title: The signal peptide-like segment of hpaXm is required for its association to the cell wall in transgenic tobacco plants.
abstract: Harpins, encoded by hrp (hypersensitive response and pathogenicity) genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR). HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum-a synonym of X. campestris pv. malvacearum (Smith 1901-1978). A putative signal peptide (1-MNSLNTQIGANSSFL-15) of hpaXm was predicted in the nitroxyl-terminal (N-terminal)by SignalP (SignalP 3.0 server). Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.). Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV) showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1-45 bp) in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall.
authors.affiliation: College of Environment and Plant Protection, Hainan University, Haikou, Hainan Province, China.|Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresource, Haikou, Hainan Province, China.
authors.forename: Le
authors.initials: L
authors.lastname: Li
authors.affiliation: College of Environment and Plant Protection, Hainan University, Haikou, Hainan Province, China.|Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresource, Haikou, Hainan Province, China.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Miao
authors.affiliation: College of Environment and Plant Protection, Hainan University, Haikou, Hainan Province, China.|Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresource, Haikou, Hainan Province, China.
authors.forename: Wenbo
authors.initials: W
authors.lastname: Liu
authors.affiliation: U.S. Horticultural Research Laboratory, USDA-ARS, Fort Pierce, Florida, United States of America.
authors.forename: Shujian
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D015139:Blotting, Southern; D002473:Cell Wall / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D018745:Genome, Plant; D050505:Mutant Proteins / Q000378:metabolism; D018515:Plant Leaves / Q000382:microbiology / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals; D017384:Sequence Deletion; D012995:Solubility; D013329:Structure-Activity Relationship; D014026:Nicotiana / Q000235:genetics* / Q000382:microbiology* / Q000821:virology; D014027:Tobacco Mosaic Virus; D014158:Transcription, Genetic; D014974:Xanthomonas
chemical_list: D001425:Bacterial Outer Membrane Proteins; D050505:Mutant Proteins; D021382:Protein Sorting Signals