Overexpression of thaumatin-like protein in transgenic tomato plants confers enhanced resistance to Alternaria solani

Abstract

A cDNA encoding thaumatin-like protein (TLP) from rice was cloned into the binary vector pMON410 under the control of the CaMV 35S promoter for Agrobacterium-mediated transformation of tomato. All putative transformants were tested for the integration and expression of the chimeric gene by polymerase chain reaction (PCR) for hygromycin resistance gene (hph) and enzyme-linked immunosorbent assay (ELISA) for TLP respectively. Constitutive, high-level expression of TLP was observed in transgenic plants. The transgenic lines exhibited increased resistance to Alternaria solani, the early blight pathogen compared to non-transgenic tomato plants.     

Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility

Polyphenol oxidases (PPOs; EC 1.14.18.1 or EC 1.10.3.2) catalyze the oxidation of phenolics to quinones, highly reactive intermediates whose secondary reactions are responsible for much of the oxidative browning that accompanies plant senescence, wounding, and responses to pathogens. To assess the impact of PPO expression on resistance to Pseudomonas syringae pv. tomato we introduced a chimeric antisense potato PPO cDNA into tomato (Lycopersicon esculentum L.). Oxidation of caffeic acid, the dominant o-diphenolic aglycone of tomato foliage, was decreased ca. 40-fold by antisense expression of PPO. All members of the PPO gene family were downregulated: neither immunoreactive PPO nor PPO-specific mRNA were detectable in the transgenic plants. In addition, the antisense PPO construct suppressed inducible increases in PPO activity. Downregulation of PPO in antisense plants did not affect growth, development, or reproduction of greenhouse-grown plants. However, antisense PPO expression dramatically increased susceptibility to P. syringae expressing the avirulence gene avrPto in both Pto and pto backgrounds. In a compatible (pto) interaction, plants constitutively expressing an antisense PPO construct exhibited a 55-fold increase in bacterial growth, three times larger lesion area, and ten times more lesions cm^–2 than nontransformed plants. In an incompatible (Pto) interaction, antisense PPO plants exhibited 100-fold increases in bacterial growth and ten times more lesions cm^–2 than nontransformed plants. Although it is not clear whether hypersusceptibility of antisense plants is due to low constitutive PPO levels or failure to induce PPO upon infection, these findings suggest a critical role for PPO-catalyzed phenolic oxidation in limiting disease development. As a preliminary effort to understand the role of induced PPO in limiting disease development, we also examined the response of PPO promoter::-glucuronidase constructs when plants are challenged with P. syringae in both Pto and pto backgrounds. While PPO B inducibility was the same in both compatible and incompatible interactions, PPO D, E and F were induced to higher levels and with different expression patterns in incompatible interactions.     

Overexpression of the plasma membrane-localized NDR1 protein results in enhanced bacterial disease resistance in Arabidopsis thaliana

Previous studies have established that mutations in the NDR1 gene in Arabidopsis thaliana suppress the resistance response of three resistance proteins, RPS2, RPM1, and RPS5, to Pseudomonas syringae pv. tomato (Pst) strain DC3000 containing the cognate effector genes, avrRpt2, avrRpm1, and avrpPhB, respectively. NDR1 is a plasma membrane (PM)-localized protein, and undergoes several post-translational modifications including carboxy-terminal processing and N-linked glycosylation. Expression of NDR1 under the NDR1 native promoter complements the ndr1-1 mutation, while overexpression of NDR1 results in enhanced resistance to virulent Pst. Sequence analysis and mass spectrometry suggest that NDR1 is localized to the PM via a C-terminal glycosylphosphatidyl-inositol (GPI) anchor. GPI modification would potentially place NDR1 on the outer surface of the PM, perhaps allowing NDR1 to act as a transducer of pathogen signals and/or interact directly with the pathogen.     

Enhanced resistance to bacterial pathogen in transgenic tomato plants expressing cathelicidin antimicrobial peptide

A human cathelicidin antimicrobial peptide (hCAP18/LL-37) is the only member of the mammalian cathelicidin family of proteins that is present in humans. The LL-37 gene was fused to the secretory signal peptide sequence (sp) and a new construct, pSP1-spLL-37, was transformed into tomato. Integration of single copy of the LL-37 was confirmed by TaqMan-PCR and its expression was confirmed by RT-PCR and ELISA assay. The transgenic tomato plants exhibited significant resistance to bacterial soft rot and bacterial spot where it showed strong concerted expression of PR-protein, LTP and AFP1 genes. In vitro screening of protein extracts isolated from the leaves of transgenic plants delimited the growth of P. carotovorum ssp. carotovorum to 15%, and that of X. campestris pv. vesicatoria to 35%. The subcellular localization of LL-37::GFP fusion protein was mainly localized in the cell membrane and cytoplasm. LL-37 expressing-tomato could therefore provide reliable bio-protection against bacterial soft rot and bacterial spot.     

