EphB3 as a potential mediator of developmental and reparative osteogenesis

The ephrin-B family of membrane-bound ligands is involved in skeletal patterning, osteogenesis, and bone homeostasis. Yet, despite the increasing collection of data affirming their importance in bone, the Eph tyrosine kinases that serve as the receptors for these ephrins in osteoblast stem cell niches remain unidentified. Here we report the expression of EphB3 at sites of bone growth in the embryo, especially at the calvaria suture fronts, periosteum, chondrocytes and trabeculae of developing long bones. Strong EphB3 expression persisted in the adult calvarial sutures and in the proliferative chondrocytes of long bones, both of which are documented niches for osteoblastic stem cells. We observed EphB3-positive cells in the tissue filling a created calvarial injury, further implying EphB3 involvement in bone healing. Genetic knockout of EphB3 caused an increase in the bone tissue volume as a fraction of total volume in six-week old calvaria and in femoral trabecular density, compared to wild type controls. This difference resolved by twelve weeks of age, when we instead observed an increase in the bone volume of femoral trabeculae and in trabecular thickness. Our data identify EphB3 as a candidate regulator of osteogenesis either alone or in combination with other bone-expressed Ephs, and indicate that it appears to function as a limiter of bone growth.

Plasma Membrane H+-ATPase SmPHA4 Negatively Regulates the Biosynthesis of Tanshinones in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge has been widely used in the treatment of cardiovascular and cerebrovascular diseases, due to the pharmacological action of its active components such as the tanshinones. Plasma membrane (PM) H+-ATPase plays key roles in numerous physiological processes in plants. However, little is known about the PM H+-ATPase gene family in S. miltiorrhiza (Sm). Here, nine PM H+-ATPase isoforms were identified and named SmPHA1–SmPHA9. Phylogenetic tree analysis showed that the genetic distance of SmPHAs was relatively far in the S. miltiorrhiza PM H+-ATPase family. Moreover, the transmembrane structures were rich in SmPHA protein. In addition, SmPHA4 was found to be highly expressed in roots and flowers. HPLC revealed that accumulation of dihydrotanshinone (DT), cryptotanshinone (CT), and tanshinone I (TI) was significantly reduced in the SmPHA4-OE lines but was increased in the SmPHA4-RNAi lines, ranging from 2.54 to 3.52, 3.77 to 6.33, and 0.35 to 0.74 mg/g, respectively, suggesting that SmPHA4 is a candidate regulator of tanshinone metabolites. Moreover, qRT-PCR confirmed that the expression of tanshinone biosynthetic-related key enzymes was also upregulated in the SmPHA4-RNAi lines. In summary, this study highlighted PM H+-ATPase function and provided new insights into regulatory candidate genes for modulating secondary metabolism biosynthesis in S. miltiorrhiza.

Expression Profiles of Alkaloid-Related Genes across the Organs of Narrow-Leafed Lupin (Lupinus angustifolius L.) and in Response to Anthracnose Infection

The main restraint obstructing the wider adoption of lupins as protein crops is the presence of bitter and toxic quinolizidine alkaloids (QAs), whose contents might increase under exposure to stressful environmental conditions. A poor understanding of how QAs accumulate hinders the breeding of sweet varieties. Here, we characterize the expression profiles of QA-related genes, along with the alkaloid content, in various organs of sweet and bitter narrow-leafed lupin (NLL, Lupinus angustifolius L.). Special attention is paid to the RAP2-7 transcription factor, a candidate regulator of the QA pathway. We demonstrate the upregulation of RAP2-7 and other QA-related genes, across the aerial organs of a bitter cultivar and the significant correlations between their expression levels, thus supporting the role of RAP2-7 as an important regulatory gene in NLL. Moreover, we showed that the initial steps of QA synthesis might occur independently in all aerial plant organs sharing common regulatory mechanisms. Nonetheless, other regulatory steps might be involved in RAP2-7-triggered QA accumulation, given its expression pattern in leaves. Finally, the examination of QA-related gene expression in plants infected with Colletotrichum lupini evidenced no connection between QA synthesis and anthracnose resistance, in contrast to the important role of polyamines during plant–pathogen interactions.

Rsp activates expression of the Cnt system in Staphylococcus aureus

Background The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Rsp Activates Expression of the Cnt System in Staphylococcus aureus

Background: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results: A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Rsp Activates Expression of the Cnt System in Staphylococcus aureus

Background: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results: A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Rsp Activates Expression of the Cnt System in Staphylococcus aureus

Background: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results: A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Rsp Activates Expression of the Cnt System in Staphylococcus aureus

Background: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results: A twofold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Rsp Activates Expression of the Cnt System in Staphylococcus aureus

Background The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps and other exporters in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE exporter gene. Results A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in both low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. A decrease in the expression of cntK (1.5-fold) and cntA (1.6-fold) was seen for Δfur Δrsp compared to Δfur strain. Similarly decreased expression of cntK (1.3-fold), cntA (1.6-fold) and cntE (1.6-fold) genes was seen comparing Δzur Δrsp and Δzur mutants but statistically not significant, indicating that Rsp is dependent on Fur and Zur. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for both the cntK and cntA promoters. Conclusions Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Weighted gene co-expression network analysis unveils gene networks associated with the Fusarium head blight resistance in tetraploid wheat

Background Fusarium head blight (FHB) resistance in the durum wheat breeding gene pool is rarely reported. Triticum turgidum ssp. carthlicum line Blackbird is a tetraploid relative of durum wheat that offers partial FHB resistance. Resistance QTL were identified for the durum wheat cv. Strongfield × Blackbird population on chromosomes 1A, 2A, 2B, 3A, 6A, 6B and 7B in a previous study. The objective of this study was to identify the defense mechanisms underlying the resistance of Blackbird and report candidate regulator defense genes and single nucleotide polymorphism (SNP) markers within these genes for high-resolution mapping of resistance QTL reported for the durum wheat cv. Strongfield/Blackbird population. Results Gene network analysis identified five networks significantly (P < 0.05) associated with the resistance to FHB spread (Type II FHB resistance) one of which showed significant correlation with both plant height and relative maturity traits. Two gene networks showed subtle differences between Fusarium graminearum-inoculated and mock-inoculated plants, supporting their involvement in constitutive defense. The candidate regulator genes have been implicated in various layers of plant defense including pathogen recognition (mainly Nucleotide-binding Leucine-rich Repeat proteins), signaling pathways including the abscisic acid and mitogen activated protein (MAP) kinase, and downstream defense genes activation including transcription factors (mostly with dual roles in defense and development), and cell death regulator and cell wall reinforcement genes. The expression of five candidate genes measured by quantitative real-time PCR was correlated with that of RNA-seq, corroborating the technical and analytical accuracy of RNA-sequencing. Conclusions Gene network analysis allowed identification of candidate regulator genes and genes associated with constitutive resistance, those that will not be detected using traditional differential expression analysis. This study also shed light on the association of developmental traits with FHB resistance and partially explained the co-localization of FHB resistance with plant height and maturity QTL reported in several previous studies. It also allowed the identification of candidate hub genes within the interval of three previously reported FHB resistance QTL for the Strongfield/Blackbird population and associated SNPs for future high resolution mapping studies.