The role of post-Golgi transport pathways and sorting motifs in the plasmodesmal targeting of the movement protein (MP) of Ourmia melon virus (OuMV)

Natali Ozber; Paolo Margaria; Charles T. Anderson; Massimo Turina; Cristina Rosa

The presence of MP mutants in upper uninoculated leaves of N. benthamiana at 14 dpi was tested by western blot analysis; only MPwt and MP_D/G could be detected Virion accumulation was also determined in upper uninoculated leaves at 14 dpi by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using α-CP antiserum. All mutants, except the one carrying MP_D/G, failed to move systemically in both N. benthamiana and Arabidopsis (Table 1) . We also analyzed the coding sequence of MP in OuMV mutants by RT-PCR and sequencing of the PCR product to determine whether mutations in Y and LL motifs were reversed, or whether additional mutations were introduced during infection assays. The reversion of MP_D to wild-type was observed only in one Arabidopsis plant (Table 1) The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/724716 doi: bioRxiv preprint CP, and pBin61-GFP and collected samples from the infiltrated leaves at 24 hpi and 48 hpi. GFP signal was monitored at 36 hpi to confirm that all plant cells at the infiltrated areas were infected (Fig. S2 ). Quantification of RNA1 as proxy for replication was performed by qRT-PCR in all samples. At 24 hpi, all wild-type OuMV expressing samples displayed similar levels of RNA1 and all MP mutant expressing samples showed slightly higher amount of RNA1; however, this difference is not statistically significant, suggesting that all mutant constructs were similarly expressed via agrobacterium transient expression (Fig. S3 ). While all mutants were able to replicate at high levels at 48 hpi, we observed a 3.5-to 7-fold decrease in the replication of all mutants compared to the wild-type (Fig. 2E ). This result suggests that mutations in the Y and LL motifs of OuMV MP have also an effect in virus replication.