like (TAL) effectors of Xanthomonas oryzae pv oryzae (Xoo). Our goal here was to elucidate the molecular mechanisms controlling the induction of immunity by Xa1. We used yeast two-hybrid assays to screen for host factors that interact with Xa1 and identified the AP2/ERF-type transcription factor OsERF101/OsRAP2.6. Molecular complementation assays were used to confirm the interactions among Xa1, OsERF101 and two TAL effectors. We created OsERF101-overexpressing and knockout mutant lines in rice and identified genes differentially regulated in these lines, many of which are predicted to be involved in the regulation of response to stimulus. Xa1 interacts in the nucleus with the TAL effectors and OsERF101 via the BED domain. Unexpectedly, both the overexpression and the knockout lines of OsERF101 displayed Xa1-dependent, enhanced resistance to an incompatible Xoo strain. Different sets of genes were up- or downregulated in the overexpression and knockout lines. Our results indicate that OsERF101 regulates the recognition of TAL effectors by Xa1, and functions as a positive regulator of Xa1-mediated immunity. Furthermore, an additional Xa1-mediated immune pathway is negatively regulated by OsERF101.

Cooperative regulation of PBI1 and MAPKs controls WRKY45 transcription factor in rice immunity.

The U-box type ubiquitin ligase PUB44 positively regulates pattern-triggered immunity in rice. Here, we identify PBI1, a protein that interacts with PUB44. Crystal structure analysis indicates that PBI1 forms a four-helix bundle structure. PBI1 also interacts with WRKY45, a master transcriptional activator of rice immunity, and negatively regulates its activity. PBI1 is degraded upon perception of chitin, and this is suppressed by silencing of PUB44 or expression of XopP, indicating that PBI1 degradation depends on PUB44. These data suggest that PBI1 suppresses WRKY45 activity when cells are in an unelicited state, and during chitin signaling, PUB44-mediated degradation of PBI1 leads to activation of WRKY45. In addition, chitin-induced MAP kinase activation is required for WRKY45 activation and PBI1 degradation. These results demonstrate that chitin-induced activation of WRKY45 is regulated by the cooperation between MAP kinase-mediated phosphorylation and PUB44-mediated PBI1 degradation.

Effects of physical therapy on blood pressure in daily clinical practice-a pilot study.

[Purpose] Most exercise therapy procedures induce hemodynamic changes and could be a cardiovascular risk. This pilot study investigated factors that induce an exaggerated increase in blood pressure during exercise therapy. [Participants and Methods] We measured the blood pressure and pulse rate before and after exercise therapy for ambulation on days 1, 2, and 7 of the exercise therapy in patients (n=23; age, 69 ± 11 years) who were hospitalized for a stroke or an orthopedic surgery. [Results] Each participant's blood pressure and pulse rate were significantly increased after the exercise therapy. Regression analysis demonstrated that the increase in systolic blood pressure was independently predicted by body weight and pulse rate before the exercise therapy. In the logistic regression analysis, age and body weight were independent predictors of the exaggerated increase in systolic blood pressure (fourth quartile). [Conclusion] A significant increase in blood pressure was induced by exercise therapy. There was a correlation between systolic blood pressure increase and pulse rate before the exercise therapy. Old age or increased body weight predicts exaggerated increase in blood pressure during exercise therapy.

OsDRE2 contributes to chitin-triggered response through its interaction with OsRLCK185.

The rice receptor-like cytoplasmic kinase 185 (OsRLCK185) interacts with the chitin receptor complex OsCERK1/CEBiP and positively regulates chitin-induced immune responses including MAP kinase activation, ROS production and defense gene expression. To elucidate the regulatory mechanisms of OsRLCK185-mediated immunity, we searched for interactors of OsRLCK185. OsDRE2a, rice homologs of the yeast Dre2 protein, were identified as novel interactors of OsRLCK185. OsDRE2a interacted with OsRLCK185 at plasma membrane. The conserved cysteine residues in CIAPIN1 domain of OsDRE2a were essential for tight interaction of OsRLCK185. OsDRE2a was phosphorylated by OsRLCK185. The expression of OsDRE2a and OsDRE2b was induced after chitin treatment. Reduction of OsDRE2a and OsDRE2b mRNA levels by RNA interference resulted in the decreased chitin-induced ROS production. Thus, it is likely that OsDRE2 regulates OsRLCK185-mediated immune responses.

