Koji Dohi

Long-distance, graft-transmissible action of Arabidopsis FLOWERING LOCUS T protein to promote flowering

Day length perceived by a leaf is a major environmental factor that controls the timing of flowering. It has been believed that a mobile, long-distance signal called florigen is produced in the leaf under inductive day length conditions, and is transported to the shoot apex where it triggers floral morphogenesis. Grafting experiments have shown that florigen is transmissible from a donor plant that has been subjected to inductive day length to an uninduced recipient plant. However, the nature of florigen has long remained elusive. Arabidopsis FLOWERING LOCUS T (FT) is expressed in cotyledons and leaves in response to inductive long days (LDs). FT protein, with a basic region/leucine zipper (bZIP) transcription factor FD, acts in the shoot apex to induce target meristem identity genes such as APETALA1 (AP1) and initiates floral morphogenesis. Recent studies have provided evidence that the FT protein in Arabidopsis and corresponding proteins in other species are an important part of florigen. Our work shows that the FT activity, either from overexpressing or inducible transgenes or from the endogenous gene, to promote flowering is transmissible through a graft junction, and that an FT protein with a T7 tag is transported from a donor scion to the apical region of recipient stock plants and becomes detectable within a day or two. The sequence and structure of mRNA are not of critical importance for the long-distance action of the FT gene. These observations led to the conclusion that the FT protein, but not mRNA, is the essential component of florigen.

Membrane-Bound Tomato Mosaic Virus Replication Proteins Participate in RNA Synthesis and Are Associated with Host Proteins in a Pattern Distinct from Those That Are Not Membrane Bound

ABSTRACT Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.

Saccharomyces cerevisiae SSD1 orthologues are essential for host infection by the ascomycete plant pathogens Colletotrichum lagenarium and Magnaporthe grisea

Fungal plant pathogens have evolved diverse strategies to overcome the multilayered plant defence responses that confront them upon host invasion. Here we show that pathogenicity of the cucumber anthracnose fungus, Colletotrichum lagenarium, and the rice blast fungus, Magnaporthe grisea, requires a gene orthologous to Saccharomyces cerevisiae SSD1, a regulator of cell wall assembly. Screening for C. lagenarium insertional mutants deficient in pathogenicity led to the identification of ClaSSD1. Following targeted gene replacement, appressoria of classd1 mutants retained the potential for penetration but were unable to penetrate into host epidermal cells. Transmission electron microscopy suggested that appressorial penetration by classd1 mutants was restricted by plant cell wall‐associated defence responses, which were observed less frequently with the wild‐type strain. Interestingly, on non‐host onion epidermis classd1 mutants induced papilla formation faster and more abundantly than the wild type. Similarly, colonization of rice leaves by M. grisea was severely reduced after deletion of the orthologous MgSSD1 gene and attempted infection by the mutants was accompanied by the accumulation of reactive oxygen species within the host cell. These results suggest that appropriate assembly of the fungal cell wall as regulated by SSD1 allows these pathogens to establish infection by avoiding the induction of host defence responses.

Inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells

Summary.Although suspension-cultured plant cells have many potential merits as sources of useful proteins, the lack of an efficient expression system has prevented using this approach. In this study, we established an inducible tomato mosaic virus (ToMV) infection system in tobacco BY-2 suspension-cultured cells to inducibly and efficiently produce a foreign protein. In this system, a modified ToMV encoding a foreign protein as replacement of the coat protein is expressed from stably transformed cDNA under the control of an estrogen-inducible promoter in transgenic BY-2 cells. Estrogen added to the culture activates an estrogen-inducible transactivator expressed constitutively from the transgene and induces transcription and replication of viral RNA. In our experiments, accumulation of viral RNA and expression of green fluorescent protein (GFP) encoded in the virus were observed within 24 h after induction. The amount of GFP reached approximately 10% of total soluble protein 4 d after induction. In contrast, neither viral RNA nor GFP were detected in uninduced cells. The inducible virus infection system established here should be utilized not only for the expression of foreign proteins, but also for investigations into the viral replication process in cultured plant cells.

Recombinant expression and characterization of N-acetylglucosaminyltransferase I derived from Nicotiana tabacum.

The C-terminal catalytic domain of tobacco N-acetylglucosaminyltransferase I fused to maltose-binding protein was produced in Escherichia coli as a soluble form with significant activity. The protein was affinity-purified using amylose resin, and its enzymatic properties were investigated, including its divalent cation requirements, optimal temperature, optimal pH, and substrate specificity.

Resistance in leaf blades assessed by counting conidia correlates with whole-plant-specific resistance in leaf sheaths in a compatible rice– Magnaporthe oryzae interaction

Resistance in the leaf blades of rice plants against a virulent race of the rice blast fungus Magnaporthe oryzae was quantitatively examined using a modified spot inoculation method. Numbers of conidia produced in the lesions were affected by plant age and paralleled the frequency of resistance infection types, which is indicative of whole-plant-specific resistance (WPSR), in the inoculated leaf sheaths of the corresponding plants. Exogenous abscisic acid treatment, which suppresses WPSR, also increased the susceptibility of the leaf blades. These results indicate a correlation between the resistance of the leaf blades and the WPSR in the leaf sheaths.

The “resurrection method” for modification of specific proteins in higher plants

We describe a new method designated “the resurrection method” by which a modified protein is expressed in higher plants in place of the original protein. The modified gene constructed by introducing synonymous codon substitutions throughout the original gene to prevent the sequence‐specific degradation of its mRNA during RNA silencing is expressed while the expression of the original gene is suppressed. Here, we report the successful alteration of the biochemical properties of green fluorescent protein expressed in transgenic Nicotiana benthamiana, suggesting that this method could be useful for gene control in living plants.

Improved expression and characterization of recombinant human Golgi α1,2-mannosidase I isoforms (IA2 and IC) by Escherichia coli.

Golgi α1,2-mannosidase I is involved in the N-linked oligosaccharide processing pathway. In this study, two truncated genes encoding for human Golgi α1,2-mannosidase I (hManIA2: amino acids 127-626 and hManIC: amino acids 118-617) were expressed in Escherichia coli to characterize the enzymes. These genes were fused to a T7 protein tag and a histidine tag at the N- and C-terminal ends, respectively, and purified using Co(2+) affinity chromatography. The properties including optimal temperature, optimal pH, and substrate specificity of the purified enzymes were investigated by HPLC using pyridylamino (PA)-labeled oligosaccharides as substrates. The stability of hManIA2 was dependent on the presence of Ca(2+), which was also required for its activity. On the other hand, hManIC was stable in the absence of Ca(2+), even though Ca(2+) was also effective for the activity of hManIC. While the similarity of the amino acid sequences is over 60%, hManIA2 and hManIC showed different substrate specificities particularly toward M9A and M8C.