Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression

In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells [45, 46]. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5 regions were translationally fused with the -D-glucuronidase reporter gene (GUS). The GNT1 5 region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5 regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.Department of Plant Molecular Biology, Leiden University     

Ergosterol treatment leads to the expression of a specific set of defence-related genes in tobacco

Ergosterol is the main sterol of most fungi. Production of reactive oxygen species after the treatment of tobacco and tomato cells by nano-molar concentrations of ergosterol was previously observed as well as the activation of some stress activated mitogen-activated protein kinases on alfalfa cells. In this paper, the expression of some defence-related genes after the ergosterol treatment of tobacco Nicotiana tabacum plants is reported. The gene expression of pathogenesis related proteins of families PR1, PR3, PR5 and proteinase inhibitors of class I and II together with enzymes participating in the defence response, such as phenylalanine-ammonia lyase and sesquiterpene cyclase, were monitored by RT-qPCR. In addition, the concentrations of salicylic acid, an important signalling molecule, increased in time due to the enzyme activation. On the other hand, ergosterol did not provoke tissue necrosis and the possible cross-talk between the signalling pathways of salicylate and jasmonate was observed. Collected data shows that ergosterol is able to activate the expression of a number of defence genes and could increase resistance against pathogens.     

A second mutation enhances resistance of a tobacco mutant to sulfonylurea herbicides

Summary Cultures of Nicotiana tabacum cells homozgous for a mutation (S4) at the SuRB locus that confers resistance to the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl (Chaleff and Ray 1984; Chaleff and Bascomb 1987) were used to isolate a doubly mutant cell line (S4 Hra/S4+) resistant to even higher herbicide concentrations. Growth of cells homozygous for both the S4 and Hra mutations (S4 Hra/S4 Hra) was uninhibited by a herbicide concentration 500-fold higher than a concentration by which growth of S4+/S4+ callus was inhibited by 75%. Plants homozygous for both mutations were at least five-fold more resistant to foliar applications of chlorsulfuron than were singly mutant S4+/S4+ plants. This enhanced resistance was inherited as a single, semidominant, nuclear trait that is genetically linked to the S4 mutation. Acetolactate synthase (ALS) activity in extracts of leaves of doubly mutant (S4 Hra/S4 Hra) plants was approximately 20-fold more resistant to inhibition by chlorsulfuron and sulfometuron methyl than was ALS activity in singly mutant (S4+/ S4+) leaf extracts, which was in turn more resistant to inhibition by these compounds than was the normal enzyme. Extracts prepared from plants of these three genotypes possessed the same ALS specific activities. Therefore, Hra represents a second independent mutation at or near the SuRB locus that reduces the sensitivity of tobacco ALS activity to inhibition by sulfonylurea herbicides.     

Molecular tagging of the tobacco chromosome carrying the TMV-resistance gene (N gene) by Agrobacterium-mediated transformation

Summary The hypersensitive response of tobacco to inoculation with tobacco mosaic virus (TMV) is controlled by a single dominant gene, the N gene. As a first step in localizing and transferring the N gene, we have prepared a line of tobacco plants in which the kanamycin-resistance (Km^r) gene is closely linked to the N gene. Nicotiana tabacum plants heterozygous for the N gene were transformed to Km^r by Agrobacterium carrying pMON200. Eighty-nine independent transformed clones were regenerated and were backcrossed with nontransformed, TMV-sensitive plants. Progeny from these crosses were screened first for Km^r; then the Km^r progeny were inoculated with TMV and scored for the hypersensitive response. Of the initial 89 clones, 68 appeared to have integrated a single functional Km^r gene. Initial tests for TMV resistance indicated possible linkage between Km^r and the N gene in 11 plants. With further testing, linkage has been established for two of these plant lines. In one of these lines, the two genes were 30–40 map units apart, and evidence of somatic instability in the linkage was obtained. However, in the second line, linkage between Km^r and the N gene was tight, and recombination between the genes in this case was only 5%. Southern hybridization revealed that this plant contained only a single copy of the Km^r gene. Linkage between Km^r and the N gene in this plant line has been verified in each of two additional backcross generations.Abbreviations nptII Neomycin phosphotransferase gene - Km^r kanamycin resistant - Km^s kanamycin sensitive - TMV tobacco mosaic virus - TMV-R TMV resistant - TMV-S TMV sensitive     

Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization,T-DNA methylation and conditional expression of opine genes

Summary Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut⁺), shoot regeneration (Reg⁺) and octopine synthesis (Ocs⁺)), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut⁺Reg⁺ but shows little or no octopine synthesis activity (Ocs^-). Subclones of TSO38, however, are either Ocs^- or Ocs⁺. Ocs^- shoots become Ocs⁺ under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs^- subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs⁺ and an Ocs^- subclone of TSO38, which were negative for the presence of mannopine (Mas^-) and agropine (Ags^-), became Mas⁺Ags⁺ after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs^- line and in the Ocs⁺ line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs^- and an Ocs⁺ TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs⁺ and an Ocs^- subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs^- and the Ocs⁺ TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.     

