Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H(c)): antigen E) by Pichia pastoris

A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.     

Leafhopper-induced plant resistance enhances predation risk in a phytophagous beetle

Many herbivores elicit biochemical, physiological, or morphological changes in their host plants that render them more resistant to co-occurring herbivores. Yet, despite the large number of studies that investigate how induced resistance affects herbivore preference and performance, very few have simultaneously explored the cascading effects of induction on higher trophic levels and consequences for prey suppression. In our study system, early-season herbivory by leafhoppers elevated plant resistance to subsequent attack by chrysomelid beetles sharing the same host plant. Notably, beetles feeding on leafhopper-damaged plants incurred developmental penalties (e.g., prolonged time in early larval instars) that rendered them more susceptible to predation by natural enemies. As a result, the combined bottom-up effect of leafhopper-induced resistance and the top-down effect of enhanced predation resulted in the synergistic suppression of beetle populations. These results emphasize that higher trophic level dynamics should be considered in conjunction with induced resistance to better understand how plants mediate interspecific interactions in phytophagous insect communities.     

Red to far-red ratio correction in plant growth chambers - growth responses and influence of thermal load on garden pea

Plant growth chambers are commonly used to minimize environmental variation but the light sources used vary considerably from natural light and from each other. Incandescent globes are often used to add more far-red light, with the aim of producing a more natural red to far-red ratio (R:FR), but also add to thermal load. High-intensity discharge lamps are often used to produce higher irradiances, more akin to natural light, but the thermal implications are rarely considered because air temperature is controlled. This paper examines the spectral properties and thermal implications of growth chamber light sources and takes a whole-plant physiology approach, by examining growth responses of a photoperiodic pea line ( Pisum sativum L. cv. Torsdag) in the same growth chamber type under different light sources – in essence using plants to study the controlled environments rather than vice-versa. High R:FR delayed flowering and inhibited internode extension in pea. However, the addition of far-red-rich incandescent globes in the proportions provided in the growth chambers (400–500 W) did little to reduce R:FR, did not induce earlier flowering and actually further inhibited internode length. Leaflet size and yield were significantly reduced. While air temperature was maintained at 20°C in all experiments, radiant temperature was significantly higher under high irradiance and/or with incandescent added, and soil temperatures were elevated. Growth responses under these lights were similar to the effect caused by elevating the air temperature. An alternative method of controlling R:FR, without thermal load implications, using light-emitting diodes is described.     

Does carbon partitioning in ectomycorrhizal pine seedlings under elevated CO2 vary with fungal species?

Enhanced soil respiration in response to elevated atmospheric CO₂ has been demonstrated, and ectomycorrhizal (ECM) fungi are of particular interest since they partition host-derived photoassimilates belowground. Although a strong response of ECM fungi to elevated CO₂ has been shown, little is still known about the functional diversity among species. We studied carbon (C) partitioning in mycorrhizal Scots pine seedlings in response to short-term CO₂ enrichment, using seven ECM species with different ecological strategies. Mycorrhizal associations were synthesised and seedlings grown in large Petri dishes containing peat:vermiculite and nutrient solution for 10–15 weeks, after which half of the microcosms were exposed to elevated CO₂ treatment (710 ppm) for 15 days and the other half were kept in ambient CO₂ treatment. Partitioning of C was quantified by pulse labelling the seedlings with ¹⁴CO₂ and examining the distribution of labelled assimilates in shoot, root and extraradical mycelial compartments by destructive harvest and liquid scintillation counting. Fungal biomass was determined with PLFA analysis. The respiratory loss of ¹⁴CO₂ was on average greater in the elevated CO₂ treatment for most species compared to the ambient CO₂ treatment. More label was retrieved in the shoots in the ambient CO₂ treatment compared to elevated CO₂ (significant for P. involutus and P. fallax). Greater amounts of label were found in the extraradical mycelial compartment in all species (except P. involutus) in elevated CO₂ compared to ambient CO₂ (significant for L. bicolor, P. byssinum, P. fallax and R. roseolus). Fungal biomass production increased significantly with elevated CO₂ for two species (H. velutipes and A. muscaria); three species (P. fallax, P. involutus and R. roseolus) showed a similar but non-significant trend, whereas L. bicolor and P. byssinum produced less biomass in elevated CO₂ compared to ambient CO₂. When ¹⁴C in the mycelial compartment and respiration was expressed per unit fungal PLFA the difference between CO₂ treatments disappeared. We demonstrated that different ECM fungal isolates respond differently in C partitioning in response to CO₂ enrichment. These results suggest that under certain growth conditions, when nutrients are not limiting, ECM fungi respond rapidly to increasing C-availability through changed biomass production and respiration.     

Constitutive polymorphic cyanogenesis in the Australian rainforest tree, Ryparosa kurrangii (Achariaceae)

Cyanogenesis, the liberation of volatile hydrogen cyanide from endogenous cyanide-containing compounds, is a proven plant defence mechanism and the particular cyanogens involved have taxonomic utility. The cyclopentenoncyanhydrin glycoside gynocardin was the only cyanogen isolated from foliar tissue of the rare Australian rainforest tree, Ryparosa kurrangii (Achariaceae). Mechanical damage simulating foliar herbivory did not induce a significant increase in the expression of cyanogenesis over a 24h period, indicating cyanogenic herbivore defence in R. kurrangii is constitutive. The cyanogenic potential of mature leaves was quantitatively polymorphic between trees in a natural population, ranging from 0.54 to 4.77 mg CN g(-1) dry wt leaf tissue.     

A Markov chain Monte Carlo strategy for sampling from the joint posterior distribution of pedigrees and population parameters under a Fisher-Wright model with partial selfing

A simple population genetic model is presented for a hermaphrodite annual species, allowing both selfing and outcrossing. Those male gametes (pollen) responsible for outcrossing are assumed to disperse much further than seeds. Under this model, the pedigree of a sample from a single locality is loop-free. A novel Markov chain Monte Carlo strategy is presented for sampling from the joint posterior distribution of the pedigree of such a sample and the parameters of the population genetic model (including the selfing rate) given the genotypes of the sampled individuals at unlinked marker loci. The computational costs of this Markov chain Monte Carlo strategy scale well with the number of individuals in the sample, and the number of marker loci, but increase exponentially with the age (time since colonisation from the source population) of the local population. Consequently, this strategy is particularly suited to situations where the sample has been collected from a population which is the result of a recent colonisation process.