ABSTRACT
Eukaryotic translation initiation factors (eIFs) are important for mRNA translation but also pivotal for plant-virus interaction. Most of these plant-virus interactions were found between plant eIFs and the viral protein genome-linked (VPg) of potyviruses. In case of lost interaction due to mutation or deletion of eIFs, the viral translation and subsequent replication within its host is negatively affected, resulting in a recessive resistance. Here we report the identification of the Beta vulgaris Bv-eIF(iso)4E as a susceptibility factor towards the VPg-carrying beet chlorosis virus (genus Polerovirus). Using yeast two-hybrid and bimolecular fluorescence complementation assays, the physical interaction between Bv-eIF(iso)4E and the putative BChV-VPg was detected, while the VPg of the closely related beet mild yellowing virus (BMYV) was found to interact with the two isoforms Bv-eIF4E and Bv-eIF(iso)4E. These VPg-eIF interactions within the polerovirus-beet pathosystem were demonstrated to be highly specific, as single mutations within the predicted cap-binding pocket of Bv-eIF(iso)4E resulted in a loss of interaction. To investigate the suitability of eIFs as a resistance resource against beet infecting poleroviruses, B. vulgaris plants were genome edited by CRISPR/Cas9 resulting in knockouts of different eIFs. A simultaneous knockout of the identified BMYV-interaction partners Bv-eIF4E and Bv-eIF(iso)4E was not achieved, but Bv-eIF(iso)4EKO plants showed a significantly lowered BChV accumulation and decrease in infection rate from 100% to 28.86%, while no influence on BMYV accumulation was observed. Still, these observations support that eIFs are promising candidate genes for polerovirus resistance breeding in sugar beet.
ABSTRACT
In the field of plant virology, the usage of reverse genetic systems has been reported for multiple purposes. One is understanding virus-host interaction by labelling viral cDNA clones with fluorescent protein genes to allow visual virus tracking throughout a plant, albeit this visualization depends on technical devices. Here we report the first construction of an infectious cDNA full-length clone of beet mosaic virus (BtMV) that can be efficiently used for Agrobacterium-mediated leaf inoculation with high infection rate in Beta vulgaris, being indistinguishable from the natural virus isolate regarding symptom development and vector transmission. Furthermore, the BtMV clone was tagged with the genes for the monomeric red fluorescent protein or the Beta vulgaris BvMYB1 transcription factor, which activates the betalain biosynthesis pathway. The heterologous expression of BvMYB1 results in activation of betalain biosynthesis genes in planta, allowing visualization of the systemic BtMV spread with the naked eye as red pigmentation emerging throughout beet leaves. In the case of BtMV, the BvMYB1 marker system is stable over multiple mechanical host passages, allows qualitative as well as quantitative virus detection and offers an excellent opportunity to label viruses in plants of the order Caryophyllales, allowing an in-depth investigation of virus-host interactions on the whole plant level.
