Novel System for the Simultaneous Analysis ofGeminivirus DNA Replication and Plant Interactions inNicotianabenthamiana

Abstract

The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based on Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replicationassociated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R 2 , R 5 , R 7 and K 11 or H 56 , L 57 and H 58 prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.

An RGG sequence in the replication-associated protein (Rep) of Tomato yellow leaf curl Sardinia virus is involved in transcriptional repression and severely impacts resistance in Rep-expressing plants

“…The karyophilic property of Rep of begomoviruses is consistent with its role during virus replication. Our results are in accordance with previous immunocytochemical staining of the Tomato golden mosaic virus (TGMV) Rep in infected plants (Nagar et al, 1995) and nuclear targeting of African cassava mosaic virus Rep-GFP fusion proteins (Hong et al, 2003). Our work further shows that a large Cterminal deletion (aa 131-359) in TYLCSV Rep does not prevent nuclear accumulation and suggests that the RGG sequence is not involved in this process.…”

A single amino acid change in a geminiviral Rep protein differentiates between triggering a plant defence response and initiating viral DNA replication

“…Moreover, efficient viral DNA replication also depends on interactions between Rep and the virus-encoded replication-enhancing protein and coat protein (Malik et al, 2005;Settlage et al, 2005), as well as with several other host factors (Bagewadi et al, 2004;Castillo et al, 2003;Kong & Hanley-Bowdoin, 2002;Luque et al, 2002;Settlage et al, 2001;Singh et al, 2007;Xie et al, 1999Xie et al, , 1995. Second, Rep of ACMV and related begomoviruses can elicit HR (Hong et al, 2003;Selth et al, 2004;van Wezel et al, 2002a). ACMV Rep and its homologue in tomato yellow leaf curl virus can also interact with the sumoylation system, which may participate in plant defence against pathogen infections (Castillo et al, 2004).…”

“…PCR detection using primers P1 and P2 (Hong et al, 2003) (Fig. 2a) demonstrated that Rep-GFP or Rep*-GFP expressed from PVX/Rep-GFP or PVX/Rep*-GFP, respectively, could mobilize the episomal replicon in pOri-2 plants to produce the diagnostic 1.64 kb DNA fragment, which could only be generated from the circular molecules (Fig.…”

“…ACMV Rep also plays an important role in host-virus interactions as it triggers the hypersensitive response (HR) although the question remains as to whether this is a classical virulence factor/R-gene interaction (Dixon et al, 1994;Greenberg, 1997). Expression of Rep in Nicotiana benthamiana causes rapid cell death and a systemic burst of H 2 O 2 production (Hong et al, 2003;van Wezel et al, 2002a). Thus, while Rep is vital for viral DNA replication, Rep also acts as an elicitor of the HR which is detrimental to the virus life cycle.…”

“…To test this hypothesis, we used an artificial system to produce ACMV Rep mutants to investigate their functions in inducing HR and/or initiating viral DNA replication in transgenic N. benthamiana line pOri-2. This line contains a direct repeat of the ACMV origin of replication (ori) flanking a non-viral DNA fragment including the b-glucuronidase (GUS) coding sequence and a polyadenylation region, in which initiation of transreplication occurs in the presence of the ACMV Rep protein (Hong et al, 2003).…”