Plum pox virus (PPV) is a member of the genus Potyvirus that is able to infect a large variety of plant species, including trees of the genus Prunus, its natural host. When some PPV isolates are propagated for an extended time in herbaceous plants, their ability to infect trees is reduced. The molecular basis of this change in host infectivity is poorly understood. We report the construction of hybrid viruses from cDNA clones of two D-strain isolates of PPV, PPV-D and PPV-R, which differ in their host range. PPV-D can infect GF305 peach seedlings efficiently, however, it is unable to infect Nicotiana clevelandii plants. Conversely, PPV-R infects N. clevelandii, but not GF305 peach seedlings. The analyses of the hybrid viruses showed that, although determinants of PPV pathogenicity are extensively spread throughout the PPV genome, the 3' terminal region of the PPV-R genome, including the 3' noncoding region and the coding regions for the coat protein (CP), NIb, and part of NIa protein, is sufficient to confer infectivity of N. clevelandii in a PPV-D background. Our data demonstrate a high concentration of amino acid substitutions in the CP and a host-specific effect of a deletion at the N terminus of this protein in PPV pathogenicity in peach and N. clevelandii infectivity experiments. These results suggest that relevant host specificity determinants are located in the N-terminal region of the CP. The analyses of the PPV-R and PPV-D chimeras also showed that key host-specific pathogenicity determinants lie in the 5' terminal third of the PPV genome, a region that spans proteins P1, HCPro, and P3. The selection of mutations in only a few specific residues in proteins P1, P3, and 6K1 after partial adaptation of a chimeric virus (BD-GFP) to N. clevelandii further suggests a relevant role for these proteins in host adaptation.

Lipid droplets (LDs) are ubiquitous organelles in plant cells, but their physiological roles are largely unknown. To gain insight into the function of LDs in plants, we have characterized the Arabidopsis homologs of SEIPIN proteins, which are crucial factors for LD biogenesis in yeast and animals. is expressed almost exclusively in embryos, while and have broader expression profiles with maximal levels in embryos and pollen, where LDs accumulate most abundantly. Genetic analysis demonstrates that all three contribute to proper LD biogenesis in embryos, whereas in pollen, only and play a significant role. The double and triple mutants accumulate extremely enlarged LDs in seeds and pollen, which hinders their subsequent mobilization during germination. Interestingly, electron microscopy analysis reveals the presence of nuclear LDs attached to type I nucleoplasmic reticulum in triple mutant embryos, supporting that SEIPINs are essential for maintaining the correct polarity of LD budding at the nuclear envelope, restricting it to the outer membrane. In pollen, the perturbations in LD biogenesis and turnover are coupled to reduced germination in vitro and with lower fertilization efficiency in vivo. In seeds, germination per se is not affected in and triple mutants, but there is a striking increase in seed dormancy levels. Our findings reveal the relevance of SEIPIN-dependent LD biogenesis in pollen transmission and in adjusting the timing of seed germination, two key adaptive traits of great importance in agriculture.

The potyviruses Plum pox virus (PPV) and Tobacco vein mottling virus (TVMV) have distinct host ranges and induce different symptoms in their common herbaceous hosts. To test the relevance of the P1 protein in host compatibility and pathogenicity, hybrid viruses were constructed in which the P1 coding sequence of PPV was completely or partially replaced by the corresponding sequences from TVMV. Infections induced by these chimeric viruses revealed that the TVMV P1 and a PPV/TVMV hybrid P1 proteins are functionally equivalent in herbaceous plants to the P1 protein of a PPV isolate adapted to these hosts, in spite of having high sequence divergence. Moreover, the presence of TVMV P1 sequences enhanced the competence of a low-infectivity PPV-D-derived chimera in Nicotiana clevelandii. Conversely, all PPV/TVMV hybrids were unable to infect Prunus persicae, a specific host for PPV, suggesting that TVMV P1 is not functionally competent in this plant. Together, these data highlight the importance of the P1 protein in defining the virus host range.

The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1. Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.