Functional expression of Cf9 and Avr9 genes in Brassica napus induces enhanced resistance to Leptosphaeria maculans.
The tomato Cf9 resistance gene induces an Avr9-dependent hypersensitive response (HR) in tomato and transgenic Solanaceae spp. We studied whether the Cf9 gene product responded functionally to the corresponding Avr9 gene product when introduced in a heterologous plant species. We successfully expressed the Cf9 gene under control of its own promoter and the Avr9 or Avr9R8K genes under control of the p35S1 promoter in transgenic oilseed rape. We demonstrated that the transgenic oilseed rape plants produced the Avr9 elicitor with the same specific necrosis-inducing activity as reported for Cladosporium fulvum. An Avr9-dependent HR was induced in Cf9 oilseed rape upon injection of intercellular fluid containing Avr9. We showed Avr9-specific induction of PR1, PR2, and Cxc750 defense genes in oilseed rape expressing CJ9. Cf9 x Avr9 oilseed rape did not result in seedling death of the F1 progeny, independent of the promoters used to express the genes. The F1 (Cf9 x Avr9) plants, however, were quantitatively more resistant to Leptosphaeria maculans. Phytopathological analyses revealed that disease development of L. maculans was delayed when the pathogen was applied on an Avr9-mediated HR site. We demonstrate that the CJ9 and Avr9 gene can be functionally expressed in a heterologous plant species and that the two components confer an increase in disease resistance. (+info)
Recombinant pronapin precursor produced in Pichia pastoris displays structural and immunologic equivalent properties to its mature product isolated from rapeseed.
2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability. (+info)
Toxic oil syndrome: the perspective after 20 years.
Toxic oil syndrome burst upon the scene in Spain in May of 1981, draining the resources of a newly evolving political and social medicine system. The vehicle of the causative toxic agent was identified as an illicit oil that had been diverted from industrial use and refined in order to remove the aniline denaturant, and that was sold in unlabeled 5-liter containers by itinerant salesmen. Over 20,000 people were ultimately affected, and over 1,200 deaths from all causes have been recorded in the affected cohort. The epidemiologic investigation of toxic oil syndrome involved all facets of investigative and analytical work; from visits to factories and interviewing workers, to sophisticated chemical and statistical analytical techniques. This investigation serves as a further illustration that data and information of all types, and from a wide range of fields, need to be systematically collected and evaluated in order to best resolve an epidemiologic mystery. Astute clinical observation of the patients, however, led to the hypothesis that toxic oil syndrome was a result of a toxic exposure. In this and other epidemics of unknown etiology, clinical observation and the intense scrutiny of patients' histories, signs, and symptoms by treating clinicians have often led to hypotheses that could be tested epidemiologically. When there are medical unknowns, the role of the astute clinician continues to be crucial. The toxic oil syndrome epidemic is an example of how even a developed country can be affected by a massive epidemic of environmental origin if failures occur in the systems that control and regulate the food supply or other consumer products. However, such failures could occur anywhere that large commercial networks operate on the regulatory edge, and if these business lack an in depth knowledge of the consequences of alterations in manufacturing conditions. Such was the case with eosinophilia-myalgia syndrome as well, when apparently minor alterations in manufacturing conditions of L-tryptophan led to an increase in impurities in the product that were later associated with the illness. These risks are even greater in countries with few or inconsistent control systems, making the food and drug supply potential portals of entry for serious health hazards, as is further exemplified by the tragic episode of pediatric renal failure in Haiti associated with a legitimate consumer product, paracetamol elixir, that had been manufactured using a fraudulently supplied toxic ingredient, diethylene glycol (81). The potential toxicants in the adulterated rapeseed oil were present in extremely small amounts. If fatty acid anilides or related compounds are indeed the etiologic agents in toxic oil syndrome, then these compounds must be extremely toxic at the parts per million concentrations at which they were found. Further, the roles of causative agents in the development of disorders such as scleroderma, eosinophilic fasciitis, eosinophilic perimyositis, and other similar diseases are unknown, but scientists can speculate that some sort of low level environmental agent may play a role if such extremely small quantities of contaminants are indeed capable of causing disease. Although the exact identity of the etiologic agent in toxic oil syndrome remains unknown, work on toxic oil syndrome continues. Follow-up clinical studies and long-term mortality studies are under way. Investigation of the mechanisms involved in toxic oil syndrome continues. The identification of suspect chemical compounds, their characterization, and effects will hopefully one day contribute to the prevention of other similar diseases. (+info)
Male fitness of oilseed rape (Brassica napus), weedy B. rapa and their F(1) hybrids when pollinating B. rapa seeds.
