LaCl3 treatment improves Agrobacterium-mediated immature embryo genetic transformation frequency of maize.

KEY MESSAGE: We report an optimized transformation system that uses a LaCl3 pretreatment (a Ca2+ channel blocker) for enhancing Agrobacterium-mediated infection of immature embryos and improving the genetic transformation frequency of maize. Agrobacterium-mediated genetic transformation of immature embryos is important for gene-function studies and molecular breeding of maize. However, the relatively low genetic transformation frequency remains a bottleneck for applicability of this method, especially on commercial scale. We report that pretreatment of immature embryos with LaCl3 (a Ca2+ channel blocker) improves the infection frequency of Agrobacterium tumefaciens, increases the proportion of positive callus, yields more positive regenerated plantlets, and increases the transformation frequency from 8.40 to 17.60% for maize. This optimization is a novel method for improving the frequency of plant genetic transformations mediated by Agrobacterium tumefaciens.

Molecular characterization for food safety assessment of a genetically modified late blight resistant potato: an unusual case.

Standard food safety assessments of genetically modified crops require a thorough molecular characterization of the novel DNA as inserted into the plant that is intended for commercialization, as well as a comparison of agronomic and nutritional characteristics of the genetically modified to the non-modified counterpart. These characterization data are used to identify any unintended changes in the inserted DNA or in the modified plant that would require assessment for safety in addition to the assessment of the intended modification. An unusual case of an unintended effect discovered from the molecular characterization of a genetically modified late blight resistant potato developed for growing in Bangladesh and Indonesia is presented here. Not only was a significant portion of the plasmid vector backbone DNA inserted into the plant along with the intended insertion of an R-gene for late blight resistance, but the inserted DNA was split into two separate fragments and inserted into two separate chromosomes. One fragment carries the R-gene and the other fragment carries the NPTII selectable marker gene and the plasmid backbone DNA. The implications of this for the food safety assessment of this late blight resistant potato are considered.

Genetic transfection, hairy root induction and solasodine accumulation in elicited hairy root clone of Solanum erianthum D. Don.

An efficient genetic transfection technique has been established using A4 strain of Agrobacterium rhizogenes for the first time in a medicinally valuable plant Solanum erianthum D. Don. The explants were randomly pricked with sterile needle, inoculated with bacterial suspension. The infected leaves were then washed and transferred to MS basal medium fortified with cefotaxime for hairy root induction. A maximum transformation efficiency of 72 % has been recorded after two days of co-cultivation period. The transfer of rolA and rolB genes from the bacterium to the plant genome has been confirmed in five transformed hairy rootlines by standard Polymerase Chain Reaction technique. On the basis of growth analysis and secondary metabolite study two potential rhizoclones (A4-HR-A and A4-HR-B) were selected. Rhizoclone A4-HR-A can produce highest amount of alkaloid, phenolic and flavonoid, whereas A4-HR-B was observed to be highest tannin producer. Alkaloid like solasodine, commercially important for steroidal drug synthesis, was quantified from leaf and A4-HR-A clone by an improved High Performance Liquid Chromatography method. This showed a sustainable increase (1.33 fold) in production of solasodine in hairy rootline.

Transgenic expression of Hyp-1 gene from Hypericum perforatum L. alters expression of defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens.

MAIN CONCLUSION: Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Landrace maize varieties differ from conventional and genetically modified hybrid maize in response to inoculation with arbuscular mycorrhizal fungi.

Land area planted with genetically modified (GM) crops has grown rapidly, and Brazil has the second largest area with those plants. There is, however, limited information on the possible effects of that technology on non-target organisms, especially root symbionts, such as arbuscular mycorrhizal fungi (AMF). We evaluated AMF symbiosis development in five maize genotypes: one landrace, two conventional hybrids (DKB 240 and Formula), and two GM hybrids (DKB 240-VT Pro and Formula TL). We evaluated symbiosis response in two separate experiments: one in autumn and the other in summer. Plants were inoculated with Rhizophagus clarus (Rc) and Gigaspora margarita (Gm) and compared to plants without inoculation. We evaluated root colonization, spore number, and plant biomass and phosphorous accumulation 30 and 60 days after inoculation. There were no consistent effects of GM crops, but AMF species and maize genotype affected symbiosis development. Formula genotype (isoline and GM) had a negative response to inoculation, with a decrease of around 30% in biomass and P concentration in Rc-inoculated plants. The maize landrace had a positive response, with increases of 17% and 14% in the same variables. DKB genotype (isoline and GM) showed negative, positive, and neutral effects. The results show that plant genetic identity is a determinant factor in symbiosis performance, suggesting that plants selected in low P availability can make better use of mycorrhizal symbiosis. Given the role that AMF play in different ecosystem processes, use of landrace maize may contribute to agrobiodiversity conservation.

