Volatile communication in plants relies on a KAI2-mediated signaling pathway.

Plants are constantly exposed to volatile organic compounds (VOCs) that are released during plant-plant communication, within-plant self-signaling, and plant-microbe interactions. Therefore, understanding VOC perception and downstream signaling is vital for unraveling the mechanisms behind information exchange in plants, which remain largely unexplored. Using the hormone-like function of volatile terpenoids in reproductive organ development as a system with a visual marker for communication, we demonstrate that a petunia karrikin-insensitive receptor, PhKAI2ia, stereospecifically perceives the (-)-germacrene D signal, triggering a KAI2-mediated signaling cascade and affecting plant fitness. This study uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).

Age associated susceptibility to SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still an ongoing global health crisis. Clinical data indicate that the case fatality rate (CFR) is age dependent, with a higher CFR percentage in the elderly population. We compared the pathogenesis of SARS-CoV-2 in young and aged K18-hACE2 transgenic mice. We evaluated morbidity, mortality, viral titers, immune responses, and histopathology in SARS-CoV-2-infected young and old K18-hACE2 transgenic mice. Within the limitation of having a low number of mice per group, our results indicate that SARS-CoV-2 infection resulted in slightly higher morbidity, mortality, and viral replication in the lungs of old mice, which was associated with an impaired IgM response and altered cytokine and chemokine profiles. Results of this study increase our understanding of SARS-CoV-2 infectivity and immuno-pathogenesis in the elderly population.

Mitochondrial Transplantation promotes protective effector and memory CD4+ T cell response during Mycobacterium tuberculosis infection and diminishes exhaustion and senescence in elderly CD4+ T cells.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M.tb), remains a significant health concern worldwide, especially in populations with weakened or compromised immune systems, such as the elderly. Proper adaptive immune function, particularly a CD4+ T cell response, is central to host immunity against M.tb. Chronic infections, such as M.tb, as well as aging promote T cell exhaustion and senescence, which can impair immune control and promote progression to TB disease. Mitochondrial dysfunction contributes to T cell dysfunction, both in aging and chronic infections and diseases. Mitochondrial perturbations can disrupt cellular metabolism, enhance oxidative stress, and impair T-cell signaling and effector functions. This study examined the impact of mitochondrial transplantation (mito-transfer) on CD4+ T cell differentiation and function using aged mouse models and human CD4+ T cells from elderly individuals. Our study revealed that mito-transfer in naïve CD4+ T cells promoted the generation of protective effector and memory CD4+ T cells during M.tb infection in mice. Further, mito-transfer enhanced the function of elderly human T cells by increasing their mitochondrial mass and modulating cytokine production, which in turn reduced exhaustion and senescence cell markers. Our results suggest that mito-transfer could be a novel strategy to reestablish aged CD4+ T cell function, potentially improving immune responses in the elderly and chronic TB patients, with a broader implication for other diseases where mitochondrial dysfunction is linked to T cell exhaustion and senescence.

Extracellular Delivery of Functional Mitochondria Rescues the Dysfunction of CD4+ T Cells in Aging.

Mitochondrial dysfunction alters cellular metabolism, increases tissue oxidative stress, and may be principal to the dysregulated signaling and function of CD4+ T lymphocytes in the elderly. In this proof of principle study, it is investigated whether the transfer of functional mitochondria into CD4+ T cells that are isolated from old mice (aged CD4+ T cells), can abrogate aging-associated mitochondrial dysfunction, and improve the aged CD4+ T cell functionality. The results show that the delivery of exogenous mitochondria to aged non-activated CD4+ T cells led to significant mitochondrial proteome alterations highlighted by improved aerobic metabolism and decreased cellular mitoROS. Additionally, mito-transferred aged CD4+ T cells showed improvements in activation-induced TCR-signaling kinetics displaying markers of activation (CD25), increased IL-2 production, enhanced proliferation ex vivo. Importantly, immune deficient mouse models (RAG-KO) showed that adoptive transfer of mito-transferred naive aged CD4+ T cells, protected recipient mice from influenza A and Mycobacterium tuberculosis infections. These findings support mitochondria as targets of therapeutic intervention in aging.

IL-10 Modulation Increases Pyrazinamide's Antimycobacterial Efficacy against Mycobacterium tuberculosis Infection in Mice.

