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Plants generally possess a strong ability to regenerate organs; for example, in tissue culture, shoots can regenerate from callus, a clump of actively proliferating, undifferentiated cells. Processing of pre-mRNA and ribosomal RNAs is important for callus formation and shoot regeneration. However, our knowledge of the roles of RNA quality control via the nonsense-mediated mRNA decay (NMD) pathway in shoot regeneration is limited. Here, we examined the shoot regeneration phenotypes of the low-beta-amylase1 (lba1)/upstream frame shift1-1 (upf1-1) and upf3-1 mutants, in which the core NMD components UPF1 and UPF3 are defective. These mutants formed callus from hypocotyl explants normally, but this callus behaved abnormally during shoot regeneration: the mutant callus generated numerous adventitious root structures instead of adventitious shoots in an auxin-dependent manner. Quantitative RT-PCR and microarray analyses showed that the upf mutations had widespread effects during culture on shoot-induction medium. In particular, the expression patterns of early auxin response genes, including those encoding AUXIN/INDOLE ACETIC ACID (AUX/IAA) family members, were significantly affected in the upf mutants. Also, the upregulation of shoot apical meristem-related transcription factor genes, such as CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, was inhibited in the mutants. Taken together, these results indicate that NMD-mediated transcriptomic regulation modulates the auxin response in plants and thus plays crucial roles in the early stages of shoot regeneration.

The herb Ruta chalepensis L. exhibits medical effects, such as anti-inflammatory, central nervous system depressant, and antipyretic activities. However, a genetic transformation method has not yet been developed for this species. In this paper, a simple and efficient tissue culture and genetic transformation system for R. chalepensis is reported. An amyloid β-peptide (Aβ) gene, which is considered to be a causative agent of Alzheimer's disease (AD), fused with green-fluorescent protein (GFP), was introduced into R. chalepensis. When the leaves of R. chalepensis expressing Aβ-GFP were administered orally to C57BL/6J mice, serum anti-Aβ antibody titers of several mice were elevated without the use of an adjuvant. These results indicated that an oral vaccine against AD using R. chalepensis may be feasible. R. chalepensis is rich in bioactive compounds that may have synergistic effects with the vaccine for AD. Plant-derived vaccines are safer and cheaper than those produced from animal cells or microbes, because plants can serve as biofactories at low cost and with high biosynthetic capacity.

While Marchantia polymorpha has been utilized as a model system to investigate fundamental biological questions for over almost two centuries, there is renewed interest in M. polymorpha as a model genetic organism in the genomics era. Here we outline community guidelines for M. polymorpha gene and transgene nomenclature, and we anticipate that these guidelines will promote consistency and reduce both redundancy and confusion in the scientific literature.

MicroRNAs (miRNAs) have important roles in gene regulation during plant development. Previous studies revealed that some miRNAs are highly shared by most land plants. Recently, the liverwort, Marchantia polymorpha, has been studied by molecular genetic approaches, and sequencing of its genome is currently underway. The expression pattern and the detailed functions of miRNAs during Marchantia development are unknown. Here, we profiled the small RNAs expressed in thalli, antheridiophores and archegoniophores of M. polymorpha using high-throughput RNA sequencing. We revealed that a limited number of miRNAs are shared between M. polymorpha and the moss, Physcomitrella patens, and that a number of miRNAs are M. polymorpha specific. Like other land plants, cognate target genes corresponding to conserved miRNAs could be found in the genome database and were experimentally confirmed to guide cleavage of target mRNAs. The results suggested that two genes in the SPL (SQUAMOSA PROMOTER BINDING-LIKE) transcription factor family, which are regulated by miR156 in most land plants, were instead targeted by two distinct miRNAs in M. polymorpha. In order to demonstrate the physiological roles of miRNAs in M. polymorpha, we constructed an miRNA ectopic expression system to establish overexpression transformants for conserved miRNAs, miR166 and miR319. Ectopic expression of these miRNAs induced abnormal development of the thallus and gemma cups, suggesting that balanced expression of miRNA/target mRNAs has a crucial role in developmental regulation in M. polymorpha. Profiling data on miRNA together with the ectopic expression system would provide new information on the liverwort small RNA world and evolutionary divergence/conservation of small RNA function among land plants.

Summary In angiosperms, the phase transition from vegetative to reproductive growth involves the de-repression of the squamosa promoter-binding-protein-like (SPL) class of transcription factors, which is negatively regulated by the specific microRNAs (miRNAs/miRs) miR156/529 [ 1 ]. Non-vascular land plants also undergo growth-phase transition to the reproductive state, but knowledge regarding the controlling mechanisms is limited. Here, we investigate the reproductive transition in the liverwort Marchantia polymorpha, focusing on the roles of miR529c [ 2 , 3 , 4 ] and MpSPL2. First, we established mir529c-null mutants using CRISPR/Cas9. Even in the absence of far-red light-supplemented long-day condition, which is usually needed to induce reproductive development [ 5 , 6 ], the mutant thalli developed sexual reproductive organs (gametangia) and produced gametes. Transgenic plants expressing a miR529-resistant MpSPL2 transgene also showed a similar phenotype of reproductive transition in the absence of inductive far-red light signals. In these mutants and transgenic plants, the MpSPL2 mRNA abundance was elevated. Mpspl2ko mutant plants showed successful gamete development and fertilization, which suggests that MpSPL2 is involved in, but not essential for, sexual reproduction in M. polymorpha. Furthermore, analysis of Mpspl2ko mutant and its complemented lines suggests that MpSPL2 may have a role in promotion of reproductive transition. These findings support the notion that the transition to reproductive development in liverworts is controlled by a system similar to that in angiosperms, and the miR156/529-SPL module has common significance in the control of the vegetative-to-reproductive transition during development in many land plants, including liverworts.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant., 陸上植物の祖先の特徴をもつ苔類ゼニゴケの全ゲノム構造を解明. 京都大学プレスリリース. 2017-10-06.