Stacking three late blight resistance genes from wild species directly into African highland potato varieties confers complete field resistance to local blight races.

Considered responsible for one million deaths in Ireland and widespread famine in the European continent during the 1840s, late blight, caused by Phytophthora infestans, remains the most devastating disease of potato (Solanum tuberosum L.) with about 15%-30% annual yield loss in sub-Saharan Africa, affecting mainly smallholder farmers. We show here that the transfer of three resistance (R) genes from wild relatives [RB, Rpi-blb2 from Solanum bulbocastanum and Rpi-vnt1.1 from S. venturii] into potato provided complete resistance in the field over several seasons. We observed that the stacking of the three R genes produced a high frequency of transgenic events with resistance to late blight. In the field, 13 resistant transgenic events with the 3R-gene stack from the potato varieties 'Desiree' and 'Victoria' grew normally without showing pathogen damage and without any fungicide spray, whereas their non-transgenic equivalent varieties were rapidly killed. Characteristics of the local pathogen population suggest that the resistance to late blight may be long-lasting because it has low diversity, and essentially consists of the single lineage, 2_A1, which expresses the cognate avirulence effector genes. Yields of two transgenic events from 'Desiree' and 'Victoria' grown without fungicide to reflect small-scale farm holders were estimated to be 29 and 45 t/ha respectively. This represents a three to four-fold increase over the national average. Thus, these late blight resistant potato varieties, which are the farmers' preferred varieties, could be rapidly adopted and bring significant income to smallholder farmers in sub-Saharan Africa.

Probing active microbes involved in Bt-containing rice straw decomposition.

Transgenic Bacillus thuringiensis (Bt) rice extends significant protection against insect pests and meets the increasing demands for food and energy. Many studies have been conducted investigating the impacts of Bt rice to the agricultural ecosystem, but much less attention has been given to efforts attempting to determine how the presence of Bt rice influences and shapes the microbial community, especially the active microbes. Stable isotope probing and high-throughput sequencing were employed to explore the active microbes involved in Bt-containing straw decomposition. Compared to its near isoline, the Bt straw contained higher contents of total N, total P, total K, lignin, cellulose, and Cry1Ab toxin protein. These chemical differences did not affect the decomposition rate but significantly changed the active microbial decomposer communities. During the decomposition of Bt-containing straw, fungi were more affected than bacteria. Agromyces, Terrabacter, Microbacterium, Glycomyces, and Kribbella were the most representative unique (existed only in the Bt treatments and appeared at the early stage) bacterial genera, and Trichoderma was the most representative unique fungal genus in the Bt straw decomposition. By using similarity index calculation and function prediction, the significant differences between Bt straw and non-Bt straw treatments were found to be transient for both microbial taxa and functional traits. These results suggested that Bt rice has a significant but transient impact on soil microbes in terms of microbial straw decomposition.

Coexpression of octopine and succinamopine Agrobacterium virulence genes to generate high quality transgenic events in maize by reducing vector backbone integration.

Agrobacterium-mediated transformation is a complex process that is widely utilized for generating transgenic plants. However, one of the major concerns of this process is the frequent presence of undesirable T-DNA vector backbone sequences in the transgenic plants. To mitigate this deficiency, a ternary strain of A. tumefaciens was modified to increase the precision of T-DNA border nicking such that the backbone transfer is minimized. This particular strain supplemented the native succinamopine VirD1/VirD2 of EHA105 with VirD1/VirD2 derived from an octopine source (pTi15955), the same source as the binary T-DNA borders tested here, residing on a ternary helper plasmid containing an extra copy of the succinamopine VirB/C/G operons and VirD1. Transformation of maize immature embryos was carried out with two different test constructs, pDAB101556 and pDAB111437, bearing the reporter YFP gene and insecticidal toxin Cry1Fa gene, respectively, contained in the VirD-supplemented and regular control ternary strains. Molecular analyses of ~ 700 transgenic events revealed a significant 2.6-fold decrease in events containing vector backbone sequences, from 35.7% with the control to 13.9% with the VirD-supplemented strain for pDAB101556 and from 24.9% with the control to 9.3% with the VirD-supplemented strain for pDAB111437, without compromising transformation efficiency. In addition, while the number of single copy events recovered was similar, there was a 24-26% increase in backbone-free events with the VirD-supplemented strain compared to the control strain. Thus, supplementing existing VirD1/VirD2 genes in Agrobacterium, to recognize diverse T-DNA borders, proved to be a useful tool to increase the number of high quality events in maize.

