Multiple functional self-association interfaces in plant TIR domains.

The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.

TaDrAp1 and TaDrAp2, Partner Genes of a Transcription Repressor, Coordinate Plant Development and Drought Tolerance in Spelt and Bread Wheat.

Down-regulator associated protein, DrAp1, acts as a negative cofactor (NC2α) in a transcription repressor complex together with another subunit, down-regulator Dr1 (NC2ß). In binding to promotors and regulating the initiation of transcription of various genes, DrAp1 plays a key role in plant transition to flowering and ultimately in seed production. TaDrAp1 and TaDrAp2 genes were identified, and their expression and genetic polymorphism were studied using bioinformatics, qPCR analyses, a 40K Single nucleotide polymorphism (SNP) microarray, and Amplifluor-like SNP genotyping in cultivars of bread wheat (Triticum aestivum L.) and breeding lines developed from a cross between spelt (T. spelta L.) and bread wheat. TaDrAp1 was highly expressed under non-stressed conditions, and at flowering, TaDrAp1 expression was negatively correlated with yield capacity. TaDrAp2 showed a consistently low level of mRNA production. Drought caused changes in the expression of both TaDrAp1 and TaDrAp2 genes in opposite directions, effectively increasing expression in lower yielding cultivars. The microarray 40K SNP assay and Amplifluor-like SNP marker, revealed clear scores and allele discriminations for TaDrAp1 and TaDrAp2 and TaRht-B1 genes. Alleles of two particular homeologs, TaDrAp1-B4 and TaDrAp2-B1, co-segregated with grain yield in nine selected breeding lines. This indicated an important regulatory role for both TaDrAp1 and TaDrAp2 genes in plant growth, ontogenesis, and drought tolerance in bread and spelt wheat.

Comparative Analysis of the Flax Immune Receptors L6 and L7 Suggests an Equilibrium-Based Switch Activation Model.

NOD-like receptors (NLRs) are central components of the plant immune system. L6 is a Toll/interleukin-1 receptor (TIR) domain-containing NLR from flax (Linum usitatissimum) conferring immunity to the flax rust fungus. Comparison of L6 to the weaker allele L7 identified two polymorphic regions in the TIR and the nucleotide binding (NB) domains that regulate both effector ligand-dependent and -independent cell death signaling as well as nucleotide binding to the receptor. This suggests that a negative functional interaction between the TIR and NB domains holds L7 in an inactive/ADP-bound state more tightly than L6, hence decreasing its capacity to adopt the active/ATP-bound state and explaining its weaker activity in planta. L6 and L7 variants with a more stable ADP-bound state failed to bind to AvrL567 in yeast two-hybrid assays, while binding was detected to the signaling active variants. This contrasts with current models predicting that effectors bind to inactive receptors to trigger activation. Based on the correlation between nucleotide binding, effector interaction, and immune signaling properties of L6/L7 variants, we propose that NLRs exist in an equilibrium between ON and OFF states and that effector binding to the ON state stabilizes this conformation, thereby shifting the equilibrium toward the active form of the receptor to trigger defense signaling.

Animal NLRs continue to inform plant NLR structure and function.

Plant NLRs share many of the structural hallmarks of their animal counterparts. At a functional level, the central nucleotide-binding pocket appears to have binding and hydrolysis activities, similar to that of animal NLRs. The TIR domains of plant NLRs have been shown to self-associate, and there is emerging evidence that full-length plant NLRs may do so as well. It is therefore tempting to speculate that plant NLRs may form higher-order complexes similar to those of the mammalian inflammasome. Here we review the available knowledge on structure-function relationships in plant NLRs, focusing on how the information available on animal NLRs informs the mechanism of plant NLR function, and highlight the evidence that innate immunity signalling pathways in multicellular organisms often require the formation of higher-order protein complexes.

The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins.

Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world's most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.

Measuring extinction with digital holography: nonspherical particles and experimental validation.

Through simulations and experiment, this Letter shows how a particle's extinction cross section can be extracted from a digital hologram. Spherical and nonspherical particles are considered covering a range of cross-sectional values of nearly five orders of magnitude. The extracted cross sections are typically less than 10% in error from the true values. It is also shown that holograms encompassing a sufficiently large angular range of scattered light yield an estimate for the absorption cross section.

Animal NLRs provide structural insights into plant NLR function.