To characterize leaf cell wall proteins relating the architectural changes of leaves, we analyzed Nicotiana tabacum leaf cell wall proteins that were extracted by the treatment with lithium chloride. Some of these proteins showed amino acid sequence homology to some germin-like proteins (GLP). Based of those sequences, we isolated the cDNA encoding the GLPs (NtGLP1-1, NtGLP2-1). Phylogenetic analysis including de novo assembled tobacco GLPs using EST clones, revealed that tobacco GLPs belong to at least 5 different subgroups of GLP and both NtGLP1 and NtGLP2 belong to GLP subfamily 3. We showed that the NtGLP1 actually localizes to cell wall and revealed that it predominantly localized at specific sites on the leaf cell wall where intercellular attachment was just bifurcated. Expression of the NtGLP1 mRNA was mainly detected in leaves especially at elongating stage. NtGLP1 is possibly relevant to development of leaf intercellular space.

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.

Leaf primordia are generated at the periphery of the shoot apex, developing into flat symmetric organs with adaxial-abaxial polarity, in which the indeterminate state is repressed. Despite the crucial role of the ASYMMETRIC LEAVES1 (AS1)-AS2 nuclear-protein complex in leaf adaxial-abaxial polarity specification, information on mechanisms controlling their downstream genes has remained elusive. We systematically analyzed transcripts by microarray and chromatin immunoprecipitation assays and performed genetic rescue of as1 and as2 phenotypic abnormalities, which identified a new target gene, ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), which encodes an abaxial factor acting downstream of the AS1-AS2 complex. While the AS1-AS2 complex represses ETT by direct binding of AS1 to the ETT promoter, it also indirectly activates miR390- and RDR6-dependent post-transcriptional gene silencing to negatively regulate both ETT and ARF4 activities. Furthermore, AS1-AS2 maintains the status of DNA methylation in the ETT coding region. In agreement, filamentous leaves formed in as1 and as2 plants treated with a DNA methylation inhibitor were rescued by loss of ETT and ARF4 activities. We suggest that negative transcriptional, post-transcriptional and epigenetic regulation of the ARFs by AS1-AS2 is important for stabilizing early leaf partitioning into abaxial and adaxial domains.

Decapping 1 (DCP1), Decapping 2 (DCP2) and VARICOSE (VCS) are components of the decapping complex that removes the 7-methyl-guanosine 5'-diphosphate from the 5' end of mRNAs. In animals, the decapping proteins are involved in miRNA-mediated gene silencing, whereas in plants the roles of the decapping proteins in the miRNA pathway are not well understood. Here we demonstrated that the accumulation of miRNAs decreased in dcp1, dcp2 and vcs mutants, indicating that DCP1, DCP2 and VCS are important for the miRNA pathway in Arabidopsis thaliana. The primary miRNAs (pri-miRNAs) did not increase and miRNA biogenesis components did not decrease in these mutants, suggesting that the miRNA decrease in decapping mutants is not due to the defect of pri-miRNA processing. We showed that the accumulation of miRNA targets increased concomitantly with the decrease of miRNA in the decapping mutants. Our results suggested that the seedling lethal phenotypes in the dcp1, dcp2 and vcs mutants are caused not only by the defect in decapping, but also by the disruption of miRNA-mediated gene regulation.

Stress granules, a type of cytoplasmic RNA granules in eukaryotic cells, are induced in response to various environmental stresses, including high temperature. However, how high temperatures induce the formation of these stress granules in plant cells is largely unknown. Here, we characterized the process of stress granule formation in Arabidopsis thaliana by combining live imaging and electron microscopy analysis. In seedlings grown at 22 °C, stress granule formation was induced at temperatures above a critical threshold level of 34 °C in the absence of transpiration. The threshold temperature was the same regardless of whether the seedlings were grown at 22 °C or 4 °C. High-resolution live imaging microscopy revealed that stress granule formation is not correlated with the sizes of pre-existing RNA processing bodies (P-bodies) but that the two structures often associated rapidly. Immunoelectron microscopy revealed a previously unidentified characteristic of the fine structures of Arabidopsis stress granules and P-bodies: the lack of ribosomes and the presence of characteristic electron-dense globular and filamentous structures. These results provide new insights into the universal nature of stress granules in eukaryotic cells.

The genome of the nematode Caenorhabditis elegans possesses an orthologous sequence to the Drosophila muscleblind (mbl) and mammalian muscleblind-like genes (MBNLs). This ortholog, K02H8.1, which has a high degree of homology (about 50%) to human MBNLs, encodes two zinc finger domains, as does the sequence of the Drosophila mbl gene. This distinguishes it from human MBNLs, which encode four zinc finger domains. In this study, we cloned six major isoforms of K02H8.1 using cDNA generated from C. elegans total RNA. All six of the cloned isoforms had an SL1 leader sequence at the 5'-position. Interestingly, one of the isoforms lacked a zinc finger domain-encoding sequence. To understand better the function of K02H8.1, we performed yeast three-hybrid experiments to characterize the binding of K02H8.1 to bait RNAs. K02H8.1 exhibited strong binding affinity for CUG and CCUG repeats, and the binding affinity was very similar to that of MBNLs. In addition, promoter analysis was performed using promoter-green fluorescent protein (GFP) fusion constructs. The expression of GFP driven by the K02H8.1 promoter was absent in muscle; however, significant GFP expression was detected in the neurons around the pharynx.