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     

Function of polyphenol oxidase in higher plants

Recent evidence has supported the folllowing views:
1. Polyphenol oxidase (PPO) is a plastidic enzyme that is unclear-coded, but is inactive until incorporated into the plastid.
2. In healthy green tissues PPO exists in a latent form on the thylakoid membrane and is not involved in synthesis of phenolic compounds. In leucoplasts, proplastids, or amyloplasts PPO is often present in a latent form in rudimentary thylakoids.
3. PPO normally functions as a phenol oxidase in vivo only in sencent or damaged cells.
4. In the functional chloroplast, PPO may be involved in some aspect of oxygen chemistry – pherhaps mediation of pseudocyclic photophosphorylation.     

Overexpression of SlGGP-LIKE gene enhanced the resistance of tomato to salt stress

Ascorbic acid (AsA) plays an important role in scavenging reactive oxygen species (ROS) and reducing photoinhibition in plants, especially under stress. The function of SlGGP which encodes the key enzyme GDP-L-galactose phosphorylase in AsA synthetic pathway is relatively clear. However, there is another gene SlGGP-LIKE that encodes this enzyme in tomato, and there are few studies on it, especially under salt stress. In this study, we explored the function of this gene in tomato salt stress response using transgenic lines overexpressing SlGGP-LIKE (OE). Under normal conditions, overexpressing SlGGP-LIKE can increase the content of reduced AsA and the ratio of AsA/ DHA (dehydroascorbic acid), as well as the level of xanthophyll cycle. Under salt stress, compared with the wild-type plants (WT), the OE lines can maintain higher levels of reduced AsA. In addition, OE lines also have higher levels of reduced GSH (glutathione) and total GSH, higher ratios of AsA/DHA and GSH/oxidative GSH (GSSR), and higher level of xanthophyll cycle. Therefore, the OE lines are more tolerant to salt stress, with higher photosynthetic activity, higher antioxidative enzyme activities, higher content of D1 protein, lower production rate of ROS, and lighter membrane damage. These results indicate that overexpressing SlGGP-LIKE can enhance tomato resistance to salt stress through promoting the synthesis of AsA.

     

Kanamycin resistance during in vitro development of pollen from transgenic tomato plants

Effects of kanamycin on pollen germination and tube growth of pollen from non-transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin, however, severely affected tube growth of pollen from non-transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200–400 mgl-1. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development.Abbreviations ATW Agrobacterium Tomato Wageningen - KAN Kanamycin - KANr Kanamycin resistant - KANs Kanamycin sensitive - mRNA messenger RNA - NPT Neomycin phosphotransferase     

Molecular mechanisms involved in bacterial speck disease resistance of tomato

An important recent advance in the field of plant-microbe interactions has been the cloning of genes that confer resistance to specific viruses, bacteria, fungi or nematodes. Disease resistance (R) genes encode proteins with predicted structural motifs consistent with them having roles in signal recognition and transduction. The future challenge is to understand how R gene products specifically perceive defence-eliciting signals from the pathogen and transduce those signals to pathways that lead to the activation of plant defence responses. In tomatoes, the Pto kinase (product of the Pto R gene) confers resistance to strains of the bacterial speck pathogen, Pseudomonas syringae pv. tomato, that carry the corresponding avirulence gene avrPto. Resistance to bacterial speck disease is initiated by a mechanism involving the physical interaction of the Pto kinase and the AvrPto protein. This recognition event initiates signalling events that lead to defence responses including an oxidative burst, the hypersensitive response and expression of pathogenesis-related genes. Pto-interacting (Pti) proteins have been identified that appear to act downstream of the Pto kinase and our current studies are directed at elucidating the roles of these components.     

Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants

Selectable markers of bacterial origin such as the neomycin phosphotransferase type II gene, which can confer kanamycin resistance to transgenic plants, represent an invaluable tool for plant engineering. However, since all currently used antibiotic-resistance genes are of bacterial origin, there have been concerns about horizontal gene transfer from transgenic plants back to bacteria, which may result in antibiotic resistance. Here we characterize a plant gene, Atwbc19, the gene that encodes an Arabidopsis thaliana ATP binding cassette (ABC) transporter and confers antibiotic resistance to transgenic plants. The mechanism of resistance is novel, and the levels of resistance achieved are comparable to those attained through expression of bacterial antibiotic-resistance genes in transgenic tobacco using the CaMV 35S promoter. Because ABC transporters are endogenous to plants, the use of Atwbc19 as a selectable marker in transgenic plants may provide a practical alternative to current bacterial marker genes in terms of the risk for horizontal transfer of resistance genes.     

