Increased killing of Bacillus subtilis on the hair roots of transgenic T4 lysozyme-producing potatoes

Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium Erwinia carotovora subsp. carotovora have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to associate the cells of Bacillus subtilis with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Désirée and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed B. subtilis cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5- to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growth-sustaining effects of plants on bacteria.     

Increased Killing of Bacillus subtilis on the Hair Roots of Transgenic T4 Lysozyme-Producing Potatoes

Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium Erwinia carotovora subsp. carotovora have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to associate the cells of Bacillus subtilis with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Désirée and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed B. subtilis cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5- to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growth-sustaining effects of plants on bacteria.     

Differential Patterns of Bacteriolytic Activities in Potato in Comparison to Bacteriophage T4 and Hen Egg White Lysozymes

A comparison of the enzymatic activities of endogenous potato bacteriolytic enzymes with bacteriophage T4 and hen egg white lysozyme has been performed. Using Erwinia carotovora atroseptica and Pseudomonas solanacearum as substrates in, comparison to Micrococcus lysodeikticus a differential pattern of bacteriolytic activities could be detected. The expression pattern of endogenous potato lysozymes suggests that their functional activity against phytopathogenic bacteria in planta is unlikely. Antibacterial activities in transformed, T4 lysozyme expressing and non–transformed potato plants are evaluated.     

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.     

A plant transformation vector with a minimal T-DNA II. Irregular integration patterns of the T-DNA in the plant genome

A minimal T-DNA binary vector was used for Agrobacterium-mediated transfer of a chimeric T4 lysozyme gene located next to the left border, and transgenic potato plants which expressed T4 lysozyme protein were identified and further analysed. Frequent rearrangements of T4 lysozyme transgenes were detected. A vector derivative containing two matrix associated regions (MARs) flanking its multiple cloning site was constructed. In transgenic potato plants, reduced variability in gene expression due to position effects was detected. When either the donor vector contained MAR sequences, or when vector pPCV701 which contains a pBR322 fragment next to the left border were used, only relatively few rearrangements were observed. However, when the T4 lysozyme gene was driven by a CaMV 35S promoter modified by multiplied enhancer region carrying either 2 or 4 elements, frequent rearrangements were again obtained.     

Fungal and bacterial disease resistance in transgenic plants expressing human lysozyme

The human lysozyme gene, which is assembled by the stepwise ligation of chemically synthesized oligonucleotides, was introduced into tobacco (Nicotiana tabacum cv `SR1') by the Agrobacterium-mediated method. The introduced human lysozyme gene was highly expressed under the control of the cauliflower mosaic virus 35S promoter, and the gene product accumulated in the transgenic tobacco plants. The transgenic tobacco plants showed enhanced resistance against the fungus Erysiphe cichoracearum – both conidia formation and mycelial growth were reduced, and the size of the colony was diminished. Microscopic observation revealed that the transgenic tobacco plants carried the resistant phenotype, analogous to that of the resistant cultivar `Kokubu' which had been selected by conventional breeding. Growth of the phytopathogenic bacterium Pseudomonas syringae pv. tabaci was also strongly retarded in the transgenic tobacco, and the chlorotic halo of the disease symptom was reduced to 17% of that observed in the wild-type tobacco. Thus, the introduction of a human lysozyme gene is an effective approach to protect crops against both fungal and bacterial diseases. Received: 9 September 1996 / Revision received: January 9 1997 / Accepted: 20 February 1997     

Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.     

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

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.     

Plants genetically modified to produce N-acylhomoserine lactones communicate with bacteria.

N-acylhomoserine lactones (AHLs) play a critical role in plant/microbe interactions. The AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), induces exoenzymes that degrade the plant cell wall by the pathogenic bacterium Erwinia carotovora. Conversely, the antifungal activity of the biocontrol bacterium Pseudomonas aureofaciens 30-84 is due (at least in part) to phenazine antibiotics whose synthesis is regulated by N-hexanoylhomoserine lactone (HHL). Targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in plants of the cognate AHL signaling molecules (OHHL and HHL). The AHLs produced by the transgenic plants were sufficient to induce target gene expression in several recombinant bacterial AHL biosensors and to restore biocontrol activity to an HHL-deficient P. aureofaciens strain. In addition, pathogenicity was restored to an E. carotovora strain rendered avirulent as a consequence of a mutation in the OHHL synthase gene, carI. The ability to generate bacterial quorum-sensing signaling molecules in the plant offers novel opportunities for disease control and for manipulating plant/microbe interactions.     

