Bioactive beads-mediated transformation of rice with large DNA fragments containing Aegilops tauschii genes.

Transformation with large DNA molecules enables multiple genes to be introduced into plants simultaneously to produce transgenic plants with complex phenotypes. In this study, a large DNA fragment (ca. 100 kb) containing a set of Aegilops tauschii hardness genes was introduced into rice plants using a novel transformation method, called bioactive beads-mediated transformation. Nine transgenic rice plants were obtained and the presence of transgenes in the rice genome was confirmed by PCR and FISH analyses. The results suggested that multiple transgenes were successfully integrated in all transgenic plants. The expression of one of the transgenes, puroindoline b, was confirmed at the mRNA and protein levels in the T(2) generation. Our study clearly demonstrates that the bioactive bead method is capable of producing transgenic rice plants carrying large DNA fragments. This method will facilitate the production of useful transgenic plants by introducing multiple genes simultaneously.

Over-expression of putative transcriptional coactivator KELP interferes with Tomato mosaic virus cell-to-cell movement.

Tomato mosaic virus (ToMV) encodes a movement protein (MP) that is necessary for virus cell-to-cell movement. We have demonstrated previously that KELP, a putative transcriptional coactivator of Arabidopsis thaliana, and its orthologue from Brassica campestris can bind to ToMV MP in vitro. In this study, we examined the effects of the transient over-expression of KELP on ToMV infection and the intracellular localization of MP in Nicotiana benthamiana, an experimental host of the virus. In co-bombardment experiments, the over-expression of KELP inhibited virus cell-to-cell movement. The N-terminal half of KELP (KELPdC), which had been shown to bind to MP, was sufficient for inhibition. Furthermore, the over-expression of KELP and KELPdC, both of which were co-localized with ToMV MP, led to a reduction in the plasmodesmal association of MP. In the absence of MP expression, KELP was localized in the nucleus and the cytoplasm by the localization signal in its N-terminal half. It was also shown that ToMV amplified normally in protoplasts prepared from leaf tissue that expressed KELP transiently. These results indicate that over-expressed KELP interacts with MP in vivo and exerts an inhibitory effect on MP function for virus cell-to-cell movement, but not on virus amplification in individual cells.

Improvement of transformation efficiency by bioactive-beads-mediated gene transfer using DNA-lipofectin complex as entrapped genetic material.

The efficiency of bioactive-beads-mediated plant transformation was improved using DNA-lipofectin complex as the entrapped genetic material instead of naked DNA used in the conventional method. In the improved method, beads aggregated and formed clusters around the protoplasts resulting in a 4-fold higher transformation efficiency than that by the conventional method.

Transformation of yeast using bioactive beads with surface-immobilized yeast artificial chromosomes.

Yeast artificial chromosomes (YACs) are useful cloning vectors with the capacity to carry large DNA inserts. The largest barrier using such large DNA molecules in transformation experiments has been their physical instability in a solution. We developed a new method for transforming yeast with chromosome-sized DNA. The method uses bioactive beads composed of calcium alginate to immobilize yeast chromosomal DNAs. Chromosomal DNA immobilized on bioactive beads is physically stable when compared with naked chromosomal DNAs. The bead-mediated transformation performed well, not only with respect to the transformation frequency, but also in successful transformation using split chromosomal DNA that exceeded 450 kb in size. In this chapter we introduce a new method for transforming yeast using bioactive beads. In conjunction with genomic YAC libraries and the yeast chromosome-splitting method, this technique will pave the way to stable and effective transfer of YACs into yeast cells.

Domains of tobacco mosaic virus movement protein essential for its membrane association.

A series of deletion mutants of tobacco mosaic virus movement protein (TMV-MP) was used to identify domains of the protein necessary for membrane association. A membrane fraction was isolated from tobacco BY-2 protoplasts infected with wild-type and mutant TMV that produce MP carrying a 3 aa deletion. Deletions that affected membrane association were clustered around the two major hydrophobic regions of MP that are predicted to be transmembrane. Deletions in other hydrophobic regions also reduced membrane association. In addition, a non-functional mutant of MP, in which one of the known phosphorylation sites was eliminated, was not associated with cellular membranes, while a functional second site revertant restored membrane association. This indicates that MP function requires interaction with membrane; however, membrane association was not sufficient for function. Results are consistent with the hypothesis that TMV-MP is an integral or tightly associated membrane protein that includes two hydrophobic transmembrane domains.

Stimulated parametric emission microscopy.

We propose a novel microscopy technique based on the four-wave mixing (FWM) process that is enhanced by two-photon electronic resonance induced by a pump pulse along with stimulated emission induced by a dump pulse. A Ti:sapphire laser and an optical parametric oscillator are used as light sources for the pump and dump pulses, respectively. We demonstrate that our proposed FWM technique can be used to obtain a one-dimensional image of ethanol-thinned Coumarin 120 solution sandwiched between a hole-slide glass and a cover slip, and a two-dimensional image of a leaf of Camellia sinensis.

Tobacco mosaic virus infection spreads cell to cell as intact replication complexes.

Plant viruses encode movement proteins (MPs) that facilitate cell-cell transport of infection through plasmodesmata. Intracellular and intercellular spread of virus replication complexes (VRCs) of tobacco mosaic virus was followed in intact leaf tissue from 12 to 36 h post infection (hpi) by using confocal microscopy. From 12 hpi, VRCs in primary infected cells were associated with cortical endoplasmic reticulum, and at 14 hpi, exhibited high intracellular mobility ( approximately 160 nm/sec); mobility was slowed between 14 and 16 hpi ( approximately 40 nm/sec), and by 18 hpi, VRCs were stationary, adjacent to plasmodesmata. VRCs traversed the plasmodesmata between 18 and 20 hpi. The process of formation and movement of VRCs was repeated in adjacent cells in 3-4 h vs. 20 h from primary infected cells. The rapid intracellular movement of the VRCs and the spread to adjacent cells was blocked by inhibitors of filamentous actin and myosin, but not by inhibitors of microtubules. We propose a model whereby cell-cell spread of tobamovirus infection is accomplished by subviral replication complexes that initiate TMV replication immediately after entry to adjacent cells.

Defective tobamovirus movement protein lacking wild-type phosphorylation sites can be complemented by substitutions found in revertants.

We reported previously that the movement protein (MP) of tomato mosaic tobamovirus is phosphorylated, and we proposed that MP phosphorylation is important for viral pathogenesis. Experimental data indicated that phosphorylation enhances the stability of MP in vivo and enables the protein to assume the correct intracellular location to perform its function. A mutant virus designated 37A238A was constructed; this virus lacked two serine residues within the MP, which prevented its phosphorylation. In the present study, we inoculated plants with the 37A238A mutant, and as expected, it was unable to produce local lesions on the leaves. However, after an extended period, we found that lesions did occur, which were due to revertant viruses. Several revertants were isolated, and the genetic changes in their MPs were examined together with any changes in their in vivo characteristics. We found that reversion to virulence was associated first with increased MP stability in infected cells and second with a shift in MP intracellular localization over time. In one case, the revertant MP was not phosphorylated in vivo, but it was functional.

In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.