Developing a rapid and highly efficient cowpea regeneration,transformation and genome editing system using embryonic axis explants

Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.     

Stress profiling of longevity mutants identifies Afg3 as a mitochondrial determinant of cytoplasmic mRNA translation and aging

Although environmental stress likely plays a significant role in promoting aging, the relationship remains poorly understood. To characterize this interaction in a more comprehensive manner, we examined the stress response profiles for 46 long‐lived yeast mutant strains across four different stress conditions (oxidative, ER, DNA damage, and thermal), grouping genes based on their associated stress response profiles. Unexpectedly, cells lacking the mitochondrial AAA protease gene AFG3 clustered strongly with long‐lived strains lacking cytosolic ribosomal proteins of the large subunit. Similar to these ribosomal protein mutants, afg3Δ cells show reduced cytoplasmic mRNA translation, enhanced resistance to tunicamycin that is independent of the ER unfolded protein response, and Sir2‐independent but Gcn4‐dependent lifespan extension. These data demonstrate an unexpected link between a mitochondrial protease, cytoplasmic mRNA translation, and aging.     

CD18 is required for intestinal T cell responses at multiple immune checkpoints

The intestinal immune response to oral Ags involves a complex multistep process. The requirements for optimal intestinal T cell responses in this process are unclear. LFA-1 plays a critical role in peripheral T cell trafficking and activation, however, its role in intestinal immune responses has not been precisely defined. To dissect the role of LFA-1 in intestinal immune responses, we used a system that allows for segregation of T cell migration and activation through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 TCR transgenic mice into wild-type BALB/c mice. We find that wild-type mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate decreases in the numbers of Ag-specific T cells in the intestinal lamina propria after oral Ag administration. We also find that in addition to its role in trafficking to intestinal secondary lymphoid organs, LFA-1 is required for optimal CD4(+) T cell proliferation in vivo upon oral Ag immunization. Furthermore, CD18(-/-) DO11.10 CD4(+) T cells primed in the intestinal secondary lymphoid organs demonstrate defects in up-regulation of the intestinal-specific trafficking molecules, alpha(4)beta(7) and CCR9. Interestingly, the defect in trafficking of CD18(-/-) DO11.10 CD4(+) T cells to the intestinal lamina propria persists even under conditions of equivalent activation and intestinal-tropic differentiation, implicating a role for CD18 in the trafficking of activated T cells into intestinal tissues independent of the earlier defects in the intestinal immune response. This argues for a complex role for CD18 in the early priming checkpoints and ultimately in the trafficking of T cells to the intestinal tissues during an intestinal immune response.     

TLR4 Deters Perfusion Recovery and Upregulates Toll-like Receptor 2 (TLR2) in Ischemic Skeletal Muscle and Endothelial Cells

Toll-like receptors (TLRs) play an important role in regulating muscle regeneration and angiogenesis in response to ischemia. TLR2 knockout mice exhibit pronounced skeletal muscle necrosis and abnormal vessel architecture after femoral artery ligation, suggesting that TLR2 signaling is protective during ischemia. TLR4, an important receptor in inflammatory signaling, has been shown to regulate TLR2 expression in other systems. We hypothesize that a similar relationship between TLR4 and TLR2 may exist in hindlimb ischemia in which TLR4 upregulates TLR2, a mediator of angiogenesis and perfusion recovery. We examined the expression of TLR2 in unstimulated and in TLR-agonist treated endothelial cells (ECs). TLR2 expression (low in control ECs) was upregulated by lipopolysaccharide, the danger signal high mobility group box-1, and hypoxia in a TLR4-dependent manner. Endothelial tube formation on Matrigel as well as EC permeability was assessed as in vitro measures of angiogenesis. Time-lapse imaging demonstrated that ECs lacking TLR4 formed more tubes, whereas TLR2 knockdown ECs exhibited attenuated tube formation. TLR2 also mediated EC permeability, an initial step during angiogenesis, in response to high-mobility group box-1 (HMGB1) that is released by cells during hypoxic injury. In vivo, ischemia-induced upregulation of TLR2 required intact TLR4 signaling that mediated systemic inflammation, as measured by local and systemic IL-6 levels. Similar to our in vitro findings, vascular density and limb perfusion were both enhanced in the absence of TLR4 signaling, but not if TLR2 was deleted. These findings indicate that TLR2, in the absence of TLR4, improves angiogenesis and perfusion recovery in response to ischemia.     

Molecular mechanisms underlying genotype‐dependent responses to dietary restriction

Dietary restriction (DR) increases lifespan and attenuates age‐related phenotypes in many organisms; however, the effect of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here, we describe a large‐scale effort to define molecular mechanisms that underlie genotype‐specific responses to DR. The effect of DR on lifespan was determined for 166 single gene deletion strains in Saccharomyces cerevisiae. Resulting changes in mean lifespan ranged from a reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR), which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processes that underlie genotype‐dependent effects of DR that may be important modulators of DR in higher organisms.     