Summary To study cellular morphogenesis genetically, we isolated loss-of-function mutants of Arabidopsis thaliana, designated ibo1. The ibo1 mutations cause local outgrowth in the middle of epidermal cells of the hypocotyls and petioles, resulting in the formation of a protuberance. In Arabidopsis, the hypocotyl epidermis differentiates into two alternate cell files, the stoma cell file and the non-stoma cell file, by a mechanism involving TRANSPARENT TESTA GLABRA1 (TTG1) and GLABRA2 (GL2). The ectopic protuberances of the ibo1 mutants were preferentially induced in the non-stoma cell files, which express GL2. TTG1-dependent epidermal patterning is required for protuberance formation in ibo1, suggesting that IBO1 functions downstream from epidermal cell specification. Pharmacological and genetic analyses demonstrated that ethylene promotes protuberance formation in ibo1, implying that IBO1 acts antagonistically to ethylene to suppress radial outgrowth. IBO1 is identical to NEK6, which encodes a Never In Mitosis A (NIMA)-related protein kinase (Nek) with sequence similarity to Neks involved in microtubule organization in fungi, algae, and animals. The ibo1-1 mutation, in which a conserved Glu residue in the activation loop is substituted by Arg, completely abolishes its kinase activity. The intracellular localization of GFP-tagged NEK6 showed that NEK6 mainly accumulates in cytoplasmic spots associated with cortical microtubules and with a putative component of the γ-tubulin complex. The localization of NEK6 is regulated by the C-terminal domain, which is truncated in the ibo1-2 allele. These results suggest that the role of NEK6 in the control of cellular morphogenesis is dependent on its kinase action and association with the cortical microtubules.

There has been much recent research on the contribution of microRNA (miRNA) in plant organogenesis and hormone action. In plants, it has been reported that Dicer-like 1 (DCL1), HYPONASTIC LEAVES1 (HYL1) and SERRATE (SE) are involved in the production of miRNAs. The means by which miRNAs are processed and transported is not well understood in detail, however. In this study, we investigated the intracellular localization and intermolecular interaction of these molecules using imaging techniques, including bimolecular fluorescence complementation and fluorescence resonance energy transfer techniques, making use of various enhanced fluorescent proteins. We found that DCL1, HYL1 and SE formed bodies which localized in the nuclei. We were also able to locate the miRNA primary transcript using an MS2-tagged method on these bodies. It appears very likely that the observed DCL1-HYL1-SE nuclear body is involved in miRNA production. Co-expression of SmD3 or SmB proteins revealed the localization of DCL1-HYL1-SE complexes in the SmD3/SmB nuclear bodies.

The sequence profiles of small interfering RNAs (siRNAs) in Arabidopsis infected with the crucifer tobamovirus tobacco mosaic virus (TMV)-Cg were determined by using a small RNA cloning technique. The majority of TMV-derived siRNAs were 21 nt in length. The size of the most abundant endogenous small RNAs in TMV-infected plants was 21 nt, whilst in mock-inoculated plants, it was 24 nt. Northern blot analysis revealed that some microRNAs (miRNAs) accumulated more in TMV-infected plants than in mock-inoculated plants. The question of whether the TMV-Cg-encoded 126K replication protein, an RNA-silencing suppressor, caused small RNA enrichment was examined. Transient expression of the replication protein did not change the pattern of miRNA processing. However, miRNA, miRNA* (the opposite strand of the miRNA duplex) and hairpin-derived siRNA all co-immunoprecipitated with the replication protein. Gel mobility-shift assays indicated that the replication protein binds small RNA duplexes. These results suggest that the tobamovirus replication protein functions as a silencing suppressor by binding small RNA duplexes, changing the small RNA profile in infected plants.

Virus vectors have been used to study plant gene function by transiently overexpressing specific gene products, or conversely, silencing specific endogenous gene functions. Previously, we reported the establishment of tobamovirus vectors and its applications. In this study, we observed that tobamovirus vector could drive ectopic expression of green fluorescent protein (GFP) in roots after infecting leaves, indicating successful invasion into underground tissues. Subsequently, we attempted to apply the tobamovirus vector system to suppress the expression of putrescine N-methyltransferase (PMT), which is the first committed key enzyme for the biosynthesis of nicotine and is active in the underground parts of plants. Two or three weeks after inoculation with the vector harboring a partial fragment of PMT cDNA, we observed a reduction in the PMT mRNA level in the root and in the nicotine content of the aerial parts of the plant Nicotina benthamiana. The possibilities and limitations of the virus vector for the analysis of metabolic pathways are discussed.

Virus infection is established when viral proteins can interact with host factors to execute replication and/or cell-to-cell movement. Even after the virus infection has started, host resistance reactions, if trigged, would suppress further virus propagation. We would like to introduce what we understand about host factors as determinants of infection establishment and as key resistance molecules. Genome-wide information of Arabidopsis is providing us much information about such host factors involved in virus infection.

A novel hypernodulation mutant line was isolated from Lotus japonicus Miyakojima MG-20 by irradiation with a helium ion beam. This mutant, named klavier (klv), had roots that were densely covered with small nodules. The nodulation zone of klv was significantly wider than that of the wild type. Grafting experiments showed that klv is impaired in the long-distance shoot-to-root autoregulatory mechanism. Thus the shoot genotype was found to be responsible for the negative regulation of nodule development by KLV. Nodulation of klv showed a higher tolerance to nitrogen (KNO3) than the wild type, which is a common feature of hypernodulating mutants. In addition to an increased number of nodules, the klv mutant showed convex leaf veins on the adaxial leaf surface, markedly delayed flowering and dwarf phenotypes. Microscopic examination of the leaf veins revealed that they were discontinuous. Other phenotypes such as fasciated stems, increased number of flowers and bifurcated pistils were also frequently observed in the klv mutant. Among these phenotypes, hypernodulation, aberrant leaf vein formation and significantly delayed flowering were all linked in a monogenic and recessive manner, indicating that these phenotypes are caused by either a single mutation, or tightly linked mutations. KLV was mapped within 0.29 cM on the long arm of chromosome 1.