A potato STRUBBELIG-RECEPTOR FAMILY member, StLRPK1, associates with StSERK3A/BAK1 and activates immunity.

Plant STRUBBELIG (SUB)-RECEPTOR FAMILY (SRF) genes encode putative leucine-rich repeat transmembrane receptor-like kinases. SRFs have been reported to play essential roles in tissue morphogenesis in many plant organs. Here, we show that a potato SRF family gene, StLRPK1, is involved in plant immunity. StLRPK1 is located at the cell plasma membrane and is strongly induced by culture filtrate from in vitro growth of the late blight pathogen Phytophthora infestans. Overexpression of StLRPK1 in stable transgenic potato or ectopic expression in Nicotiana benthamiana plants enhances P. infestans disease resistance, whereas RNA interference (RNAi) of StLRPK1 in potato decreases disease resistance. We found that StLRPK1 constitutively interacts with a pivotal co-receptor, SERK3A/BAK1, which plays a central role in plant immunity. Virus-induced gene silencing of SERK3A/BAK1 in N. benthamiana lines expressing StLRPK1 attenuated P. infestans resistance, indicating that SERK3A/BAK1 is required for StLRPK1-mediated immunity. Finally, we show that StLRPK1-triggered late blight resistance depends on the mitogen-activated protein kinase kinase MEK2 and mitogen-activated protein kinase WIPK. We propose a model in which StLRPK1 associates with SERK3A/BAK1 to positively regulate plant immunity to P. infestans through a MAPK cascade. These data provide new insights into our understanding of SRF function in plant immunity.

Potato snakin-1 gene enhances tolerance to Rhizoctonia solani and Sclerotinia sclerotiorum in transgenic lettuce plants.

Snakin-1 is a cysteine-rich antimicrobial peptide (AMP) isolated from potato tubers, with broad-spectrum activity. It belongs to the Snakin/GASA family, whose members have been studied because of their diverse roles in important plant processes, including defense. To analyze if this defensive function may lead to disease tolerance in lettuce, one of the most worldwide consumed leafy vegetable, we characterized three homozygous transgenic lines overexpressing Snakin-1. They were biologically assessed by the inoculation with the fungal pathogens Rhizoctonia solani and Sclerotinia sclerotiorum both in vitro and in planta at the greenhouse. When in vitro assays were performed with R. solani on Petri dishes containing crude plant extracts it was confirmed that the expressed Snakin-1 protein has antimicrobial activity. Furthermore, transgenic lines showed a better response than wild type in in vivo challenges against R. solani both in chamber and in greenhouse. In addition, two of these lines showed significant in vivo protection against the pathogen S. sclerotiorum in challenge assays on adult plants. Our results show that Snakin-1 is an interesting candidate gene for the selection/breeding of lettuce plants with increased fungal tolerance.

WRKY1 acts as a key component improving resistance against Alternaria solani in wild tomato, Solanum arcanum Peralta.

Early blight (EB), caused by Alternaria solani, is a major threat to global tomato production. In comparison with cultivated tomato (Solanum lycopersicum), a wild relative, S. arcanum exhibits strong resistance against EB. However, molecular cascades operating during EB resistance in wild or cultivated tomato plants are largely obscure. Here, we provide novel insight into spatio-temporal molecular events in S. arcanum against A. solani. Transcriptome and co-expression analysis presented 33-WRKYs as promising candidates of which 12 SaWRKYs displayed differential expression patterns in resistant and susceptible accessions during EB disease progression. Among these, SaWRKY1 exhibited induced expression with significant modulation in xyloglucan endotrans hydrolase 5 (XTH5) and MYB2 expressions that correlated with the disease phenotypes. Electro-mobility shift assay confirmed physical interaction of recombinant SaWRKY1 to SaXTH5 and SaMYB2 promoters. Comparative WRKY1 promoter analysis between resistant and susceptible plants revealed the presence of crucial motifs for defence mechanism exclusively in resistant accession. Additionally, many defence-related genes displayed significant expression variations in both the accessions. Further, WRKY1 overexpressing transgenic plants exhibited higher levels of EB resistance while RNAi silencing lines had increased susceptibility to A. solani with altered expression of XTH5 and MYB2. Overall, these findings demonstrate the positive influence of WRKY1 in improving EB resistance in wild tomato and this could be further utilized as a potential target through genetic engineering to augment protection against A. solani in crop plants.

Nicotiana benthamiana Matrix Metalloprotease 1 (NMMP1) gene confers disease resistance to Phytophthora infestans in tobacco and potato plants.

