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

Abstract

Truncated versions of the replication-associated protein (Rep) of Tomato yellow leaf curl Sardinia virus (TYLCSV) can interfere with various viral functions and the N-terminal 130 aa are sufficient for strongly inhibiting C1-gene transcription and virus replication and confer resistance in transgenic plants. In this work, we analysed the relevance of an RGG sequence at aa 124-126, highly conserved in begomoviruses, in these inhibitory functions as well as in the subcellular localization of Rep. Although no role of this RGG sequence was detected by cell fractionation and immunogold labelling in Rep localization, this sequence appears relevant for the transcriptional control of the C1-gene and for the inhibition of viral replication and dramatically impacts resistance in transgenic plants. These results are discussed in the context of the model of Repmediated resistance against TYLCSV.Begomoviruses (family Geminiviridae) are ssDNA viruses infecting a wide range of crops and causing severe diseases worldwide. Plants resistant to begomoviruses have been obtained by genetic engineering, and ORF C1/AL1/L1 encoding the replication-associated protein (Rep) has been successfully used for this purpose (Vanderschuren et al., 2007;Prins et al., 2008). Rep is a 40-41 kDa multitask protein necessary for virus replication, specifically cleaving the viral genome within a conserved stem-loop structure (Heyraud-Nitschke et al., 1995; Laufs et al., 1995). During virus multiplication, Rep binds to upstream untranslated viral sequences, known as iterons, repressing its own expression (Sunter et al., 1993;Eagle et al., 1994; HanleyBowdoin et al., 1999). Rep forms homo-oligomers of various aggregation orders (Orozco et al., 1997(Orozco et al., , 2000 through central oligomerization domains (Orozco et al., 2000;Choudhury et al., 2006), which in the case of Tomato yellow leaf curl Sardinia virus (TYLCSV) lies between aa 120 and 180 Clérot & Bernardi, 2006). Begomoviruses multiply in the nuclei of infected cells (Hanley-Bowdoin et al., 1999) and perturb the host cell cycle to favour their replication (Ascencio-Ibáñez et al., 2008). In this context, Rep was shown to interact with cell cycle factors such as proliferating cell nuclear antigen or homologues of the retinoblastoma family (pRBR) (Xie et al., 1995;Kong et al., 2000).Nicotiana benthamiana and tomato plants expressing a truncated TYLCSV Rep (Rep-210) are resistant to TYLCSV (Noris et al., 1996;Brunetti et al., 1997) by using a mechanism involving the transcriptional inhibition of the C1-gene promoter and/or the formation of dysfunctional oligomers between Rep-210 and the viral Rep (Brunetti et al., 2001;Lucioli et al., 2003). Further work showed that Rep-130 is the minimal sequence tightly inhibiting C1-gene transcription and virus replication in protoplasts, conferring high levels of virus resistance in transgenic plants (Lucioli et al., , 2008. Since a further deletion of 10 aa (121-RSARGGQQTA-130) consistently reduced the inhibitory activity , we decided to investigate thi...

Tomato yellow leaf curl Sardinia virus‐resistant tomato plants expressing the multifunctional N‐terminal domain of the replication‐associated protein show transcriptional changes resembling stress‐related responses

“…In a previous study, we showed that Rep 130 , when ectopically expressed in N. benthamiana plants, retains some of the biochemical and biological functions described for the full‐length Rep. In particular, Rep 130 was shown to tightly repress TYLCSV C1 gene transcription (Lucioli et al ., ) and to be specifically targeted to the nucleus (Sardo et al ., ), both functions being essential for its ability to inhibit TYLCSV replication in single cells and whole plants (Lucioli et al ., ; Sardo et al ., ). In addition, the ability of TYLCSV Rep 130 to inhibit TYLCSV replication, but not that of the Portuguese strain of Tomato yellow leaf curl virus TYLCV‐Mld (AF105975) (Lucioli et al ., ), suggests that either Rep 130 is inactive in recognizing and cutting the conserved nonanucleotide sequence of geminiviruses or that this activity is not able to confer TYLCV‐Mld resistance.…”

“…In the case of TYLCSV, plant-expressed Rep130 retains the ability to be compartmentalized within the nucleus/nucleolus and to inhibit C1 gene transcription, whereas a further deletion of 10 amino acids from its C-terminus abolishes both functions (Lucioli et al, 2003;Sardo et al, 2011). In addition, a recent deletion analysis conducted on the Rep of Tomato golden mosaic virus (TGMV), a bipartite begomovirus, has shown that the Rep region responsible for the binding to the small ubiquitin-related modifier (SUMO)-conjugating enzyme (SCE1) lies inside this N-terminal domain between amino acids 85 and 114 (Sánchez-Durán et al, 2011).…”

Artificial microRNA-mediated resistance against Oman strain of tomato yellow leaf curl virus

“…It was shown that the expression of the Rep protein of tomato yellow leaf curl Sardinia virus (TYLCSV) lacking a C-terminal end resulted in TYLCSV resistance in tomato ( Brunetti et al., 1997 ). Sardo et al. (2011) have used a C-terminal-truncated Rep transgene to develop TYLCSV resistance in N .…”

Multifunctional roles of geminivirus encoded replication initiator protein

“…The expression of the AC2 ORF brings about the suppression of plant defence responses and the expression of late viral genes in the later phase of geminiviral infection. Apart from this in Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep protein contains a highly conserved RGG sequence at amino acids located at 124-126 positons which when mutated alters the subcellular localization and inhibits its transcriptional autoregulation [66].…”