BACKGROUND: The plant immune system employs intracellular NLRs (nucleotide binding [NB], leucine-rich repeat [LRR]/nucleotide-binding oligomerization domain [NOD]-like receptors) to detect effector proteins secreted into the plant cell by potential pathogens. Activated plant NLRs trigger a range of immune responses, collectively known as the hypersensitive response (HR), which culminates in death of the infected cell. Plant NLRs show structural and functional resemblance to animal NLRs involved in inflammatory and innate immune responses. Therefore, knowledge of the activation and regulation of animal NLRs can help us understand the mechanism of action of plant NLRs, and vice versa. SCOPE: This review provides an overview of the innate immune pathways in plants and animals, focusing on the available structural and biochemical information available for both plant and animal NLRs. We highlight the gap in knowledge between the animal and plant systems, in particular the lack of structural information for plant NLRs, with crystal structures only available for the N-terminal domains of plant NLRs and an integrated decoy domain, in contrast to the more complete structures available for animal NLRs. We assess the similarities and differences between plant and animal NLRs, and use the structural information on the animal NLR pair NAIP/NLRC4 to derive a plausible model for plant NLR activation. CONCLUSIONS: Signalling by cooperative assembly formation (SCAF) appears to operate in most innate immunity pathways, including plant and animal NLRs. Our proposed model of plant NLR activation includes three key steps: (1) initially, the NLR exists in an inactive auto-inhibited state; (2) a combination of binding by activating elicitor and ATP leads to a structural rearrangement of the NLR; and (3) signalling occurs through cooperative assembly of the resistosome. Further studies, structural and biochemical in particular, will be required to provide additional evidence for the different features of this model and shed light on the many existing variations, e.g. helper NLRs and NLRs containing integrated decoys.

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity.

Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.

Does intracytoplasmic sperm injection pose an increased risk of genitourinary congenital malformations in offspring compared to in vitro fertilization? A systematic review and meta-analysis.

PURPOSE: We systematically reviewed and performed a meta-analysis of the genitourinary congenital malformation rate after conception by intracytoplasmic sperm injection compared to in vitro fertilization. MATERIALS AND METHODS: We systematically reviewed studies to compare perinatal outcomes between children conceived by intracytoplasmic sperm injection vs in vitro fertilization. Studies showing genitourinary congenital malformation rates were included. We assessed the risk of bias, focusing on the quality of genitourinary congenital malformation reporting and analysis targeted at singletons. Meta-analysis was done using a random effects model for 3 outcomes, including overall genitourinary congenital malformation, hypospadias and cryptorchidism. Sensitivity analysis was also performed in only studies at low risk for bias. RESULTS: The initial search yielded 1,482 articles. We performed a full text review of 111 of these studies, of which 22 met inclusion criteria for systematic review. Meta-analysis of intracytoplasmic sperm injection and in vitro fertilization in 12,270 and 24,240 cases, respectively, revealed that intracytoplasmic sperm injection was associated with a significantly higher rate of overall genitourinary congenital malformation compared to in vitro fertilization (OR 1.27, 95% CI 1.02-1.59, p = 0.04). However, when including only 4 studies at low risk for bias with a total of 7,727 and 14,308 intracytoplasmic sperm injection and in vitro fertilization cases, respectively, the difference was not significant (OR 1.28, 95% CI 1.00-1.64, p = 0.05). There was no statistically significant difference in the rate of hypospadias (OR 1.21, 95% CI 0.87-1.69) or cryptorchidism (OR 1.39, 95% CI 0.97-2.00) between males conceived by intracytoplasmic sperm injection vs in vitro fertilization. On all analyses there was no significant statistical heterogeneity between studies (I(2) = 0). CONCLUSIONS: Intracytoplasmic sperm injection is associated with a slightly higher risk of genitourinary malformation in offspring than in vitro fertilization. However, when only higher quality studies were analyzed, the difference was not significant. The hypospadias and cryptorchidism rates in offspring are similar for the 2 conception methods.

Using holography to measure extinction.

This work presents a new concept to measure the extinction cross section for a single particle in situ. The concept involves recording the hologram produced by the interference of a particle's forward-scattered light with the incident light. This interference pattern is fundamentally connected to the energy flow that gives rise to extinction, and, by integrating this measured pattern, one obtains an approximation for the cross section. Mie theory is used to show that this approximation can be as little as 1% in error of the true value for many cases of practical interest. Moreover, since an image of the particle can be computationally reconstructed from a measured hologram using the Fresnel-Kirchhoff diffraction theory, one can obtain the cross section simultaneously with the particle shape and size.

Necrophytoremediation of phenanthrene and pyrene in contaminated soil.

