PHYTO MEDIZIN Mitteilungen der Deutschen ... - Die DPG

hesperis and PVM particles were detected by electron microscopy. In addition
PVM coat protein was shown by ELISA as well as by Western-Blot and
PVM-RNA was amplified by Reverse –Transcriptase – PCR.
Characterisation of proteins involved in translocation of the geminivirus
Watermelon chlorotic stunt virus in its vector Bemisia tabaci (Genn.)
A Gadelseed 1 , I Abdullahi 1 , GA Dafalla 2 and S Winter 1; 1 DSMZ Plant Virus Division,
BBA, Braunschweig and 2 Plant Pathology Center, Fac. of Agric. Sci, Gezira University,
Wadmedani, Sudan
Begomoviruses rely on Bemisia tabaci vector insects for horizontal transmission.
For this process, the virus passages the vector by crossing the midgut
and the salivary glands (PSG & ASG) representing epithelial barriers for
vector translocation. At each barrier, the transcytosis (endocytosis/exocytosis)
event is believed to be based on a receptor-mediated mechanism
where vector receptors interact with virions. To investigate the
biochemistry of whitefly vector / begomovirus interaction leading to successful
virus transmission, proteins extracted from B. tabaci insects were reacted
with purified WmCSV protein preparations. Upon SDS-PAGE of total B.
tabaci protein extracts followed by virus overlay assay (far western analysis),
five polypeptide species, 63, 51.3, 29.5, 26.3, and 25.7 kDa, were identified
specifically reacting with purified WmCSV. None of these proteins reacted
with preparations of a purified tombusvirus or with antibodies against the 65
kDa GroEl protein, a protein produced by insect endosymbiotic bacteria often
found in B. tabaci and also implicated in virus transmission. Similar proteins
to those from B. tabaci, were also identified in non vector insects (Trialeurodes
vaporariorum and Aphis craciphora), however, the
29.5 and 26.3 kDa protein reactions were only found in B. tabaci. Using
differential protein purification protocols, soluble and membrane bound
whitefly proteins were purified, separated by 2D electrophoresis and analysed
in far western assays. Protein spots of the insoluble protein fractions separated
by 2D electrophoresis and characterised by a specific reaction in far
western analysis with WmCSV virion preparations, were further subjected to
in-gel trypsin digestion and analysed by mass spectrometry and MS-MS mass
measurement in Q-TOF. Results of the preliminary protein analysis will be
presented.
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