Accumulation of genes for susceptibility to rust fungi for which barley is nearly a nonhost results in two barley lines with extreme multiple susceptibility

Nonhost resistance is the most common type of resistance in plants. Understanding the factors that make plants susceptible or resistant may help to achieve durably effective resistance in crop plants. Screening of 109 barley (Hordeum vulgare L.) accessions in the seedling stage indicated that barley is a complete nonhost to most of the heterologous rust fungi studied, while it showed an intermediate status with respect to Puccinia triticina, P. hordei-murini, P. hordei-secalini, P. graminis f. sp. lolii and P. coronata ff. spp. avenae and holci. Accessions that were susceptible to a heterologous rust in the seedling stage were much more or completely resistant at adult plant stage. Differential interaction between barley accessions and heterologous rust fungi was found, suggesting the existence of rust-species-specific resistance. In particular, many landrace accessions from Ethiopia and Asia, and naked-seeded accessions, tended to be susceptible to several heterologous rusts, suggesting that some resistance genes in barley are effective against more than one heterologous rust fungal species. Some barley accessions had race-specific resistance against P. hordei-murini. We accumulated genes for susceptibility to P. triticina and P. hordei-murini in two genotypes called SusPtrit and SusPmur, respectively. In the seedling stage, these accessions were as susceptible as the host species to the target rusts. They also showed unusual susceptibility to other heterologous rusts. These two lines are a valuable asset to further experimental work on the genetics of resistance to heterologous rust fungi.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-004-1319-1Abbreviations ff. spp Formae speciales - RIL Recombinant inbred line - DC Double cross - DC-S Progeny produced by selfing of double-cross plants     

A multi-generation analysis of the stability of transgenic virus resistance in doubled-haploid tobacco lines

Plants can be genetically engineered for virus resistance by transformation with a viral gene. We transformed tobacco with the tomato spotted wilt virus (TSWV) nucleocapsid gene from the Hawaiian L isolate in order to obtain TSWV resistant breeding lines. Doubled-haploid lines were produced from primary transgenic plants that were selected for resistance to the virus. Several of these lines showed very high levels of resistance and were symptomless after inoculation with the Hawaiian L isolate of TSWV. The accumulation of only low levels of full-length transgene RNA and protein observed in these lines is consistent with an RNA-mediated mechanism of resistance. The lines that were highly resistant to the Hawaiian L isolate of TSWV were also found to be highly resistant to several other isolates of TSWV, while lines that were only moderately resistant to the Hawaiian L isolate were often susceptible to the other isolates. The highly resistant lines were advanced over several generations by self-pollination. Although these lines were fully homozygous, several lines lost resistance in later generations, indicating that the resistance was unstable. Selection for resistance in these unstable lines did not prevent the occurrence of susceptible progeny in subsequent generations. Therefore, testing over several generations is required to determine the stability of resistance when breeding crops with transgenic virus resistance.     

High meiotic stability of a foreign gene introduced into tobacco by Agrobacterium-mediated transformation

Summary Two lines of transgenic Nicotiana tabacum transformed to kanamycin resistance by means of a binary Agrobacterium vector containing a nos-npt gene were investigated over three generations. Southern hybridization and crossing analyses revealed that a single copy of T-DNA had integrated in each line and that the kanamycin resistance was regularly transmitted to the progeny as a monogenic dominant trait. Homozygous transgenic plants were fully fertile, morphologically normal and did not significantly differ from wild-type plants in the quantitative characters examined (plant height, flowering time). The two lines showed very low, but significantly different levels of meiotic instability: kanamycin-sensitive plants occurred among backcross progeny from homozygous transgenic plants with frequencies of 6/45,000 and 25/45,000, respectively. The sensitive plants arose independently of each other and thus resulted from meiotic rather than mitotic events. These findings demonstrate for the first time that integrated foreign genes can be transmitted to progeny with the high degree of meiotic stability required for commercial varieties of crop plants. They emphasize the importance of non-homologous integration and of avoiding co-integration of inactive gene copies for achieving meiotically stable transformants.     

Characterisation of basal resistance (BR) by expression patterns of newly isolated representative genes in tobacco

Increasing evidence indicates that plants, like animals, use basal resistance (BR), a component of the innate immune system, to defend themselves against foreign organisms. Contrary to the hypersensitive reaction (HR)-type cell death, recognition in the case of BR is unspecific, as intruders are recognised based on their common molecular patterns. Induction of BR is not associated with visible symptoms, in contrast to the HR-type cell death. To analyse the early events of BR in tobacco plants we have carried out a subtractive hybridisation between leaves treated with the HR-negative mutant strain Pseudomonas syringae pv. syringae 61 hrcC and non-treated control leaves. Random sequencing from the 304 EBR clones yielded 20 unique EST-s. Real-time PCR has proved that 8 out of 10 clones are activated during BR. Six of these EST-s were further analyzed. Gene expression patterns in a time course showed early peaks of most selected genes at 3–12 h after inoculation (hpi), which coincided with the development-time of BR. Upon treatments with different types of bacteria we found that incompatible pathogens, their hrp mutants, as well as non-pathogens induce high levels of expression while virulent pathogens induce only a limited gene-expression. Plant signal molecules like salicylic acid, methyl jasmonate, ethylene and spermine, known to be involved in plant defense were not able to induce the investigated genes, therefore, an unknown signalling mechanism is expected to operate in BR. In summary, we have identified representative genes associated with BR and have established important features of BR by analysing gene-expression patterns.     

