Suspension-cultured Tobacco BY-2 Cells Research Articles

Functional Analysis of Two Chromosomal Virulence Genes chvA and acvB of Agrobacterium tumefaciens Using Avirulent Mutants with Transposo 5 Insertion in the Respective Gene.

A mutant (M3 strain) of Agrobacterium tumefaciens was isolated by transposon (Tn5) mutagenesis. The M3 strain was avirulent on all host plants tested, Daucus carota, Solanum tuberosum and Kalanchoe pinnata. The avirulent phenotype of M3 strain was due to an insertion of Tn5 into the chvA chromosomal virulence gene. Using this M3 strain (chvA:: Tn5) and two other strains, B119 strain (acvB: Tn5) and A208 strain (parent, virulent), the functions of the chvA and acvB chromosomal virulence genes of A. tumefaciens were analyzed. When M3, B119 and A208 strains were co-cultured with tobacco BY2 suspension-culture cells in the presence and absence of acetosyringone (As), efficient T-DNA transfer from an Agrobacterium cell to the nucleus of a tobacco cell occurred only with A208 strain in the presence of As. However, the T-strand (an intermediate form of T-DNA for transfer) was produced normally in all three strains in the presence of As. Analyses with tricine-SDS-polyacrylamide gel electrophoresis and electron microscopy demonstrated that pili, which are assumed to mediate T-DNA transfer into the host cell, were not produced on the cell surface of the M3 strain in either media with or without As. On the other hand, perfect pili were produced on the cell surface of the A208 strain in the presence of As. On the cell surface of the B119 strain, “imperfect” pili which were thinner than those on the A208 strain were produced in the presence of As. Taken together, these results indicate that chvA and acvB likely play roles in virulence by influencing formation of the pili through which the T-strands are thought to be transfer into host cells.

Oilseed isocitrate lyases lacking their essential type 1 peroxisomal targeting signal are piggybacked to glyoxysomes.

Isocitrate lyase (IL) is an essential enzyme in the glyoxylate cycle, which is a pathway involved in the mobilization of stored lipids during postgerminative growth of oil-rich seedlings. We determined experimentally the necessary and sufficient peroxisome targeting signals (PTSs) for cottonseed, oilseed rape, and castor bean ILs in a well-characterized in vivo import system, namely, suspension-cultured tobacco (Bright Yellow) BY-2 cells. Results were obtained by comparing immunofluorescence localizations of wild-type and C-terminal-truncated proteins transiently expressed from cDNAs introduced by microprojectile bombardment. The tripeptides ARM-COOH (on cottonseed and castor bean ILs) and SRM-COOH (on oilseed rape IL) were necessary for targeting and actual import of these ILs into glyoxysomes, and ARM-COOH was sufficient for redirecting chloramphenicol acetyltransferase (CAT) from the cytosol into the glyoxysomes. Surprisingly, IL and CAT subunits without these tripeptides were still acquired by glyoxysomes, but only when wild-type IL or CAT-SKL subunits, respectively, were simultaneously expressed in the cells. These results reveal that targeting signal-depleted subunits are being piggybacked as multimers to glyoxysomes by association with subunits possessing a PTS1. Targeted multimers are then translocated through membrane pores or channels to the matrix as oligomers or as subunits before reoligomerization in the matrix.

C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells

The potential of tobacco BY-2 suspension-cultured cells for examining in vivo targeting and import of proteins into plant peroxisomes was shown recently in our laboratory. In the current study, the necessity and sufficiency of putative C-terminal targeting signals on cottonseed malate synthase and bacterial chloramphenicol acetyl-transferase (CAT) were examined in BY-2 cells. Cotton suspension cells also were evaluated as another in vivo peroxisome targeting system. Ultrastructural views of BY-2 cells showed that the peroxisomes were relatively small (0.1-0.3 μm diameter), a characteristic of so-called “unspecialized” peroxisomes, Peroxisomes in cotton and tobacco cells were identified with anti-cottonseed catalase IgGs as distinct immunofluorescent particles clearly distinguishable from abundant immunofluorescent mitochondria and plastids, marked with antibodies to β-ATPase and stearoyl-ACP Δ 9 desaturase, respectively. The C-terminal ser-lys-leu (SKL) motif is a well-established peroxisome targeting signal (PTS 1) for mammals and yeasts, but not for plants. Antiserum raised against SKL peptides recognized proteins only in peroxisomes in cotton and tobacco cells. The necessity of SKL-COOH for targeting of proteins to plant peroxisomes had not been demonstrated; we showed that SKL-COOH was necessary for directing cottonseed malate synthase to BY-2 peroxisomes. KSRM-COOH, a conservative modification of SKL-COOH, was shown by others to be sufficient for redirecting CAT in stably-transformed Arabidopsis plants to the leaf peroxisomes. Here we show with the same CAT constructs (e.g., pMON316CAT-KSRM) that KSRM is sufficient for targeting transiently-expressed passenger proteins to unspecialized BY-2 peroxisomes. These results provide new direct evidence for the necessity of SKL-COOH (a type 1 PTS) and sufficiency of a conservative modification of the PTS 1 (KSRM-COOH) for in vivo, heterologous targeting of proteins to plant peroxisomes.

Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants.

Cell plate formation in tobacco root tips and synchronized dividing suspension cultured tobacco BY-2 cells was examined using cryofixation and immunocytochemical methods. Due to the much improved preservation of the cells, many new structural intermediates have been resolved, which has led to a new model of cell plate formation in higher plants. Our electron micrographs demonstrate that cell plate formation consists of the following stages: (1) the arrival of Golgi-derived vesicles in the equatorial plane, (2) the formation of thin (20 +/- 6 nm) tubes that grow out of individual vesicles and fuse with others giving rise to a continuous, interwoven, tubulo-vesicular network, (3) the consolidation of the tubulo-vesicular network into an interwoven smooth tubular network rich in callose and then into a fenestrated plate-like structure, (4) the formation of hundreds of finger-like projections at the margins of the cell plate that fuse with the parent cell membrane, and (5) cell plate maturation that includes closing of the plate fenestrae and cellulose synthesis. Although this is a temporal chain of events, a developing cell plate may be simultaneously involved in all of these stages because cell plate formation starts in the cell center and then progresses centrifugally towards the cell periphery. The "leading edge" of the expanding cell plate is associated with the phragmoplast microtubule domain that becomes concentrically displaced during this process. Thus, cell plate formation can be summarized into two phases: first the formation of a membrane network in association with the phragmoplast microtubule domain; second, cell wall assembly within this network after displacement of the microtubules. The phragmoplast microtubules end in a filamentous matrix that encompasses the delicate tubulo-vesicular networks but not the tubular networks and fenestrated plates. Clathrin-coated buds/vesicles and multivesicular bodies are also typical features of the network stages of cell plate formation, suggesting that excess membrane material may be recycled in a selective manner. Immunolabeling data indicate that callose is the predominant lumenal component of forming cell plates and that it forms a coat-like structure on the membrane surface. We postulate that callose both helps to mechanically stabilize the early delicate membrane networks of forming cell plates, and to create a spreading force that widens the tubules and converts them into plate-like structures. Cellulose is first detected in the late smooth tubular network stage and its appearance seems to coincide with the flattening and stiffening of the cell plate.