An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

Population structure of South African and Australian Pyrenophora teres isolates.

There are two recognized forms of the disease net blotch of barley: the net form caused by Pyrenophora teres f. teres (PTT) and the spot form caused by P. teres f. maculata (PTM). In this study, amplified fragment length polymorphism analysis was used to investigate the genetic diversity and population structure of 60 PTT and 64 PTM isolates collected across Australia (66 isolates) and in the south-western Cape of South Africa (58 isolates). For comparison, P. tritici-repentis, Exserohilum rostratum and Bipolaris sorokiniana samples were also included in the analyses. Both distance-and model-based cluster analyses separated the PTT and PTM isolates into two strongly divergent genetic groups. Significant variation was observed both among the South African and Australian populations of PTT and PTM and among sampling locations for the PTT samples. Results suggest that sexual reproduction between the two forms is unlikely and that reproduction within the PTT and PTM groups occurs mainly asexually.

Biological control of bellyache bush (Jatropha gossypiifolia) in Australia: South America as a possible source of natural enemies

Bellyache bush (Jatropha gossypiifolia, Euphorbiaceae), a deciduous shrub introduced as an ornamental from tropical America, is a major and expanding weed of rangelands and riparian zones in northern Australia. Biological control is the most economically viable and long-term management solution for this weed. Surveys for potential biological control agents for J gossypiifolia in Mexico,Central America and the Caribbean resulted in release of the seed-feeding jewel bug Agonosoma trilineatum (Hemiptera: Scutelleridae), which failed to establish, and prioritisation of a leaf-rust Phakopsora arthuriana (Puccineales: Phakopsoraceae) for host-specificity testing, which is ongoing. With poor prospects for new agents from Mexico and Central America and the Caribbean, the search for candidate agents on J gossypiifolia shifted to localities south of the equator. Surveys were conducted on the purple-leaf form of J gossypiifolia, Jatropha excisa, Jatropha clavuligera and Jatropha curcas in Peru, Bolivia and Paraguay in 2012 and 2013. A total of 11 insect species, one mite species and the leaf-rust (P. arthuriana) were observed. These include a yet to be described leafmining moth (Stomphastis sp.) (Lepidoptera: Gracillaridae), a shoot and leaf-galling midge Prodiplosis longifila, and leaf-feeding midge Prodiplosis sp. near longifila (both Diptera:Cecidomyiidae) and an unidentified leaf-feeding moth larva (Lepidoptera: Notodontidae). The leafminer is widespread and damaging and has a field host range restricted to the genus Jatropha in Peru and Bolivia, holds the greatest promise as a biological control agent in Australia. Phakopsora arthuriana was recorded for the first time ever from Bolivia and Peru. Further exploration will be conducted in Peru and Bolivia during the wet season to confirm the field host range of collected agents,and to look for more new agents. Promising agents with field host-range restricted to Jatropha spp. will be imported into a quarantine facility in Australia for host-specificity testing.