Imbalanced Expression of Tau and Tubulin Induces Neuronal Dysfunction in C. elegans Models of Tauopathy.

Tauopathy is a type of dementia defined by the accumulation of filamentous tau inclusions in neural cells. Most types of dementia in the elderly, including Alzheimer's disease, are tauopathies. Although it is believed that tau protein abnormalities and/or the loss of its functions results in neurodegeneration and dementia, the mechanism of tauopathy remains obscure. Loss of microtubules and/or tubulin is a known consequence of tau accumulating in neurons in Alzheimer's disease. In other words, there is an excess level of tau relative to tubulin in tauopathy neurons. To test whether this imbalance of tau and tubulin expression results in the neurotoxicity of tau, we developed several transgenic C. elegans lines that express human tau at various levels in pan-neurons. These worms showed behavioral abnormalities in a tau expression-dependent manner. The knockdown of a tubulin-specific chaperon, or a subset of tubulin, led to enhanced tau toxicity even in low-expressing tau-transgenic worms that showed no abnormal behaviors. In addition, the suppression of tau expression in tubulin knockdown worms rescued neuronal dysfunction. Thus, not only the overexpression of tau but also a reduction in tubulin can trigger the neurotoxicity of tau. Tau expressed in worms was also highly phosphorylated and largely bound to tubulin dimers rather than microtubules. Relative amount of tubulin-unbound tau was increased in high-expressing tau-transgenic worms showing tau toxicity. We further demonstrated that tau aggregation was inhibited by co-incubation of purified tubulin in vitro, meaning sufficient amounts of tubulin can protect against the formation of tau inclusions. These results suggest that the expression ratio of tau to tubulin may be a determinant of the tauopathy cascade.

Chitin receptor-mediated activation of MAP kinases and ROS production in rice and Arabidopsis.

Rapid induction of plant immune responses is essential to inhibit colonization and invasion by pathogens. Plants can recognize pathogen-associated molecular patterns (PAMPs) including fungal chitin and bacterial flagellin using pattern-recognition receptors (PRRs), which trigger the intracellular activation of mitogen-activated protein kinase (MAPK) cascades and the production of reactive oxygen species (ROS). MAPK activation and ROS production play pivotal roles in the induction of robust immune responses. Recent investigation of chitin- and flagellin-induced immune signaling revealed that receptor-like cytoplasmic kinases (RLCKs) connect PRR-mediated pathogen recognition to MAPK activation and ROS production. In addition, although the MAPK cascade is mediated by 3 sequentially activated protein kinases, MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK, how MAPKKKs are activated downstream of PRRs in plants has not been identified until recently. In this review, we summarize recent findings of RLCK-mediated MAPK activation and ROS production in rice and Arabidopsis.

Conservation of Chitin-Induced MAPK Signaling Pathways in Rice and Arabidopsis.

