fourth joint meeting of dutch and german

Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus
resistance
K Boonrod 1 , D Galetzka 1,2 , PD Nagy 3 , U Conrad 4 and G Krczal 1
1 Centrum Grüne Gentechnik, RLP AgroScience GmbH, Neustadt, 2 Klinikum der Johannes
Gutenberg Universität Mainz, Institut für Humangenetik, Mainz, 3 Department of Plant Pathology,
University of Kentucky, Lexington, USA and 4 Institute of Plant Genetics and Crop Research
Gatersleben (IPK), Gatersleben
E-mail: gabi.krczal@dlr.rlp.de
Crop loss due to viral diseases is still a major problem for agriculture today. We present a strategy
to achieve virus resistance based on the expression of single-chain Fv fragments (scFvs) against a
conserved domain in a plant viral RNA-dependent RNA polymerase (RdRp), a key enzyme in virus
replication. The selected scFvs inhibited complementary RNA synthesis of different plant virus
RdRps in vitro and virus replication in planta. Moreover, the scFvs also bound to the RdRp of the
distantly related hepatitis C virus. T1 and T2 progeny of transgenic lines of Nicotiana benthamiana
expressing different scFvs either in the cytosol or in the endoplasmic reticulum showed varying
degrees of resistance against four plant viruses from different genera, three of which belong to the
Tombusviridae family. Virus resistance based on antibodies to RdRps adds another tool to the
repertoire for combating plant viruses.
Translocation and immunolocalisation of Watermelon chlorotic stunt virus, WmCSV, in its
vector Bemisia tabaci (Genn.)
I Fahmy Farouk 1 , D-E Lesemann 2 and S Winter 1
1 2
DSMZ Plant Virus Division, Braunschweig and BBA, Dept. of Plant Virology, Microbiology and
Biosafety, Braunschweig
E-mail: s.winter@bba.de
To investigate the mechanisms of begomovirus transmission by Bemisia tabaci, acquisition and
translocation of Watermelon chlorotic stunt virus, WmCSV, in the insect was studied. A whitefly
transmissible WmCSV isolate from Sudan and 4 non transmissible mutants carrying a single amino
acid mutation in the core region of the capsid protein were used for the translocation experiments.
B. tabaci fed on virus infected watermelon plants were transferred to non host plants for virus
discharge and subsequently used to infect watermelons. Transmission was taken as evidence for a
functional interaction between virions, the gut membrane and the primary or accessory salivary
glands. Fresh, dissected organs from viruliferous whiteflies feeding on wild type or mutant virus
were examined by PCR to determine the presence of virus. WmCSV was detected in the midgut,
hemocoel and salivary glands of B. tabaci for the transmissible and the non transmissible virus
mutants. However, in Trialeurodes vaporariorum (a non vector of geminiviruses), wild-type
WmCSV was detected in the midgut only, thus suggesting that the virus was not capable of
crossing the gut wall. In contrast, in B. tabaci, the accessory and/or the primary salivary glands
apparently present the significant epithelial barrier to virus transmission. Hence, for the
begomovirus WmCSV and its whitefly vector, a pathway similar to the luteovirus / aphid
translocation is expected. In immunolocalization studies with organs excised from viruliferous
insects carrying wild type or non transmissible virus mutants, a specific labelling with WmCSV
antiserum was obtained at the microvilli region of the epithelial cells of the gut wall food canal,
indicating for putative virus adsorption sites or, for a putative receptor-mediated delivery to the
hemoceol. Respective experiments carried out with primary and accessory salivary glands so far
did not indicate for specific adhesion/entry sites into these organs.
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