A 38-amino-acid sequence encompassing the arm domain of the cucumber necrosis virus coat protein functions as a chloroplast transit Peptide in infected plants.
Experiments to determine the subcellular location of the coat protein (CP) of the tombusvirus Cucumber necrosis virus (CNV) have been conducted. By confocal microscopy, it was found that an agroinfiltrated CNV CP-green fluorescent protein (GFP) fusion targets chloroplasts in Nicotiana benthamiana leaves and that a 38-amino-acid (aa) region that includes the complete CP arm region plus the first 4 amino acids of the shell domain are sufficient for targeting. Western blot analyses of purified and fractionated chloroplasts showed that the 38-aa region directs import to the chloroplast stroma, suggesting that the CNV arm can function as a chloroplast transit peptide (TP) in plants. Several features of the 38-aa region are similar to features typical of chloroplast TPs, including (i) the presence of an alanine-rich uncharged region near the N terminus, followed by a short region rich in basic amino acids; (ii) a conserved chloroplast TP phosphorylation motif; (iii) the requirement that the CNV 38-aa sequence be present at the amino terminus of the imported protein; and (iv) specific proteolytic cleavage upon import into the chloroplast stroma. In addition, a region just downstream of the 38-aa sequence contains a 14-3-3 binding motif, suggesting that chloroplast targeting requires 14-3-3 binding, as has been suggested for cellular proteins that are targeted to chloroplasts. Chloroplasts of CNV-infected plants were found to contain CNV CP, but only the shell and protruding domain regions were present, indicating that CNV CP enters chloroplasts during infection and that proteolytic cleavage occurs as predicted from agroinfiltration studies. We also found that particles of a CNV CP mutant deficient in externalization of the arm region have a reduced ability to establish infection. The potential biological significance of these findings is discussed.
Xiang
Yu
Y
Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia V0H 1Z0, Canada.
Kakani
Kishore
K
Reade
Ron
R
Hui
Elizabeth
E
Rochon
D'Ann
D
D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000737:chemistry / Q000378:metabolism* / Q000821:virology; D017799:Cucumovirus / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000821:virology*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D014026:Nicotiana / Q000737:chemistry / Q000378:metabolism
D036022:Capsid Proteins; D060365:Chloroplast Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C078737:chloroplast transit peptides; D049452:Green Fluorescent Proteins