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 (beta-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.     

Identification and characterization of Nip, necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora is a gram-negative bacterium that causes soft rot disease of many cultivated crops. When a collection of E. carotovora subsp. carotovora isolates was analyzed on a Southern blot using the harpin-encoding gene hrpN as probe, several harpinless isolates were found. Regulation of virulence determinants in one of these, strain SCC3193, has been characterized extensively. It is fully virulent on potato and in Arabidopsis thaliana. An RpoS (SigmaS) mutant of SCC3193, producing elevated levels of secreted proteins, was found to cause lesions resembling the hypersensitive response when infiltrated into tobacco leaf tissue. This phenotype was evident only when bacterial cells had been cultivated on solid minimal medium at low pH and temperature. The protein causing'the cell death was purified and sequenced, and the corresponding gene was cloned. The deduced sequence of the necrosis-inducing protein (Nip) showed homology to necrosis- and ethylene-inducing elicitors of fungi and oomycetes. A mutant strain of E. carotovora subsp. carotovora lacking the nip gene showed reduced virulence in potato tuber assay but was unaffected in virulence in potato stem or on other tested host plants.     

Transgenic Potato with PVY Coat Protein Gene and Its Small-Scale Field Test

Potato virus Y (PVY) infection may cause a severe yield depression up to 80%. To develop the potato (Solanum tuberosum L. ) cultivars that resist PVY infection is very crucial in potato production. The authors have been cloned the coat protein gene of PVY from its Chinese isolate. A chimaeric gene containing the cauliflower mosaic virus 35S promoter and PVY coat protein coding region was introduced into the potato cultivars “Favorita”, “Tiger head” and “K4” via Agrobacterium tumefaciens. Results from PCR and Southern blot analysis confirmed that the foreign gene has integrated into the potato chromosomes. These transgenic potato plants were mechanically inoculated with PVY virus (20 mg/L). The presence of the virus in the potato plants was determined by ELISA and method of back inoculation into tobacco. The authors observed a drastic reduction in the accumulation of virus in some transgenic potato lines. Furthermore, some transgenic potato lines produced more tubers per plant than the untransformed potato did, and the average weight of these transgenic plant tubers was also increased. In the field test, the morphology and development of these transgenic potato plants were normal, 3 transgenic lines of “Favorita” exhibited a higher yield than the untrasformed virus-free potato with an increase ranged from 20% to 30%. From these transgenic lines, it will be very hopeful to develop a potato cultivar which not only has a significant resistance to PVY infection, but also a good harvest in potato production.     

Expression of a bacterial flagellin gene triggers plant immune responses and confers disease resistance in transgenic rice plants

Flagellin is a component of bacterial flagella and acts as a proteinaceous elicitor of defence responses in organisms. Flagellin from a phytopathogenic bacterium, Acidovorax avenae strain N1141, induces immune responses in suspension-cultured rice cells. To analyse the function of flagellin in rice, we fused the N1141 flagellin gene to the cauliflower mosaic virus 35S promoter and introduced it into rice. Many of the resulting transgenic rice plants accumulated flagellin at various levels. The transgenic rice developed pale spots in the leaves. The expression of a defence-related gene for phenylalanine ammonia-lyase was induced in the transgenic plants, and H(2)O(2) production and cell death were observed in some plants with high levels of gene expression, suggesting that the flagellin triggers immune responses in the transgenic rice. Transgenic plants inoculated with Magnaporthe grisea, the causal agent of rice blast, showed enhanced resistance to blast, suggesting that the flagellin production confers disease resistance in the transgenic rice.     