In this study, the effect of necrophytoremediation, using pea and wheat straws on the remediation soil contaminated with two common polycyclic aromatic hydrocarbons (PAHs), phenanthrene and pyrene alone or in combination was investigated. In addition, monitoring of the population of PAH-utilising microorganisms together with PCR-Denaturing Gradient Gel Electrophoresis (DGGE)-sequencing methods were used to further elucidate the effect of straw addition on the bacterial, fungal and nidA gene (a functional gene involved in the degradation of PAHs) communities. The addition of pea straw had a positive effect on the degradation of PAHs, especially for pyrene. For example, the addition of pea straw to pyrene-contaminated soil resulted in an increase in the degradation of pyrene from 15% (66 mg kg(-1)) in the corresponding control to 70% (301 mg kg(-1)). The results from the most probable number (MPN) of PAH-utilising microorganisms and PCR-DGGE-sequencing methods indicated that the addition of straw led to an increase in microbial hydrocarbonoclastic biomass rather than changes in microbial diversity. For example, in pyrene-contaminated soil, the number of PAH-utilising microorganisms in the soil amended with pea straw reached 5.6 log10 CFU g(-1) dry soil, ~13-fold increase when compared with the numbers present in the control soil (4.5 log10 CFU g(-1) dry soils); however, the Shannon diversity index did not increase significantly. The sequencing of bands of interest from DGGE profiles revealed the presence of Actinobacteria, Firmicutes and Proteobacteria in the bacterial community. For fungi, sequenced bands belonged to Ascomycota, Basidiomycota and Mucoromycotina. In summary, this study has shown that necrophytoremediation using pea straw represents a promising biostimulation and cost effective agent which can be used for the bioremediation of PAH-contaminated soils.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties.

Human aquaporin 1 (hAQP1) forms homotetrameric channels that facilitate fluxes of water and small solutes across cell membranes. In addition to water channel activity, hAQP1 displays non-selective monovalent cation-channel activity gated by intracellular cyclic GMP. Dual water and ion-channel activity of hAQP1, thought to regulate cell shape and volume, could offer a target for novel therapeutics relevant to controlling cancer cell invasiveness. This study probed properties of hAQP1 ion channels using proteoliposomes, which, unlike conventional cell-based systems such as Xenopus laevis oocytes, are relatively free of background ion channels. Histidine-tagged recombinant hAQP1 protein was synthesized and purified from the methylotrophic yeast, Pichia pastoris, and reconstituted into proteoliposomes for biophysical analyses. Osmotic water channel activity confirmed correct folding and channel assembly. Ion-channel activity of hAQP1-Myc-His6 was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His6 channels displayed coordinated gating, a single-channel conductance of approximately 75 pS, and multiple subconductance states. Applicability of this method for structure-function analyses was tested using hAQP1-Myc-His6 D48A/D185A channels modified by site-directed mutations of charged Asp residues estimated to be adjacent to the central ion-conducting pore of the tetramer. No differences in conductance were detected between mutant and wild-type constructs, suggesting the open-state conformation could differ substantially from expectations based on crystal structures. Nonetheless, the method pioneered here for AQP1 demonstrates feasibility for future work defining structure-function relationships, screening pharmacological inhibitors, and testing other classes in the broad family of aquaporins for previously undiscovered ion-conducting capabilities.

Shared decision-making for pediatric elective penile surgery.

INTRODUCTION: In pediatric surgery, proxy decision-makers are frequently involved in treatment planning and may experience decisional conflict (DC). Shared decision-making (SDM) approaches may be effective to remedy DC. This study investigates DC and SDM involvement in elective pediatric penile surgery. METHODS: Forty-four parents of children aged <8 years undergoing elective penile surgery consultations at a tertiary pediatric hospital were prospectively enrolled. Patient and physician questionnaires were used to assess the SDM process and the SURE (Sure of myself; Understand information; Risk-benefit ratio; Encouragement) screening test was used to assess DC. RESULTS: Thirty-seven (84.1%) mothers and seven (15.9%) fathers were enrolled for circumcision (n=33, 75.0%) and distal hypospadias repair (n=11, 25.0%) consultations, with 21 (47.7%) choosing to proceed with surgery. Seven (15.9%) participants experienced clinically significant DC. Participant gender was not associated with higher levels of DC (p=0.318). The average patient and physician SDM scores were 88.2±10.0 and 85.3±7.4, respectively, with no correlation found between participant and physician perception of SDM involvement (p=0.168, p=0.276). DC was significantly associated with lower participant and physician ratings of SDM. CONCLUSIONS: There was a high perception of SDM involvement by both parents and pediatric urologists regarding elective penile surgery. Of the 15% of parents experiencing DC, there was an association with lower participant and physician levels of SDM involvement. Despite high SDM scores overall, discrepancies exist between the perceived physician and participant SDM involvement.

