A high-throughput system for two-hybrid screening based on growth curve analysis in microtiter plates

The yeast two-hybrid system is a powerful tool for identifying novel protein-protein interactions. In general, biochemical marker genes such as lacZ are exploited for indirect quantification of the interaction, and commonly involve the conduct of rather laborious β-galactosidase assays. This paper describes a simple alternative method based on growth curve analysis of yeast cultures that is amenable to microtiter plate format, and therefore allows the quantification of large numbers of yeast two-hybrid combinations. The analyzed results of yeast cultures grown in microtiter plates were compared with those obtained from the classical β-galactosidase assay. We conclude that the method presented here is reproducible, of equal or greater sensitivity than the β-galactosidase assay, and can be further adapted for application to the conduct of large-scale, automated yeast two-hybrid experiments

Formation of an SCFZTL complex is required for proper regulation of circadian timing

The circadian timing system involves an autoregulatory transcription/translation feedback loop that incorporates a diverse array of factors to maintain a 24-h periodicity. In Arabidopsis a novel F-box protein, ZEITLUPE (ZTL), plays an important role in the control of the free-running period of the circadian clock. As a class, F-box proteins are well-established components of the Skp/Cullin/F-box (SCF) class of E3 ubiquitin ligases that link the target substrates to the core ubiquitinating activity of the ligase complex via direct association with the Skp protein. Here we identify and characterize the SCFZTL complex in detail. Yeast two-hybrid tests demonstrate the sufficiency and necessity of the F-box domain for Arabidopsis Skp-like protein (ASK) interactions and the dispensability of the unique N-terminal LOV domain in this association. Co-immunoprecipitation of full-length (FL) ZTL with the three known core components of SCF complexes (ASK1, AtCUL1 and AtRBX1) demonstrates that ZTL can assemble into an SCF complex in vivo. F-box-containing truncated versions of ZTL (LOV-F and F-kelch) can complex with SCF components in vivo, whereas stably expressed LOV or kelch domains alone cannot. Stable expression of F-box-mutated FL ZTL eliminates the shortened period caused by mild ZTL overexpression and also abolishes ASK1 interaction in vivo. Reduced levels of the core SCF component AtRBX1 phenocopy the long period phenotype of ztl loss-of-function mutations, demonstrating the functional significance of the SCFZTL complex. Taken together, our data establish SCFZTL as an essential SCF class E3 ligase controlling circadian period in plants

Characterization of <i>Triticum aestivum</i> Abscisic Acid Receptors and a Possible Role for These in Mediating Fusairum Head Blight Susceptibility in Wheat

<div><p>Abscisic acid (ABA) is a well-characterized plant hormone, known to mediate developmental aspects as well as both abiotic and biotic stress responses. Notably, the exogenous application of ABA has recently been shown to increase susceptibility to the fungal pathogen <i>Fusarium graminearum</i>, the causative agent of <i>Fusarium</i> head blight (FHB) in wheat and other cereals. However roles and mechanisms associated with ABA’s modulation of pathogen responses remain enigmatic. Here the identification of putative ABA receptors from available genomic databases for <i>Triticum aestivum</i> (bread wheat) and <i>Brachypodium distachyon</i> (a model cereal) are reported. A number of these were cloned for recombinant expression and their functionality as ABA receptors confirmed by <i>in vitro</i> assays against protein phosphatases Type 2Cs. Ligand selectivity profiling of one of the wheat receptors (Ta_PYL2DS_FL) highlighted unique activities compared to <i>Arabidopsis</i> AtPYL5. Mutagenic analysis showed Ta_PYL2DS_FL amino acid D180 as being a critical contributor to this selectivity. Subsequently, a virus induced gene silencing (VIGS) approach was used to knockdown wheat Ta_PYL4AS_A (and similar) <i>in planta</i>, yielding plants with increased early stage resistance to FHB progression and decreased mycotoxin accumulation. Together these results confirm the existence of a family of ABA receptors in wheat and <i>Brachypodium</i> and present insight into factors modulating receptor function at the molecular level. That knockdown of Ta_PYL4AS_A (and similar) leads to early stage FHB resistance highlights novel targets for investigation in the future development of disease resistant crops.</p></div

Effect of the VIGS knockdown of Wheat Ta_PYL4AS_A on <i>F</i>. <i>graminearum</i> infection of <i>T</i>. <i>aestivum</i> cv. ‘Fielder’.

<p>A) Relative expression of Ta_PYL4AS_A in various VIGS treated wheat plants. Quantification of the gene expression of Ta_PYL4AS_A was performed 12 days post BSMV:Ta_PYL4AS_A application (+/- <i>F</i>. <i>graminearum</i> (Fg) application 7 days after the BMSV rub), compared to controls treated with BMSV:GFP, with Ta_actin used as an endogenous control. Uninfected plants, black bars; Fg infected plants, white bars. The expression level of the BMSV:GFP control was set to a value of 1 (n = 3). BSMV treated plants were treated with <i>F</i>. <i>graminearum</i> spores 7 days after the BMSV rub and assessed for <i>F</i>. <i>graminearum</i> infection in the B) spikelets and C) rachis at days 3, 5, 7 9 and 13 post—<i>F</i>. <i>graminearum</i> infection in BSMV:GFP (black bars) and BSMV:Ta_PYL4AS_A (white bars). Data were tested using 2-way ANOVA, and for each day separately using a t-test. (n = 9). D) Accumulation of DON in BSMV:Ta_PYL4AS_A (black bars) and BSMV:GFP (white bars) treated wheat heads. Samples were analyzed by LC/MS at 5 and 13 days post <i>F</i>. <i>graminearum</i> infection (n = 3). For all panels, error bars indicate standard error of the mean. Significant differences between samples are indicated with asterisks, (* p < 0.05, ** p < 0.01, *** p < 0.001).</p

Evolutionary relationships of wheat, rice, <i>B</i>. <i>distachion</i> and <i>A</i>. <i>thaliana</i> ABA receptors.

