Abstract
Potato virus Y (PVY) is an important plant virus and causes great losses every year. Viral infection often leads to abnormal chloroplasts. The first step of chloroplast division is the formation of FtsZ ring (Z-ring), and the placement of Z-ring is coordinated by the Min system in both bacteria and plants. In our lab, the helper-component proteinase (HC-Pro) of PVY was previously found to interact with the chloroplast division protein NtMinD through a yeast two-hybrid screening assay and a bimolecular fluorescence complementation (BiFC) assay in vivo. Here, we further investigated the biological significance of the NtMinD/HC-Pro interaction. We purified the NtMinD and HC-Pro proteins using a prokaryotic protein purification system and tested the effect of HC-Pro on the ATPase activity of NtMinD in vitro. We found that the ATPase activity of NtMinD was reduced in the presence of HC-Pro. In addition, another important chloroplast division related protein, NtMinE, was cloned from the cDNA of Nicotiana tabacum. And the NtMinD/NtMinE interaction site was mapped to the C-terminus of NtMinD, which overlaps the NtMinD/HC-Pro interaction site. Yeast three-hybrid assay demonstrated that HC-Pro competes with NtMinE for binding to NtMinD. HC-Pro was previously reported to accumulate in the chloroplasts of PVY-infected tobacco and we confirmed this result in our present work. The NtMinD/NtMinE interaction is very important in the regulation of chloroplast division. To demonstrate the influence of HC-Pro on chloroplast division, we generated HC-Pro transgenic tobacco with a transit peptide to retarget HC-Pro to the chloroplasts. The HC-Pro transgenic plants showed enlarged chloroplasts. Our present study demonstrated that the interaction between HC-Pro and NtMinD interfered with the function of NtMinD in chloroplast division, which results in enlarged chloroplasts in HC-Pro transgenic tobacco. The HC-Pro/NtMinD interaction may cause the formation of abnormal chloroplasts in PVY-infected plants.
Highlights
Chloroplasts are believed to have prokaryotic ancestors engulfed by a heterotrophic eukaryotic host cell [1]
When the same amount of helper-component proteinase (HC-Pro) was added to various amounts of NtMinD, the ATPase activity of NtMinD was reduced in each case (Fig 1A)
On the other hand, when various amounts of HC-Pro were added to the same amount of NtMinD protein, a dose-dependent effect was observed: more HC-Pro led to more reduction of the ATPase activity of NtMinD (Fig 1B)
Summary
Chloroplasts are believed to have prokaryotic ancestors engulfed by a heterotrophic eukaryotic host cell [1]. They proliferate from pre-existing chloroplasts by binary fission [2]. Chloroplast division in plants is much more complicated and is mediated by several proteins, both of prokaryotic origin and from the eukaryotic host [8,9,10]. The ATPase activity is critical to release the AtMinD-mediated inhibition of Z-ring formation. AtMinE is a homologue of the topological specificity factor in bacteria [6] that stimulates the ATPase activity of AtMinD at the mid-point of chloroplasts to ensure symmetric division
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