The likelihood that two species hybridise and backcross may depend strongly on environmental conditions, and possibly on competitive interactions between parents and hybrids. We studied the paternity of seeds produced by weedy Brassica rapa growing in mixtures with oilseed rape (B. napus) and their F(1) hybrids at different frequencies and densities. Paternity was determined by the presence of a transgene, morphology, and AFLP markers. In addition, observations of flower and pollen production, and published data on pollen fertilisation success, zygote survival, and seed germination, allowed us to estimate an expected paternity. The frequency and density of B. napus, B. rapa, and F(1) plants had a strong influence on flower, pollen, and seed production, and on the paternity of B. rapa seeds. Hybridisation and backcrossing mostly occurred at low densities and at high frequencies of B. napus and F(1), respectively. F(1) and backcross offspring were produced mainly by a few B. rapa mother plants. The observed hybridisation and backcrossing frequencies were much lower than expected from our compilation of fitness components. Our results show that the male fitness of B. rapa, B. napus, and F(1) hybrids is strongly influenced by their local frequencies, and that male fitness of F(1)hybrids, when pollinating B. rapa seeds, is low even when their female fitness (seed set) is high. (+info)
Supplementing barley or rapeseed meal to dairy cows fed grass-red clover silage: I. Rumen degradability and microbial flow.
The present study was conducted to measure the flow of microbial and nonmicrobial N fractions entering the omasal canal of lactating dairy cows fed grass-red clover silage supplemented with barley and rapeseed meal. Four ruminally cannulated Finnish Ayrshire dairy cows were fed, in a 4 x 4 Latin square design, grass-red clover silage alone or supplemented with (on DM basis) 5.1 kg/d of barley, 1.9 kg/d of rape-seed meal or 5.1 kg/d of barley and 1.9 kg/d rapeseed meal. Nonammonia N flow entering the omasal canal was fractionated into microbial and nonmicrobial N using 15N. Microbial N was fractionated into N associated with liquid-associated bacteria, particle-associated bacteria, and protozoa. Supplementation of diets with barley increased microbial N flow entering the omasal canal (P < 0.01) but had no effect on nonmicrobial N flow. Increased microbial N flow was attributed to liquid-associated bacteria and protozoa. Barley had no effect on apparent ruminal N degradability, but increased true ruminal N degradability (P < 0.01). Barley had no effect on urinary N excretion, but increased daily N retention (P = 0.03). Furthermore, barley supplementation decreased ruminal (P = 0.02) and total tract (P < 0.01) NDF digestibility. Supplementation of diets with rapeseed meal increased apparent ruminal N degradability (P < 0.01) and nonmicrobial N flow entering the omasal canal (P < 0.01), but had no effect on true ruminal N degradability. Despite higher N excretion in urine, rapeseed meal improved daily N retention (P < 0.01). Milk yield was increased (P < 0.01) by barley and rapeseed meal supplements, with the responses being additive. Responses attained with barley were primarily due to increased energy supply for ruminal microbes and improvements in energy and protein supply for the animal. However, provision of readily digestible carbohydrates in barley did not improve microbial capture of ruminal ammonia. Benefits associated with rapeseed meal supplementation were explained as an increase in the supply of ruminally undegradable protein. (+info)
Supplementing barley or rapeseed meal to dairy cows fed grass-red clover silage: II. Amino acid profile of microbial fractions.