Enhanced resistance to<Emphasis Type="Italic"> Phytophthora infestans</Emphasis> and<Emphasis Type="Italic"> Alternaria solani</Emphasis> in leaves and tubers,respectively, of potato plants with decreased activity of the plastidic ATP/ADP transporter

Recently, it has been reported that tubers of transgenic potato ( Solanum tuberosum L.) plants with decreased activity of the plastidic ATP/ADP transporter (AATP1) contain less starch, despite having an increased glucose level [P. Geigenberger et al. (2001) Plant Physiol 125:1667-1678]. The metabolic alterations correlated with enhanced resistance to the bacterium Erwinia carotovora. Here it is shown that transgenic potato tubers, possessing less starch yet increased glucose levels due to the expression of a cytoplasm-localized yeast invertase, exhibit drastic susceptibility to E. carotovora. In addition, it is demonstrated that AATP1 anti-sense tubers show an increased capacity to ward off the pathogenic fungus Alternaria solani. In contrast to AATP1 anti-sense tubers, the corresponding leaf tissue does not show changes in carbohydrate accumulation. However, upon elicitor treatment, AATP1 anti-sense leaves possess an increased capacity to release H(2)O(2) and activate various defence-related genes, reactions that are associated with substantially delayed appearance of disease symptoms caused by Phytophthora infestans. Grafting experiments between AATP1 anti-sense plants and wild-type plants indicate the presence of a signal that is generated in AATP1 rootstocks and primes wild-type scions for potentiated activation of cellular defence responses in leaves. Together, the results suggest that (i) the enhanced pathogen tolerance of AATP1 anti-sense tubers is not due to "high sugar resistance", (ii) the increased disease resistance of AATP1 anti-sense tubers is effective against different types of pathogen and (iii) a systemic signal induced by antisensing the plastidic ATP/ADP transporter in potato tubers confers increased resistance to pathogens.     

Control of virulence gene expression by plant calcium in the phytopathogen Erwinia carotovora

Plant calcium can modulate a particular plant–pathogen interaction and have a decisive role in disease development. Enhanced resistance to the phytopathogenic enterobacterium Erwinia carotovora , the causal agent of bacterial soft rot disease, is observed in high-calcium plants. One of the main virulence determinants of E. carotovora , the PehA endopolygalacturonase, is specifically required in the early stages of the infection. Production of PehA was found to be dependent on the calcium concentration in the bacterial environment. An increase in extracellular calcium to mM concentrations repressed pehA gene expression without reducing or even enhancing expression of other extracellular enzyme-encoding genes of this pathogen. An increase in plant calcium levels could be correlated to enhanced resistance to E. carotovora infection and to an inhibition of in planta production of PehA. Ectopic expression of pehA from a calcium-insensitive promoter allowed E. carotovora to overcome this calcium-induced resistance. The results imply that plant calcium can constitute an important signal molecule in plant–pathogen interaction, which acts by modulating the expression of virulence genes of the pathogen.     

Effects of T4 lysozyme release from transgenic potato roots on bacterial rhizosphere communities are negligible relative to natural factors

Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for Bacteria, Actinomycetales, or alpha- or beta-Proteobacteria. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.     

Potato plants genetically modified to produce N-acylhomoserine lactones increase susceptibility to soft rot erwiniae

Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing [QS]). In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid. We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant. In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E. carotovora. Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.     

Phenotypic and genotypic characterization of antagonistic bacteria associated with roots of transgenic and non-transgenic potato plants

Rhizobacteria obtained during a risk assessment study from parental and transgenic T4 lysozyme-expressing potato plants were investigated to determine whether or not the strains could be grouped based on the source of isolation, transgenic or non-transgenic plants, respectively. A total of 68 representative bacterial strains of the group of enterics and pseudomonads were investigated by phenotypic profiling (the antagonistic activity towards bacterial and fungal plant pathogens, the production of the plant growth hormone indole-3-acetic acid [auxin], and the sensitivity to T4 lysozyme in vitro) and genotypic profiling by PCR fingerprints using BOX primers. All isolates were identified by fatty acid methyl ester (FAME) analysis. Computer-based analysis of the phenotypic characteristics showed that both, enterics and Pseudomonas strains clustered into six to seven groups at an Euclidian distance of 10. According to their BOX-PCR-generated fingerprints the Pseudomonas strains clustered into seven groups and the enterobacteria into two groups at the same genetic distance level of 10. The majority of groups were heterogeneous and contained isolates from all plant lines. In conclusion, cluster analysis of the phenotypic and genotypic features did not reveal correlations between bacterial isolates and transgenic character of plants.     

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.