Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell derived antigen

The innate immune system detects pathogens and initiates adaptive immune responses. Inflammasomes are central components of the innate immune system, but whether inflammasomes provide sufficient signals to activate adaptive immunity is unclear. In intestinal epithelial cells (IECs), inflammasomes activate a lytic form of cell death called pyroptosis, leading to epithelial cell expulsion and the release of cytokines. Here we employed a genetic system to show that simultaneous antigen expression and inflammasome activation specifically in IECs is sufficient to activate CD8+ T cells. By genetic elimination of direct T cell priming by IECs, we found that IEC-derived antigens are cross-presented to CD8+ T cells. However, cross-presentation of IEC-derived antigen to CD8+ T cells only partially depended on IEC pyroptosis. In the absence of inflammasome activation, cross-priming of CD8+ T cells required Batf3+ dendritic cells (cDC1), whereas cross-priming in the presence of pyroptosis required a Zbtb26+ but Batf3-independent cDC population. These data suggest the existence of parallel pyroptosis-dependent and pyroptosis-independent pathways for cross-presentation of IEC-derived antigens.

Comprehensive Analysis of Plastid Gene Expression During Fruit Development and Ripening of Kiwifruit

A serious limitation in the application of plastid biotechnology is the low-level expression of transgene in non-green plastids like chromoplasts compared with photosynthetically active chloroplasts. Unlike other fruits, not all chloroplasts are transformed into chromoplast during ripening of red-fleshed kiwifruit ( Actinidia chinensis vs Hongyang) fruits, which may make kiwifruit as an ideal horticultural plant for oral vaccine production by plastid engineering. To identify cis -elements potentially triggering high-level transgene expression in edible tissues of the ‘Hongyang’ kiwifruit, here we report a comprehensive analysis of kiwifruit plastid gene transcription in the green leaves and fruits at three different developing stages. While transcripts of a few photosynthesis-related genes and most genetic system genes were substantially upregulated in green fruits compared with leaves, nearly all plastid genes were significantly downregulated at the RNA level during fruit development. Expression of a few genes remained unchanged, including psbA , the gene encoding the D1 polypeptide of photosystem II. However, PsbA protein accumulation decreased continuously during chloroplast-to-chromoplast differentiation. Analysis of post-transcriptional steps in mRNA maturation, including intron splicing and RNA editing, revealed that splicing and editing may contribute to regulating plastid gene expression. Altogether, 40 RNA editing sites were verified, and five of them were newly discovered. Taken together, this study has generated a valuable resource for the analysis of plastid gene expression, and provides cis -elements for future efforts to engineer the plastid genome of kiwifruit.

Derivation of pb(II)-sensing Escherichia coli cell-based biosensors from arsenic responsive genetic systems

Heavy metal-responsive operons were used for the generation of Escherichia coli cell-based biosensors. The selectivity and specificity of the biosensors were determined based on the interaction between heavy metals and regulatory proteins; thereby, the modulating target selectivity of biosensors could be achieved by changing target sensing properties of regulatory proteins. The results of this study demonstrated that Pb(II)-sensing biosensors could be generated from an arsenic-responsive genetic system, which was originally used for arsenic-sensing biosensors. The amino acids around to As(III)-binding sites of ArsR were mutated and cysteine residues were relocated to modulate the metal selectivity. In addition, genes encoding metal ion-translocating P-type ATPases, such as copA and zntA, were deleted to enhance the specificity by increasing the intercellular levels of divalent metal ions. Based on the results, channel protein deleted E. coli cells harboring a pair of recombinant genes, engineered ArsR and arsAp::egfp, showed enhanced responses upon Pb exposure and could be used to quantify the amount of Pb(II) in artificially contaminated water and plants grown in media containing Pb(II). Although we focused on generating Pb(II)-specific biosensors in this study, the proposed strategy has a great potential for the generation of diverse heavy metal-sensing biosensors and risk assessment of heavy metals in environmental samples as well as in plants.

Diploid mint (M. longifolia) can produce spearmint type oil with a high yield potential

Mint oil is a key source of natural flavors with wide industrial applications. Two unbalanced polyploid cultivars named Native (Mentha Spicata L) and Scotch (M. × gracilis Sole) are the main producers of spearmint type oil, which is characterized by high levels of the monoterpenes (−)-carvone and (−)-limonene. These cultivars have been the backbone of spearmint oil production for decades, while breeding and improvement remained largely unexplored, in part, due to sterility in cultivated lines. Here we show that sexual breeding at the diploid level can be leveraged to develop new varieties that produce spearmint type oil, along with the improvement of other important traits. Using field trials and GC-FID oil analysis we characterized plant materials from a public germplasm repository and identified a diploid accession that exhibited 89.5% increase in oil yield, compared to the industry standard, and another that produces spearmint type oil. Spearmint-type oil was present at high frequency in a segregating F2 population (32/160) produced from these two accessions. Field-testing of ten of these F2 lines showed segregation for oil yield and confirmed the production of spearmint-type oil profiles. Two of these lines combined high yield and spearmint-type oil with acceptable analytic and sensory profiles. These results demonstrate that spearmint-type oil can be produced in a diploid background with high yield potential, providing a simpler genetic system for the development of improved spearmint varieties.

Rolling Circle RNA Synthesis Catalysed by RNA

RNA-catalysed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5′ and 3′ ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

Delayed DNA replication in haploid human embryonic stem cells

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

A nuclear cAMP microdomain suppresses tumor growth by Hippo pathway inactivation

cAMP signaling pathways are critical for both oncogenesis and tumor suppression. cAMP signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. We developed a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncovered a previously unappreciated nuclear cAMP microdomain that functionally activates a tumor suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397, export of YAP from the nucleus, without YAP protein degradation. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer, and since Hippo pathway deregulation can lead to oncogenesis and chemotherapeutic resistance, drugs directed at the nuclear cAMP microdomain may provide new avenues for the treatment of cancer.

Characterization and reverse genetic establishment of cattle derived Akabane virus in China

Background Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. Results An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. Conclusion We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Characterization and Use in Wheat Breeding of Leaf Rust Resistance Genes from Durable Varieties

Leaf rust is one of the most significant diseases of wheat worldwide. In Argentina, it is one of the main reasons for variety replacement that becomes susceptible after large-scale use. Some varieties showed durable resistance to this disease, including Buck Manantial and Sinvalocho MA. RILs (Recombinant Inbred Lines) were developed for each of these varieties and used in genetics studies to identify components of resistance, both in greenhouse inoculations using leaf rust races, and in field evaluations under natural population infections. In Buck Manantial, the APR gene LrBMP1 was associated with resistance in field tests. In crosses involving Sinvalocho MA, four genes were previously identified and associated with resistance in field testing: APR (Adult Plant Resistance) gene LrSV1, the APR genetic system LrSV2 + LrcSV2 and the ASR (All Stage Resistance) gene LrG6. Using backcrosses, LrBMP1 was introgressed in four commercial susceptible varieties and LrSV1, LrSV2 + LrcSV2 and LrG6 were simultaneously introgressed in three susceptible commercial varieties. The use of molecular markers for recurrent parent background selection allowed us to select resistant lines with more than 80% similarity to commercial varieties. Additionally, progress towards positional cloning of the genetic system LrSV2 + LrcSV2 for leaf rust APR is reported.