An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP.

Resistance (R) proteins are key regulators of the plant innate immune system and are capable of pathogen detection and activation of the hypersensitive cell death immune response. To understand the molecular mechanism of R protein activation, we undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain of M were shown to affect R protein function. When purified using a yeast expression system and assayed for ATP and ADP, these mutated proteins exhibited marked differences in the quantity and identity of the bound nucleotide. ADP was bound to recombinant wild-type M protein, while the nonfunctional P-loop mutant did not have any nucleotides bound. In contrast, ATP was bound to an autoactive M protein mutated in the highly conserved MHD motif. These data provide direct evidence supporting a model of R protein function in which the "off" R protein binds ADP and activation of R protein defense signaling involves the exchange of ADP for ATP.

Engineering Saccharomyces cerevisiae to release 3-Mercaptohexan-1-ol during fermentation through overexpression of an S. cerevisiae Gene, STR3, for improvement of wine aroma.

Sulfur-containing aroma compounds are key contributors to the flavor of a diverse range of foods and beverages. The tropical fruit characters of Vitis vinifera L. cv. Sauvignon blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexan-1-ol-acetate, and 4-mercapto-4-methylpentan-2-one (4MMP). These volatile thiols are found in small amounts in grape juice and are formed from nonvolatile cysteinylated precursors during fermentation. In this study, we overexpressed a Saccharomyces cerevisiae gene, STR3, which led to an increase in 3MH release during fermentation of a V. vinifera L. cv. Sauvignon blanc juice. Characterization of the enzymatic properties of Str3p confirmed it to be a pyridoxal-5'-phosphate-dependent cystathionine ß-lyase, and we demonstrated that this enzyme was able to cleave the cysteinylated precursors of 3MH and 4MMP to release the free thiols. These data provide direct evidence for a yeast enzyme able to release aromatic thiols in vitro that can be applied in the development of self-cloned yeast to enhance wine flavor.

Transition of care to an adult spina bifida clinic: patient perspectives and medical outcomes.

PURPOSE: Transitioning care of patients with spina bifida to adult centers poses a challenge. We sought patient and parent perspectives on the transition process at our center and correlated these perspectives with medical outcomes. MATERIALS AND METHODS: Patients who attended the adult spina bifida clinic at our institution were invited to complete SF-36®, the Ambulatory Care Experience Survey and a Transition of Care Survey. Urological and neurosurgical medical outcomes were correlated with health status, clinic experience and perspectives on the transition process. Statistical analysis was done using SPSS®, version 16.0. RESULTS: Of 105 patients with spina bifida 24 participated in the study. SF-36 results showed that the physical health domain correlated with lack of employment (p = 0.006). Patients whose parents completed the surveys on their behalf were more likely to have physical limitations than the patients who completed surveys (p = 0.011). Urological and neurosurgical outcomes did not significantly affect SF-36, Ambulatory Care Experience Survey or Transition of Care Survey scores in this patient population. Patients and caregivers identified similar key elements and barriers in the transition process. Satisfaction with care at the pediatric center was higher than at the adult center. CONCLUSIONS: Transitioning care of patients with spina bifida from pediatric to adult care poses challenges for the health care system. Medical outcomes do not impact the patient perspective of the transition process. To optimize the transition of care we must address the barriers identified by patients and their caregivers.

Crystallization and X-ray diffraction analysis of the C-terminal domain of the flax rust effector protein AvrM.

The flax rust effector AvrM is a secreted protein of unknown fold that is recognized by the M resistance protein in flax. In order to investigate the structural basis of the AvrM-M interaction and possible virulence-associated functions of AvrM, the C-terminal domains of two different AvrM variants (AvrM-A and avrM) were crystallized. Crystals of native AvrM-A were obtained using pentaerythritol ethoxylate (15/4 EO/OH) as a precipitant and diffracted X-rays to 2.9 Å resolution. Selenomethionine-derivative crystals of similar quality were obtained using PEG 1500 as a precipitant. Both the native and selenomethionine-labelled AvrM-A crystals had symmetry of space group C222(1) with eight molecules in the asymmetric unit. Crystals of avrM had symmetry of space group P2(1)2(1)2(1) and diffracted X-rays to 2.7 Å resolution. Initial AvrM-A phases were calculated using the single-wavelength anomalous dispersion (SAD) method and a partial model was built. Phases for avrM were obtained by molecular replacement using the partial AvrM-A model.

Modified "Allele-Specific qPCR" Method for SNP Genotyping Based on FRET.

The proposed method is a modified and improved version of the existing "Allele-specific q-PCR" (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.