Positive feedbacks to growth of an invasive grass through alteration of nitrogen cycling

Understanding the mechanisms by which invasive plants maintain dominance is essential to achieving long-term restoration goals. While many reports have suggested invasive plants alter resource availability, experimental tests of feedbacks between invasive plants and soil resources are lacking. We used field observations and experimental manipulations to test if the invasive grass Microstegium vimineum both causes and benefits from altered soil nitrogen (N) cycling. To quantify M. vimineum effects on N dynamics, we compared inorganic N pools and nitrification rates in 20 naturally invaded and uninvaded plots across a range of mixed hardwood forests, and in experimentally invaded and uninvaded common garden plots. Potential nitrification rates were 142 and 63?% greater in invaded than uninvaded plots in forest and common garden soils, respectively. As a result, soil nitrate was the dominant form of inorganic N during peak M. vimineum productivity in both studies. To determine the response of M. vimineum to altered nitrogen availability, we manipulated the dominant N form (nitrate or ammonium) in greenhouse pots containing M. vimineum alone, M. vimineum with native species, and native species alone. M. vimineum productivity was highest in monocultures receiving nitrate; in contrast, uninvaded native communities showed no response to N form. Notably, the positive response of M. vimineum to nitrate was not apparent when grown in competition with natives, suggesting an invader density threshold is required before positive feedbacks occur. Collectively, our results demonstrate that persistence of invasive plants can be promoted by positive feedbacks with soil resources but that the magnitude of feedbacks may depend on interspecific interactions.     

Histology and microscopic anatomy of the thyroid gland during the larval development of Pseudis platensis (Anura,Hylidae)

Several hormones regulate anuran larval development, most notably thyroid hormones (THs). In anurans, metamorphosis fails when the thyroid gland is absent or inactivated, resulting in giant tadpoles. Larval gigantism occurs naturally in neotropical frogs of the genus Pseudis as a result of a prolonged larval period. Its thyroid function is poorly investigated and the focus of this study. We describe qualitative and quantitative variations in larval development for field-captured specimens of Pseudis platensis and compare those to the development of two sympatric species, Phyllomedusa sauvagii and Pithecopus azureus, which have small tadpoles and a shorter larval period. We describe morphological changes in the thyroid glands of larval and adult specimens. In contrast to other species with similar ecological requirements, P. platensis exhibits distinct glandular activity. During premetamorphosis, there was little or no thyroid activity, a period in which the tadpole reached 70% of its maximum size. Development and degree of activity of the thyroid gland determine the duration of the early stages of the larval period. Thyroid gland histology in tadpoles appears to correlate with the TH activity, and in turn with the diversity in anuran life history transitions.     

Species delineation in the Capitella species complex (Annelida: Capitellidae): geographic and genetic variation in the northern Gulf of Mexico

Capitella capitata was traditionally used as a biological indicator species due to its ubiquitous distribution and high densities in disturbed and polluted marine and estuarine sediments. Based on allozyme and developmental studies, it is now clear that C. capitata is a species complex consisting of multiple distinct lineages worldwide, including the recently described C. teleta, a model species for spiralian development. The coast of the northern Gulf of Mexico, with its numerous bays and estuaries and frequently occurring natural and anthropogenic disturbances, provides an appropriate region for such studies. We sequenced a fragment of the mitochondrial cytochrome c oxidase subunit I gene for individuals of C. cf. capitata and C. cf. aciculata (distinguished by acicular spines on the first two chaetigers) collected from Texas and Florida coasts and analyzed them in conjunction with data available in GenBank. Our results indicate the presence of a Gulf of Mexico clade that is distinct from populations in Canada and the Indo‐Pacific. Populations in the northern Gulf of Mexico are structured geographically, with support for Texas and Florida clades, and there do not seem to be clear boundaries between C. cf. capitata and C. cf. aciculata. This is corroborated by the fact that multiple specimens were morphologically intermediate between the two species. In future studies, we aim to clarify whether the intermediate morphologies represent ontogenetic stages, neutral morphological variation, phenotypic plasticity, or sexual dimorphism in a single species or whether several lineages with incomplete reproductive barriers are present.     

Widespread expression of the Supv3L1 mitochondrial RNA helicase in the mouse

Supv3L1 is an evolutionarily conserved helicase that plays a critical role in the mitochondrial RNA surveillance and degradation machinery. Conditional ablation of Supv3L1 in adult mice leads to premature aging phenotypes including loss of muscle mass and adipose tissue and severe skin abnormalities. To get insights into the spatial and temporal expression of Supv3L1 in the mouse, we generated knock-in and transgenic strains in which an EGFP reporter was placed under control of the Supv3L1 native promoter. During development, expression of Supv3L1 begins at the blastocyst stage, becomes widespread and strong in all fetal tissues and cell types, and continues during postnatal growth. In mature animals reporter expression is only slightly diminished in most tissues and continues to be highly expressed in the brain, peripheral sensory organs, and testis. Together, these data confirm that Supv3L1 is an important developmentally regulated gene, which continues to be expressed in all mature tissues, particularly the rapidly proliferating cells of testes, but also in the brain and sensory organs. The transgenic mice and cell lines derived from them constitute a valuable tool for the examination of the spatial and temporal aspects of Supv3L1 promoter activity, and should facilitate future screens for small molecules that regulate Supv3L1 expression.