Geminiviruses, like all viruses, rely on the host cell machinery to establish a successful infection, but the identity and
function of these required host proteins remain largely unknown. Tomato yellow leaf curl Sardinia virus (TYLCSV), a
monopartite geminivirus, is one of the causal agents of the devastating Tomato yellow leaf curl disease (TYLCD). The
transgenic 2IRGFP N. benthamiana plants, used in combination with Virus Induced Gene Silencing (VIGS), entail an
important potential as a tool in reverse genetics studies to identify host factors involved in TYLCSV infection. Using these
transgenic plants, we have made an accurate description of the evolution of TYLCSV replication in the host in both space
and time. Moreover, we have determined that TYLCSV and Tobacco rattle virus (TRV) do not dramatically influence each
other when co-infected in N. benthamiana, what makes the use of TRV-induced gene silencing in combination with TYLCSV
for reverse genetic studies feasible. Finally, we have tested the effect of silencing candidate host genes on TYLCSV infection,
identifying eighteen genes potentially involved in this process, fifteen of which had never been implicated in geminiviral
infections before. Seven of the analyzed genes have a potential anti-viral effect, whereas the expression of the other eleven
is required for a full infection. Interestingly, almost half of the genes altering TYLCSV infection play a role in postranslational
modifications. Therefore, our results provide new insights into the molecular mechanisms underlying geminivirus infections,
and at the same time reveal the 2IRGFP/VIGS system as a powerful tool for functional reverse genetics studies.
