A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus‐like particles

Plant expression systems based on nonreplicating virus‐based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant‐based production and assembly of Bluetongue virus‐like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus–based HyperTrans (CPMV‐HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV‐8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.     

The major economic field diseases of cowpea in the humid agro-ecologies of south-western Nigeria

Cowpea, which has now become an important protein source for the teeming populace of Nigerians, especially those living in the humid agro-ecological zones of south-western Nigeria, is severely attacked by diseases. The causal agents of these diseases find the environment more conducive for survival and hence induced disease conditions in the host plant, causing significant yield reduction. This paper reviews the present situation of the cowpea diseases in the humid forest agro-ecologies of south-western Nigeria.     

In Vitro toxicity of oil extracted from neem seeds collected from different locations across savanna agro-ecological zones of Nigeria on seed and soil-borne pathogens of cowpea (Vigna unguiculata L. Walp)

Graded extracts of neem seed oil extracted across the savanna agro-ecological zones (AEZs) of Nigeria were tested on mycelial growth of Colletotrichum capsici and Macrophomina phaseolina of cowpea in Nigeria. Minimum inhibition concentrations (MIC) and effectiveness levels of oil from AEZs were determined. Data were subject to ANOVA. Biplot was employed to access variation and interactions among the AEZs. Across the AEZs, fungitoxicity, MIC and effectiveness level varies; concentrations of 0.1 and 1.0% reduced the growth of C. capsici, while M. phaseolina was reduced at 10% concentration. On M. phaseolina, neem oil extracted from Ilorin, Ogbomosho (derived savanna) and Mokwa (southern guinea savanna) performed similarly, while a strong positive association existed between Ogbomosho (derived savanna) and Hadejia (Sahel savanna) samples. On C. capsici, samples from Bida (southern guinea savanna) and Ilorin (Derived savanna) were similar. This result could be a possible link between ecology, biodiversity and toxic principles in plant materials.     

Effect of tissue phosphorus concentration on the mineralisation of nitrogen from stylo and cowpea residues

Laboratory studies were conducted to investigate the effect of phosphorus concentration in residues of cowpea (t Vigna unguiculata, L. Walp) and stylo (t Stylosanthes hamata, L., cv Verano) on their rate of nitrogen mineralisation when incubated in a soil whose P status was deficient for plant growth. Residues with a range of P concentrations were obtained by applying varying rates of P to soil in which the plants were grown in the field or the glasshouse. Variations in P concentration of field- or glasshouse-grown residues were not accompanied by variations in other chemical components (C:N ratio, lignin and polyphenol concentrations). Both lignin and polyphenol concentrations were higher in the field-grown than in the glasshouse-grown residues. Lignin concentration was greater in cowpea than in stylo, but polyphenols were higher in stylo. Cowpea residues mineralised N less rapidly than stylo. N mineralisation from residues with low P concentration was consistently less than from those of higher P concentration; reduced mineralisation was observed for P concentration in the residues below 1.6 g kg^–1. When inorganic P was added to the residue-soil systems, N mineralisation from the residues was increased, though no interaction between the effects of adding inorganic P and P concentration in the residues was observed.     

Callose formation as parameter for assessing genotypical plant tolerance of aluminium and manganese

Callose ((1,3)--glucan) formation in plant tissues is induced by excess Al and Mn. In the present study callose was spectrophotometrically quantified in order to evaluate whether it could be used as a parameter to identify genotypical differences in Al and Mn tolerance. Mn leaf-tissue tolerance of cowpea and linseed genotypes was assessed using the technique of isolated leaf tissue floating on Mn solution. Genotypical differences in the density of brown speckles on the leaf tissue (Mn toxicity symptoms) correlated closely with the concentrations of callose for both plant species. In cell suspension cultures Mn excess also induced callose formation. However, differences in tolerance of cowpea genotypes using callose formation as a parameter could only be found in cultured cowpea cells if controls cultured at optimum Mn supply showed low background callose. As soon as after 1 h, Al supply (50 M) induced callose formation predominantly in the 5-mm root tip of soybean seedlings. Callose concentration in the 0–30 mm root tips was inversely related to the root elongation rate when roots were subjected to an increasing Al supply above 10 M. Three soybean genotypes differed in inhibition of root-elongation rate and induction of callose formation when treated with 50 M Al for 8 h. Relative callose concentrations and relative root-elongation rates for these genotypes were significantly negatively correlated.     

