Infection of cowpea mesophyll protoplasts with cowpea mosaic virus

Hibi, Tadaaki; Rezelman, G.; Kammen, A. van

doi:10.1016/0042-6822(75)90107-5

Cited by 76 publications

(50 citation statements)

References 21 publications

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“…After 30 min, they were collected by centrifugation, washed with 0.6 M-mannitol and incubated (1 x 106/ml) at 25 °C under continuous illumination as described by Rottier et al (1979). The percentage of protoplasts infected with CPMV was determined by fluorescent staining with anti-CPMV serum and fluorescein isothiocyanate-conjugated goat anti-rabbit Ig (Nordic) 24 h after inoculation (Hibi et al, 1975;Maule et al, 1980). This inoculation method was very efficient; using only 0-5 to 1 btg CPMV RNA, approximately 80~ of the protoplasts became infected (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Expression of Cowpea Mosaic Virus M RNA in Cowpea Protoplasts

Rezelman

Kammen

Wellink

1989

Journal of General Virology

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SUMMARYCowpea mosaic virus (CPMV) M RNA is translated in vitro into two polyproteins of Mr values 105000 (105K) and 95K. Using antiserum against the small capsid protein VP23, these proteins have now been detected in cowpea protoplasts, a few hours after inoculation with CPMV. These proteins could also be detected at later stages of infection, but only when proteolytic processing was inhibited by the addition of ZnCI> Using antiserum against a synthetic peptide, corresponding to a part of the overlapping C-terminal ends of the 58K and 48K proteins, the 58K protein, which is the aminoterminal cleavage product of the 105K protein, was found in the cytoplasmic fraction of infected protoplasts. The 48K protein, derived from the 95K protein, was detected in both the cytoplasmic and membrane fractions of protoplasts. The presence of the 105K, 95K, 58K and 48K proteins in CPMV-infected protoplasts indicates that separate initiation codons on the M RNA are used in vivo to produce the 105K and 95K polyproteins, as demonstrated in vitro.

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Section: Methodsmentioning

confidence: 99%

Expression of Cowpea Mosaic Virus M RNA in Cowpea Protoplasts

Rezelman

Kammen

Wellink

1989

Journal of General Virology

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“…Cowpea plants were grown and mesophyll protoplasts isolated from primary leaves of 10-day-old plants, essentially as described by Hibi et al (1975) and modified by van Beek et al (1985). All media contained 0.6 M-mannitol and 2.5 mM-MES, and were adjusted to pH 5-7.…”

Section: Methodsmentioning

confidence: 99%

Tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts

Lent¹,

Storms²,

Meer³

et al. 1991

Journal of General Virology

166

104

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In cowpea plant cells infected with cowpea mosaic virus, tubular structures containing virus particles are formed in the plasmodesmata between adjacent cells; these structures are supposedly involved in cell-to-cell spread of the virus. Here we show that similar tubular structures are also formed in cowpea protoplasts, from which the cell wall and plasmodesmata are absent. Between 12 and 21 h post-inoculation, tubule formation starts in the periphery of the protoplast at the level of the plasma membrane. Upon assembly, the viruscontaining tubule is enveloped by the plasma membrane and extends into the culture medium. This suggests that the tubule has functional polarity and makes it likely that a tubule 'grows' into a neighbouring cell in vivo. On average, 75 % of infected protoplasts were shown to possess tubular structures extending from their surface. The tubule wall was 3 to 4 nm thick and they were up to 20 gm in length, as shown by fluorescent light microscopy and negative staining electron microscopy. By analogy to infected plant cells, both the viral 58K/48K movement and capsid proteins were located in these tubules, as determined by immunofluorescent staining and immunogold labelling using specific antisera against these proteins. These results demonstrate that the formation of tubules is not necessarily dependent on the presence of plasmodesmata or the cell wall, and that they are composed, at least in part, of virus-encoded components.

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“…Poly-D-lysine concentrations higher than 1/~g/ml caused a marked decrease of the number of protoplasts surviving the infection procedure. The detrimental effect of poly-cations in cowpea protoplasts has been also observed by Hibi et al (1975) and Rao & Hiruki (1978). Poly-cations were also required for the effective infection of cowpea protoplasts with clover yellow mosaic virus (CYMV) (Rao & Hiruki, 1978), whereas infection of such protoplasts with cowpea mosaic virus (CPMV) (Hibi et al, 1975) and cucumber mosaic virus (Koike et al, 1976) was achieved in the absence of poly-cations.…”

Section: Discussionmentioning

confidence: 61%

“…For the inoculation of cowpea protoplasts with TMV or C-TMV, 0.1 M-potassium phosphate buffer pH 5.4 at 0 °C was better than 0.01 M-potassium citrate pH 5.2 at 25 °C, used for the infection of such protoplasts with CPMV (Hibi et al, 1975) and CYMV (Rao & Hiruki, 1978). The amount of virus synthesized in the infected protoplasts 24 h after inoculation was at least three times higher whereas the frequency of infection was similar.…”

Section: Discussionmentioning

confidence: 99%

“…After inoculation, the protoplasts were washed four times with sterile 0.6 M-mannitol, 10 mM-CaCI z to remove poly-D-lysine and unadsorbed virus and were then incubated for appropriate periods in the medium of Hibi et al (1975) containing 5 #g/ml aureomycin instead of 1 /tg/ml cephaloridin. The determination of the frequency of infection of the cowpea protoplasts was as described by Otsuki & Takebe (1969).…”

Section: Discussionmentioning

confidence: 99%

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