Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpin(pss)-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS).
The hypersensitive response (HR) is a form of cell death associated with plant resistance to pathogen infection. Harpin(pss), an elicitor from the bacterium Pseudomonas syringae pv. syringae, induces a HR in non-host plants. Previously, we reported an amphipathic protein from sweet pepper interfering with harpin(pss)-mediated HR. In this report, we isolated and characterized a cDNA clone encoded that amphipathic protein from sweet pepper. This protein is designated as PFLP (plant ferredoxin-like protein) by virtue of its high homology with plant ferredoxin protein containing an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain responsible for redox activity. Recombinant PFLP obtained from Escherichia coli was able to significantly increase active oxygen species (AOS) generation when mixed with harpin(pss) in tobacco suspension cells. It also showed enhanced HR when co-infiltrated with harpin(pss) in tobacco leaves. We used a transgenic tobacco suspension cells system that constitutively expresses the Pflp gene driven by the CaMV 35S promoter to study the function of PFLP in enhancing harpin(pss)-mediated hypersensitive cell death in vivo. In response to harpin(pss), suspension cells derived from Pflp transgenic tobacco showed a significant increase both in the generation of AOS and in cell death as compared to the wild type. AOS inhibitors diphenylene iodonium chloride (DPI) and lanthanum chlorate (LaCl3) were used to study the involvement of AOS in harpin(pss)-induced cell death. Our results demonstrate enhanced generation of AOS is necessary to cause enhanced hypersensitive cell death in Pflp transgenic tobacco cells and it is plasma membrane-bound NADPH-oxidase-dependent. Sub-cellular localization studies showed that PFLP is present in the cytoplasm and chloroplast of Pflp transgenic tobacco cells, but only in the chloroplast, not in the cytoplasm, of wild-type tobacco cells. It is possible that PFLP can change the redox state of the cell upon harpin(pss) inoculation to increase AOS generation and hypersensitive cell death. Overall, this study will provide a new insight in the functional properties of ferredoxin in hypersensitive cell death.
Dayakar
Badri Venkata
BV
Institute of Botany, Academia Sinica, Taipei, 115 29, Taiwan.
Lin
Hao-Jan
HJ
Chen
Cheng-Hsien
CH
Ger
Mang-Jye
MJ
Lee
Bor-Heng
BH
Pai
Chia-Hwei
CH
Chow
David
D
Huang
Hsiang-En
HE
Hwang
Shaw-Yhi
SY
Chung
Mei-Chu
MC
Feng
Teng-Yung
TY
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000494:pharmacology*; D001483:Base Sequence; D002212:Capsicum / Q000235:genetics*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002736:Chloroplasts / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004357:Drug Synergism; D005288:Ferredoxins / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology*; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000166:cytology / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D018076:DNA, Complementary; D005288:Ferredoxins; D010940:Plant Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C474922:plant ferredoxin-like protein, Capsicum annuum; C087295:HrpZ protein, Pseudomonas syringae