Teng-Yung Feng

The Sweet Pepper Ferredoxin-Like Protein (pflp) Conferred Resistance Against Soft Rot Disease in Oncidium Orchid

Genetic engineering to date has not been used to introduce disease resistance genes into the orchid gene pool. The ferredoxin-like protein gene originally isolated from sweet pepper is thought to function as a natural defense against infection due to its antimicrobial properties. Hence it was reasoned that introduction of this gene might produce Oncidium plants resistant to Erwinia carotovora, the causal agent for the soft rot disease. An expression vector containing sweet pepper ferredoxin-like protein (pflp) cDNA, hph and gusA coding sequence was successfully transformed into protocorm-like bodies (PLBs) of Oncidium orchid, using Agrobacterium tumefaciens strain EHA105. A total of 17 independent transgenic orchid lines was obtained, out of which six transgenic lines (β-glucuronidase (GUS) positive) were randomly selected and confirmed by Southern, northern and western blot analyses. A bioassay was conducted on the transgenic lines. Transgenic plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest that pflp may be an extremely useful gene for genetic engineering strategies in orchids to confer resistance against soft rot disease.

Sweet pepper ferredoxin-like protein (pflp) gene as a novel selection marker for orchid transformation

A novel method for selection of transgenic plants utilizing the sweet pepper (Capsicum annuum L.) ferredoxin-like protein (pflp) gene as selection marker and Erwinia carotovora as the selection agent has been developed. An expression vector containing a pflp cDNA driven by a cauliflower mosaic virus 35S promoter was successfully transformed into protocorm-like bodies of Oncidium orchid by Agrobacterium tumefaciens and particle bombardment, respectively. Erwinia carotovora was used as a selection agent to screen transformants, thereby obtaining transgenic plants without the use of an antibiotic selection agent. A total of 32 independent transgenic orchid lines were obtained, out of which 9 transgenic lines (β-glucuronidase positive) were randomly selected and confirmed by Southern and northern blot analyses. The transgenic orchid plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest the novel use of the pflp gene as a resistance selection marker in plant genetic engineering strategies. In the future, the use of the pflp gene as a selection marker may facilitate the use of smaller gene constructs due to removal of bulky antibiotic selection and reporter genes. These constructs can then be used to incorporate additional genes of choice.

Transgenic rice plants expressing the ferredoxin-like protein (AP1) from sweet pepper show enhanced resistance to Xanthomonas oryzae pv. oryzae

We used particle bombardment to cotransform mature seed-derived rice callus (Oryza sativa L., ssp. japonica, cv. Eyi 105) with plasmids containing the linked marker genes gusA and hpt, and the ap1 gene encoding an amphipathic protein previously shown to delay the hypersensitive response induced in non-host plants by the pathogen Pseudomonas syringae pv. syringae (Pss). Thirty-two independent lines of transgenic rice plants were regenerated, and 27 of these lines carried all three transgenes as shown by molecular analysis. A bacterial blight inoculation test was carried out on ten lines. In each case, plants carrying the ap1 gene showed enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo) race 6 at various levels. This suggests the ap1 gene could be a useful candidate for genetic engineering strategies in rice to provide bacterial blight resistance.

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. Harpinpss, 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 harpinpss-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 harpinpss in tobacco suspension cells. It also showed enhanced HR when co-infiltrated with harpinpss 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 harpinpss-mediated hypersensitive cell death in vivo. In response to harpinpss, 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 harpinpss-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 harpinpss 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.

Constitutive expression of hrap gene in transgenic tobacco plant enhances resistance against virulent bacterial pathogens by induction of a hypersensitive response

Hypersensitive response-assisting protein (HRAP) has been previously reported as an amphipathic plant protein isolated from sweet pepper that intensifies the harpin(Pss)-mediated hypersensitive response (HR). The hrap gene has no appreciable similarity to any other known sequences, and its activity can be rapidly induced by incompatible pathogen infection. To assess the function of the hrap gene in plant disease resistance, the CaMV 35S promoter was used to express sweet pepper hrap in transgenic tobacco. Compared with wild-type tobacco, transgenic tobacco plants exhibit more sensitivity to harpin(Pss) and show resistance to virulent pathogens (Pseudomonas syringae pv. tabaci and Erwinia carotovora subsp. carotovora). This disease resistance of transgenic tobacco does not originate from a constitutive HR, because endogenous level of salicylic acid and hsr203J mRNA showed similarities in transgenic and wildtype tobacco under noninfected conditions. However, following a virulent pathogen infection in hrap transgenic tobacco, hsr203J was rapidly induced and a micro-HR necrosis was visualized by trypan blue staining in the infiltration area. Consequently, we suggest that the disease resistance of transgenic plants may result from the induction of a HR by a virulent pathogen infection.

cDNA cloning and characterization of a plant protein that may be associated with the harpinPSS-mediated hypersensitive response.