It has been reported that eukaryotic organisms have a nonsense-mediated mRNA decay (NMD) system to exclude aberrant mRNAs that produce truncated proteins. NMD is an RNA surveillance pathway that degrades mRNAs possessing premature translation termination codons (PTCs), thus avoiding production of possibly toxic truncated proteins. Three interacting proteins, UPF1, UPF2 and UPF3, are required for NMD in mammals and yeasts, and their amino acid sequences are well conserved among most eukaryotes, including plants. In this study, 'The Arabidopsis Information Resource' database was searched for mRNAs with premature termination codons. We selected five of these mRNAs and checked for the presence of PTCs in these mRNAs when translated in vivo. As a result we identified aberrant mRNAs produced by alternative splicing for each gene. These genes produced at least one alternative splicing variant including a PTC (PTC+) and another variant without a PTC (PTC-). We analyzed their PTC+/PTC- ratios in wild-type Arabidopsis and upf3 mutant plants and showed that the PTC+/PTC- ratios were higher in atupf3 mutant plants than wild-type plants and that the atupf3 mutant was less able to degrade mRNAs with premature termination codons than wild-type plants. This indicated that the AtUPF3 gene is required by the plant NMD system to obviate aberrantly spliced mRNA.

The intercellular communication by plasmodesmata (PD) is important for the growth and development of plants, and the transport of macromolecules through PD is likely to be regulated by developmental signals. While PD in the apical meristem transport macromolecules such as mRNAs, the branched PD in the mature leaf do not transport large macromolecules freely. The changes in PD during development might be important for sink-to-source changes in leaves, but the molecular mechanism is still unknown. Movement proteins (MPs) of the tobacco mosaic virus localize in the branched PD and increase the size exclusion limit, allowing transport of viral RNA. We developed a method for differential extraction of MP from isolated cell walls of transgenic tobacco leaves expressing MP or MP tagged with green-fluorescent protein. Lithium chloride at a concentration of 8 M removed filamentous structures in branched PD, the possible attachment site of MP. As some endogenous proteins were coeluted with MP by the treatment, this extraction method might be a powerful tool for investigating MP-interacting proteins in branched PD.

Lycopersicon esculentum cultivar Micro-Tom is a miniature tomato with many advantages for studies of the molecular biology and physiology of plants. To evaluate the suitability of Micro-Tom as a host plant for the study of pathogenesis, Micro-Tom plants were inoculated with 16 well-known fungal, bacterial, and viral pathogens of tomato. Athelia rolfsii, Botryotinia fuckeliana, Oidium sp., Phytophthora infestans, and Sclerotinia sclerotiorum caused typical symptoms and sporulated abundantly on Micro-Tom. Micro-Tom was resistant to Alternaria alternata, Corynespora cassiicola, and Fusarium oxysporum. When Micro-Tom was inoculated with 17 isolates of Ralstonia solanacearum, many isolates induced wilt symptoms. Agrobacterium tumefaciens also was pathogenic, causing crown galls on stem tissue after needle prick inoculation. In Micro-Tom sprayed with Pseudomonas syringae pv. tomato, P. s. pv. tabaci, or P. s. pv. glycinea, bacterial populations did not increase, and yellow lesions appeared only on leaves sprayed with P. s. pv. tomato. Tomato mosaic virus, Tomato aspermy virus, and Cucumber mosaic virus systemically infected Micro-Tom, which developed symptoms characteristic of other cultivars of tomato after infection with the respective virus. These results indicated that Micro-Tom was generally susceptible to most of the important tomato pathogens and developed typical symptoms, whereas certain pathogens were restricted by either hypersensitive resistance or nonhost resistance on Micro-Tom. Therefore, an assortment of Micro-Tom–pathogen systems should provide excellent models for studying the mechanism of susceptible and resistant interactions between plants and pathogens.

Salicylic acid (SA)-induced protein kinase (SIPK) in Nicotiana tabacum L. is activated in response to various stimuli. We assessed the response of SIPK in N. benthamiana. leaves to ultraviolet-C (UV-C) and SA stress. Infiltration of Agrobacterium tumefaciens EHA105 cells containing pBE2113-GST-SIPK into leaves gave transient production of glutathione-S transferase (GST)-fused SIPK. Irradiation of agroinfiltrated leaves with UV-C (254 nm) activated GST-SIPK and endogenous protein kinases with a molecular mass of about 45 kDa, close to that of SIPK simultaneously. These results suggest that SIPK is involved in stress signalling in Nicotiana plants under UV-C irradiation.

Alzheimer's disease (AD), the leading cause of dementia in the elderly population, still remains without an effective treatment. The accumulation and deposition of the amyloid-beta peptide (Abeta) in the brain is thought to be a key event in the pathogenesis of AD. Recently, a novel exciting technology has been investigated to combat AD: new immunotherapeutic approaches have been described that are based on vaccination with the Abeta peptide itself, and this has been shown to induce functionally beneficial anti-Abeta antibody responses in different transgenic animal models of AD. Here we report the high level expression of GFP-Abeta1-40 and 1-42 peptides in Capsicum annum var. angulosum (green pepper) using a new tomato mosaic tobamovirus-based hybrid replication vector. After preinoculation of Nicotiana benthamiana plants with the in vitro transcript of the vector, the isolated virions were used to inoculate green pepper, which accumulated the GFPAbeta1-40 or 1-42 fusion proteins to a level of 100 microg/g of leaves 7 days after inoculation. These results make it possible to test whether oral immunization by feeding plant samples could stimulate antibody production against Abeta peptides.

