1.13.16
IDENTIFICATION OF THE PROTEINS ASSOCIATED WITH CIRCULATIVE TRANSMISSION OF BARLEY YELLOW DWARF LUTEOVIRUSES FROM SITOBION AVENAE ANDSCHIZAPHIS GRAMINUM

WANG XIFENG and ZHOU GUANGHE

Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, China

Background and objectives
The barley yellow dwarf luteoviruses (BYDVs) are transmitted in a circulative non-propagative manner by cereal aphids. Briefly, this means that virus particles are ingested along with phloem sap from infected host plants and transcellularly transported through the hindgut into the hamocoel. Upon contacting the accessory salivary glands (ASG), they may be transported through this gland, eventually arriving at the salivary duct from which they are excreted with the saliva when the aphid feeds [1]. Luteoviruses display a high degree of vector specificity among aphid species. These well-developed specificities suggest an intimate association between a virus and its vectors in which both surface domains of the viral capsid and proteins of the aphid are involved. Studies on the identification on aphid-derived components interacting with luteoviruses have recently been initiated [2]. Here we reported the identification of the proteins associated with transmission of BYDV-GAV isolate from its vectors, Sitobion avenae and Schizaphis graminum.

Materials and methods
BYDV-GAV isolate is a MAV-Iike isolate found in China. It has a strong serological reaction with MAV antiserum from Dr Lister. Its vectors S. avenae and S. graminum, and non-vector Rhodopalosiphum padi were reared on wheat in an illuminated incubator at 201C with a photoperiod of 16 h/day. The identification of proteins associated with transmission of BYDV was conducted according to Van den Heuvel's method [2].

Results and conclusions
To detect whether BYDV showed affinity to protein components from its vector aphids, we separated whole-body extracts of the aphids by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred the proteins to nitrocellulose. The proteins were reacted with purified GAV virus and probed with MAV IgG labelled with alkaline phosphatase. In this way, two proteins of 31 kDa (p31) and 44 kDa (p44) displayed the highest virus-binding capacity, and were identified in the whole-body extracts of S. avenae and S. graminum. BYDV-binding proteins were not found in the whole-body extracts of R. padi, which do not transmit the GAV isolate. Selecting beet black scorch virus (BBSV) transmitted by soil fungus and its antiserum as a check, we did not find the proteins that showed any affinity with them in the whole-body extracts of the aphid species. So, p31 and p44 may be the proteins associated with transmission of BYDV in its vectors. Furthermore, we dissected the bodies of S. graminum, obtaining the tissues of ASG and gut respectively, and only found p31 and p44 existing in ASG from the protein extracts of them.

Isolation of p31 and p44 from S. graminum was carried out by electro-elution from gel sliccs after SDS-PAGE of whole extracts. Samples (50 mg) of purified p31 and p44, emulsified in Freund's incomplete adjuvant, were injected into two rabbits respectively at day 1, 8, 15, 22 and 29 for antiserum production. Using these antisera as the first antibody to probe the proteins of the aphids (Western blotting), p31 and p44 were also found in S. avenae and S. graminum, but not in R. padi.

References
1. Gildow FE, Gray SM, 1993. Phytopathology 83, 1293-1302.
2. Van den Heuvel JFIM, Verbeek M, van der Wilk F, 1994. Journal of General Virology 75, 2559-65.