Hypersensitive response-assisting protein (HRAP) is a novel plant protein that can intensify the harpinPSS-mediated hypersensitive response (HR) in harpinPSS-insensitive plants, such as the vegetative stage of sweet pepper. In this report, we identified a HRAP cDNA clone from sweet pepper (Capsicum annuum cv. ECW). The sequence of this cDNA clone showed no appreciable similarity to any other known sequences. However, it contained three positively charged regions, a typical signal peptide and a cAMP-dependent phosphorylation site. The hrap mRNA accumulated preferentially during the incompatible interaction of sweet pepper leaves with a pathogenic bacterium, Pseudomonas syringae pv. syringae. When the hrap gene transcription level was high, the sweet pepper leaves readily expressed the harpinPSS-mediated HR. The hrap gene transcription level in sweet pepper was also higher during the reproductive stage than during the vegetative stage. The HRAP distribution in an individual plant and different plant species was investigated. We found that all the organs of sweet pepper, except fruit, could express two different forms of HRAP. Moreover, the hrap gene was presented in many plant species including tobacco, Arabidopsis, and rice. In conclusion, our results suggest that the hrap gene is widely distributed throughout the plant world and its transcription level correlates with plant sensitivity to harpinPSS. The interaction between HRAP and harpinPSS reveals a novel way to interpret the interaction mechanism between plants and bacterial pathogens.

Resistance Enhancement of Transgenic Tomato to Bacterial Pathogens by the Heterologous Expression of Sweet Pepper Ferredoxin-I Protein

ABSTRACT Expression of a foreign gene to enhance plant disease resistance to bacterial pathogens is a favorable strategy. It has been demonstrated that expressing sweet pepper ferredoxin-I protein (PFLP) in transgenic plants can enhance disease resistance to bacterial pathogens that infect leaf tissue. In this study, PFLP was applied to protect tomato (Lycopersicon esculentum cv. cherry Cln1558a) from the root-infecting pathogen, Ralstonia solanacearum. Independent R. solanacearum resistant T(1) lines were selected and bred to produce homozygous T(2) generations. Selected T(2) transgenic lines 24-18-7 and 26-2-1a, which showed high expression levels of PFLP in root tissue, were resistant to disease caused by R. solanacearum. In contrast, the transgenic line 23-17-1b and nontransgenic tomato, which showed low expression levels of PFLP in root tissue, were not resistant to R. solanacearum infection. The expansion of R. solanacearum populations in stem tissue of transgenic tomato line 24-18-7 was limited compared with the nontransgenic tomato Cln1558a. Using a detached leaf assay, transgenic line 24-18-7 was also resistant to maceration caused by E. carotovora subsp. carotovora; however, resistance to E. carotovora subsp. carotovora was less apparent in transgenic lines 26-2-1a and 23-17-1b. These results demonstrate that PFLP is able to enhance disease resistance at different levels to bacterial pathogens in individual tissue of transgenic tomato.

Expression of the hypersensitive response-assisting protein in Arabidopsis results in harpin-dependent hypersensitive cell death in response to Erwinia carotovora

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpinPss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H2O2 and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN− mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.

Disease resistance to bacterial pathogens affected by the amount of ferredoxin-I protein in plants.

SUMMARY Ferredoxin-I (Fd-I) is a fundamental protein that is involved in several metabolic pathways. The amount of Fd-I found in plants is generally regulated by environmental stress, including biotic and abiotic events. In this study, the correlation between quantity of Fd-I and plant disease resistance was investigated. Fd-I levels were increased by inoculation with Pseudomonas syringae pv. syringae but were reduced by Erwinia carotovora ssp. carotovora. Transgenic tobacco over-expressing Fd-I with the sense sweet pepper Fd-I gene (pflp) was resistant to E. carotovora ssp. carotovora and the saprophytic bacterium P. fluorescens. By contrast, transgenic tobacco with reduced total Fd-I and the antisense pflp gene was susceptible to E. carotovora ssp. carotovora and P. fluorescens. Both of these transgenic tobaccos were resistant to P. syringae pv. syringae. By contrast, the mutated E. carotovora ssp. carotovora, with a defective harpin protein, was able to invade the sense-pflp transgenic tobacco as well as the non-transgenic tobacco. An in vitro kinase assay revealed that harpin could activate unidentified kinases to phosphorylate PFLP. These results demonstrate that Fd-I plays an important role in the disease defence mechanism.

Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells

Abstract This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently deliver the plasmid DNA into intact plant cells.