Virus-induced gene silencing (VIGS) can be used to study gene function by mediating sequence-specific mRNA degradation and suppressing the expression of endogenous target genes. We previously demonstrated that the TocJ vector based on the tomato mosaic tobamoviruses (ToMV) was able to multiply, spread systemically and express green fluorescence protein in Solanaceous plants. TocJ harbouring fragments of endogenous genes could induce VIGS of the parental gene expression, but also induced viral infection symptoms. In this study, an attenuated strain of ToMV, L11A, was used to construct a ToMV vector in order to reduce the virus-induced symptoms. This new vector, named LcJ, was able to spread systemically and mediated VIGS of endogenous genes without visible symptoms. We propose that the use of this attenuated strain for the construction of virus vectors is beneficial for the induction of VIGS.

SUMMARY Cross-protection is a procedure that has been utilized to protect crops against virulent strains of viruses by pre-treatment with closely related attenuated strains of the virus. We constructed a mutant of crucifer tobamovirus Cg, which is analogous to L(11)A, an attenuated strain of Tomato mosaic virus-L (ToMV-L). This mutant, named CgYD, caused few disease symptoms and could spread throughout Arabidopsis thaliana Col-0 plants. Initial infection with CgYD was shown to efficiently cross-protect against a challenge with wild-type Cg. Thus, we have established in Arabidopsis a powerful system for investigating mechanisms of cross-protection. Using this system, we showed that cross-protection was not overcome, even if a higher concentration of the virion, or purified virion RNA, were used in the challenge. We also demonstrated that cross-protection requires that the second virus be very similar in sequence to Cg, which is a characteristic of RNA silencing. However the RNA dependent RNA polymerase SDE1/SGS2 associated with post-transcriptional gene silencing was not required for cross-protection.

In present-day biology, a facile gene delivery system is required to study gene function in a high throughput way. The use of plant virus vectors that can introduce foreign or extra endogenous genes into plants attracts much attention. It is advantageous in that researchers need only minimal experience and results can be obtained in a short period of time. Here we describe infectious cDNA clones of the tomato mosaic tobamoviruses (ToMV) that were modified for facile insertion of foreign genes and which retained the ability to multiply in plants. This vector, named TocJ, was able to harbor the GFP gene from Aequorea victoria and systemically spread in some Solanaceous plants. TocJ would be suitable for the overexpression of foreign genes and the analysis of gene functions in various Solanaceous plants.

Troglitazone glucuronidation in human liver and intestine microsomes and recombinant UDP-glucuronosyltransferases (UGTs) were thoroughly characterized. All recombinant UGT isoforms in baculovirus-infected insect cells (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) exhibited troglitazone glucuronosyltransferase activity. Especially UGT1A8 and UGT1A10, which are expressed in extrahepatic tissues such as stomach, intestine, and colon, showed high catalytic activity, followed by UGT1A1 and UGT1A9. The kinetics of the troglitazone glucuronidation in the recombinant UGT1A10 and UGT1A1 exhibited an atypical pattern of substrate inhibition when the substrate concentration was over 200 micro M. With a Michaelis-Menten equation at 6 to 200 micro M troglitazone, the K(m) value was 11.1 +/- 5.8 micro M and the V(max) value was 33.6 +/- 3.7 pmol/min/mg protein in recombinant UGT1A10. In recombinant UGT1A1, the K(m) value was 58.3 +/- 29.2 micro M and the V(max) value was 12.3 +/- 2.5 pmol/min/mg protein. The kinetics of the troglitazone glucuronidation in human liver and jejunum microsomes also exhibited an atypical pattern. The K(m) value was 13.5 +/- 2.0 micro M and the V(max) value was 34.8 +/- 1.2 pmol/min/mg for troglitazone glucuronidation in human liver microsomes, and the K(m) value was 8.1 +/- 0.3 micro M and the V(max) was 700.9 +/- 4.3 pmol/min/mg protein in human jejunum microsomes. When the intrinsic clearance was estimated with the in vitro kinetic parameter, microsomal protein content, and weight of tissue, troglitazone glucuronidation in human intestine was 3-fold higher than that in human livers. Interindividual differences in the troglitazone glucuronosyltransferase activity in liver microsomes from 13 humans were at most 2.2-fold. The troglitazone glucuronosyltransferase activity was significantly (r = 0.579, p0.05) correlated with the beta-estradiol 3-glucuronosyltransferase activity, which is mainly catalyzed by UGT1A1. The troglitazone glucuronosyltransferase activity in pooled human liver microsomes was strongly inhibited by bilirubin (IC(50) = 1.9 micro M), a typical substrate of UGT1A1. These results suggested that the troglitazone glucuronidation in human liver would be mainly catalyzed by UGT1A1. Interindividual differences in the troglitazone glucuronosyltransferase activity in S-9 samples from five human intestines was 8.2-fold. The troglitazone glucuronosyltransferase activity in human jejunum microsomes was strongly inhibited by emodin (IC(50) = 15.6 micro M), a typical substrate of UGT1A8 and UGT1A10, rather than by bilirubin (IC(50) = 154.0 micro M). Therefore, it is suggested that the troglitazone glucuronidation in human intestine might be mainly catalyzed by UGT1A8 and UGT1A10.