Mechanisms to shorten the duration of tuberculosis (TB) treatment include new drug formulations or schedules and the development of host-directed therapies (HDTs) that better enable the host immune system to eliminate Mycobacterium tuberculosis. Previous studies have shown that pyrazinamide, a first-line antibiotic, can also modulate immune function, making it an attractive target for combinatorial HDT/antibiotic therapy, with the goal to accelerate clearance of M. tuberculosis. In this study, we assessed the value of anti-IL-10R1 as an HDT along with pyrazinamide and show that short-term anti-IL-10R1 blockade during pyrazinamide treatment enhanced the antimycobacterial efficacy of pyrazinamide, resulting in faster clearance of M. tuberculosis in mice. Furthermore, 45 d of pyrazinamide treatment in a functionally IL-10-deficient environment resulted in sterilizing clearance of M. tuberculosis. Our data suggest that short-term IL-10 blockade with standard TB drugs has the potential to improve clinical outcome by reducing the treatment duration.

Aerosolized sulfated hyaluronan derivatives prolong the survival of K18 ACE2 mice infected with a lethal dose of SARS-CoV-2.

Despite several vaccines that are currently approved for human use to control the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent medical need for therapeutic and prophylactic options. SARS-CoV-2 binding and entry in human cells involves interactions of its spike (S) protein with several host cell surface factors, including heparan sulfate proteoglycans (HSPGs), transmembrane protease serine 2 (TMPRSS2), and angiotensin-converting enzyme 2 (ACE2). In this paper we investigated the potential of sulphated Hyaluronic Acid (sHA), a HSPG mimicking polymer, to inhibit the binding of SARS-CoV-2 S protein to human ACE2 receptor. After the assessment of different sulfation degree of sHA backbone, a series of sHA functionalized with different hydrophobic side chains were synthesized and screened. The compound showing the highest binding affinity to the viral S protein was further characterized by surface plasmon resonance (SPR) towards ACE2 and viral S protein binding domain. Selected compounds were formulated as solutions for nebulization and, after being characterized in terms of aerosolization performance and droplet size distribution, their efficacy was assessed in vivo using the K18 human (h)ACE2 transgenic mouse model of SARS-CoV-2 infection.

Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173-initiated trans-acting cascade silencing.

Small RNAs (sRNAs) such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) are short 20-24-nucleotide non-coding RNAs. They are key regulators of gene expression in plants and other organisms. Several 22-nucleotide miRNAs trigger biogenesis cascades of trans-acting secondary siRNAs, which are involved in various developmental and stress responses. Here we show that Himalayan Arabidopsis thaliana accessions having natural mutations in the miR158 locus exhibit robust cascade silencing of the pentatricopeptide repeat (PPR)-like locus. Furthermore, we show that these cascade sRNAs trigger tertiary silencing of a gene involved in transpiration and stomatal opening. The natural deletions or insertions in MIR158 led to improper processing of miR158 precursors, thereby blocking synthesis of mature miR158. Reduced miR158 levels led to increased levels of its target, a pseudo-PPR gene that is targeted by tasiRNAs generated by the miR173 cascade in other accessions. Using sRNA datasets derived from Indian Himalayan accessions, as well as overexpression and knockout lines of miR158, we show that absence of miR158 led to buildup of pseudo-PPR-derived tertiary sRNAs. These tertiary sRNAs mediated robust silencing of a gene involved in stomatal closure in Himalayan accessions lacking miR158 expression. We functionally validated the tertiary phasiRNA that targets NHX2, which encodes a Na+ -K+ /H+ antiporter protein, thereby regulating transpiration and stomatal conductance. Overall, we report the role of the miRNA-TAS-siRNA-pseudogene-tertiary phasiRNA-NHX2 pathway in plant adaptation.

An inducible potato (E,E)-farnesol synthase confers tolerance against bacterial pathogens in potato and tobacco.

