Characterization of Urtica dioica agglutinin isolectins and the encoding gene family

Urtica dioica agglutinin (UDA) has previously been found in roots and rhizomes of stinging nettles as a mixture of UDA- isolectins. Protein and cDNA sequencing have shown that mature UDA is composed of two hevein domains and is processed from a precursor protein. The precursor contains a signal peptide, two in-tandem hevein domains, a hinge region and a carboxyl-terminal chitinase domain. Genomic fragments encoding precursors for UDA-isolectins have been amplified by five independent polymerase chain reactions on genomic DNA from stinging nettle ecotype Weerselo. One amplified gene was completely sequenced. As compared to the published cDNA sequence, the genomic sequence contains, besides two basepair substitutions, two introns located at the same positions as in other plant chitinases. By partial sequence analysis of 40 amplified genes, 16 different genes were identified which encode seven putative UDA- isolectins. The deduced amino acid sequences share 78.9–98.9% identity. In extracts of roots and rhizomes of stinging nettle ecotype Weerselo six out of these seven isolectins were detected by mass spectrometry. One of them is an acidic form, which has not been identified before. Our results demonstrate that UDA is encoded by a large gene family.     

Isolectin composition of individual clones of Urtica dioica: Evidence for phenotypic differences

Analysis of the isolectin composition of 102 individual nettle ( Urtica dioica L.) clones by ion-exchange chromatography revealed the occurrence of at least 11 different isolectins, which all had the same molecular structure and exhibited identical carbohydrate-binding specificity and agglutination properties. All 11 isolectins, however, did not occur simultaneously; 34 combinations of either 1, 2, 3, 4 or 5 isolectins were found. Since the occurrence of multiple molecular forms of the nettle agglutinin cannot be explained by the (partial) autotetraploid character of stinging nettle it is postulated to rely on the expression of a family of closely related lectin genes.     

The vacuolar targeting signal of the 2S albumin from Brazil nut resides at the C terminus and involves the C-terminal propeptide as an essential element.

Genetic constructs in which different N- and C-terminal segments of Brazil nut (Bertholletia excelsa H.B.K.) 2S albumin were fused to secretory yeast invertase were transformed into tobacco (Nicotiana tabacum) plants to investigate the vacuolar targeting signal of the 2S albumin. None of the N-terminal segments, including the complete precursor containing all propeptides, was able to direct the invertase to the vacuoles. However, a short C-terminal segment comprising the last 20 amino acids of the precursor was sufficient for efficient targeting of yeast invertase to the vacuoles of the transformed tobacco plants. Further analyses showed that peptides of 16 and 13 amino acids of the C-terminal segment were still sufficient, although they had slightly lower efficiency. When segments of 9 amino acids or shorter were analyzed, a decrease to approximately 30% was observed. These segments included the C-terminal propeptide of four amino acids (Ile-Ala-Gly-Phe). When the 2S albumin was expressed in tobacco, it was also localized to the vacuoles of mesophyll cells. If the C-terminal propeptide was deleted from the 2S albumin precursor, all of this truncated 2S albumin was secreted from the tobacco cells. These results indicate that the C-terminal propeptide is necessary but not sufficient for vacuolar targeting. In addition, an adjacent segment of at least 12 amino acids of the mature protein is needed to form the complete signal for efficient targeting.     

Antimicrobial peptides fromMirabilis jalapa andAmaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco

The cDNAs encoding the seed antimicrobial peptides (AMPs) fromMirabilis jalapa (Mj-AMP2) andAmaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolleet al., Plant Mol Biol 28:713–721 (1995) and 22:1187–1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195–1206 (1991)]; an Ac-AMP2wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing eitherwild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. Thein vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with eitherBotrytis einerea orAlternaria longipes.     

Resistance ofUrtica dioica to mycorrhizal colonization: a possible involvement ofUrtica dioica agglutinin

Roots of stinging nettle (Urtica dioica L.) were sampled at different sites around Basel (Switzerland) and examined under the microscope. They were completely devoid of mycorrhizal structures. Similarly, stinging nettle plants grown in the greenhouse in the presence of the arbuscular mycorrhizal fungusGlomus mosseae did not show any signs of mycorrhiza formation. Spread ofG. mosseae through the rhizosphere of stinging nettle plants was inhibited, and application of extracts of stinging nettle roots and rhizomes to hyphal tips ofG. mosseae reduced hyphal growth.Urtica dioica agglutinin, an antifungal protein present in the rhizomes of stinging nettle, inhibited hyphal growth in a similar way as the crude root extract. The possibility thatUrtica dioica agglutinin is at least partially responsible for the inability of stinging nettle to form the arbuscular mycorrhizal symbiosis withG. mosseae is discussed.     

A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco.

Barley lectin is synthesized as a preproprotein with a glycosylated carboxyl-terminal propeptide (CTPP) that is removed before or concomitant with deposition of the mature protein in vacuoles. Expression of a cDNA clone encoding barley lectin in transformed tobacco plants results in the correct processing, maturation, and accumulation of active barley lectin in vacuoles [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. The glycan of the propeptide is not essential for vacuolar sorting, but may influence the rate of post-translational processing [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. To investigate the functional role of the CTPP in processing, assembly, and sorting of barley lectin to vacuoles, a mutant barley lectin cDNA clone lacking the 15-amino acid CTPP was prepared. The CTPP deletion mutant of barley lectin was expressed in tobacco protoplasts, suspension-cultured cells, and transgenic plants. In all three systems, the wild-type barley lectin was sorted to vacuoles, whereas the mutant barley lectin was secreted to the incubation media. Therefore, we conclude that the carboxyl-terminal domain of the barley lectin proprotein is necessary for the efficient sorting of this protein to plant cell vacuoles.     