The decapping enzymes DCP1 and DCP2 are components of a decapping complex that degrades mRNAs. DCP2 is the catalytic core and DCP1 is an auxiliary subunit. It has been assumed that DCP1 and DCP2 are consistently co-localized in cytoplasmic RNA granules called processing bodies (P-bodies). However, it has not been confirmed whether DCP1 and DCP2 co-localize in Arabidopsis thaliana. In this study, we generated DCP1-green fluorescent protein (GFP) and DCP2-GFP transgenic plants that complemented dcp1 and dcp2 mutants, respectively, to see whether localization of DCP2 is identical to that of DCP1. DCP2 was present throughout the cytoplasm, whereas DCP1 formed P-body-like foci. Use of DCP1-GFP/DCP2-red fluorescent protein (RFP) or DCP1-RFP/DCP2-GFP plants showed that heat treatment induced DCP2 assembly into DCP1 foci. In contrast, cold treatment did not induce DCP2 assembly, while the number of DCP1 foci increased. These changes in DCP1 and DCP2 localization during heat and cold treatments occurred without changes in DCP1 and DCP2 protein abundance. Our results show that DCP1 and DCP2 respond differently to environmental changes, indicating that P-bodies have diverse DCP1 and DCP2 proportions depending on environmental conditions. The localization changes of DCP1 and DCP2 may explain how specific mRNAs are degraded during changes in environmental conditions.

Tobacco mosaic virus (TMV) encodes a 30-kDa movement protein (MP) which enables viral movement from cell to cell. It is, however, unclear whether the 126- and 183-kDa replicase proteins are involved in the cell-to-cell movement of TMV. In the course of our studies into TMV-R, a strain with a host range different from that of TMV-U1, we have obtained an interesting chimeric virus, UR-hel. The amino acid sequence differences between UR-hel and TMV-U1 are located only in the helicase-like domain of the replicase. Interestingly, UR-hel has a defect in its cell-to-cell movement. The replication of UR-hel showed a level of replication of the genome, synthesis, and accumulation of MP similar to that observed in TMV-U1-inoculated protoplasts. Such observations support the hypothesis that the replicase coding region may in some fashion be involved in cell-to-cell movement of TMV.

Introduction of a single amino acid substitution (Gln979 to Ile) into the 130- and 180-kDa proteins of tomato mosaic tobamovirus (ToMV) rendered the mutant virtually unable to replicate in tomato cells while it replicated perfectly in tobacco cells. The mechanism of this change in host specificity is discussed.

The N gene-mediated hypersensitive response (HR) in tobacco provides a high degree of resistance against most tobamoviruses by halting the progress of infection at the site of inoculation. A previous report indicated a role for the 126/183-kDa replicase in induction of the HR in tobacco containing the N gene (H. S. Padgett and R. N. Beachy, Plant Cell 5:577-586, 1993). Chimeric virus genomes were constructed in which the genes encoding the 126/183-kDa proteins of the HR-eliciting pathogen, tobacco mosaic virus (TMV), and the resistance breaking tobamovirus, Ob, were exchanged. Inoculation of the chimeric viruses to leaves of Nicotiana tabacum cv. Xanthi NN confirmed that either the replicase protein of TMV or its mRNA was responsible for induction of HR. An expression vector based on the Ob virus was used to express fragments of various replicase genes. With this approach, it was determined that the HR is caused by a portion of the replicase protein extending from amino acid 692 to 1116. Consistent with this result, Ob mutants that induce the HR on NN tobacco were found to carry mutations within the same portion of the replicase gene. The N gene-mediated HR is inactive at high temperatures, yet these mutants were able to overcome the HR at significantly lower temperatures than could TMV, indicating that the temperature sensitivity of the N gene response is manifested at the level of interaction between the virus and the defense response mechanism.

The rakkyo strain (TMV-R) and the common strain (TMV-U1) of tobacco mosaic virus exhibit distinct host range differences. TMV-R infects rakkyo plants, a monocot host that TMV-U1 is unable to infect. However, TMV-R causes only latent infection in Nicotiana tabacum cv. Bright Yellow (BY) in inoculated leaves, whereas TMV-U1 infects BY systemically and induces mosaic symptoms. Complete nucleotide sequencing of the TMV-R genomic RNA revealed amino acid changes in the 130K/180K replicase proteins, the 30K protein, and the coat protein and nucleotide changes in the 5′ and 3′ noncoding regions compared to TMV-U1. To identify viral components involved in determination of differences in host range, we have mapped determinants for the differential infection phenotype in BY plants by constructing chimeric viruses between the two strains in the present study. Examination of the infection phenotypes of the 14 constructed chimeric viruses in BY showed that determinants defining the differential infection phenotype in BY reside in the 130K/180K replicase proteins and the 3′ noncoding region. Cognate combination of the 130K/180K replicase proteins and the 3′ noncoding region of TMV-U1 origin is required to produce systemic infection in BY plants.

The intercellular and intracellular distribution of the movement protein (MP) of the Ob tobamovirus was examined in infected leaf tissues using an infectious clone of Ob in which the MP gene was translationally fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria. In leaves of Nicotiana tabacum and N. benthamiana, the modified virus caused fluorescent infection sites that were visible as expanding rings. Microscopy of epidermal cells revealed subcellular patterns of accumulation of the MP:GFP fusion protein which differed depending upon the radial position of the cells within the fluorescent ring. Punctate, highly localized fluorescence was associated with cell walls of all of the epidermal cells within the infection site, and apparently represents association of the fusion protein with plasmodesmata; furthermore, fluorescence was retained in cell walls purified from infected leaves. Within the brightest region of the fluorescent ring, the MP:GFP was observed in irregularly shaped inclusions in the cortical regions of infected cells. Fluorescent filamentous structures presumed to represent association of MP:GFP with microtubules were observed, but were distributed differently within the infection sites on the two hosts. Within cells containing filaments, a number of fluorescent bodies, some apparently streaming in cytoplasmic strands, were also observed. The significance of these observations is discussed in relation to MP accumulation, targeting to plasmodesmata, and degradation.