Terpene synthases (TPSs) have diverse biological functions in plants. Though the roles of TPSs in herbivore defense are well established in many plant species, their role in bacterial defense has been scarce and is emerging. Through functional genomics, here we report the in planta role of potato (Solanum tuberosum) terpene synthase (StTPS18) in bacterial defense. Expression of StTPS18 was highest in leaves and was induced in response to Pseudomonas syringae and methyl jasmonate treatments. The recombinant StTPS18 exhibited bona fide (E,E)-farnesol synthase activity forming a sesquiterpenoid, (E,E)-farnesol as the sole product, utilising (E,E)-farnesyl diphosphate (FPP). Subcellular localization of GFP fusion protein revealed that StTPS18 is localized to the cytosol. Silencing and overexpression of StTPS18 in potato resulted in reduced and enhanced tolerance, respectively, to bacterial pathogens P. syringae and Ralstonia solanacearum. Bacterial growth assay using medium containing (E,E)-farnesol significantly inhibited P. syringae growth. Moreover, StTPS18 overexpressing transgenic potato and Nicotiana tabacum leaves, and (E,E)-farnesol and P. syringae infiltrated potato leaves exhibited elevated expression of sterol pathway and members of pathogenesis-related genes with enhanced phytosterol accumulation. Interestingly, enhanced phytosterols in 13 C3 -(E,E)-farnesol infiltrated potato leaves were devoid of any noticeable 13 C labeling, indicating no direct utilization of (E,E)-farnesol in phytosterols formation. Furthermore, leaves of StTPS18 overexpressing transgenic lines had no detectable (E,E)-farnesol similar to the control plant, and emitted lower levels of sesquiterpenes than the control. These findings point towards an indirect involvement of StTPS18 and its product (E,E)-farnesol in bacterial defense through upregulation of phytosterol biosynthesis and defense genes.

IL-10 Receptor Blockade Delivered Simultaneously with Bacillus Calmette-Guérin Vaccination Sustains Long-Term Protection against Mycobacterium tuberculosis Infection in Mice.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) immunization still remains the best vaccination strategy available to control the development of active tuberculosis. Protection afforded by BCG vaccination gradually wanes over time and although booster strategies have promise, they remain under development. An alternative approach is to improve BCG efficacy through host-directed therapy. Building upon prior knowledge that blockade of IL-10R1 during early Mycobacterium tuberculosis infection improves and extends control of M. tuberculosis infection in mice, we employed a combined anti-IL-10R1/BCG vaccine strategy. An s.c. single vaccination of BCG/anti-IL10-R1 increased the numbers of CD4+ and CD8+ central memory T cells and reduced Th1 and Th17 cytokine levels in the lung for up to 7 wk postvaccination. Subsequent M. tuberculosis challenge in mice showed both an early (4 wk) and sustained long-term (47 wk) control of infection, which was associated with increased survival. In contrast, protection of BCG/saline-vaccinated mice waned 8 wk after M. tuberculosis infection. Our findings demonstrate that a single and simultaneous vaccination with BCG/anti-IL10-R1 sustains long-term protection, identifying a promising approach to enhance and extend the current BCG-mediated protection against TB.

6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro.

The emergence of SARS-CoV-2 has led to a global health crisis that, in addition to vaccines and immunomodulatory therapies, calls for the identification of antiviral therapeutics. The papain-like protease (PLpro) activity of nsp3 is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG), an orally available and widely available generic drug, inhibits SARS-CoV-2 replication in Vero-E6 cells with an EC50 of approximately 2 µM. 6-TG also inhibited PLpro-catalyzed polyprotein cleavage and de-ISGylation in cells and inhibited proteolytic activity of the purified PLpro domain in vitro. We therefore propose that 6-TG is a direct-acting antiviral that could potentially be repurposed and incorporated into the set of treatment and prevention options for COVID-19.

Antiviral activity of oleandrin and a defined extract of Nerium oleander against SARS-CoV-2.