地表球囊霉诱发番茄抗早疫病的机理

丛枝菌根可以改善植物营养状况,提高宿主植物的抗病性.本文研究了番茄幼苗预先接种丛枝菌根真菌(AMF)地表球囊霉后对番茄植株保护酶活性和防御反应基因表达,以及对番茄早疫病抗性的影响.结果表明:被AMF侵染的番茄植株在接种早疫病病原菌茄链格孢菌后,其叶片内的超氧化物歧化酶(SOD)和过氧化物酶(POD)活性迅速提高.其中SOD酶活性在接种后18h达到最高,比只接种地表球囊霉(G)、茄链格孢菌(A)以及未接种AMF和病原菌的对照(CK)分别高28.6％、79.2％和82.8％;POD酶活性在接种后65 h达到最高,分别比G、A处理和CK高762％、18.3％和1710％.经荧光定量PCR检测表明,AMF侵染后的番茄植株再接种病原菌,其叶片中PR1(病程相关蛋白基因)、PR2(β-1,3-葡聚糖酶基因)和PR3(几丁质酶基因)基因的最高转录水平达到CK的9.67、8.54和13.4倍.与CK相比,先接种地表球囊霉再接种茄链格孢菌的番茄植株(GA)的早疫病发病率和病情指数分别降低了36.3％和61.4％.预先接种AMF的番茄植株在遇到病原菌袭击时诱导的防御反应强而迅速,诱发(priming)可能是菌根真菌提高宿主植物抗病性的重要机制.     

Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants.

Synthesis of the phytohormone ethylene is believed to be essential for many plant developmental processes. The control of ripening in climacteric fruits and vegetables is among the best characterized of these processes. One approach to reduce ethylene synthesis in plants is metabolism of its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). Soil bacteria containing an enzyme, ACC deaminase, were identified by their ability to grow on ACC as a sole nitrogen source. The gene encoding ACC deaminase was cloned and introduced into tomato plants. Reduction in ethylene synthesis in transgenic plants did not cause any apparent vegetative phenotypic abnormalities. However, fruits from these plants exhibited significant delays in ripening, and the mature fruits remained firm for at least 6 weeks longer than the nontransgenic control fruit. These results indicated that ACC deaminase is useful for examining the role of ethylene in many developmental and stress-related processes in plants as well as for extending the shelf life of fruits and vegetables whose ripening is mediated by ethylene.     

Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors

Protease inhibitors have been proposed as potential defense molecules for increased insect resistance in crop plants. Compensatory over-production of insensitive proteases in the insect, however, has limited suitability of these proteins in plant protection, with very high levels of inhibitor required for increased plant resistance. In this study we have examined whether combined used of two inhibitors is effective to prevent this compensatory response. We show that leaf-specific over-expression of the potato PI-II and carboxypeptidase inhibitors (PCI) results in increased resistance to Heliothis obsoleta and Liriomyza trifolii larvae in homozygote tomato lines expressing high levels (#62;1 the total soluble proteins) of the transgenes. Leaf damage in hemizygous lines for these transformants was, however, more severe than in the controls, thus evidencing a compensation response of the larvae to the lower PI concentrations in these plants. Development of comparable adaptive responses in both insects suggests that insect adaptation does not entail specific recognition of the transgene, but rather represents a general adaptive mechanism triggered in response to the nutritional stress imposed by sub-lethal concentrations of the inhibitors. Combined expression of defense genes with different mechanisms of action rather than combinations of inhibitors may then offer a better strategy in pest management as it should be more effective in overcoming this general adaptive response in the insect.     

Proteolytic processing of polyphenol oxidase from plants and fungi

Polyphenol oxidase (PPO), a metalloenzyme containing a type-3 copper center, is produced by many species of plants, fungi, and bacteria. There is great variability in the subunit molecular mass reported for PPO, even from a single species. In some cases, experimental evidence (usually protein sequencing by Edman degradation) indicates that the variability in molecular mass for PPO from a given species is the result of proteolytic processing at the N and/or C-termini of the protein. In order to identify specific sequence regions where proteolysis occurs in PPO from most species, the experimentally established N and C-termini of these proteolyzed enzymes were compared to the protein sequences of other PPOs for which the N and C-termini have not been established by protein sequencing methods. In all cases the N-terminal proteolysis sites were located prior to a conserved arginine residue, and the C-terminal proteolysis sites were located following a conserved tyrosine motif. Based on the sites of proteolysis, molecular masses were calculated for the enzymes, and the calculated values were used to rationalize the varying molecular masses reported in the literature. To determine the structural implications of N and C-terminal proteolysis, the proteolysis sites were related to the two available PPO structures: Ipomoea batatas catechol oxidase and Streptomyces castaneoglobisporus tyrosinase. A structural “core” region that appears to be essential for structural stability and enzymatic activity was identified.