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.     

Coordinated Activation of Programmed Cell Death and Defense Mechanisms in Transgenic Tobacco Plants Expressing a Bacterial Proton Pump

In plants, programmed cell death is thought to be activated during the hypersensitive response to certain avirulent pathogens and in the course of several differentiation processes. We describe a transgenic model system that mimics the activation of programmed cell death in higher plants. In this system, expression of a bacterial proton pump in transgenic tobacco plants activates a cell death pathway that may be similar to that triggered by recognition of an incompatible pathogen. Thus, spontaneous lesions that resemble hypersensitive response lesions are formed, multiple defense mechanisms are apparently activated, and systemic resistance is induced in the absence of a pathogen. Interestingly, mutation of a single amino acid in the putative channel of this proton pump renders it inactive with respect to lesion formation and induction of resistance to pathogen challenge. This transgenic model system may provide insights into the mechanisms involved in mediating cell death in higher plants. In addition, it may also be used as a general agronomic tool to enhance disease protection.     

HSR203 antisense suppression in tobacco accelerates development of hypersensitive cell death

Activation of the tobacco gene hsr203 is rapid, highly localized, specific for incompatible plant-pathogen interactions, and strongly correlated with programmed cell death occurring in response to diverse pathogens. Functional characterization of hsr203 gene product has shown that HSR203 is a serine hydrolase that displays esterase activity. We show here that transgenic tobacco plants deficient in HSR203 protein exhibit an accelerated hypersensitive response when inoculated with an avirulent strain of Ralstonia solanacearum. This response was accompanied by a maximal level of cell death and a drastic inhibition of in planta bacterial growth. Transgenic plants deficient in HSR203 were also found to show increased resistance in a dosage-dependent manner to Pseudomonas syringae pv. pisi, another avirulent bacterial pathogen, and to virulent and avirulent races of Phytophthora parasitica, a fungal pathogen of tobacco, but not to different virulent bacteria. Surprisingly, expression of another hsr gene, hsr515, and that of the defence genes PR1-a and PR5, was strongly reduced in the transgenic lines. Our results suggest that hsr203 antisense suppression in tobacco can have pleiotropic effects on HR cell death and defence mechanisms, and induces increased resistance to different pathogens.     

Activation of Ntf4, a tobacco mitogen-activated protein kinase, during plant defense response and its involvement in hypersensitive response-like cell death

Mitogen-activated protein kinase (MAPK) cascades are important signaling modules in eukaryotic cells. They function downstream of sensors/receptors and regulate cellular responses to external and endogenous stimuli. Recent studies demonstrated that SIPK and WIPK, two tobacco (Nicotiana spp.) MAPKs, are involved in signaling plant defense responses to various pathogens. Ntf4, another tobacco MAPK that shares 93.6% and 72.3% identity with SIPK and WIPK, respectively, was reported to be developmentally regulated and function in pollen germination. We found that Ntf4 is also expressed in leaves and suspension-cultured cells. Genomic analysis excluded the possibility that Ntf4 and SIPK are orthologs from the two parental lines of the amphidiploid common tobacco. In vitro and in vivo phosphorylation and activation assays revealed that Ntf4 shares the same upstream MAPK kinase, NtMEK2, with SIPK and WIPK. Similar to SIPK and WIPK, Ntf4 is also stress responsive and can be activated by cryptogein, a proteinaceous elicitin from oomycetic pathogen Phytophthora cryptogea. Tobacco recognition of cryptogein induces rapid hypersensitive response (HR) cell death in tobacco. Transgenic Ntf4 plants with elevated levels of Ntf4 protein showed accelerated HR cell death when treated with cryptogein. In addition, conditional overexpression of Ntf4, which results in high cellular Ntf4 activity, is sufficient to induce HR-like cell death. Based on these results, we concluded that Ntf4 is multifunctional. In addition to its role in pollen germination, Ntf4 is also a component downstream of NtMEK2 in the MAPK cascade that regulates pathogen-induced HR cell death in tobacco.     

Pathogen-induced expression of cyclo-oxygenase homologue in hot pepper (Capsicum annuum cv. Pukang).

