Abstract
Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) protein is involved in viral movement. AltMV TGB3 subcellular localization was previously shown to be distinct from that of Potato virus X (PVX) TGB3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculation were observed in Nicotiana benthamiana when AltMV TGB3 was over-expressed from PVX. Plants with over-expressed TGB3 showed more lethal damage under dark conditions than under light. Yeast-two-hybrid analysis and bimolecular fluorescence complementation (BiFC) reveal that Arabidopsis thaliana PsbO1 has strong interactions with TGB3; N. benthamiana PsbO (NbPsbO) also showed obvious interaction signals with TGB3 through BiFC. These results demonstrate an important role for TGB3 in virus cell-to-cell movement and virus-host plant interactions. The Photosystem II oxygen-evolving complex protein PsbO interaction with TGB3 is presumed to have a crucial role in symptom development and lethal damage under dark conditions. In order to further examine interactions between AtPsbO1, NbPsbO, and TGB3, and to identify the binding domain(s) in TGB3 protein, BiFC assays were performed between AtPsbO1 or NbPsbO and various mutants of TGB3. Interactions with C-terminally deleted TGB3 were significantly weaker than those with wild-type TGB3, and both N-terminally deleted TGB3 and a TGB3 mutant previously shown to lose chloroplast interactions failed to interact detectably with PsbO in BiFC. To gain additional information about TGB3 interactions in AltMV-susceptible plants, we cloned 12 natural AltMV TGB3 sequence variants into a PVX expression vector to examine differences in symptom development in N. benthamiana. Symptom differences were observed on PVX over-expression, with all AltMV TGB3 variants showing more severe symptoms than the WT PVX control, but without obvious correlation to sequence differences.
Highlights
The chloroplast has for many years been recognized as susceptible to damage during plant virus infections
YEAST TWO-HYBRID INTERACTIONS Several proteins were identified by screening of an Arabidopsis cDNA library with Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) as bait; because the Photosystem II (PS II) Oxygen-evolving complex (OEC) protein, A. thaliana PsbBO1 (AtPsbO1), showed the strongest interaction, and because we had previously shown that TGB3 localizes to the chloroplast (Lim et al, 2010a), we selected PsbO for detailed examination
BIMOLECULAR FLUORESCENCE COMPLEMENTATION ASSAYS Reciprocal interactions between AltMV TGB3 and AtPsbO1 were detected by bimolecular fluorescence complementation (BiFC) only when both constructs were expressed with the enhanced Yellow Fluorescent Protein (eYFP) fragment fused to the N-terminus of the test protein
Summary
The chloroplast has for many years been recognized as susceptible to damage during plant virus infections. Infection with Tobacco mosaic virus (TMV) was shown to interfere with starch mobilization in local lesions on tobacco (Holmes, 1931). TMV particles were observed in association with chloroplasts (Esau and Cronshaw, 1967; Granett and Shalla, 1970), while Reinero and Beachy (1986) showed that TMV coat protein (CP) accumulated within chloroplasts of infected tobacco. Schoelz and Zaitlin (1989) demonstrated that TMV genomic RNA, but not subgenomic RNA, enters tobacco chloroplasts, and suggested that the CP detected may be translated by chloroplast ribosomes from the genomic RNA due to the presence of a Shine–Dalgarno sequence upstream of the CP initiation codon. Other viruses have been shown to associate with chloroplasts. Both CP and the HC-Pro of Potato virus Y (PVY) were detected in chloroplasts (Gunasinghe and Berger, 1991).
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