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Faba bean necrotic yellows virus (FBYNV), Milk vetch dwarf virus (MDV), and Subterranean clover stunt virus (SCSV) are nanoviruses that infect leguminous plants. From MDV- and SCSV-infected tissue we identified viral DNAs that encode a replication initiator protein (Rep), essential for replication of the multiple circular single-stranded DNAs of these viruses. These previously undescribed Rep proteins of MDV and SCSV are strikingly similar in sequence and functionally equivalent to the master Rep protein of FBYNV. Moreover, we demonstrated that the master Rep proteins of the three viruses are able to trigger replication of heterologous nanovirus DNAs. Such cross-species replication may reflect a considerable potential for genetic reassortment among nanoviruses in nature and be of significance for their evolution.

Tomato yellow leaf curl virus (TYLCV) is a whitefly-transmitted geminivirus with a monopartite genome. We have investigated the functions of the V1, V2, and C2 ORFs by mutational analysis. We analyzed the ability of TYLCV mutants containing disrupted ORFs V1, V2, or C2 to replicate, spread, and cause symptoms in Nicotiana benthamiana and tomato plants. All the mutants retained the capability of autonomous replication in protoplast-derived cells of tomato and leaf discs of N. benthamiana, although both V1 and V2 gene products appeared to play a role in the accumulation of viral single-stranded DNA. In contrast, none of the mutants was able to systemically infect tomato plants, demonstrating that the V1, V2, and C2 gene products are all required for a successful infection process in this host. The effect of the mutation in ORF C2 appeared to be host-specific, since N. benthamiana plants were systemically infected, although symptom development was attenuated.

Faba bean necrotic yellows virus (FBNYV) belongs to the nanoviruses, plant viruses whose genome consists of multiple circular single-stranded DNA components. Eleven distinct DNAs, 5 of which encode different replication initiator (Rep) proteins, have been identified in two FBNYV isolates. Origin-specific DNA cleavage and nucleotidyl transfer activities were shown for Rep1 and Rep2 proteins in vitro, and their essential tyrosine residues that catalyze these reactions were identified by site-directed mutagenesis. In addition, we showed that Rep1 and Rep2 proteins hydrolyze ATP, and by changing the key lysine residue in the proteins’ nucleoside triphosphate binding sites, demonstrated that this ATPase activity is essential for multiplication of virus DNA in vivo. Each of the five FBNYV Rep proteins initiated replication of the DNA molecule by which it was encoded, but only Rep2 was able to initiate replication of all the six other genome components. Furthermore, of the fiverep components, only the Rep2-encoding DNA was always detected in 55 FBNYV samples from eight countries. These data provide experimental evidence for a master replication protein encoded by a multicomponent single-stranded DNA virus.

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In inside-out vesicles, a large difference is observed in oxygen yield activity between pH 6.5 and 7.6 which is in contrast to right-side-out vesicles. The light saturation behaviour of the transition Sz + S1 also shows a pH dependence. In inside-out thylakoids, at pH 6.5, the light saturation curve of the S2 + SJ transitions exhibits 2 different light saturation processes whereas at pH 7.6 it resembles the light saturation behaviour of the other S-state transitions. This result is explained by the existence of a second donor in the S2 state (diffe~nt from the main donor), which cont~but~ to about 30% of the Sz + S3 transitions at pH 6.5. This second donor needs to be protonated and thus, is not active at pH 7.6 in inside-out thylakoids.pH effect Oxygen evolution Photosystem II Inside-out thylakoid Secondary donor