Previously we made a series of deletion mutants in the 5′ non-coding region of tomato mosaic tobamo-virus (ToMV) RNA and checked their ability to replicate in tobacco protoplasts. Long deletions in this region caused the virus to lose the ability to replicate. Several mutants with deletions of about 10 nucleotides (short deletion mutants; SDM) retained the ability to replicate. In this study, we inoculated SDMs onto systemic host plants and observed their symptoms. One mutant (19/32) caused severe mosaic symptoms on some tobacco plants (Nicotiana tabacum cv. Samsun) but no symptoms on others. Virus accumulation in 19/32-inoculated plants paralleled the severity of symptoms. Four CAA repeat sequences were deleted in 19/32. Progeny 19/32 from plants showing severe systemic mosaic symptoms had acquired additional nucleotides in this region. We conclude that the CAA repeat sequence is related to the fitness of the virus population to replicate in whole plants rather than to translation of ToMV replicase genes.

The complete nucleotide sequence of the genome of a rakkyo strain of tobacco mosaic virus (TMV-R), which exhibits distinct host range differences from the common strain of TMV, was determined. The overall nucleotide sequence homology with TMV-U1 (a common strain of TMV) is 94.2%. The amino acid sequence homologies of the four encoded proteins (180K, 130K, 30K, coat protein) are from 95.9% to 98.0% compared with TMV-U1. To facilitate the analysis of the novel host range of TMV-R, a full-length clone of the genome containing a bacteriophage T7 RNA polymerase promoter was assembled from two cDNA clones and designated pRF3. In vitro transcripts derived from pRF3 were highly infectious. The infections of RF3, wild-type TMV-R, and U3/12-4 (derived from pU3/12-4, an infectious clone of TMV-U1) were compared on Nicotiana tabacum cv. Bright Yellow (BY) plants. No systemic mosaic symptoms were observed on plants inoculated with RF3 and TMV-R, while BY plants inoculated with U3/12-4 developed distinct mosaic symptoms on the upper leaves 8-9 days post-inoculation. The green fluorescent protein (GFP) gene was introduced into pRF3 and pU3/12-4 by replacing the coat protein gene to get two GFP expressing chimeric virus clones: pR-GFP or pU1-GFP. Transcripts from pU1-GFP produced strong fluorescence when inoculated onto BY leaves, while those from pR-GFP produced only very faint fluorescence.

Many plant viruses have single-stranded RNAs as their genomes. During the course of replication, genomic RNAs and their complementary template RNAs are likely to form double-stranded (ds) RNA at least transiently. To attack replication intermediates of many plant RNA viruses specifically, we introduced a yeast-derived ds-RNA specific RNase gene called pac 1 into plants. The transformed plants showed a decrease in lesion numbers when they were challenged with tomato mosaic virus, and a delay in the appearance of symptoms when inoculated with cucumber mosaic virus or potato virus Y.

By using a new tobacco mosaic virus (TMV) vector [Hamamoto, H., et al. (1993) Bio/Technology, 11, 930–932], we have constructed TMV particles which present three different kinds of epitopes, two of them from influenza virus hemagglutinin (HA), and one from human immunodeficiency virus type I (HIV-I) envelope protein, on the surface of the particles. Each of these TMV particles reacted with each anti-peptide antiserum. These results suggest that this TMV vector can be used as an antigen presentation system.

The genomic RNA sequence of tobamovirus Ob (Ob), which can spread systemically in tobacco carrying the N gene, was determined. It consists of 6507 nucleotides and contains four open reading frames, exactly corresponding to the genomic organization of tobamoviruses known so far, i.e. encoding the 130K, 180K, 30K and coat proteins. There were no nucleotide overlaps between any open reading frames. The Ob nucleic acid sequence, predicted protein sequences and gene organization were compared with those of other tobamoviruses reported previously. This virus was originally reported as a tomato mosaic virus; however, the nucleotide sequence data given here refute this classification. The determinants that allow tobamovirus Ob to overcome the N gene, a feature peculiar to this virus, were not identified apart from sequence data. This virus should be regarded as a new tobamovirus. The determinants interacting with the tentative N gene product have not yet been analysed.

Various vaccine therapies for Alzheimer's disease (AD) have been investigated. Here we report transgenic rice expressing amyloid β-peptide (Aβ). The Aβ42 gene fused with a green fluorescent protein gene was introduced into rice using the Agrobacterium method. When transgenic brown rice expressing Aβ was orally administered to mice, serum anti-Aβ antibody titers were elevated. The same results were observed when mice were fed boiled, transgenic brown rice. The results indicate that an edible vaccine against AD using rice may be feasible. A vaccine derived from rice would be far cheaper than existing medical vaccines.

