Use of agroclones towards understanding the interactions between Beet soil-borne virus and Polymyxa betae

Beet soil-borne virus (BSBV) is a virus infecting the Chenopodiaceae, a family to which belong the sugar beet (Beta vulgaris) and the spinach (Spinacia oleracea). It is transmitted by a soil-borne plasmodiophorid, Polymyxa betae Keskin, and is generally associated in the field with other viruses such as Beet virus Q (BVQ) or Beet necrotic yellow vein virus (BNYVV). Although BSBV seems to be asymptomatic and does not have any economical importance, its small genome with separated encoded functions makes it an ideal candidate for the study of the interactions between P. betae and its related viruses (BSBV, BVQ, BNYVV, etc.). The recent breakthroughs in viral inoculations by means of Agrobacterium tumefaciens enable the simultaneous inoculation of a wide number of plants. This technique is based on agroclones which are A. tumefaciens carrying a binary vector which encodes a full cDNA clone of a virus. Different techniques can be used to inoculate the bacteria to the plant. Among those, the sprout-vacuum infiltration (SVI) seems to be suitable for B. vulgaris while the leaf infiltration is a convenient method to apply to S. oleracea. The project of this Master thesis was to provide tools for studying the interaction between BSBV and its vector. Three main objectives were set: the development of an efficient SVI for B. vulgaris, the search of a systemic spinach cultivar to create a secondary pathosystem based on S. oleracea, and the creation of a reporter agroclone by means of a fluorescent marker (phiLOV2.1). On the one hand, 21 cultivars of sugar beet were assessed under different SVI protocols in order to select suitable hosts for the pathosystem while 12 spinach cultivars were leaf infiltrated at different optical densities. On the other hand, the BSBV capsid readthrough protein was replaced by the coding sequence of phiLOV2.1. The results obtained by (q)RT-PCR did not meet all the expectations. The SVI gave very weak or no detection, depending on the protocol used while even in the systemic spinach cultivar, no roots systemicity was found, making S. oleracea an unsuitable host for a secondary pathosystem of study. As for the reporter agroclone, the protein expressed was probably toxic or its fluorescence was not detectable. However, throughout the experiments performed, some very interesting features related to irregular BSBV detection were also pointed out. The silencing and spreading of the virus in the plant were probably involved and complementary experiments may be worth carrying out.