Tomato yellow leaf curl virus (TYLCV) (Geminiviridae: Begomovirus) is exclusively vectored by the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). TYLCV transmission depends upon a 63-kDa GroEL protein produced by the vector's endosymbiotic bacteria. B. tabaci is a species complex comprising several genetically distinct biotypes that show different secondary-symbiont fauna. In Israel, the B biotype harbors Hamiltonella, and the Q biotype harbors Wolbachia and Arsenophonus. Both biotypes harbor Rickettsia and Portiera (the obligatory primary symbionts). The aim of this study was to determine which B. tabaci symbionts are involved in TYLCV transmission using B. tabaci populations collected in Israel. Virus transmission assays by B. tabaci showed that the B biotype efficiently transmits the virus, while the Q biotype scarcely transmits it. Yeast two-hybrid and protein pulldown assays showed that while the GroEL protein produced by Hamiltonella interacts with TYLCV coat protein, GroEL produced by Rickettsia and Portiera does not. To assess the role of Wolbachia and Arsenophonus GroEL proteins (GroELs), we used an immune capture PCR (IC-PCR) assay, employing in vivo- and in vitro-synthesized GroEL proteins from all symbionts and whitefly artificial feeding through membranes. Interaction between GroEL and TYLCV was found to occur in the B biotype, but not in the Q biotype. This assay further showed that release of virions protected by GroEL occurs adjacent to the primary salivary glands. Taken together, the GroEL protein produced by Hamiltonella (present in the B biotype, but absent in the Q biotype) facilitates TYLCV transmission. The other symbionts from both biotypes do not seem to be involved in transmission of this virus.