Perception of microbe-associated molecular patterns (MAMPs) including chitin by pattern recognition receptors (PRRs) rapidly induces activation of mitogen-activated protein kinase (MAPK) cascades. However, how PRRs transmit immune signals to the MAPK cascade is largely unknown. Recently, Arabidopsis receptor-like cytoplasmic kinase PBL27 has been reported to activate MAPKs through phosphorylation of AtMAPKKK5 in the chitin signaling pathway. In this study, we found that OsRLCK185, a rice ortholog of PBL27, regulates chitin-induced MAPK activation in a similar fashion to PBL27 in rice. Upon chitin perception, OsRLCK185 is phosphorylated by OsCERK1, a component of the chitin receptor complex. OsRLCK185 interacted with OsMAPKKK11 and OsMAPKKK18, rice orthologs of AtMAPKKK5, in yeast two-hybrid assays. Silencing of both OsMAPKKK11 and OsMAPKKK18 significantly reduced chitin-induced activation of OsMPK3 and OsMPK6. Expression levels of OsMAPKKK18 were much higher than that of OsMAPKKK11 in rice cells, which was consistent with the fact that the Osmapkkk11 single mutation did not affect MAPK activation. This result suggested that OsMAPKKK18 plays a more important role than OsMAPKKK11 in the chitin-induced activation of OsMPK3 and OsMPK6. The bimolecular fluorescence complementation (BiFC) experiment indicated that OsRLCK185 interacted with OsMAPKKK18 at the plasma membrane in planta. In vitro phosphorylation experiments showed that OsRLCK185 directly phosphorylates OsMAPKKK18. Furthermore, OsMAPKKK18 interacted with the MAPKK OsMKK4, the upstream component of OsMPK3/6. These results suggested that OsRLCK185 connects the chitin receptor to the MAPK cascade consisting of OsMAPKKK18-OsMKK4-OsMPK3/6. Our data revealed that chitin-induced MAPK activation in rice and Arabidopsis is regulated by common homologous elements.

Curcumin improves tau-induced neuronal dysfunction of nematodes.

Tau is a key protein in the pathogenesis of various neurodegenerative diseases, which are categorized as tauopathies. Because the extent of tau pathologies is closely linked to that of neuronal loss and the clinical symptoms in Alzheimer's disease, anti-tau therapeutics, if any, could be beneficial to a broad spectrum of tauopathies. To learn more about tauopathy, we developed a novel transgenic nematode (Caenorhabditis elegans) model that expresses either wild-type or R406W tau in all the neurons. The wild-type tau-expressing worms exhibited uncoordinated movement (Unc) and neuritic abnormalities. Tau accumulated in abnormal neurites that lost microtubules. Similar abnormalities were found in the worms that expressed low levels of R406W-tau but were not in those expressing comparative levels of wild-type tau. Biochemical studies revealed that tau is aberrantly phosphorylated but forms no detergent-insoluble aggregates. Drug screening performed in these worms identified curcumin, a major phytochemical compound in turmeric, as a compound that reduces not only Unc but also the neuritic abnormalities in both wild-type and R406W tau-expressing worms. Our observations suggest that microtubule stabilization mediates the antitoxicity effect of curcumin. Curcumin is also effective in the worms expressing tau fragment, although it does not prevent the formation of tau-fragment dimers. These data indicate that curcumin improves the tau-induced neuronal dysfunction that is independent of insoluble aggregates of tau.

Bacterial effector modulation of host E3 ligase activity suppresses PAMP-triggered immunity in rice.

Pathogen effector proteins are delivered to host cells to suppress plant immunity. However, the mechanisms by which effector proteins function are largely unknown. Here we show that expression of XopP(Xoo), an effector of rice pathogen Xanthomonas oryzae pv. oryzae, in rice strongly suppresses peptidoglycan (PGN)- and chitin-triggered immunity and resistance to X. oryzae. XopP(Xoo) targets OsPUB44, a rice ubiquitin E3 ligase with a unique U-box domain. We find that XopP(Xoo) directly interacts with the OsPUB44 U-box domain and inhibits ligase activity. Two amino-acid residues specific for the OsPUB44 U-box domain are identified, which are responsible for the interaction with XopP(Xoo). Silencing of OsPUB44 suppresses PGN- and chitin-triggered immunity and X. oryzae resistance, indicating that OsPUB44 positively regulates immune responses. Thus, it is likely that XopP(Xoo) suppresses immune responses by directly interacting with and inhibiting a positive regulator of plant immunity.