With continued expansion of the coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome 2 (SARS-CoV-2), both antiviral drugs as well as effective vaccines are desperately needed to treat patients at high risk of life-threatening disease. Here, we present in vitro evidence for significant inhibition of SARS-CoV-2 by oleandrin and a defined extract of N. oleander (designated as PBI-06150). Using Vero cells, we found that prophylactic (pre-infection) oleandrin (as either the pure compound or as the active principal ingredient in PBI-06150) administration at concentrations as low as 0.05 µg/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1 µg/ml concentration resulted in a greater than 3000-fold reduction in infectious virus production. The half maximal effective concentration (EC50) values were 11.98 ng/ml when virus output was measured at 24 h post-infection, and 7.07 ng/ml measured at 48 h post-infection. Therapeutic (post-infection) treatment up to 24 h after SARS-CoV-2 infection of Vero cells also reduced viral titers, with 0.1 µg/ml and 0.05 µg/ml concentrations causing greater than 100-fold reduction as measured at 48 h, and the 0.05 µg/ml concentration resulting in a 78-fold reduction. Concentrations of oleandrin up to 10 µg/ml were well tolerated in Vero cells. We also present in vivo evidence of the safety and efficacy of defined N. oleander extract (PBI-06150), which was administered to golden Syrian hamsters in a preparation containing as high as 130 µg/ml of oleandrin. In comparison to administration of control vehicle, PBI-06150 provided a statistically significant reduction of the viral titer in the nasal turbinates (nasal conchae). The potent prophylactic and therapeutic antiviral activities demonstrated here, together with initial evidence of its safety and efficacy in a relevant hamster model of COVID-19, support the further development of oleandrin and/or defined extracts containing this molecule for the treatment of SARS-CoV-2 and associated COVID-19 disease and potentially also for reduction of virus spread by persons diagnosed early after infection.

Functional characterization of a defense-responsive bulnesol/elemol synthase from potato.

Terpene synthases (TPSs) produce a variety of terpenoids that play numerous functional roles in primary and secondary metabolism, as well as in ecological interactions. Here, we report the functional characterization of an inducible potato TPS gene encoding bulnesol/elemol synthase (StBUS/ELS). The expression of StBUS/ELS in potato leaves was significantly induced in response to both bacterial (Pseudomonas syringae) and fungal (Alternaria solani) infection as well as methyl jasmonate treatment, indicating its role in defense. The leaves exhibited the highest StBUS/ELS expression followed by the stem with least and similar expression in tuber, sprout and root. Recombinant StBUS/ELS catalyzed the formation of different sesquiterpenes by utilizing farnesyl diphosphate as substrate, and the monoterpene geraniol from geranyl diphosphate. Among the sesquiterpenes formed by StBUS/ELS, elemol was the predominant product followed by α-bulnesene, bulnesol and ß-elemene. Further gas chromatography-mass spectrometry (GC-MS) analysis of StBUS/ELS assay products at different injection temperatures revealed elemol and bulnesol as the major products at 275 and 200/150°C, respectively, without much change in the levels of minor products. This indicated thermal rearrangement of bulnesol into elemol at higher temperatures. Transient overexpression of StBUS/ELS in potato leaves conferred tolerance against the growth of bacteria P. syringae and Ralstonia solanacearum, and the fungus A. solani. Further, expression analysis of pathogenesis-related (PR) genes in StBUS/ELS overexpressing leaves showed no significant change in comparison to control, indicating a direct involvement of StBUS/ELS enzymatic products against pathogens. Overall, our study suggested that StBUS/ELS is a pathogen-inducible TPS encoding bulnesol/elemol synthase and could provide a direct role in defense against biotic stress in potato.

Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

A plastid-localized bona fide geranylgeranyl diphosphate synthase plays a necessary role in monoterpene indole alkaloid biosynthesis in Catharanthus roseus.

In plants, geranylgeranyl diphosphate (GGPP, C20 ) synthesized by GGPP synthase (GGPPS) serves as precursor for vital metabolic branches including specialized metabolites. Here, we report the characterization of a GGPPS (CrGGPPS2) from the Madagascar periwinkle (Catharanthus roseus) and demonstrate its role in monoterpene (C10 )-indole alkaloids (MIA) biosynthesis. The expression of CrGGPPS2 was not induced in response to methyl jasmonate (MeJA), and was similar to the gene encoding type-I protein geranylgeranyltransferase_ß subunit (CrPGGT-I_ß), which modulates MIA formation in C. roseus cell cultures. Recombinant CrGGPPS2 exhibited a bona fide GGPPS activity by catalyzing the formation of GGPP as the sole product. Co-localization of fluorescent protein fusions clearly showed CrGGPPS2 was targeted to plastids. Downregulation of CrGGPPS2 by virus-induced gene silencing (VIGS) significantly decreased the expression of transcription factors and pathway genes related to MIA biosynthesis, resulting in reduced MIA. Chemical complementation of CrGGPPS2-vigs leaves with geranylgeraniol (GGol, alcoholic form of GGPP) restored the negative effects of CrGGPPS2 silencing on MIA biosynthesis. In contrast to VIGS, transient and stable overexpression of CrGGPPS2 enhanced the MIA biosynthesis. Interestingly, VIGS and transgenic-overexpression of CrGGPPS2 had no effect on the main GGPP-derived metabolites, cholorophylls and carotenoids in C. roseus leaves. Moreover, silencing of CrPGGT-I_ß, similar to CrGGPPS2-vigs, negatively affected the genes related to MIA biosynthesis resulting in reduced MIA. Overall, this study demonstrated that plastidial CrGGPPS2 plays an indirect but necessary role in MIA biosynthesis. We propose that CrGGPPS2 might be involved in providing GGPP for modifying proteins of the signaling pathway involved in MIA biosynthesis.