Differences in calcium efficiency between cowpea (Vigna unguiculata (L.) Walp.) cultivars

The cowpea (Vigna unguiculata (L.) Walp.) cultivars TVu 354 and Solojo were grown in solution culture at 10 to 1000 M Ca supply. The Ca supply did not vary by more than 10% during the experiment. The pH value was kept constant within 0.1 units at 4.0 by automatic titration. The cultivar TVu 354 proved to be much more Ca-efficient than Solojo. At 10 M Ca supply Solojo died, whereas TVu 354 was hardly affected in dry matter production. The differences in Ca efficiency were independent of the P supply. They could not be explained by differences in Ca uptake or Ca concentrations in the plant tissue. Short-term studies using ⁴⁵Ca, both in the dark and in the light, indicated better transport of Ca from the roots to the shoots and within the shoots to the younger leaves in the Ca-efficient cultivar TVu 354. However, the main reason for the differences between the cultivars in sensitivity to low Ca supply were differences in the Ca requirement of the plant tissue to maintain tissue organization and function. Sequential fractionation of the freeze-dried leaf tissue with hot water, 0.5 M NaCl, 1 M CH₃COOH, and 2 M HCl did not reveal cultivar differences in Ca binding state. The results clearly show that considerable genetic potential in tolerance to low Ca supply exists in cowpea. However, a better understanding of the physiological/biochemical reasons for low internal Ca requirement is needed.     

The management of populations of vesicular-arbuscular mycorrhizal fungi in acid-infertile soils of a savanna ecosystem

A field trial was conducted at two sites in the savanna ecosystem of eastern Colombia to compare the effects of inoculation with vesicular-arbuscular mycorrhizal fungi (VAMF) ofBrachiaria dictyoneura (a tropical grass), cassava (Manihot esculenta), the tropical forage legume kudzu (Pueraria phaseoloides) andSorghum sp., and two phosphate sources. The second stage of the trial studied the effect of these pre-crop treatments on the subsequent growth, nutrition and VAM status of cowpea (Vigna unguiculata) andStylosanthes capitata in the following season, compared with both crops sown in native savanna. Inoculation significantly increased the levels of VAM and plant yields in the early growth stages of all crops during the first season, particularly with the rock phosphate (RP) source. The most significant increases were observed in the mycorrhiza-dependent cassava and kudzu crops up to 15 weeks after sowing, and were associated with increased foliar uptake of P and Mg. The effectiveness of the introduced inoculum was greater at the field site with a sandier soil. In the second season the levels of VAM in roots of cowpea andS. capitata were all increased significantly in pre-cropped plots compared with a savanna control. The increased presence of VAM was associated with significantly increased yields on plots previously sown to cassava, kudzu andSorghum sp. The data support the idea that increasing the VAMF inoculum potential of these acid-infertile soils by inoculation or pre-crops can greatly increase the rate of establishment of mycorrhiza-dependent host plants.     

Interrelations between Meloidogyne javanica,Rotylenchulus reniformis,and Rhizobium Sp. on Vigna sinensis

The interactions of Meloidogyne javanica, Rotylenchulus reniformis, and Rhizobium sp. on cowpea seedlings were investigated. Upon simultaneous inoculation with the two nematode species, M. javanica invaded first but did not affect root invasion by R. reniformis. M. javanica populations increased less in competition with R. reniformis than when present alone. Preinvasion by R. renilormis significantly suppressed the number of M. javanica in the roots. Inoculation of M. javanica and/or R. reniformis with rhizobia did not affect nodulation. Nodule formation was hindered only when R. reniformis infection preceded rhizobial inoculation. Nitrogen nodules were formed on M. javanica galls. Nodules and M. javanica galls served as infection sites for both nematodes. Although R. reniIormis on the roots reduced the space for M. javanica infection, M. javanica is more competitive than R. reniformis and ultimately predominates as a result of its higher reproductive potential and shorter time spent in the soil before infection.     

Low-temperature limitations of photosynthesis in three tropical Vigna species: A chlorophyll fluorescence study

The temperature-dependence of photosynthesis and chlorophyll (Chl) fluorescence quenching components was studied between 0 and 45°C in three tropical, chilling-sensitive Vigna species and in chilling-tolerant pea. Photosynthesis of the Vigna spp. was approx. 20% more reduced by temperatures between 7 and 30°C than in pea. The latter revealed significant changes in Chl fluorescence parameters at much lower temperature than the Vigna spp. Below 15°C, the reduction state of Q_A increased quickly in pea, while in Vigna already below 30°C, an increase of reduced Q_A was obtained. The analysis of different components of non-photochemical Chl fluorescence quenching (q_N) revealed, that in pea photoinhibitory quenching (q_I) occurred below 13°C. Below ca. 7°C, a sudden breakdown of both q_P and the fast relaxing component of q_N was observed in pea.In Vigna, susceptibility of LHC II phosphorylation or limitation of electron flow by damage to PS I, the PS II reaction centre or the water-splitting system were not responsible for the chilling-sensitivity of photosynthesis between 5 and 30°C. Instead, photosynthesis was gradually limited by an inefficient use of reduction equivalents. This, in turn may increase susceptibilty to photoinhibition, which occurred below 20°C in Vigna. The combined study of q_P and of the different components of q_N allowed the demonstration of the subsequent occurrence of different limiting processes with decreasing temperature in the chilling-sensitive Vigna species.