Holger Jeske

Geminiviruses manage the transport of their DNA within plants with the help of three proteins, the coat protein (CP), the nuclear shuttle protein (NSP), and the movement protein (MP). The DNA-binding capabilities of CP, NSP, and MP of Abutilon mosaic virus (AbMV; family Geminiviridae ; genus Begomovirus ) were scrutinized using gel mobility shift assays and electron microscopy. CP and NSP revealed a sequence-independent affinity for both double-stranded and single-stranded DNA, as has been previously reported for other begomoviruses. MP interacted selectively with dimeric supercoiled plasmid DNA in the electrophoretic assay. Further apparent size- and form-selective binding capacities of MP have been previously reported for another geminivirus ( Bean dwarf mosaic virus ), but in the case of AbMV, they have been identified as the result of electrophoretic interference rather than of complex formation. Without these complications, electron microscopy confirmed the assembly of double-stranded supercoiled DNA with NSP and MP into conspicuous structures and provided the first direct evidence for cooperative interaction of MP, NSP, and DNA. Based on these results and previous ones, a transport model of geminiviruses is discussed in which NSP packages DNA and MP anchors this complex to the protoplasmic leaflets of plasma membranes and microsomes for cell-to-cell movement.

The single-stranded circular DNA ofAbutilon mosaic geminivirionsis complemented to double-stranded DNA by host proteins after infecting cells. This double-stranded DNA serves as a template for replication as well as transcription and is assembled into host nucleosomes, yielding circular viral minichromosomes. Their chromatin structure was analyzed by use of isolated nuclei combining nuclease sensitivity assays with ligation-mediated PCR, evaluating nucleosomal ladders and topoisomer distributions in one- and two-dimensional gels by blot hybridization. Viral minichromosomes were found to exist in at least two defined structures covered with 11 or 12 nucleosomes, leaving open gaps accessible for interactions with other host factors. Nucleosome-free gaps were colocalized with promoter structures and the origin of replication in both components of genomic DNA (DNA A and DNA B). Nucleosomes were positioned over the entire viral DNA in at least two alternative phases with different periodicities. The distribution of topoisomers of monomeric viral circular double-stranded DNA confirmed the presence of variable chromatin structures revealing maximum frequencies of molecules with either 11, 12, or 13 superhelical turns (corresponding to respective numbers of nucleosomes) at maximal frequency at different stages during leaf development of infected plants. The role of variable chromatin structures for gene regulation of geminiviruses is discussed.

Geminiviruses package single-stranded circular DNA and replicate via double-stranded DNA intermediates. During the past decade, increasing evidence has led to the general acceptance that their replication follows a rolling-circle replication mechanism like bacteriophages with single-stranded DNA. In a recent study, we showed that this is also true forAbutilon mosaic geminivirus(AbMV), but that this particular virus may also use a recombination-dependent replication (RDR) route in analogy to T4 phages. Because AbMV is a special case, since it has been propagated on ornamental plants for more than a hundred years, it was interesting to determine whether RDR is common among other geminiviruses. We analyzed geminiviruses from different genera and geographic origins by using BND cellulose chromatography in combination with an improved high resolution two-dimensional gel electrophoresis, and we conclude that multitasking in replication is widespread, at least forAfrican cassava mosaic,Beet curly top,Tomato golden mosaic, andTomato yellow leaf curl virus.

Geminiviruses have spread worldwide and have become increasingly important in crop plants during recent decades. Recombination among geminiviruses was one major source of new variants. Geminiviruses replicate via rolling circles, confirmed here by electron microscopic visualization and two‐dimensional gel analysis of Abutilon mosaic virus (AbMV) DNA. However, only a minority of DNA intermediates are consistent with this model. The majority are compatible with recombination‐dependent replication (RDR). During development of naturally infected leaves, viral intermediates compatible with both models appeared simultaneously, whereas agro‐infection of leaf discs with AbMV led to an early appearance of RDR forms but no RCR intermediates. Inactivation of viral genes ac2 and ac3 delayed replication, but produced the same DNA types as after wild‐type infection, indicating that these genes were not essential for RDR in leaf discs. In conclusion, host factors alone or in combination with the viral AC1 protein are necessary and sufficient for the production of RDR intermediates. The consequences of an inherent geminiviral recombination activity for the use of pathogen‐derived resistance traits are discussed.

Abutilonmosaic virus (AbMV), a bipartite geminivirus of the genus Begomovirus, has been vegetatively propagated for many years inAbutilon sellovianumin which it is strictly phloem‐restricted. Usingin situhybridization and immunological analyses, the tissue tropism of AbMV in the laboratory hostNicotiana benthamianawas compared with that of two other bipartite begomoviruses, African cassava mosaic virus (ACMV) and tomato golden mosaic virus (TGMV). Analysis of the first systemically infected leaves and longitudinal sections of axillary and flower buds revealed that all three viruses are initially confined to the vascular traces, although both ACMV and TGMV are later detectable in nearly all tissue types. In contrast, AbMV remained strictly phloem‐limited in this host throughout the course of infection. The ability of ACMV and TGMV to move out ofN. benthamianaphloem tissues is correlated with the development of severe symptoms in comparison with the mild symptoms associated with AbMV infection. It was also demonstrated thatSida micranthamosaic virus, a virus that is closely related to AbMV, is phloem‐limited inMalva parvifloraeven though it induces severe leaf curl, stunting and necrosis in this host. The present data demonstrate that bipartite begomoviruses can exhibit strikingly different patterns of tissue tropism.