Selective delipidation of Mycobacterium bovis BCG enables direct pulmonary vaccination and enhances protection against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is the leading killer due to an infectious organism. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine approved against TB, however, its efficacy against pulmonary TB is poor. While BCG is currently inoculated intradermally, the natural route of M.tb infection is through the lung. Excessive lung pathology caused by pulmonary inoculation of BCG has prevented the use of this immunization route. Here, we show that selective chemical treatment of BCG with petroleum ether removes inflammatory lipids from the bacterial surface while keeping BCG viable. Pulmonary vaccination using this modified BCG attenuated inflammatory responses, prevented immunopathology of the lung, and significantly increased protection against M.tb infection in mice. We further directly linked IL-17A as the responsible contributor of improved immunity against M.tb infection. These results provide evidence that selective removal of cytotoxic lipids from the BCG surface attenuates inflammation and offers a safer and superior vaccine against TB causing less damage post-infectious challenge with M.tb.

Cytomegalovirus immunoglobulin G titers do not predict reactivation risk in immunocompetent hosts.

Cytomegalovirus (CMV) reactivation occurs in roughly one-third of immunocompetent patients during critical illness, and is associated with worse outcomes. These outcomes have prompted consideration of early antiviral prophylaxis, but two-third of patients would receive unnecessary treatment. Tissue viral load has been associated with risk of reactivation in murine models, and recent work has suggested a relationship between immune responses to CMV and underlying viral load. We, therefore, sought to confirm the hypothesis that serum CMV-specific immunoglobulin G (IgG) correlates with tissue viral load, and might be used to predict the risk of reactivation during critical illness. We confirm that there is a good correlation between tissue viral load and serum CMV-specific IgG after laboratory infection of inbred mice. Further, we show that naturally infected outbred hosts have variable tissue viral DNA loads that do not correlate well with serum IgG. Perhaps as a consequence, CMV-specific IgG was not predictive of reactivation events in immunocompetent humans. When reactivation did occur, those with the lowest IgG levels had longer durations of reactivation, but IgG quartiles were not associated with differing peak DNAemia. Together our data suggest that CMV-specific IgG titers diverge from tissue viral loads in outbred immunocompetent hosts, and their importance for the control of reactivation events remains unclear.

Altered monocyte phenotypes but not impaired peripheral T cell immunity may explain susceptibility of the elderly to develop tuberculosis.

Tuberculosis (TB) is the leading killer due to a single infectious disease worldwide. With the aging of the global population, the case rate and deaths due to TB are highest in the elderly population. While general immunosenescence associated with old age is thought to contribute to the susceptibility of the elderly to develop active TB disease, very few studies of immune function in elderly individuals with Mycobacterium tuberculosis (M.tb) infection or disease have been performed. In particular, impaired adaptive T cell immunity to M.tb is one proposed mechanism for the elderly's increased susceptibility primarily on the basis of the decreased delayed type hypersensitivity response to tuberculin-purified protein derivative in the skin of elderly individuals. To investigate immunological reasons why the elderly are susceptible to develop active TB disease, we performed a cross-sectional observational study over a five year period (2012-2016) enrolling participants from 2 age groups (adults: 25-44 years; elderly: 65 and older) and 3 M.tb infection statuses (active TB, latent TB infection, and healthy controls without history of M.tb infection). We hypothesized that impaired peripheral T cell immunity plays a role in the biological susceptibility of the elderly to TB. Contrary to our hypothesis, we observed no evidence of impaired M.tb specific T cell frequency or altered production of cytokines implicated in M.tb control (IFN-γ, IL-10) in peripheral blood in the elderly. Instead, we observed alterations in monocyte proportion and phenotype with age and M.tb infection that suggest their potential role in the susceptibility of the elderly to develop active TB. Our results suggest a potential link between the known widespread low-grade systemic inflammation of old age, termed "inflammaging," with the elderly's specific susceptibility to developing active TB. Moreover, our results highlight the need for further research into the biological reasons why the elderly are more susceptible to disease and death from TB, so that public health systems can be better equipped to face the present and future problem of TB in an aging global population.

