1.13.2S
A SECOND HELPER PROTEIN IS REQUIRED FOR THE APHID TRANSMISSION OF CAULIFLOWER MOSAIC VIRUS

V LEH1, E JACQUOT1, A GELDREICH1, D LECLERC2, M CERUTTI3, P YOT1, M KELLER1 and S BLANC3

1IBMP, CNRS, 67084 Strasbourg cedex, France; 2FMI, PO Box 2543, Basel, Switzerland; 3Unite BCM, INRA-CNRS, 30380 Saint Christol-les-Aids, France

Background and objectives
Helper-dependency is a common feature of plant viruses transmitted by vectors in a non-circulative manner [1]. Helpers are virus-encoded, non-structural proteins which are predicted to be capable of interacting, via two distinct domains, with both the virus coat protein (CP) and a putative receptor in the insect mouthparts, thus creating a reversible 'bridge' between the two. Several lines of evidences are consistent with this bridge hypothesis. However, recent observations indicate that the mode of action of helpers in the potyvirus and caulimovirus groups may be significantly different. Two genes (CP and helper genes) have been demonstrated to be involved in potyvirus aphid transmission whereas in addition to virus particle and helper, the requirement of a third factor for the aphid transmission of cauliflower mosaic virus (CaMV) could not be ruled out [2]. Here we demonstrate that this third factor is a 15 kDa protein (P15) encoded by the virus gene III which can interact in vitro with the CaMV helper protein (P18, product of gene II). The correlation between P18-P15 binding and the success of aphid transmission is being investigated and the mechanisms by which P15 participates in the virus/vector interaction will be discussed.

Materials and methods
P18-P15 binding assay: a protein blotting-protein overlay technique was used. P18 was expressed in insect cells via a baculovirus recombinant while P15 was expressed and purified using a bacterial expression system.
Aphid transmission experiments: P18, P15 and purified virions were acquired sequentially (singly or mixed in various combinations) by aphids through parafilm membranes. Aphids were then transferred onto healthy plants (10 aphids per test plant) and symptom appearance was noted three weeks later.

Results and conclusions
When aphids were fed P18 and purified virions, very little or no virus transmission was observed (transmission rate below 5%). In contrast, the addition of P15 to the solution acquired by aphids increased the transmission rate up to nearly 60%. This result demonstrated that P15 plays a key role in the molecular mechanisms of CaMV aphid transmission. Furthermore, the N-terminal domain of P15 was demonstrated to interact, in vitro, with the C-terminal domain of P18. Whether this interaction is a part of the P15 mode of action is currently being investigated and will be discussed. The involvement of three virus-encoded proteins in regulating vector transmission is a unique phenomenon among non-circulative plant viruses. This demonstrates that the nature of the 'bridge' is not uniform and supports the hypothesis that different viruses have evolved their own molecular means to converge to a helper-strategy for regulating vector transmssion.

References
1. Pirone TP, Blanc S, 1996. Annual Review of Phytopathology 34, 227-247.
2. Blanc S, Cerutti M, Usmany M, Viak JM, Hull R, 1993. Virology 192, 643-650.