The homologous carboxyl-terminal domains of microtubule-associated protein 2 and TAU induce neuronal dysfunction and have differential fates in the evolution of neurofibrillary tangles.

Microtubule-associated protein 2 (MAP2) and Tau are abundant neuronal microtubule-associated proteins. Both proteins have highly homologous carboxyl-terminal sequences that function as microtubule-binding domains. Whereas Tau is widely accepted as a pathoetiological factor in human tauopathies, including Alzheimer's disease (AD), it is not known whether there is a relationship between MAP2 and tauopathy. To better understand the pathological roles of MAP2 and Tau, we compared their behaviors in transgenic Caenorhabditis elegans in which MAP2 or Tau was expressed pan-neuronally. Both MAP2 and Tau elicited severe neuronal dysfunction and neuritic abnormalities, despite the absence of detergent-insoluble aggregates in worm neurons. Biochemical analysis revealed that the expressed MAP2 or Tau in worms was highly phosphorylated and did not bind to microtubules. Newly raised antibodies to MAP2 that effectively distinguished between the highly homologous carboxyl-terminal sequences of MAP2 and Tau showed that MAP2 was not involved in the growth process of neurofibrillary tangles in the AD brain. These results indicate that Tau and MAP2 have different fates in the inclusion formation and raise the possibility that MAP2 plays a significant role in neurotoxicity in the AD brain despite the absence of MAP2-aggregates.

A receptor-like cytoplasmic kinase targeted by a plant pathogen effector is directly phosphorylated by the chitin receptor and mediates rice immunity.

CERK1 is a lysine motif-containing plant pattern recognition receptor for chitin and peptidoglycan. Chitin recognition by OsCERK1 triggers rapid engagement of a rice MAP kinase cascade, which leads to defense response activation. How the MAP kinase cascades are engaged downstream of OsCERK1 remains obscure. Searching for host proteins that interact with Xoo1488, an effector of the rice pathogen Xanthomonas oryzae, we identified the rice receptor-like cytoplasmic kinase, OsRLCK185. Silencing OsRLCK185 suppressed peptidoglycan- and chitin-induced immune responses, including MAP kinase activation and defense-gene expression. In response to chitin, OsRLCK185 associates with, and is directly phosphorylated by, OsCERK1 at the plasma membrane. Xoo1488 inhibits peptidoglycan- and chitin-induced immunity and pathogen resistance. Additionally, OsCERK1-mediated phosphorylation of OsRLCK185 is suppressed by Xoo1488, resulting in the inhibition of chitin-induced MAP kinase activation. These data support a role for OsRLCK185 as an essential immediate downstream signaling partner of OsCERK1 in mediating chitin- and peptidoglycan-induced plant immunity.

Overexpression of 1-Deoxy-D-xylulose-5-phosphate reductoisomerase gene in chloroplast contributes to increment of isoprenoid production.

Plants synthesize a large number of isoprenoid compounds that are of industrial, nutritional and medicinal importance. 1-Deoxy-D-xylulose reductoisomerase (DXR) catalyzes the first committed step of plastidial isoprenoid-precursor biosynthesis. In the present study, we generated transplastomic tobacco plants that overproduced DXR from Synechosystis sp. strain PCC6803. The transformants showed increase in the content of various isoprenoids such as chlorophyll a, beta-carotene, lutein, antheraxanthin, solanesol and beta-sitosterol, indicating that the DXR reaction is one of the key steps controlling isoprenoid level in tobacco leaves. A qualitative change in isoprenoid composition was also observed. The growth phenotype of the transplastomic plants was similar to that of wild-type plants. These results showed that plastid metabolic engineering is useful in manipulating the yield of isoprenoids in plants.

Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering.