Four ruminally cannulated dairy cows were used to examine the effect of diet on the AA composition of rumen bacteria and protozoa, and the flow of microbial and nonmicrobial AA entering the omasal canal. Cows were offered grass-red clover silage alone, or that supplemented with 5.1 kg DM of barley, 1.9 kg DM of rapeseed meal, or 5.1 kg DM of barley and 1.9 kg DM of rapeseed meal according to a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments. During the first 10 d of each period, cows had free access to silage and, thereafter intake was restricted to 95% of ad libitum intake. Postruminal digesta flow was assessed using the omasal canal sampling technique in combination with a triple marker method. Liquid- (LAB) and particle- (PAB) associated bacteria were isolated from digesta in the reticulorumen and protozoa from digesta entering the omasal canal. Microbial protein flow was determined using 15N as a microbial marker. Flows of AA entering the omasal canal were similar in cows fed silage diets supplemented with barley or rapeseed meal. However, rapeseed meal increased nonmicrobial AA flow while barley increased the flow of AA associated with LAB and protozoa. Diet had negligible effects on the AA profile of microbial fractions. Comparison of AA profiles across diets indicated differences between LAB and PAB for 10 out of 17 AA measured. Rumen bacteria and protozoa were found to be different for 14 out of 15 AA measured. For grass silage-based diets, energy and protein supplementations appear to alter postruminal AA supply through modifications in the proportionate contribution of microbial and nonmicrobial pools to total protein flow rather than as a direct result of changes in the AA profile of microbial protein. (+info)
Coevolution of the S-locus genes SRK, SLG and SP11/SCR in Brassica oleracea and B. rapa.
Brassica self-incompatibility (SI) is controlled by SLG and SRK expressed in the stigma and by SP11/SCR expressed in the anther. We determined the sequences of the S domains of 36 SRK alleles, 13 SLG alleles, and 14 SP11 alleles from Brassica oleracea and B. rapa. We found three S haplotypes lacking SLG genes in B. rapa, confirming that SLG is not essential for the SI recognition system. Together with reported sequences, the nucleotide diversities per synonymous and nonsynonymous site (pi(S) and pi(N)) at the SRK, SLG, and SP11 loci within B. oleracea were computed. The ratios of pi(N):pi(S) for SP11 and the hypervariable region of SRK were significantly >1, suggesting operation of diversifying selection to maintain the diversity of these regions. In the phylogenetic trees of 12 SP11 sequences and their linked SRK alleles, the tree topology was not significantly different between SP11 and SRK, suggesting a tight linkage of male and female SI determinants during the evolutionary course of these haplotypes. Genetic exchanges between SLG and SRK seem to be frequent; three such recent exchanges were detected. The evolution of S haplotypes and the effect of gene conversion on self-incompatibility are discussed. (+info)
Characterization and effects of the replicated flowering time gene FLC in Brassica rapa.
Functional genetic redundancy is widespread in plants and could have an important impact on phenotypic diversity if the multiple gene copies act in an additive or dosage-dependent manner. We have cloned four Brassica rapa homologs (BrFLC) of the MADS-box flowering-time regulator FLC, located at the top of chromosome 5 of Arabidopsis thaliana. Relative rate tests revealed no evidence for differential rates of evolution and the ratios of nonsynonymous-to-synonymous substitutions suggest BrFLC loci are not under strong purifying selection. BrFLC1, BrFLC2, and BrFLC3 map to genomic regions that are collinear with the top of At5, consistent with a polyploid origin. BrFLC5 maps near a junction of two collinear regions to Arabidopsis, one of which includes an FLC-like gene (AGL31). However, all BrFLC sequences are more closely related to FLC than to AGL31. BrFLC1, BrFLC2, and BrFLC5 cosegregate with flowering-time loci evaluated in populations derived by backcrossing late-flowering alleles from a biennial parent into an annual parent. Two loci segregating in a single backcross population affected flowering in a completely additive manner. Thus, replicated BrFLC genes appear to have a similar function and interact in an additive manner to modulate flowering time. (+info)