Early season root production and zoospore infection of cultivars of flue-cured tobacco that differ in level of partial resistance to Phytophthora parasitica var. nicotianae

Root production of four cultivars of flue-cured tobacco was quantified in the field, greenhouse and phytotron. The cultivars ranged in level of partial resistance to the black shank pathogen, Phytophthora parasitica var. nicotianae, from susceptible to highly resistant. In the field, root-observation plates were installed approximately 10 cm from plants, and in greenhouse and phytotron studies, plants were grown in 4-liter containers with one sloping transparent side for root observation. Root growth was determined weekly for four weeks after transplanting in the field and daily up to 14 days after transplanting in the greenhouse and phytotron. Root tracings were made on acetate sheets placed against the sloping transparent side of the containers or against the transparent observation plates in the field following removal of soil from the outside of the observation plate. Root growth was quantified by retracing the root pattern on the acetate sheets over a digitizing tablet attached to a personal computer. Numbers of roots, root length, and mean and maximum rate of root growth were determined. Cultivars Hicks (susceptible) and K-326 (low level of resistance) had significantly larger root systems than moderately resistant G-28 or highly resistant NC 82. Differences in total root length were due to increased branching that resulted in development of significantly greater numbers of roots in Hicks and K-326. For example, between day 21 and 28, Hicks produced more than three times the number of new roots as NC 82 in the field. The mean rate of root extension observed (2.17 mm hr^–1) was similar in all four cultivars. Infection efficiency on the different cultivars was determined in the field by inoculating roots with zoospores of P. p. nicotianae. Lesions were visible as water soaked areas within 24 hr of inoculation. At 48 hr after inoculation, percentages of inoculations that resulted in lesion formation were 57, 46, 23, and 16% for Hicks, K-326, G-28 and NC 82, respectively. The possible role of rooting intensity as a mechanism of avoidance to P. p. nicotianae in tobacco cultivars is discussed.     

Pathogen resistance of transgenic tobacco plants producing caffeine

Caffeine (1,3,7-trimethylxanthine) is a typical purine alkaloid, and produced by a variety of plants such as coffee and tea. Its physiological function, however, is not completely understood, but chemical defense against pathogens and herbivores, and allelopathic effects against competing plant species have been proposed. Previously, we constructed transgenic tobacco plants, which produced caffeine up to 5 microg per gram fresh weight of leaves, and showed them to repel caterpillars of tobacco cutworms (Spodoptera litura). In the present study, we found that these transgenic plants constitutively expressed defense-related genes encoding pathogenesis-related (PR)-1a and proteinase inhibitor II under non-stressed conditions. We also found that they were highly resistant against pathogens, tobacco mosaic virus and Pseudomonas syringae. Expression of PR-1a and PR-2 was higher in transgenic plants than in wild-type plants during infection. Exogenously applied caffeine to wild-type tobacco leaves exhibited the similar resistant activity. These results suggested that caffeine stimulated endogenous defense system of host plants through directly or indirectly activating gene expression. This assumption is essentially consistent with the idea of chemical defense, in which caffeine may act as one of signaling molecules to activate defense response. It is thus conceivable that the effect of caffeine is bifunctional; direct interference with pest metabolic pathways, and activation of host defense systems.     

cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

Comparison of photosystem II complexes isolated from tobacco and two chlorophyll deficient tobacco mutants

A comparative study of photosystem II complexes isolated from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contains normal stacked thylakoid membranes, and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked grana or essentially unstacked thylakoids with occasional membrane doublings, has been carried out. The corresponding photosystem II complexes had an O₂ evolving activity ranging from 290 (for the wild type) to 1100 mol O₂ x mg chlorophyll^-1 x h^-1 (for the mutant Su/su var. Aurea). The reduced photosynthetic unit size was also obvious in the mangenese and cytochrome_b559 content. The photosystem II complex from the wild type contained 4 Mn and 1 cytochrome_b559 per 200 to 280 chlorophylls, while the corresponding value for the mutant Su/su var. Aurea was 4 Mn and 1 cytochrome_b559 per 35 to 60 chlorophylls. We have also examined the polypeptide composition and show that the photosystem II complex from the wild type consisted of polypeptides of 48, 42, 33, 32, 30, 28, 23, 21, 18, 16 and 10 kDa, while the mutant complex mainly contained the polypeptides of 48, 42, 33, 32, 30, 28 and 10 kDa. In the mutant photosystem II complex the light-harvesting chlorophyll protein (peptide of 28 kDa) was reduced by a factor of 5 to 6 as compared to the wild type. With respect to the peptide composition and the photosynthetic unit size, the Triton-solubilized photosystem II complex from the mutant Su/su var. Aurea was very similar to O₂ evolving photosystem II reaction center core complexes.Abbreviations PS photosystem - chl chlorophyll - LHCP light-harvesting chlorophyll a/b protein complex