The hypersensitive reaction (HR) in plants is typified by a rapid and localized cell death at the site of pathogen infection. To understand better the molecular and cellular defence mechanism controlling HR, hot pepper leaves (Capsicum annuum cv. Pukang) were inoculated with the soybean pustule pathogen Xanthomonas campestris pv. glycine 8ra. By using the DD-PCR technique, a cDNA fragment was identified that exhibited a sequence similarity to the recently identified tobacco pathogen-induced oxygenase (PIOX) with homology to animal cyclo-oxygenase (COX). Subsequently, the full-length cDNA clone, pCa-COX1, encoding the COX homologue from the pathogen-inoculated hot pepper leaf cDNA library was isolated. The deduced amino acid sequence of Ca-COX1 shares 85.8% identity with tobacco PIOX and displays a significant degree of sequence identity (21.7-23.7%) with mammalian COXs. The expression of Ca-COX1 was markedly induced at 4-12 h after pathogen infection, while HR cell death on pepper leaves appeared at approximately 15 h post-inoculation. These results are consistent with the notion that the lipid-derived signalling pathway is involved in the initial response of hot pepper plants to pathogen infection.     

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.     

Mitochondrial alternative oxidase is not a critical component of plant viral resistance but may play a role in the hypersensitive response

Transgenic tobacco (Nicotiana tabacum) with altered levels of mitochondrial alternative oxidase (AOX) were used to examine the potential role of this electron transport chain protein in resistance to tobacco mosaic virus. We examined the effect of AOX expression on the salicylic acid-induced resistance in susceptible plants and the resistance responses of plants harboring the N-gene. A lack of AOX did not compromise the ability of salicylic acid treatment to heighten the resistance of susceptible plants. In plants with the N-gene, a lack of AOX did not compromise the ability of the hypersensitive response to restrict the virus or the ability of the plant to develop systemic acquired resistance. Overexpression of AOX did not heighten the resistance of susceptible plants, but did result in smaller hypersensitive response lesions, suggesting a link between mitochondrial function and this programmed cell death event. We conclude that AOX is not a critical component of the previously characterized salicylhydroxamic acid-sensitive pathway important in viral resistance.     

Expression of the B Subunit of E. coli Heat-labile Enterotoxin in the Chloroplasts of Plants and its Characterization

Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.     

Targeted expression of a synthetic codon optimized gene, encoding the spruce budworm antifreeze protein, leads to accumulation of antifreeze activity in the apoplasts of transgenic tobacco

A synthetic gene based on the primary sequence of the mature spruce budworm antifreeze protein (sbwAFP) was constructed by primer overlap extension. The amino acid codons were chosen to mimic those of a highly expressed tobacco nuclear gene. A DNA sequence encoding the amino-terminal leader sequence from the tobacco pathogen related protein 1b (PR), which targets the protein to the apoplastic space, was fused in frame to the synthetic sbwAFP gene. This fusion was placed downstream of the cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator in a T-DNA binary vector. Transgenic tobacco lines transcribing PR-sbwAFP were selected by RT-PCR. The apoplastic protein fractions of sbwAFP expressing tobacco lines exhibited enhanced antifreeze activity as demonstrated by the ability to inhibit ice re-crystallization and increased thermal hysteresis.     

Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects

The expression of the Acidothermus cellulolyticus endoglucanase E1 gene in transgenic tobacco (Nicotiana tabacum) was examined in this study, where E1 coding sequence was transcribed under the control of a leaf specific Rubisco small subunit promoter (tomato RbcS-3C). Targeting the E1 protein to the chloroplast was established using a chloroplast transit peptide of Rubisco small subunit protein (tomato RbcS-2A) and confirmed by immunocytochemistry. The E1 produced in transgenic tobacco plants was found to be biologically active, and to accumulate in leaves at levels of up to 1.35% of total soluble protein. Optimum temperature and pH for E1 enzyme activity in leaf extracts were 81°C and 5.25, respectively. E1 activity remained constant on a gram fresh leaf weight basis, but dramatically increased on a total leaf soluble protein basis as leaves aged, or when leaf discs were dehydrated. E1 protein in old leaves, or after 5h dehydration, was partially degraded although E1 activity remained constant. Transgenic plants exhibited normal growth and developmental characteristics with photosynthetic rates similar to those of untransformed SR1 tobacco plants. Results from these biochemical and physiological analyses suggest that the chloroplast is a suitable cellular compartment for accumulation of the hydrolytic E1 enzyme.     

Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts

FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast size and number.     

Production of the AVR9 elicitor from the fungal pathogen Cladosporium fulvum in transgenic tobacco and tomato plants

Three constructs were used to study the expression of the avirulence gene Avr9 from the fungal tomato pathogen Cladosporium fulvum in plants. They include pAVIR1, pAVIR2 and pAVIR21, encoding the wild-type AVR9 protein and two hybrid AVR9 proteins containing the signal sequences of the pathogenesis-related proteins PR-S and PR-1a, respectively. Transgenic tobacco plants obtained with the three constructs showed a normal phenotype and produced AVR9 elicitor with the same specific necrosis-inducing activity as the wild-type AVR9 elicitor produced in planta by isolates of C. fulvum containing the Avr9 gene. Level of expression was not correlated with number of T-DNA integrations, but plants homozygous for the Avr9 gene produced more elicitor protein than heterozygous plants. The amino acid sequence of the processed AVR9 peptide present in apoplastic fluid (AF) of pAVIR1 transformed plants producing the wild-type AVR9 elicitor was identical to that of the wild-type AVR9 peptide isolated from C. fulvum-infected tomato leaves. Transgenic Cf0 genotypes of tomato, obtained by transformation with construct pAVIR21, showed a normal phenotype. However, transgenic F1 plants expressing the Avr9 gene, obtained from crossing transgenic Cf0 genotypes with wild-type Cf9 genotypes, showed delayed growth, necrosis and complete plant death indicating that the AVR9 peptide produced in plants carrying the Cf9 gene is deleterious. The necrotic defence response observed in Cf9 genotypes expressing the Avr9 gene support the potential to apply avirulence genes in molecular resistance breeding.     

Chloroplast Cpn20 forms a tetrameric structure in Arabidopsis thaliana

Chloroplast chaperonin 20 (Cpn20) in higher plants is a functional homologue of the Escherichia coli GroES, which is a critical regulator of chaperonin-mediated protein folding. The cDNA for a Cpn20 homologue of Arabidopsis thaliana was isolated. It was 958 bp long, encoding a protein of 253 amino acids. The protein was composed of an N-terminal chloroplast transit peptide, and the predicted mature region comprised two distinct GroES domains that showed 42% amino acid identity to each other. The isolated cDNA was constitutively expressed in transgenic tobacco. Immunogold labelling showed that Cpn20 is accumulated in chloroplasts of transgenic tobacco. A Northern blot analysis revealed that mRNA for the chloroplast Cpn20 is abundant in leaves and is increased by heat treatment. To examine the oligomeric structure of Cpn20, a histidine-tagged construct lacking the transit peptide was expressed in E. coli and purified by affinity chromatography. Gel-filtration and cross-linking analyses showed that the expressed products formed a tetramer. The expressed products could substitute for GroES to assist the refolding of citrate synthase under non-permissive conditions. The analysis on the subunit stoichiometry of the GroEL-Cpn20 complex also revealed that the functional complex is composed of a GroEL tetradecamer and a Cpn20 tetramer.     

Threonine Overproduction in Transgenic Tobacco Plants Expressing a Mutant Desensitized Aspartate Kinase of Escherichia coli

In higher plants, the synthesis of the essential amino acid threonine is regulated primarily by the sensitivity of the first enzyme in its biosynthetic pathway, aspartate kinase, to feedback inhibition by threonine and lysine. We aimed to study the potential of increasing threonine accumulation in plants by means of genetic engineering. This was addressed by the expression of a mutant, desensitized aspartate kinase derived from Escherichia coli either in the cytoplasm or in the chloroplasts of transgenic tobacco (Nicotiana Tabacum cv Samsun NN) plants. Both types of transgenic plants exhibited a significant overproduction of free threonine. However, threonine accumulation was higher in plants expressing the bacterial enzyme in the chloroplast, indicating that compartmentalization of aspartate kinase within this organelle was important, although not essential. Threonine overproduction in leaves was positively correlated with the level of the desensitized enzyme. Transgenic plants expressing the highest leaf aspartate kinase activity also exhibited a slight increase in the levels of free lysine and isoleucine, both of which share a common biosynthetic pathway with threonine, but showed no significant change in the level of other free amino acids. The present study proposes a new molecular biological approach to increase the limiting content of threonine in higher plants.