SUMMARY: The natural pigment astaxanthin has attracted much attention because of its beneficial effects on human health, despite its expensive market price. In order to produce astaxanthin, transgenic plants have so far been generated through conventional genetic engineering of Agrobacterium-mediated gene transfer. The results of trials have revealed that the method is far from practicable because of low yields, i.e. instead of astaxanthin, large quantities of the astaxanthin intermediates, including ketocarotenoids, accumulated in the transgenic plants. In the present study, we have overcome this problem, and have succeeded in producing more than 0.5% (dry weight) astaxanthin (more than 70% of total caroteniods) in tobacco leaves, which turns their green color to reddish brown, by expressing both genes encoding CrtW (beta-carotene ketolase) and CrtZ (beta-carotene hydroxylase) from a marine bacterium Brevundimonas sp., strain SD212, in the chloroplasts. Moreover, the total carotenoid content in the transplastomic tobacco plants was 2.1-fold higher than that of wild-type tobacco. The tobacco transformants also synthesized a novel carotenoid 4-ketoantheraxanthin. There was no significant difference in the size of the aerial part of the plant between the transformants and wild-type plants at the final stage of their growth. The photosynthesis rate of the transformants was also found to be similar to that of wild-type plants under ambient CO2 concentrations of 1500 micromol photons m(-2) s(-1) light intensity.

Deactivation of aquaporins decreases internal conductance to CO2 diffusion in tobacco leaves grown under long-term drought.

We compared the diffusion conductance to CO2 from the intercellular air space to the chloroplasts (internal conductance (g i)) between tobacco leaves acclimated to long-term drought (drought-acclimated (DA)) and those grown under sufficient irrigation (well-watered (WW)), and analysed the changes in g i in relation to the leaf anatomical characteristics and a possible CO2 transporter, aquaporin. The g i, which was estimated by combined analyses of CO2 gas exchange with chlorophyll fluorescence, in the DA plants was approximately half of that in the WW plants. The mesophyll and chloroplast surface areas exposing the intercellular air space, which potentially affect g i, were not significantly different between the WW and DA plants. The amounts of plasma membrane aquaporins (PIP), immunochemically determined using radish PIP antibodies, were unrelated to g i. After treatment with HgCl2, an aquaporin inhibitor, the water permeability of the leaf tissues (measured as the weight loss of fully-turgid leaf disks without the abaxial epidermis in 1 m sorbitol) in WW plants decreased with an increase in HgCl2 concentration. The g i in the WW plants decreased to similar levels to the DA plants when the detached leaflets were fed with 0.5 mm HgCl2. In contrast, both water permeability and g i were insensitive to HgCl2 treatments in DA plants. These results suggest that deactivation of aquaporins is responsible for the significant reduction in g i observed in plants growing under long-term drought.

Effect of coding regions on chloroplast gene expression in Chlamydomonas reinhardtii.

An exogenous gene, placed between the 5'-upstream regions of the Chlamydomonas reinhardtii chloroplast genes, rbcL or psbA, and the 3'-end of the rbcL gene, do not have the same expression pattern as endogenous genes in the C. reinhardtii chloroplast. Here, we chose four chloroplast genes, rbcL, psbA, psbD and atpA, and examine the effects of chloroplast gene coding regions on gene expression in C. reinhardtii. We constructed chimeric genes composed of the promoter, 5'- and 3'-untranslated regions, varying lengths of protein coding regions of the chloroplast genes, and the bacterial beta-glucuronidase (GUS) gene (uidA) as a reporter gene, and introduced into chloroplast genomes. The transformants, which contained the rbcL-uidA and psbA-uidA chimeric genes fused to the coding region of each gene, showed high expression of uidA mRNA as compared with the previously generated transformants, RG and PG, in which uidA was only fused to the promoter and 5'-UTR of each gene. The difference in the accumulation of uidA transcripts among the transformants was the result of different rates of transcription. This result indicates that the coding region is necessary for sufficient expression of rbcL and psbA. On the other hand, the psbD and atpA coding region portions did not affect chimeric gene expression.