We previously isolated Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) from tobacco leaves. The NMMP1 gene encodes a highly conserved, Zn-containing catalytic protease domain that functions as a factor in the plant's defense against bacterial pathogens. Expression of NMMP1 was strongly induced during interactions between tobacco and one of its pathogens, Phytophthora infestans. To elucidate the role of the NMMP1 in defense of N. benthamiana against fungal pathogens, we performed gain-of-function and loss-of-function studies. NMMP1-overexpressing plants had stronger resistance responses against P. infestans infections than control plants, while silencing of NMMP1 resulted in greater susceptibility of the plants to the pathogen. This greater susceptibility correlated with fewer NMMP1 transcripts than the non-silenced control. We also examined cell death as a measure of disease. The amount of cell death induced by the necrosis-inducing P. infestans protein 1, PiNPP1, was dependent on NMMP1 in N. benthamiana. Potato plants overexpressing NMMP1 also had enhanced disease resistance against P. infestans. RT-PCR analysis of these transgenic potato plants revealed constitutive up-regulation of the potato defense gene NbPR5. NMMP1-overexpressing potato plants were taller and produced heavier tubers than control plants. We suggest a role for NMMP1in pathogen defense and development.

Environmental risk assessment of blight-resistant potato: use of a crop model to quantify nitrogen cycling at scales of the field and cropping system.

Environmental risk assessment of GM crops in Europe proceeds by step-wise estimation of effect, first in the plant, then the field plot (e.g. 10-100 m-2), field (1000-10,000 m-2) and lastly in the environment in which the crop would be grown (100-10,000 km2). Processes that operate at large scales, such as cycling of carbon (C) and nitrogen (N), are difficult to predict from plot scales. Here, a procedure is illustrated in which plot scale data on yield (offtake) and N inputs for blight resistant (both GM and non-GM) and blight-susceptible potato are upscaled by a model of crop resource use to give a set of indicators and metrics defining N uptake and release in realistic crop sequences. The greatest potential damage to environment is due to loss of N from the field after potato harvest, mainly because of the large quantity of mineral and plant matter, high in N, that may die or be left in the field. Blight infection intensifies this loss, since less fertiliser N is taken up by plants and more (as a proportion of plant mass) is returned to the soil. In a simulation based on actual crop sequences, N returns at harvest of potato were raised from 100 kg ha-1 in resistant to 150 kg ha-1 in susceptible varieties subject to a 40% yield loss. Based on estimates that blight-resistant types would require ~20% of the fungicide applied to susceptible types, introduction of resistant types into a realistic 6-year cropping sequence would reduce overall fungicide use to between 72 and 54% depending on the inputs to other crops in the sequence.

Ecological guild and enzyme activities of rhizosphere soil microbial communities associated with Bt-maize cultivation under field conditions in North West Province of South Africa.

Insecticidal proteins expressed by genetically modified Bt maize may alter the enzymatic and microbial communities associated with rhizosphere soil. This study investigated the structure and enzymatic activity of rhizosphere soil microbial communities associated with field grown Bt and non-Bt maize. Rhizosphere soil samples were collected from Bt and non-Bt fields under dryland and irrigated conditions. Samples were subjected to chemical tests, enzyme analyses, and next generation sequencing. Results showed that nitrate and phosphorus concentrations were significantly higher in non-Bt maize dryland soils, while organic carbon was significantly higher in non-Bt maize irrigated field soil. Acid phosphatase and ß-glucosidase activities were significantly reduced in soils under Bt maize cultivation. The species diversity differed between fields and Bt and non-Bt maize soils. Results revealed that Actinobacteria, Proteobacteria, and Acidobacteria were the dominant phyla present in these soils. Redundancy analyses indicated that some chemical properties and enzyme activities could explain differences in bacterial community structures. Variances existed in microbial community structures between Bt and non-Bt maize fields. There were also differences between the chemical and biochemical properties of rhizosphere soils under Bt and non-Bt maize cultivation. These differences could be related to agricultural practices and cultivar type.

Silencing of Agrobacterium tumefaciens oncogenes ipt and iaaM induces resistance to crown gall disease in plum but not in apricot.