Biological signaling processes may be mediated by complex networks in which network components and network sectors interact with each other in complex ways. Studies of complex networks benefit from approaches in which the roles of individual components are considered in the context of the network. The plant immune signaling network, which controls inducible responses to pathogen attack, is such a complex network. We studied the Arabidopsis immune signaling network upon challenge with a strain of the bacterial pathogen Pseudomonas syringae expressing the effector protein AvrRpt2 (Pto DC3000 AvrRpt2). This bacterial strain feeds multiple inputs into the signaling network, allowing many parts of the network to be activated at once. mRNA profiles for 571 immune response genes of 22 Arabidopsis immunity mutants and wild type were collected 6 hours after inoculation with Pto DC3000 AvrRpt2. The mRNA profiles were analyzed as detailed descriptions of changes in the network state resulting from the genetic perturbations. Regulatory relationships among the genes corresponding to the mutations were inferred by recursively applying a non-linear dimensionality reduction procedure to the mRNA profile data. The resulting static network model accurately predicted 23 of 25 regulatory relationships reported in the literature, suggesting that predictions of novel regulatory relationships are also accurate. The network model revealed two striking features: (i) the components of the network are highly interconnected; and (ii) negative regulatory relationships are common between signaling sectors. Complex regulatory relationships, including a novel negative regulatory relationship between the early microbe-associated molecular pattern-triggered signaling sectors and the salicylic acid sector, were further validated. We propose that prevalent negative regulatory relationships among the signaling sectors make the plant immune signaling network a “sector-switching” network, which effectively balances two apparently conflicting demands, robustness against pathogenic perturbations and moderation of negative impacts of immune responses on plant fitness., Author Summary When a plant detects pathogen attack, this information is conveyed through a molecular signaling network to turn on a large variety of immune responses. We investigated how this plant immune signaling network was organized using the model plant Arabidopsis. Wild type and mutant plants with defects in immune signaling were challenged with a pathogen. Then, expression levels of many genes were measured using microarrays. Detailed analysis of the mutation effects on gene expression allowed us to build a signaling network model composed of the genes corresponding to the mutations. This model predicted that the network components are highly interconnected and that it is very common for network components that mediate different signaling events to inhibit each other. The prevalent signaling inhibitions in the network suggest that only part of the signaling network is usually used but that if this part is attacked by pathogens, other parts kick in and back up the function of the attacked part. We speculate that plant immune signaling is highly tolerant to pathogen attack due to this backup mechanism. We also speculate use of only part of the network at any one time helps minimize negative impacts of the immune response on plant fitness.

The 30-kDa protein of tobacco mosaic virus, which is involved in cell-to-cell movement function, is phosphorylated in tobacco protoplasts. To investigate which portion of the protein is phosphorylated we inoculated several truncated 30-kDa protein mutants into protoplasts and determined whether or not those truncated proteins are phosphorylated. The results showed that amino acid residues 234–261 of the 30-kDa protein are required for this phosphorylation.

Plant microRNA (miRNA) processing requires at least two cleavage steps of respective precursors. The first cleavage step is from pri-miRNA to pre-miRNA, and the second cleavage step is from pre-miRNA to mature miRNA. Using northern blot analysis, we previously showed that the RNase III enzyme Dicer-like protein 1 (DCL1) and the double-stranded RNA-binding (DRB) protein HYL1 are involved in the processing reactions of miRNA precursors. The processing of plant miRNAs differs from that of animal miRNAs in some respects. Here, we introduce our methods for analyzing the processing of miRNA precursors through transient expression of mutated pri-miRNAs with modified stem-loop structures in Nicotiana benthamiana. The methods described here could be useful for understanding how DCL proteins and cognate DRB proteins are involved in adequate recognition and processing of substrate precursor RNA molecules.

Suppressor of gene silencing 3 (SGS3) is involved in RNA-dependent RNA polymerase 6 (RDR6)-dependent small-interfering RNA (siRNA) pathways in Arabidopsis. However, the roles of SGS3 in those pathways are unclear. Here, we show that SGS3 interacts and colocalizes with RDR6 in cytoplasmic granules. Interestingly, the granules containing SGS3 and RDR6 (named SGS3/RDR6-bodies) were distinct from the processing bodies where mRNAs are decayed and/or stored. Microscopic analyses and complementation experiments using SGS3-deletion mutants suggested that proper localization of SGS3 is important for its function. These results provide novel insights into RDR6-dependent siRNA formation in plants.Structured summaryMINT-7014710: SGS3 (uniprotkb:Q9LDX1) and RDR6 (uniprotkb:Q9SG02) physically interact (MI:0218) by bimolecular fluorescence complementation (MI:0809)MINT-7014697: RDR6 (uniprotkb:Q9SG02) and SGS3 (uniprotkb:Q9LDX1) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

MicroRNAs (miRNAs) are sequence-specific negative regulators of gene expression generated by DICER-LIKE1 (DCL1) with the assistance of a double-stranded RNA-binding protein, HYPONASTIC LEAVES1 (HYL1), in Arabidopsis. To achieve a better understanding of miRNA biogenesis, we isolated hyl1 suppressors. Our genetic screening identified a novel semidominant mutation in DCL1 (dcl1-13), which causes an amino acid substitution of Glu-395 with Lys in the ATPase/DExH-box RNA helicase domain. This mutation confers significant restoration from the developmental abnormality and a reduction in the level of miRNA in the loss-of-function mutant of HYL1. However, the dcl1-13 single mutant, exhibiting a decreased number of leaves, showed a slight decrease in miRNA accumulation. Thus, the effect of the dcl1-13 mutation is HYL1 dependent: it promotes miRNA processing in the absence of HYL1, but conversely, impairs it in the presence of HYL1. Our results suggest significant roles of the helicase domain of DCL1, which remain unclear to date, possibly in relation with HYL1.

We examined whether the 130K and 180K proteins of tobacco mosaic virus (TMV), the putative virus-encoded replicase components, produced by a replication-competent TMV mutant could complement a replication-defective mutant in a single cell. The replication-competent mutant (LDCS29) had a deletion in the coat protein gene and the replication-defective mutant (LDR28) had a large deletion in the gene encoding the 130K and 180K proteins. Neither the replication of LDR28 nor the production of the coat protein from LDR28 or LDCS29 was detected when the mutants were inoculated separately into tobacco protoplasts. However, when the two mutants were co-inoculated, the production of the LDR28 genomic RNA and the subgenomic RNA for the coat protein and accumulation of the coat protein were observed. These results show that the virus-encoded replicase components of TMV complemented the replication-defective mutant in trans.