PHYTO MEDIZIN Mitteilungen der Deutschen ... - Die DPG

construction of a BStMV full-length cDNA clone (pBStMV-FL) viral RNA
was extracted from a French BStMV isolate (11-Cal; BStMV-wt) and cDNA
fragments were amplified by RT-PCR. The fragments were cloned in succession
under control of an enhanced Cauliflower mosaic virus (CAMV) 35Spromoter
and its 3'-polyadenylation signal, resulting in pBStMV-FL. This
plasmid was transferred into plants by particle bombardment. The virus deriving
from pBStMV-FL was able to infect the plant systemically and was
detected immunologically. Comparison of the host spectrum of BStMV-FL
and BStMV-wt revealed no differences in terms of infected plants and symptom
severity. Six species were identified as host plants, at which Bromus
mollis, Hordeum vulgare and Triticum aestivum showed obvious virus symptoms.
BStMV-FL was labelled with a green fluorescent protein (GFP) at two
different positions in the genome. For this purpose the gfp gene was integrated
in-between P1 and helper component protease (pBStMV-P1/GFP/HC-
Pro) and in-between nuclear inclusion body b and coat protein (pBStMV-
NIb/GFP/CP). Four weeks after particle bombardment virus symptoms were
observed. BStMV-CP and GFP were detected by electroblot immunoassays.
Fluorescence of GFP was visualized with a confocal-laser scanning microscope.
Therefore, correct proteolytic processing of GFP occurred in BStMV-
P1/GFP/HC-Pro as well as in BStMV-NIb/GFP/CP and the released GFPs
were functional. In addition, eriophyid mites were collected from the field
and used successfully to transmit BStMV. Altogether these findings enable
further studies on infection, replication and transport of BStMV-FL and mutants
thereof in plants as well as determination of proteins and motifs involved
in vector transmission.
TSWV particle asssembly: in vivo interactions between the structural
nucleoprotein and G1 spike protein
M Snippe 1 , JW Borst 2 , R Goldbach 1 and R Kormelink 1 ; 1 Laboratory of Virology,
Wageningen University and 2 Laboratory of Biochemistry, Wageningen University
TSWV (Tomato spotted wilt virus) is a member of the Bunyaviridae family.
Virus particles are spherical and membrane bound, containing spike proteins
that consist of two glycoproteins, G1 and G2. The core contains ribonucleoproteins
(RNPs) that consists of genomic RNA tightly associated with the
nucleoprotein (N) and small amounts of the viral RNA-dependent RNA polymerase.
Enveloped virus particles arise as a result of RNP envelopment
with membranes from the Golgi apparatus containing G1 and G2. To investigate
in vivo interactions between the structural proteins involved in virus
assembly, fluorescence techniques (FRET: fluorescence resonance energy
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