BACKGROUND: In this study, two vectors with short-length chimeric transgenes were used to produce Prunus rootstocks resistant to crown gall disease through RNA-interference-mediated gene silencing of the Agrobacterium tumefaciens oncogenes ipt and iaaM. RESULTS: Transgenic plum and apricot lines were produced with efficiencies of up to 7.7 and 1.1% respectively. An in vitro evaluation method allowed identification of susceptible lines and reduction in the number of lines to be evaluated in the greenhouse. Five transgenic plum lines, expressing transgene-derived small interfering RNA (siRNA) and low levels of transgene hairpin RNA (hpRNA), showed a significant reduction in the development of the disease after infection with Agrobacterium strains C58 and A281 under greenhouse conditions. However, unexpectedly, all transgenic apricot lines were gall susceptible. The infection of apricot plants with a binary vector containing only the 6b oncogene demonstrated that the expression of this gene is involved in the induction of tumours in the apricot species. CONCLUSION: RNAi-mediated gene silencing can be used for inducing crown gall resistance in plum rootstocks. These could be used to graft non-genetically modified commercial fruit cultivars reducing, or eliminating, the disease symptoms. © 2017 Society of Chemical Industry.

Molecular cloning, functional characterization and expression of potato (Solanum tuberosum) 1-deoxy-d-xylulose 5-phosphate synthase 1 (StDXS1) in response to Phytophthora infestans.

1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the plastidial 2C-methyl-D-erythritol-4-phosphate (DOXP-MEP) pathway involved in isoprenoid biosynthesis. In this study, we cloned the complete cDNA of potato DXS gene that was designated StDXS1. StDXS1 cDNA encodes for 719 amino acid residues, with MW of 77.8 kDa, and is present in one copy in the potato genome. Phylogenetic analysis and protein sequence alignments assigned StDXS1 to a group with DXS homologues from closely related species and exhibited homodomain identity with known DXS proteins from other plant species. Late blight symptoms occurred in parallel with a reduction in StDXS1 transcript levels, which may be associated with the levels of isoprenoids that contribute to plant protection against pathogens. Subcellular localization indicated that StDXS1 targets the chloroplasts where isoprenoids are synthesized. Arabidopsis expressing StDXS1 showed a higher accumulation of carotenoids and chlorophyll as compared to wild type controls. Lower levels of ABA and GA were detected in the transgenic DXS lines as compared to control plants, which reflected on higher germination rates of the transgenic DXS lines. No changes were detected in JA or SA contents. Selected downstream genes in the DOXP-MEP pathway, especially GGPPS genes, were up-regulated in the transgenic lines.

Down-regulation of Arabidopsis DND1 orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew.

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.

Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins.

The production of biopharmaceutical proteins in plants requires efficient downstream processing steps that remove impurities such as host cell proteins (HCPs) and adventitious endotoxins produced by bacteria during transient expression. We therefore strived to develop effective routines for endotoxin removal from plant extracts and the subsequent use of the extracts to generate antibodies detecting a broad set of HCPs. At first, we depleted the superabundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) for which PEG precipitation achieved the best results, preventing a dominant immune reaction against this protein. We found that a mixture of sera from rabbits immunized with pre-depleted or post-depleted extracts detected more HCPs than the individual sera used alone. We also developed a powerful endotoxin removal procedure using Polymyxin B for extracts from wild type plants or a combination of fiber-flow filtration and EndoTrap Blue for tobacco plants infiltrated with Agrobacterium tumefaciens. The antibodies we generated will be useful for quality and performance assessment in future process development and the methods we present can easily be transferred to other expression systems rendering them useful in the field of plant molecular farming.

[Expression of plant antimicrobial peptide pro-SmAMP2 gene increases resistance of transgenic potato plants to Alternaria and Fusarium pathogens].

The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.

Germin-like protein 2 gene promoter from rice is responsive to fungal pathogens in transgenic potato plants.

Controlled transgene expression via a promoter is particularly triggered in response to pathogen infiltration. This is significant for eliciting disease-resistant features in crops through genetic engineering. The germins and germin-like proteins (GLPs) are known to be associated with plant and developmental stages. The 1107-bp Oryza sativa root GLP2 (OsRGLP2) gene promoter fused to a ß-glucuronidase (GUS) reporter gene was transformed into potato plants through an Agrobacterium-mediated transformation. The OsRGLP2 promoter was activated in response to Fusarium solani (Mart.) Sacc. and Alternaria solani Sorauer. Quantitative real-time PCR results revealed 4-5-fold increase in promoter activity every 24 h following infection. There was a 15-fold increase in OsRGLP2 promoter activity after 72 h of F. solani (Mart.) Sacc. treatment and a 12-fold increase observed with A. solani Sorauer. Our results confirmed that the OsRGLP2 promoter activity was enhanced under fungal stress. Furthermore, a hyperaccumulation of H2O2 in transgenic plants is a clear signal for the involvement of OsRGLP2 promoter region in the activation of specific genes in the potato genome involved in H2O2-mediated defense response. The OsRGLP2 promoter evidently harbors copies of GT-I and Dof transcription factors (AAAG) that act in response to elicitors generated in the wake of pathogen infection.