Correct processing of the kiwifruit protease actinidin in transgenic tobacco requires the presence of the C-terminal propeptide.

A 355 cauliflower mosaic virus promoter and a tapetum-specific promoter were used to direct the synthesis in tobacco of preproactinidin and a derivative that lacked a C-terminal extension. Preproactinidin was processed into a form that migrated identically on protein gels with mature actinidin extracted from kiwifruit. This protein was proteolytically active in vitro, and high-level accumulation of this protein appeared to be detrimental to plant growth. Plants expressing an actinidin cDNA construct that lacked the sequence encoding the C-terminal propeptide were phenotypically normal but accumulated N-proactinidin, which was proteolytically active in vitro but did not self-cleave to mature actinidin. In transgenic tobacco, the C-terminal extension of actinidin is therefore required for correct processing.     

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.     

A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin-type protease) with a C-terminal extended sequence: its purification and in vitro processing

The precursor of aqualysin I, an extracellular subtilisin-type protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65°C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS-polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C-terminal extended sequence occurs through an intermolecular self-processing mechanism.     

Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.     

Expression of recombinant trichosanthin,a ribosome-inactivating protein,in transgenic tobacco

Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.     

Vesicle transport and processing of the precursor to 2S albumin in pumpkin

Cell fractionation of pulse-chase-labeled developing pumpkin cotyledons demonstrated that proprotein precursor to 2S albumin is transported from the endoplasmic reticulum to dense vesicles and then to the vacuoles, in which pro2S albumin is processed to the mature 2S albumin. Immunocytochemical analysis showed that dense vesicles of about 300 nm in diameter mediate the transport of pro2S albumin to the vacuoles.
The primary structure of the precursor (16 578 Da) to pumpkin 2S albumin has been deduced from the nucleotide sequence of an isolated cDNA insert. The presence of a hydrophobic signal peptide at the N-terminus indicates that the precursor is a preproprotein that is converted into pro2S albumin after cleavage of the signal peptide. N-terminal sequencing of the pro2S albumin in the isolated vesicles revealed that the signal peptide is cleaved off co-translationally on the C-terminal side of alanine residue 22 of prepro2S albumin. By contrast, post-translational cleavages occur on the C-terminal sides of asparagine residues 35 and 74, which are conserved among precursors to 2S albumin from different plants. Hydropathy analysis revealed that the two asparagine residues are located in the hydrophilic regions of pro2S albumin. These findings suggest that a vacuolar processing enzyme can recognize exposed asparagine residues on the molecular surface of pro2S albumin and cleave the peptide bond on the C-terminal side of each asparagine residue to produce mature 2S albumin in the vacuoles.     

Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting

Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 mol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 mol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP-and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.     

Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin.

Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.     

Effect of the Urtica dioica agglutinin on germination and cell wall formation of Phycomyces blakesleeanus Burgeff

The lectin from stinging nettle rhizomes, Urtica dioica agglutinin (UDA), did not affect the evolution of wet and dry weight, protein, nucleic acid, ATP, cAMP and glycerol content during early germination of Phycomyces blakesleeanus spores. However, earlier investigations established a strongly reduced mycelial growth of several phytopathogenic fungi by this small plant lectin. Total uptake and incorporation of radioactive precursors showed no differences between UDA or control hyphae, but UDA significantly altered the distribution patterns of [¹⁴C]-glucose incorporated into the walls of Phycomyces blakesleeanus (more label was recovered in the chitin fraction). Moreover, a small but significant stimulation of chitin synthase and a similar inhibition of chitin deacetylase was found in cell wall preparations. These observations could lead to a better understanding of plant-pathogen interrelationships and to a further elucidation of cell wall structure in fungi.Abbreviations GlcNAc N-Acetylglucosamine - PDB potato dextrose broth - PMM Phycomyces minimal medium - UDA Urtica dioica agglutinin - TEA tri-ethyl-amine - DAB 1,4-diaminobutanone     

Controlling ‘bois noir’ disease on grapevine: does the timing of herbicide application affect vector emergence?

Bois noir is an important grapevine yellows disease that can cause serious economical losses in European grapevine production. Hyalesthes obsoletus Signoret (Hemiptera, Cixiidae) is the principal vector of bois noir in Switzerland and stinging nettle (Urtica dioica) is its favourite host plant species in vineyards. As bois noir disease can hardly be cured and direct control measures against H. obsoletus are ineffective, viticultural control practices target stinging nettle, the actual reservoir and source of both the pathogen and its vector. Currently, it is recommended to apply herbicides against stinging nettle at the end of the season to kill developing H. obsoletus nymphs. To verify if this late period of herbicide application is justified, stinging nettle patches were treated with glyphosate in the autumn, in the spring or were left untreated as a control. Herbicide applications at both dates controlled the growth of stinging nettle very well in the subsequent summer, although the autumnal treatment was slightly more efficient. To study glyphosate’s direct impact on the development of H. obsoletus nymphs, emergence traps were placed directly in the centre of treated and untreated stinging nettle patches. There was no significant difference among the three treatments in the total number of adults emerging. Thus, an aerial application of glyphosate in either spring or autumn did not inhibit the nymphs’ development on the roots of stinging nettle in the soil. Our results challenge current recommendations of applying herbicides against stinging nettle at the end of the season and suggest that stinging nettle could also be controlled in spring, alike other viticultural weeds.