Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

Using automated supplementation systems to meet growth targets for grazing sheep

Remote drafting technology now available for sheep allows targeted supplementation of individuals within a grazing flock. This paper reports results of three experiments. Experiment 1 examined the weight change of Merino wethers allowed access to either lupin grain or whole cottonseed 0, 1, 2 or 7 days/week for 6 weeks. Experiment 2 examined the weight change of Merino wethers allowed access to either lupins or a sorghum + cottonseed meal (CSM) supplement 0, 2, 4 or 7 days/week for 8 weeks. Experiment 3 investigated the relationship between five allocations of trough space at the supplement self-feeders (5–50 cm/sheep) and the weight change of Merino wethers allowed access to lupins 1 day/week for 8 weeks. In all experiments, the Merino wethers had free access as a single group to drinking water and low quality hay in a large group pen and were allowed access to supplement once per day on their scheduled days of access. No water was available in the areas containing supplement, but one-way flow gates allowed animals to return to the group pen in their own time. There was a linear response in growth rate to increased frequency of access to lupins in Experiments 1 and 2, with each additional day of access increasing liveweight gain by 26 and 21 g/day, respectively. Similarly, the response to the sorghum + CSM supplement was linear, although significantly lower (P < 0.05), at 12 g/day. Providing access to whole cottonseed resulted in no significant change in growth rate compared with the control animals. In Experiment 3, decreasing trough space from 50 to 5 cm/sheep had no effect on sheep liveweight change. It was concluded that the relationships developed here, for growth response to increased frequency of access to lupins or a sorghum + CSM supplement, could be used to indicate the most appropriate frequency of access to supplement, through a remote drafting unit, to achieve sheep weight change targets. Also, that a trough space of 5 cm/sheep appears adequate in this supplementation system.

Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG

Modulation of the immune response is an important step in the induction of protective humoral and cellular immunity against pathogens. In this study, we investigated the possibility of using a nanomaterial conjugated with the toll-like receptor (TLR) ligand CpG to modulate the immune response towards the preferred polarity. MgAl-layered double hydroxide (LDH) nanomaterial has a very similar chemical composition to Alum, an FDA approved adjuvant for human vaccination. We used a model antigen, ovalbumin (OVA) to demonstrate that MgAl-LDH had comparable adjuvant activity to Alum, but much weaker inflammation. Conjugation of TLR9 ligand CpG to LDH nanoparticles significantly enhanced the antibody response and promoted a switch from Th2 toward Th1 response, demonstrated by a change in the IgG2a:IgG1 ratio. Moreover, immunization of mice with CpG-OVA-conjugated LDH before challenge with OVA-expressing B16/F10 tumor cells retarded tumor growth. Together, these data indicate that LDH nanomaterial can be used as an immune adjuvant to promote Th1 or Th2 dominant immune responses suitable for vaccination purposes.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

Summary We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses.