Pooled effector library screening in protoplasts rapidly identifies novel Avr genes.

Crop breeding for durable disease resistance is challenging due to the rapid evolution of pathogen virulence. While progress in resistance (R) gene cloning and stacking has accelerated in recent years1-3, the identification of corresponding avirulence (Avr) genes in many pathogens is hampered by the lack of high-throughput screening options. To address this technology gap, we developed a platform for pooled library screening in plant protoplasts to allow rapid identification of interacting R-Avr pairs. We validated this platform by isolating known and novel Avr genes from wheat stem rust (Puccinia graminis f. sp. tritici) after screening a designed library of putative effectors against individual R genes. Rapid Avr gene identification provides molecular tools to understand and track pathogen virulence evolution via genotype surveillance, which in turn will lead to optimized R gene stacking and deployment strategies. This platform should be broadly applicable to many crop pathogens and could potentially be adapted for screening genes involved in other protoplast-selectable traits.

Overexpression of the WAPO-A1 gene increases the number of spikelets per spike in bread wheat.

Two homoeologous QTLs for number of spikelets per spike (SPS) were mapped on chromosomes 7AL and 7BL using two wheat MAGIC populations. Sets of lines contrasting for the QTL on 7AL were developed which allowed for the validation and fine mapping of the 7AL QTL and for the identification of a previously described candidate gene, WHEAT ORTHOLOG OF APO1 (WAPO1). Using transgenic overexpression in both a low and a high SPS line, we provide a functional validation for the role of this gene in determining SPS also in hexaploid wheat. We show that the expression levels of this gene positively correlate with SPS in multiple MAGIC founder lines under field conditions as well as in transgenic lines grown in the greenhouse. This work highlights the potential use of WAPO1 in hexaploid wheat for further yield increases. The impact of WAPO1 and SPS on yield depends on other genetic and environmental factors, hence, will require a finely balanced expression level to avoid the development of detrimental pleiotropic phenotypes.

Nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes to enhance RNAi stability in plants.

Hairpin RNA (hpRNA) transgenes are the most successful RNA interference (RNAi) method in plants. Here, we show that hpRNA transgenes are invariably methylated in the inverted-repeat (IR) DNA and the adjacent promoter, causing transcriptional self-silencing. Nucleotide substitutions in the sense sequence, disrupting the IR structure, prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine with thymine nucleotides, in a G:U hpRNA design, prevents self-silencing but still allows for the formation of hpRNA due to G:U wobble base-pairing. The G:U design induces effective RNAi in 90-96% of transgenic lines, compared to 57-65% for the traditional hpRNA design. While a traditional hpRNA transgene shows increasing self-silencing from cotyledons to true leaves, its G:U counterpart avoids this and induce RNAi throughout plant growth. Furthermore, siRNAs from G:U and traditional hpRNA show different characteristics and appear to function via different pathways to induce target DNA methylation.

Loss of interleukin-10 receptor disrupts intestinal epithelial cell proliferation and skews differentiation towards the goblet cell fate.

Intestinal epithelial cells (IEC) are crucial for maintaining proper digestion and overall homeostasis of the gut mucosa. IEC proliferation and differentiation are tightly regulated by well described pathways, however, relatively little is known about how cytokines shape these processes. Given that the anti-inflammatory cytokine interleukin (IL)-10 promotes intestinal barrier function, and insufficient IL-10 signaling increases susceptibility to intestinal diseases like inflammatory bowel disease, we hypothesized that IL-10 signaling modulates processes underlying IEC proliferation and differentiation. This was tested using in vivo and in vitro IEC-specific IL-10 receptor 1 (IL-10R1) depletion under homeostatic conditions. Our findings revealed that loss of IL-10R1 drove lineage commitment toward a dominant goblet cell phenotype while decreasing absorptive cell-related features. Diminished IL-10 signaling also significantly elevated IEC proliferation with relatively minor changes to apoptosis. Characterization of signaling pathways upstream of proliferation demonstrated a significant reduction in the Wnt inhibitor, DKK1, increased nuclear localization of ß-catenin, and increased transcripts of the proliferation marker, OLFM4, with IL-10R1 depletion. Phosphorylated STAT3 was nearly completely absent in IL-10R1 knockdown cells and may provide a mechanistic link between our observations and the regulation of these cellular processes. Our results demonstrate a novel role for IL-10 signaling in intestinal mucosal homeostasis by regulating proper balance of proliferation and IEC lineage fate.

p38 MAPK signaling mediates retinoic acid-induced CD103 expression in human dendritic cells.

Retinoic acid (RA) is an active derivative of vitamin A and a key regulator of immune cell function. In dendritic cells (DCs), RA drives the expression of CD103 (integrin αE ), a functionally relevant DC subset marker. In this study, we analyzed the cell type specificity and the molecular mechanisms involved in RA-induced CD103 expression. We show that RA treatment caused a significant up-regulation of CD103 in differentiated monocyte-derived DCs and blood DCs, but not in differentiated monocyte-derived macrophages or T cells. DC treatment with an RA receptor α (RARα) agonist led to an increase in CD103 expression similar to that in RA treatment, whereas RARA gene silencing with small interfering RNA blocked RA-induced up-regulation of CD103, pointing to a major role of RARα in the regulation of CD103 expression. To elucidate RA-induced signaling downstream of RARα, we used Western blot analysis of RA-treated DCs and showed a significant increase of p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, DCs cultured with RA and a p38 MAPK inhibitor had a significantly reduced expression of CD103 compared with DCs cultured with RA only, indicating that p38 MAPK is involved in CD103 regulation. In summary, these findings suggest that the RA-induced expression of CD103 is specific to DCs, is mediated primarily through RARα and involves p38 MAPK signaling.

Manipulating Gibberellin Control Over Growth and Fertility as a Possible Target for Managing Wild Radish Weed Populations in Cropping Systems.

Wild radish is a major weed of Australian cereal crops. A rapid establishment, fast growth, and abundant seed production are fundamental to its success as an invasive species. Wild radish has developed resistance to a number of commonly used herbicides increasing the problem. New innovative approaches are needed to control wild radish populations. Here we explore the possibility of pursuing gibberellin (GA) biosynthesis as a novel molecular target for controlling wild radish, and in doing so contribute new insights into GA biology. By characterizing ga 3-oxidase (ga3ox) mutants in Arabidopsis, a close taxonomic relative to wild radish, we showed that even mild GA deficiencies cause considerable reductions in growth and fecundity. This includes an explicit requirement for GA biosynthesis in successful female fertility. Similar defects were reproducible in wild radish via chemical inhibition of GA biosynthesis, confirming GA action as a possible new target for controlling wild radish populations. Two possible targeting approaches are considered; the first would involve developing a species-specific inhibitor that selectively inhibits GA production in wild radish over cereal crops. The second, involves making crop species insensitive to GA repression, allowing the use of existing broad spectrum GA inhibitors to control wild radish populations. Toward the first concept, we cloned and characterized two wild radish GA3OX genes, identifying protein differences that appear sufficient for selective inhibition of dicot over monocot GA3OX activity. We developed a novel yeast-based approach to assay GA3OX activity as part of the molecular characterization, which could be useful for future screening of inhibitory compounds. For the second approach, we demonstrated that a subset of GA associated sln1/Rht-1 overgrowth mutants, recently generated in cereals, are insensitive to GA reductions brought on by the general GA biosynthesis inhibitor, paclobutrazol. The location of these mutations within sln1/Rht-1, offers additional insight into the functional domains of these important GA signaling proteins. Our early assessment suggests that targeting the GA pathway could be a viable inclusion into wild radish management programs that warrants further investigation. In drawing this conclusion, we provided new insights into GA regulated reproductive development and molecular characteristics of GA metabolic and signaling proteins.

Analysis of Neutrophil Transmigration Through Epithelial Cell Monolayers.

Transmigration of neutrophils through an epithelial layer, such as in the intestine or lung, is a necessary response to a perceived attack at the mucosal surface of that tissue. This process is dynamically regulated by a number of interactive events between the neutrophil and other cell types and allows for an effective and localized neutrophil response. However, in certain inflammatory diseases, including inflammatory bowel disease and chronic obstructive pulmonary disease (COPD), persistent neutrophil accumulation can contribute to disease pathology. Elucidating the mechanisms of this aberrant neutrophil accumulation is crucial for understanding and ameliorating these disease processes. The method we describe here is a controlled model system that allows for the investigation of the interactive signals involved in neutrophil transmigration through epithelial barriers, and possible mechanisms of deregulation of this process.

Slug and Snail have differential effects in directing colonic epithelial wound healing and partially mediate the restitutive effects of butyrate.

Restitution of wounds in colonic epithelium is essential in the maintenance of health. Microbial products, such as the short-chain fatty acid butyrate, can have positive effects on wound healing. We used an in vitro model of T84 colonic epithelial cells to determine if the Snail genes Slug (SNAI2) and Snail (SNAI1), implemented in keratinocyte monolayer healing, are involved in butyrate-enhanced colonic epithelial wound healing. Using shRNA-mediated Slug/Snail knockdown, we found that knockdown of Slug (Slug-KD), but not Snail (Snail-KD), impairs wound healing in scratch assays with and without butyrate. Slug and Snail had differential effects on T84 monolayer barrier integrity, measured by transepithelial resistance, as Snail-KD impaired the barrier (with or without butyrate), whereas Slug-KD enhanced the barrier, again with or without butyrate. Targeted transcriptional analysis demonstrated differential expression of several tight junction genes, as well as focal adhesion genes. This included altered regulation of Annexin A2 and ITGB1 in Slug-KD, which was reflected in confocal microscopy, showing increased accumulation of B1-integrin protein in Slug-KD cells, which was previously shown to impair wound healing. Transcriptional analysis also indicated altered expression of genes associated with epithelial terminal differentiation, such that Slug-KD cells skewed toward overexpression of secretory cell pathway-associated genes. This included trefoil factors TFF1 and TFF3, which were expressed at lower than control levels in Snail-KD cells. Since TFFs can enhance the barrier in epithelial cells, this points to a potential mechanism of differential modulation by Snail genes. Although Snail genes are crucial in epithelial wound restitution, butyrate responses are mediated by other pathways as well.NEW & NOTEWORTHY Although butyrate can promote colonic mucosal healing, not all of its downstream pathways are understood. We show that the Snail genes Snail and Slug are mediators of butyrate responses. Furthermore, these genes, and Slug in particular, are necessary for efficient restitution of wounds and barriers in T84 epithelial cells even in the absence of butyrate. These effects are achieved in part through effects on regulation of ß1 integrin and cellular differentiation state.

Zebularine treatment is associated with deletion of FT-B1 leading to an increase in spikelet number in bread wheat.

The number of rachis nodes (spikelets) on a wheat spike is a component of grain yield that correlates with flowering time. The genetic basis regulating flowering in cereals is well understood, but there are reports that flowering time can be modified at a high frequency by selective breeding, suggesting that it may be regulated by both epigenetic and genetic mechanisms. We investigated the role of DNA methylation in regulating spikelet number and flowering time by treating a semi-spring wheat with the demethylating agent, Zebularine. Three lines with a heritable increase in spikelet number were identified. The molecular basis for increased spikelet number was not determined in 2 lines, but the phenotype showed non-Mendelian inheritance, suggesting that it could have an epigenetic basis. In the remaining line, the increased spikelet phenotype behaved as a Mendelian recessive trait and late flowering was associated with a deletion encompassing the floral promoter, FT-B1. Deletion of FT-B1 delayed the transition to reproductive growth, extended the duration of spike development, and increased spikelet number under different temperature regimes and photoperiod. Transiently disrupting DNA methylation can generate novel flowering behaviour in wheat, but these changes may not be sufficiently stable for use in breeding programs.

TEOSINTE BRANCHED1 Regulates Inflorescence Architecture and Development in Bread Wheat (Triticum aestivum).

The flowers of major cereals are arranged on reproductive branches known as spikelets, which group together to form an inflorescence. Diversity for inflorescence architecture has been exploited during domestication to increase crop yields, and genetic variation for this trait has potential to further boost grain production. Multiple genes that regulate inflorescence architecture have been identified by studying alleles that modify gene activity or dosage; however, little is known in wheat. Here, we show TEOSINTE BRANCHED1 (TB1) regulates inflorescence architecture in bread wheat (Triticum aestivum) by investigating lines that display a form of inflorescence branching known as "paired spikelets." We show that TB1 interacts with FLOWERING LOCUS T1 and that increased dosage of TB1 alters inflorescence architecture and growth rate in a process that includes reduced expression of meristem identity genes, with allelic diversity for TB1 found to associate genetically with paired spikelet development in modern cultivars. We propose TB1 coordinates formation of axillary spikelets during the vegetative to floral transition and that alleles known to modify dosage or function of TB1 could help increase wheat yields.

CD103 (αE Integrin) Undergoes Endosomal Trafficking in Human Dendritic Cells, but Does Not Mediate Epithelial Adhesion.

Dendritic cell (DC) expression of CD103, the α subunit of αEß7 integrin, is thought to enable DC interactions with E-cadherin-expressing gastrointestinal epithelia for improved mucosal immunosurveillance. In the stomach, efficient DC surveillance of the epithelial barrier is crucial for the induction of immune responses to H. pylori, the causative agent of peptic ulcers and gastric cancer. However, gastric DCs express only low levels of surface CD103, as we previously showed. We here tested the hypothesis that intracellular pools of CD103 in human gastric DCs can be redistributed to the cell surface for engagement of epithelial cell-expressed E-cadherin to promote DC-epithelial cell adhesion. In support of our hypothesis, immunofluorescence analysis of tissue sections showed that CD103+ gastric DCs were preferentially localized within the gastric epithelial layer. Flow cytometry and imaging cytometry revealed that human gastric DCs expressed intracellular CD103, corroborating our previous findings in monocyte-derived DCs (MoDCs). Using confocal microscopy, we show that CD103 was present in endosomal compartments, where CD103 partially co-localized with clathrin, early endosome antigen-1 and Rab11, suggesting that CD103 undergoes endosomal trafficking similar to ß1 integrins. Dynamic expression of CD103 on human MoDCs was confirmed by internalization assay. To analyze whether DC-expressed CD103 promotes adhesion to E-cadherin, we performed adhesion and spreading assays on E-cadherin-coated glass slides. In MoDCs generated in the presence of retinoic acid, which express increased CD103, intracellular CD103 significantly redistributed toward the E-cadherin-coated glass surface. However, DCs spreading and adhesion did not differ between E-cadherin-coated slides and slides coated with serum alone. In adhesion assays using E-cadherin-positive HT-29 cells, DC binding was significantly improved by addition of Mn2+ and decreased in the presence of EGTA, consistent with the dependence of integrin-based interactions on divalent cations. However, retinoic acid failed to increase DC adhesion, and a CD103 neutralizing antibody was unable to inhibit DC binding to the E-cadherin positive cells. In contrast, a blocking antibody to DC-expressed E-cadherin significantly reduced DC binding to the epithelium. Overall, these data indicate that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin, but is not a major driver of DC adhesion to gastrointestinal epithelia.

New alleles of the wheat domestication gene Q reveal multiple roles in growth and reproductive development.

The advantages of free threshing in wheat led to the selection of the domesticated Q allele, which is now present in almost all modern wheat varieties. Q and the pre-domestication allele, q, encode an AP2 transcription factor, with the domesticated allele conferring a free-threshing character and a subcompact (i.e. partially compact) inflorescence (spike). We demonstrate that mutations in the miR172 binding site of the Q gene are sufficient to increase transcript levels via a reduction in miRNA-dependent degradation, consistent with the conclusion that a single nucleotide polymorphism in the miRNA binding site of Q relative to q was essential in defining the modern Q allele. We describe novel gain- and loss-of-function alleles of Q and use these to define new roles for this gene in spike development. Q is required for the suppression of 'sham ramification', and increased Q expression can lead to the formation of ectopic florets and spikelets (specialized inflorescence branches that bear florets and grains), resulting in a deviation from the canonical spike and spikelet structures of domesticated wheat.

Differential regulation of CD103 (αE integrin) expression in human dendritic cells by retinoic acid and Toll-like receptor ligands.

CD103 (αE integrin) is an important dendritic cell (DC) marker that characterizes functionally distinct DC subsets in mice and humans. However, the mechanism by which CD103 expression is regulated in human DCs and the role of CD103 for DC function are not very well understood. Here, we show that retinoic acid (RA) treatment of human monocyte-derived DCs (MoDCs) increased the ability of the DCs to synthesize RA and induced MoDC expression of CD103 and ß7 at the mRNA and protein level. In contrast, RA was unable to induce the expression of CD103 in primary human DCs isolated from the gastric mucosa. Inhibition of TGF-ß signaling in MoDCs down-regulated RA-induced CD103 expression, indicating that TGF-ß-dependent pathways contribute to the induction of CD103. Conversely, when RA-treated MoDCs were stimulated with live Helicobacter pylori, commensal bacteria, LPS, or a TLR2 agonist, the RA-induced up-regulation of CD103 and ß7 integrin expression was completely abrogated. To determine whether CD103 expression impacts DC priming of CD4+ T cells, we next investigated the ability of CD103+ and CD103─ DCs to induce mucosal homing and T cell proliferation. Surprisingly, RA treatment of DCs enhanced both α4ß7 expression and proliferation in cocultured T cells, but no difference was seen between RA-treated CD103+ and CD103─ DCs. In summary, our data demonstrate that RA, bacterial products, and the tissue environment all contribute to the regulation of CD103 on human DCs and that DC induction of mucosal homing in T cells is RA dependent but not CD103 dependent.

Vascular Dysfunction in Pneumocystis-Associated Pulmonary Hypertension Is Related to Endothelin Response and Adrenomedullin Concentration.

Pulmonary hypertension subsequent to an infectious disease can be due to vascular structural remodeling or to functional alterations within various vascular cell types. In our previous mouse model of Pneumocystis-associated pulmonary hypertension, we found that vascular remodeling was not responsible for observed increases in right ventricular pressures. Here, we report that the vascular dysfunction we observed could be explained by an enhanced response to endothelin-1 (20% greater reduction in lumen diameter, P ≤ 0.05), corresponding to an up-regulation of similar magnitude (P ≤ 0.05) of the endothelin A receptor in the lung tissue. This effect was potentially augmented by a decrease in production of the pulmonary vasodilator adrenomedullin of almost 70% (P ≤ 0.05). These changes did not occur in interferon-γ knockout mice similarly treated, which do not develop pulmonary hypertension under these circumstances. Surprisingly, we did not observe any relevant changes in the vascular endothelial nitric oxide synthase vasodilatory response, which is a common potential site of inflammatory alterations to pulmonary vascular function. Our results indicate the diverse mechanisms by which inflammatory responses to prior infections can cause functionally relevant changes in vascular responses in the lung, promoting the development of pulmonary hypertension.

A genetic strategy generating wheat with very high amylose content.

Resistant starch (RS), a type of dietary fibre, plays an important role in human health; however, the content of RS in most modern processed starchy foods is low. Cereal starch, when structurally manipulated through a modified starch biosynthetic pathway to greatly increase the amylose content, could be an important food source of RS. Transgenic studies have previously revealed the requirement of simultaneous down-regulation of two starch branching enzyme (SBE) II isoforms both located on the long arm of chromosome 2, namely SBEIIa and SBEIIb, to elevate the amylose content in wheat from ~25% to ~75%. The current study revealed close proximity of genes encoding SBEIIa and SBEIIb isoforms in wheat with a genetic distance of 0.5 cM on chromosome 2B. A series of deletion and single nucleotide polymorphism (SNP) loss of function alleles in SBEIIa, SBEIIb or both was isolated from two different wheat populations. A breeding strategy to combine deletions and SNPs generated wheat genotypes with altered expression levels of SBEIIa and SBEIIb, elevating the amylose content to an unprecedented ~85%, with a marked concomitant increase in RS content. Biochemical assays were used to confirm the complete absence in the grain of expression of SBEIIa from all three genomes in combination with the absence of SBEIIb from one of the genomes.

Ppd-1 is a key regulator of inflorescence architecture and paired spikelet development in wheat.

The domestication of cereal crops such as wheat, maize, rice and barley has included the modification of inflorescence architecture to improve grain yield and ease harvesting(1). Yield increases have often been achieved through modifying the number and arrangement of spikelets, which are specialized reproductive branches that form part of the inflorescence. Multiple genes that control spikelet development have been identified in maize, rice and barley(2-5). However, little is known about the genetic underpinnings of this process in wheat. Here, we describe a modified spikelet arrangement in wheat, termed paired spikelets. Combining comprehensive QTL and mutant analyses, we show that Photoperiod-1 (Ppd-1), a pseudo-response regulator gene that controls photoperiod-dependent floral induction, has a major inhibitory effect on paired spikelet formation by regulating the expression of FLOWERING LOCUS T (FT)(6,7). These findings show that modulated expression of the two important flowering genes, Ppd-1 and FT, can be used to form a wheat inflorescence with a more elaborate arrangement and increased number of grain producing spikelets.

EARLY FLOWERING3 Regulates Flowering in Spring Barley by Mediating Gibberellin Production and FLOWERING LOCUS T Expression.

EARLY FLOWERING3 (ELF3) is a circadian clock gene that contributes to photoperiod-dependent flowering in plants, with loss-of-function mutants in barley (Hordeum vulgare), legumes, and Arabidopsis thaliana flowering early under noninductive short-day (SD) photoperiods. The barley elf3 mutant displays increased expression of FLOWERING LOCUS T1 (FT1); however, it remains unclear whether this is the only factor responsible for the early flowering phenotype. We show that the early flowering and vegetative growth phenotypes of the barley elf3 mutant are strongly dependent on gibberellin (GA) biosynthesis. Expression of the central GA biosynthesis gene, GA20oxidase2, and production of the bioactive GA, GA1, were significantly increased in elf3 leaves under SDs, relative to the wild type. Inhibition of GA biosynthesis suppressed the early flowering of elf3 under SDs independently of FT1 and was associated with altered expression of floral identity genes at the developing apex. GA is also required for normal flowering of spring barley under inductive photoperiods, with chemical and genetic attenuation of the GA biosynthesis and signaling pathways suppressing inflorescence development under long-day conditions. These findings illustrate that GA is an important floral promoting signal in barley and that ELF3 suppresses flowering under noninductive photoperiods by blocking GA production and FT1 expression.

CD4+ T cells and IFN-γ are required for the development of Pneumocystis-associated pulmonary hypertension.

Pulmonary hypertension (PH) is a disease of diverse etiology. Although primary PH can develop in the absence of prior disease, PH more commonly develops in conjunction with other pulmonary pathologies. We previously reported a mouse model in which PH occurs as a sequela of Pneumocystis infection in the context of transient CD4 depletion. Here, we report that instead of the expected Th2 pathways, the Th1 cytokine IFN-γ is essential for the development of PH, as wild-type mice developed PH but IFN-γ knockout mice did not. Because gene expression analysis showed few strain differences that were not immune-function related, we focused on those responses as potential pathologic mechanisms. In addition to dependence on IFN-γ, we found that when CD4 cells were continuously depleted, but infection was limited by antibiotic treatment, PH did not occur, confirming that CD4 T cells are required for PH development. Also, although CD8 T-cells are implicated in the pathology of Pneumocystis pneumonia, they did not have a role in the onset of PH. Finally, we found differences in immune cell phenotypes that correlated with PH, including elevated CD204 expression in lung CD11c(+) cells, but their role remains unclear. Overall, we demonstrate that a transient, localized, immune response requiring IFN-γ and CD4-T cells can disrupt pulmonary vascular function and promote lingering PH.

Inhibition of tiller bud outgrowth in the tin mutant of wheat is associated with precocious internode development.

Tillering (branching) is a major yield component and, therefore, a target for improving the yield of crops. However, tillering is regulated by complex interactions of endogenous and environmental signals, and the knowledge required to achieve optimal tiller number through genetic and agronomic means is still lacking. Regulatory mechanisms may be revealed through physiological and molecular characterization of naturally occurring and induced tillering mutants in the major crops. Here we characterize a reduced tillering (tin, for tiller inhibition) mutant of wheat (Triticum aestivum). The reduced tillering in tin is due to early cessation of tiller bud outgrowth during the transition of the shoot apex from the vegetative to the reproductive stage. There was no observed difference in the development of the main stem shoot apex between tin and the wild type. However, tin initiated internode development earlier and, unlike the wild type, the basal internodes in tin were solid rather than hollow. We hypothesize that tin represents a novel type of reduced tillering mutant associated with precocious internode elongation that diverts sucrose (Suc) away from developing tillers. Consistent with this hypothesis, we have observed upregulation of a gene induced by Suc starvation, downregulation of a Suc-inducible gene, and a reduced Suc content in dormant tin buds. The increased expression of the wheat Dormancy-associated (DRM1-like) and Teosinte Branched1 (TB1-like) genes and the reduced expression of cell cycle genes also indicate bud dormancy in tin. These results highlight the significance of Suc in shoot branching and the possibility of optimizing tillering by manipulating the timing of internode elongation.

Pneumocystis elicits a STAT6-dependent, strain-specific innate immune response and airway hyperresponsiveness.

It is widely held that exposure to pathogens such as fungi can be an agent of comorbidity, such as exacerbation of asthma or chronic obstructive pulmonary disease. Although many studies have examined allergic responses to fungi and their effects on pulmonary function, the possible pathologic implications of the early innate responses to fungal pathogens have not been explored. We examined early responses to the atypical fungus Pneumocystis in two common strains of mice in terms of overall immunological response and related pathology, such as cell damage and airway hyperresponsiveness (AHR). We found a strong strain-specific response in BALB/c mice that included recruitment of neutrophils, NK, NKT, and CD4 T cells. This response was accompanied by elevated indicators of lung damage (bronchoalveolar lavage fluid albumin and LDH) and profound AHR. This early response was absent in C57BL/6 mice, although both strains exhibited a later response associated with the clearance of Pneumocystis. We found that this AHR could not be attributed exclusively to the presence of recruited neutrophils, NKT, NK, or CD4 cells or to the actions of IFN-γ or IL-4. However, in the absence of STAT6 signaling, AHR and inflammatory cell recruitment were virtually absent. Gene expression analysis indicated that this early response included activation of several transcription factors that could be involved in pulmonary remodeling. These results show that exposure to a fungus such as Pneumocystis can elicit pulmonary responses that may contribute to morbidity, even without prior sensitization, in the context of certain genetic backgrounds.