A single cryptomonad cell harbors a complex community of organelles, bacteria, a phage, and selfish elements.

Symbiosis between prokaryotes and microbial eukaryotes (protists) has broadly impacted both evolution and ecology. Endosymbiosis led to mitochondria and plastids, the latter spreading across the tree of eukaryotes by subsequent rounds of endosymbiosis. Present-day endosymbionts in protists remain both common and diverse, although what function they serve is often unknown. Here, we describe a highly complex community of endosymbionts and a bacteriophage (phage) within a single cryptomonad cell. Cryptomonads are a model for organelle evolution because their secondary plastid retains a relict endosymbiont nucleus, but only one previously unidentified Cryptomonas strain (SAG 25.80) is known to harbor bacterial endosymbionts. We carried out electron microscopy and FISH imaging as well as genomic sequencing on Cryptomonas SAG 25.80, which revealed a stable, complex community even after over 50 years in continuous cultivation. We identified the host strain as Cryptomonas gyropyrenoidosa, and sequenced genomes from its mitochondria, plastid, and nucleomorph (and partially its nucleus), as well as two symbionts, Megaira polyxenophila and Grellia numerosa, and one phage (MAnkyphage) infecting M. polyxenophila. Comparing closely related endosymbionts from other hosts revealed similar metabolic and genomic features, with the exception of abundant transposons and genome plasticity in M. polyxenophila from Cryptomonas. We found an abundance of eukaryote-interacting genes as well as many toxin-antitoxin systems, including in the MAnkyphage genome that also encodes several eukaryotic-like proteins. Overall, the Cryptomonas cell is an endosymbiotic conglomeration with seven distinct evolving genomes that all show evidence of inter-lineage conflict but nevertheless remain stable, even after more than 4,000 generations in culture.

In vivo packaging of triacylglycerols enhances Arabidopsis leaf biomass and energy density.

Our dependency on reduced carbon for energy has led to a rapid increase in the search for sustainable alternatives and a call to focus on energy densification and increasing biomass yields. In this study, we generated a uniquely stabilized plant structural protein (cysteine [Cys]-oleosin) that encapsulates triacylglycerol (TAG). When coexpressed with diacylglycerol O-acyltransferase (DGAT1) in Arabidopsis (Arabidopsis thaliana), we observed a 24% increase in the carbon dioxide (CO2) assimilation rate per unit of leaf area and a 50% increase in leaf biomass as well as approximately 2-, 3-, and 5-fold increases in the fatty acid content of the mature leaves, senescing leaves, and roots, respectively. We propose that the coexpression led to the formation of enduring lipid droplets that prevented the futile cycle of TAG biosynthesis/lipolysis and instead created a sustained demand for de novo lipid biosynthesis, which in turn elevated CO2 recycling in the chloroplast. Fatty acid profile analysis indicated that the formation of TAG involved acyl cycling in Arabidopsis leaves and roots. We also demonstrate that the combination of Cys-oleosin and DGAT1 resulted in the highest accumulation of fatty acids in the model single-cell eukaryote, Saccharomyces cerevisiae. Our results support the notion that the prevention of lipolysis is vital to enabling TAG accumulation in vegetative tissues and confirm the earlier speculation that elevating fatty acid biosynthesis in the leaf would lead to an increase in CO2 assimilation. The Cys-oleosins have applications in biofuels, animal feed, and human nutrition as well as in providing a tool for investigating fatty acid biosynthesis and catabolism.

Production of active single-chain antibodies in seeds using trimeric polyoleosin fusion.

A variety of single-chain variable fragments (scFv) that had been previously developed to the surface epitopes of infective Trichostrongylus colubriformis L3 pathogenic gut nematodes of sheep were fused to a trimeric version of polyoleosin (three head-to-tail repeats of oleosin) and expressed in planta under the control of an Arabidopsis oleosin promoter. The fusion products were found to accumulate in oil bodies (OBs) at the range of 0.25-0.9% of the total seed protein which is comparable with the main 18 kDa isoform of Arabidopsis seed oleosin. Immunofluorescence microscopy and immuno-binding were used to demonstrate that it is possible to both purify the recombinant protein via enrichment for OBs as well as use the OBs emulsion to deliver functional recombinant scFv. This work presents a novel fusion strategy platform to boost the productivity and simplify the delivery of recombinant single chain antibodies and other like proteins.

High throughput PCR detection of Xylella fastidiosa directly from almond tissues.

Xylella fastidiosa, the causal agent of almond leaf scorch disease (ALSD), is currently re-emerging as a serious concern in California. Efficient pathogen detection is critical for ALSD epidemiological studies, particularly when a large sample size is involved. We here report a PCR procedure to detect X. fastidiosa directly from infected almond tissue without the laborious DNA extraction. Plant samples were prepared by freeze-drying and pulverized. Appropriate dilutions of the pulverized freeze-dried tissue (PFT) were determined to minimize the effect of enzyme inhibitors from plant tissue and retain PCR detection of X. fastdiosa cells at a single digit number level. This PFT-PCR procedure was evaluated by comparing to the in vitro cultivation method using 102 symptomatic samples and resulted in a predictive value of 90.8%. PFT-PCR was further applied to monitor the seasonal occurrence of X. fastidiosa from four selected almond trees in two orchards in 2005. The results matched with those of the cultivation method at 92.3%. Considering the simplicity and reliability, we conclude that PFT-PCR is a valuable option for high throughput rapid detection of X. fastidiosa.