Tomato Yellow Leaf Curl Sardinia Virus Rep-Derived Resistance to Homologous and Heterologous Geminiviruses Occurs by Different Mechanisms and Is Overcome if Virus-Mediated Transgene Silencing Is Activated

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

“…We then tested Reps mutated in the RGG sequence for their inhibitory activity on TYLCSV replication, according to Lucioli et al (2003). Rep-210SAA was strongly inhibitory, similar to Rep-210 and Rep-130 (Brunetti et al, 2001;Lucioli et al, 2003), while Rep-130SAA had only a limited effect (Fig.…”

“…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.…”

“…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.…”

“…We also analysed the impact of this mutation on TYLCSV resistance by using transgenic plants. To obtain truncated Rep with a mutation in the RGG sequence, plasmids pTOM-100 and pJTR-130 encoding Rep-210 and Rep-130, respectively (Noris et al, 1996;Lucioli et al, 2003), were mutated to pTOM-100SAA and pJTR-130SAA with the Quik Change Site-directed mutagenesis kit (Stratagene) and with primers TY-SARMUT-2225(+) (59-GTTGGCTGTCTGTTGTGCTGCACTAGC-AGATCGTCCGTC-39) and TY-SARMUT-2263(2) (59-G-ACGGACGATCTGCTAGTGCAGCACAACAGACAGCCA-AC-39). The expression cassettes from pJTR-120 , pTOM-100SAA and pJTR-130SAA were cloned into pBin19 to be used in the transformation of N. benthamiana; transgenic plants accumulating high level of Reps were self-pollinated.…”

“…To analyse the subcellular compartmentalization of the different Rep versions, nuclear and cytoplasmic fractions were prepared from transgenic lines expressing Rep-210 (line 102.22;Noris et al, 1996), Rep-130 (line 308;Lucioli et al, 2003), Rep-210SAA (lines # 1, # 2 and # 3), Rep-130SAA (line # 4) and Rep-120 (line # 7) using the CelLytic Plant Nuclei Isolation/Extraction kit (semi-pure protocol; Sigma). Four hundred nanograms of total protein quantified using the Experion Pro260 Analysis kit (Bio-Rad) was separated by SDS-PAGE and probed with Rep-antibodies, and also with antibodies against fibrillarin (kindly provided by M. Taliansky, Dundee, UK), used as nuclear control marker.…”