Ectopically expressed sweet pepper ferredoxin PFLP enhances disease resistance to Pectobacterium carotovorum subsp. carotovorum affected by harpin and protease‐mediated hypersensitive response in Arabidopsis

Plant ferredoxin‐like protein (PFLP) is a photosynthesis‐type ferredoxin (Fd) found in sweet pepper. It contains an iron–sulphur cluster that receives and delivers electrons between enzymes involved in many fundamental metabolic processes. It has been demonstrated that transgenic plants overexpressing PFLP show a high resistance to many bacterial pathogens, although the mechanism remains unclear. In this investigation, the PFLP gene was transferred into Arabidopsis and its defective derivatives, such as npr1 (nonexpresser of pathogenesis‐related gene 1) and eds1 (enhanced disease susceptibility 1) mutants and NAHG‐transgenic plants. These transgenic plants were then infected with the soft‐rot bacterial pathogen Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovora ssp. carotovora, ECC) to investigate the mechanism behind PFLP‐mediated resistance. The results revealed that, instead of showing soft‐rot symptoms, ECC activated hypersensitive response (HR)‐associated events, such as the accumulation of hydrogen peroxide (H₂O₂), electrical conductivity leakage and expression of the HR marker genes (ATHSR2 and ATHSR3) in PFLP‐transgenic Arabidopsis. This PFLP‐mediated resistance could be abolished by inhibitors, such as diphenylene iodonium (DPI), 1‐l ‐trans‐epoxysuccinyl‐leucylamido‐(4‐guanidino)‐butane (E64) and benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone (z‐VAD‐fmk), but not by myriocin and fumonisin. The PFLP‐transgenic plants were resistant to ECC, but not to its harpin mutant strain ECCAC5082. In the npr1 mutant and NAHG‐transgenic Arabidopsis, but not in the eds1 mutant, overexpression of the PFLP gene increased resistance to ECC. Based on these results, we suggest that transgenic Arabidopsis contains high levels of ectopic PFLP; this may lead to the recognition of the harpin and to the activation of the HR and other resistance mechanisms, and is dependent on the protease‐mediated pathway.     

Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpinpss-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 coliwas 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 Pflpgene 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 Pflptransgenic 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.