<p>The evolutionary history was inferred using the Neighbor-Joining method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164996#pone.0164996.ref053" target="_blank">53</a>]. The optimal tree with the sum of branch length = 4.66070389 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164996#pone.0164996.ref054" target="_blank">54</a>]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164996#pone.0164996.ref055" target="_blank">55</a>] and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated yielding a total of 79 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164996#pone.0164996.ref041" target="_blank">41</a>]. Gene locator ID’s are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164996#pone.0164996.t001" target="_blank">Table 1</a>, and for barley and rice [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164996#pone.0164996.ref021" target="_blank">21</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164996#pone.0164996.ref022" target="_blank">22</a>].</p

Amino acid reside 180 is involved in mediating the weaker effect of analog 352 against Ta_PYL2DS_FL compared to AtPYL5.

<p>A) Homology model of Ta_PYL2DS_FL (blue) was generated using the translated Genebank AK335719 sequence and a template structure of AtPYL1 (PDB ID 3NMN chain A) co-crystallized in complex with AtPP2C using the SWISS-MODEL Workspace [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164996#pone.0164996.ref059" target="_blank">59</a>]. Favorability of this model is supported based on 56% sequence identify, alignment of all substrate-binding residues to the template sequence, and a Global Model Quality Estimation scores of 0.72. This method was also applied to AtPYL5 (green aligned protein) using the NCBI NP_196163.1 sequence and yielding a GMQE score of 0.68 over the 52% identical sequences. The co-crystallized PP2C structure, At ABI1 (green) is included from the original co-crystallized template structure. B) The activity of Ta_PYL2DS_FL variants against ABA and PBI352. The activity of TaABI1 is differentially regulated by variants of Ta_PYL2DS_FL compared to WT receptor and AtPYL5, in the presence of (+)-ABA (blue bars) and analog PBI352 (red bars). The total TaABI1 activity arising in the absence of receptor stimulation was set to 100% (grey bars). The variants tested included: m1 (S86N), m2 (D180E), m3 (V183S), m4 (D180E + V183S), m5 (S86N + V183S), m6 (S86N + D180E), m7 (S86N + D180E + V183S). The protein phosphatase activity was analyzed at a constant molar ratio of receptor to TaABI1 of 10:1 in vitro at a constant analog concentration of 0.1 μM. Error bars show the standard deviation (n = 3). C) Crystal structure of AtPYL10 in complex with AtHAB1 (PDBID 3 3RTO), showing the dual hydrogen-bonding interaction of E161 (equivalent of Glu180 in AtPYL5) with AtHAB1 Q384 and AtPYL10 N158. D) Crystal structures of PYR1+HAB1 (4WVO; light teal), PYL1+AB11 (3NMN; green), PYL2+ABI2 (3UJL; yellow), PYL3+HAB1 (4DS8; pink), PYL2+HAB1 (4LG5; white) overlaid showing the interaction of the conserved ‘D180’ residues (equivalent to D180 in Ta_PYL2DS_FL) in these structures. The shorter D side chain (compared to E in AtPYL10) limits the residue to making only one hydrogen-bond interaction at a time, likely yielding a weaker overall receptor-PP2C interaction. All overlays and structure images were produced using PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.</p

The ABA Analog Activity Profiles of Ta_PYL2DS_FL are different than AtPYL5.

<p>AtABI1 and TaABI1 (AB238930.1) activity is regulated by wheat Ta_PYL2DS_FL and Arabidopsis AtPYL5 ABA receptors against various enantiomeric ABA analog pairs. A) The structures for (+)- and (-)- ABA, as well as (+)-analogs are shown. The corresponding (-)-analogs are not shown. B) Activities in the presence of <i>(S) or (+)–</i>enantiomers; C) Activities in the presence of <i>(R)</i><b>- or (-)</b> enantiomers. The protein phosphatase activity was analyzed at a constant molar ratio of receptor to PP2C of 10:1 <i>in vitro</i> at a constant analog concentration of 0.1 μM (n = 3). Blue and Red bars show activity for Ta_PYL2DS_FL against TaABI1 and AtABI1 respectively. Green and Purple bars show activity for AtPYL5 against AtABI1 and TaABI1 respectively.</p

Genomic basis of European ash tree resistance to ash dieback fungus

Populations of European ash trees (Fraxinus excelsior) are being devastated by the invasive alien fungus Hymenoscyphus fraxineus, which causes ash dieback. We sequenced whole genomic DNA from 1,250 ash trees in 31 DNA pools, each pool containing trees with the same ash dieback damage status in a screening trial and from the same seed-source zone. A genome-wide association study identified 3,149 single nucleotide polymorphisms (SNPs) associated with low versus high ash dieback damage. Sixty-one of the 192 most significant SNPs were in, or close to, genes with putative homologues already known to be involved in pathogen responses in other plant species. We also used the pooled sequence data to train a genomic prediction model, cross-validated using individual whole genome sequence data generated for 75 healthy and 75 damaged trees from a single seed source. The model’s genomic estimated breeding values (GEBVs) allocated these 150 trees to their observed health statuses with 67% accuracy using 10,000 SNPs. Using the top 20% of GEBVs from just 200 SNPs, we could predict observed tree health with over 90% accuracy. We infer that ash dieback resistance in F. excelsior is a polygenic trait that should respond well to both natural selection and breeding, which could be accelerated using genomic prediction