A WRKY transcription factor from Withania somnifera regulates triterpenoid withanolide accumulation and biotic stress tolerance through modulation of phytosterol and defense pathways.

Withania somnifera produces pharmacologically important triterpenoid withanolides that are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be elucidated. A jasmonate- and salicin-inducible WRKY transcription factor from W. somnifera (WsWRKY1) exhibiting correlation with withaferin A accumulation was functionally characterized employing virus-induced gene silencing and overexpression studies combined with transcript and metabolite analyses, and chromatin immunoprecipitation assay. WsWRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol pathway genes with corresponding reduction in phytosterols and withanolides in W. somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W. somnifera (phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover, WsWRKY1 binds to W-box sequences in promoters of W. somnifera genes encoding squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid pathway. Furthermore, while WsWRKY1 silencing in W. somnifera compromised the tolerance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco led to improved biotic stress tolerance. Together these findings demonstrate that WsWRKY1 has a positive regulatory role on phytosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its importance as a metabolic engineering tool for simultaneous improvement of triterpenoid biosynthesis and plant defense.

Transcriptomic insight into terpenoid and carbazole alkaloid biosynthesis, and functional characterization of two terpene synthases in curry tree (Murraya koenigii).

Curry tree (Murraya koenigii L.) is a rich source of aromatic terpenes and pharmacologically important carbazole alkaloids. Here, M. koenigii leaf transcriptome was generated to gain insight into terpenoid and alkaloid biosynthesis. Analysis of de novo assembled contigs yielded genes for terpene backbone biosynthesis and terpene synthases. Also, gene families possibly involved in carbazole alkaloid formation were identified that included polyketide synthases, prenyltransferases, methyltransferases and cytochrome P450s. Further, two genes encoding terpene synthases (MkTPS1 and MkTPS2) with highest in silico transcript abundance were cloned and functionally characterized to determine their involvement in leaf volatile formation. Subcellular localization using GFP fusions revealed the plastidial and cytosolic localization of MkTPS1 and MkTPS2, respectively. Enzymatic characterization demonstrated the monoterpene synthase activity of recombinant MkTPS1, which produced primarily (-)-sabinene from geranyl diphosphate (GPP). Recombinant MkTPS2 exhibited sesquiterpene synthase activity and formed (E,E)-α-farnesene as the major product from farnesyl diphosphate (FPP). Moreover, mRNA expression and leaf volatile analyses indicated that MkTPS1 accounts for (-)-sabinene emitted by M. koenigii leaves. Overall, the transcriptome data generated in this study will be a great resource and the start point for characterizing genes involved in the biosynthetic pathway of medicinally important carbazole alkaloids.

Cytomegalovirus Reinfections Stimulate CD8 T-Memory Inflation.

Cytomegalovirus (CMV) has been shown to induce large populations of CD8 T-effector memory cells that unlike central memory persist in large quantities following infection, a phenomenon commonly termed "memory inflation". Although murine models to date have shown very large and persistent CMV-specific T-cell expansions following infection, there is considerable variability in CMV-specific T-memory responses in humans. Historically such memory inflation in humans has been assumed a consequence of reactivation events during the life of the host. Because basic information about CMV infection/re-infection and reactivation in immune competent humans is not available, we used a murine model to test how primary infection, reinfection, and reactivation stimuli influence memory inflation. We show that low titer infections induce "partial" memory inflation of both mCMV specific CD8 T-cells and antibody. We show further that reinfection with different strains can boost partial memory inflation. Finally, we show preliminary results suggesting that a single strong reactivation stimulus does not stimulate memory inflation. Altogether, our results suggest that while high titer primary infections can induce memory inflation, reinfections during the life of a host may be more important than previously appreciated.