Asynchronous viral spread of two unrelated viruses determines Lettuce Big Vein Disease symptom development

Lettuce big-vein disease (LBVD) is a major disease affecting lettuce cultivation worldwide. LBVD is caused by two unrelated negative-stranded RNA viruses, that is, Mirafiori lettuce big-vein virus (MiLBVV) and Lettuce big-vein associated virus (LBVaV) both vectored by the soilborne fungus Olpidium virulentus. Despite extensive research, a synergistic effect between the two viruses has not been observed, while both viruses individually have been suggested to be the causal agent for the disease. By performing lettuce reinfections using a large soil sample collection carrying LBVD infested O. virulentus spores, the presence of LBVaV was consistently established in diseased lettuce heads, while MiLBVV infections were apparently less prevalent. Yet, aboveground infections with MiLBVV corresponded with strong disease symptoms. Strikingly, the spread of LBVaV from the root to shoot always preceded that of MiLBVV. The LBVaV systemic spread was highly synchronized between plants, while MiLBVV spread was always delayed and asynchronous. A pan-genome analysis revealed independent segment reassortments for both viruses indicative of mixed field infections over the sampled period. Yet, RNA segment abundance was highly conserved for both viruses between all re-infections, suggesting that segment abundance has a regulatory role for the two individual viruses, but that segment abundance is not impacted by the presence of the other two viruses. The pan-genome analysis also revealed different evolutionary rates of the viral ORFs suggesting that mutagenesis of certain ORFs compromises viral fitness and thus revealing a potential weak spot for both viruses. ImportanceLettuce big-vein disease (LBVD) is an important viral disease complex affecting lettuce cultivation worldwide. Here we reveal a synergistic interaction between the two principal associated segmented RNA viruses, Mirafiori lettuce big-vein virus (MiLBVV) and Lettuce big-vein associated virus (LBVaV). We show unequivocally that MiLBVV is the main virus responsible for severe disease symptoms in lettuce heads. Yet, MiLBVV root-to-shoot movement was in our conditions always preceded by LBVaV movement into the lettuce heads. Arguably, LBVaV thus facilitates the root-to-shoot movement of MiLBVV. Moreover, both viruses undergo segment reassortment increasing their genome plasticity and the reassortment events appeared to be independent events with mixed infections. Finally, we provide data that both viruses regulate gene expression via the copy number of their RNA segments, but that the genome formula does not change in dual infections. We thus provide evidence for a synergistic interaction needed for strong LBVD symptoms.