Jasmonic acid perception by COI1 involves inositol polyphosphates in Arabidopsis thaliana

Plant responses to wounding are part of their defense responses against insects, and are tightly regulated. The isoleucin conjugate of jasmonic acid (JA‐Ile) is a major regulatory molecule. We have previously shown that inositol polyphosphate signals are required for defense responses in Arabidopsis; however, the way in which inositol polyphosphates contribute to plant responses to wounding has so far remained unclear. Arabidopsis F‐box proteins involved in the perception of JA‐Ile (COI1) and auxin (TIR1) are structurally similar. Because TIR1 has recently been shown to contain inositol hexakisphosphate (InsP₆) as a co‐factor of unknown function, here we explored the possibility that InsP₆ or another inositol polyphosphate is required for COI1 function. In support of this hypothesis, COI1 variants with changes in putative inositol polyphosphate coordinating residues exhibited a reduced interaction with the COI1 target, JAZ9, in yeast two‐hybrid tests. The equivalent COI1 variants displayed a reduced capability to rescue jasmonate‐mediated root growth inhibition or silique development in Arabidopsis coi1 mutants. Yeast two‐hybrid tests using wild‐type COI1 in an ipk1Δ yeast strain exhibiting increased levels of inositol pentakisphosphate (InsP₅) and reduced levels of InsP₆ indicate an enhanced COI1/JAZ9 interaction. Consistent with these findings, Arabidopsis ipk1‐1 mutants, also with increased InsP₅ and reduced InsP₆ levels, showed increased defensive capabilities via COI1‐mediated processes, including wound‐induced gene expression, defense against caterpillars or root growth inhibition by jasmonate. The combined data from experiments using mutated COI1 variants, as well as yeast and Arabidopsis backgrounds altered in inositol polyphosphate metabolism, indicate that an inositol polyphosphate, and probably InsP₅, contributes to COI1 function.     

Time-resolved fluorescence study of excitation energy transfer in the cyanobacterium Anabaena PCC 7120

Photosynthesis Research - Excitation energy transfer (EET) and trapping in Anabaena variabilis (PCC 7120) intact cells, isolated phycobilisomes (PBS) and photosystem I (PSI) complexes have been...     

A High Force of Plasmodium vivax Blood-Stage Infection Drives the Rapid Acquisition of Immunity in Papua New Guinean Children

Background

When both parasite species are co-endemic, Plasmodium vivax incidence peaks in younger children compared to P. falciparum. To identify differences in the number of blood stage infections of these species and its potential link to acquisition of immunity, we have estimated the molecular force of blood-stage infection of P. vivax (_molFOB, i.e. the number of genetically distinct blood-stage infections over time), and compared it to previously reported values for P. falciparum.

Methods

P. vivax _molFOB was estimated by high resolution genotyping parasites in samples collected over 16 months in a cohort of 264 Papua New Guinean children living in an area highly endemic for P. falciparum and P. vivax. In this cohort, P. vivax episodes decreased three-fold over the age range of 1–4.5 years.

Results

On average, children acquired 14.0 new P. vivax blood-stage clones/child/year-at-risk. While the incidence of clinical P. vivax illness was strongly associated with _molFOB (incidence rate ratio (IRR) = 1.99, 95% confidence interval (CI95) [1.80, 2.19]), _molFOB did not change with age. The incidence of P. vivax showed a faster decrease with age in children with high (IRR = 0.49, CI95 [0.38, 0.64] p<0.001) compared to those with low exposure (IRR = 0.63, CI95[0.43, 0.93] p = 0.02).

Conclusion

P. vivax _molFOB is considerably higher than P. falciparum _molFOB (5.5 clones/child/year-at-risk). The high number of P. vivax clones that infect children in early childhood contribute to the rapid acquisition of immunity against clinical P. vivax malaria.     

Vienna RNA secondary structure server

The Vienna RNA secondary structure server provides a web interface to the most frequently used functions of the Vienna RNA software package for the analysis of RNA secondary structures. It currently offers prediction of secondary structure from a single sequence, prediction of the consensus secondary structure for a set of aligned sequences and the design of sequences that will fold into a predefined structure. All three services can be accessed via the Vienna RNA web server at http://rna.tbi.univie.ac.at/.     

Genomic analysis of cyclic-di-GMP-related genes in rhizobial type strains and functional analysis in Rhizobium etli

Rhizobia are soil bacteria that can fix nitrogen in symbiosis with leguminous plants or exist free living in the rhizosphere. Crucial to their complex lifestyle is the ability to sense and respond to diverse environmental stimuli, requiring elaborate signaling pathways. In the majority of bacteria, the nucleotide-based second messenger cyclic diguanosine monophosphate (c-di-GMP) is involved in signal transduction. Surprisingly, little is known about the importance of c-di-GMP signaling in rhizobia. We have analyzed the genome sequences of six well-studied type species (Bradyrhizobium japonicum, Mesorhizobium loti, Rhizobium etli, Rhizobium leguminosarum, Sinorhizobium fredii, and Sinorhizobium meliloti) for proteins possibly involved in c-di-GMP signaling based on the presence of four domains: GGDEF (diguanylate cyclase), EAL and HD-GYP (phosphodiesterase), and PilZ (c-di-GMP sensor). We find that rhizobia possess a high number of these proteins. Conservation analysis suggests that c-di-GMP signaling proteins modulate species-specific pathways rather than ancient rhizobia-specific processes. Two hybrid GGDEF-EAL proteins were selected for functional analysis, R. etli RHE_PD00105 (CdgA) and RHE_PD00137 (CdgB). Expression of cdgA and cdgB is repressed by the alarmone (p)ppGpp. cdgB is significantly expressed on plant roots and free living. Mutation of cdgA, cdgB, or both does not affect plant root colonization, nitrogen fixation capacity, biofilm formation, motility, and exopolysaccharide production. However, heterologous expression of the individual GGDEF and EAL domains of each protein in Escherichia coli strongly suggests that CdgA and CdgB are bifunctional proteins, possessing both diguanylate cyclase and phosphodiesterase activities. Taken together, our results provide a platform for future studies of c-di-GMP signaling in rhizobia.     

Stress Responses of Male Guinea Pigs Predict Female Preference

Mate choice is a critical part of sexual selection. One constituent of mate choice is attractiveness, which serves as a projection surface for traits signalling quality and condition to prospective reproductive partners. In guinea pigs, females generally decide whom they will select as partner and when to switch to a new one. The aim of this study was to investigate for universality in male traits possibly associated with female preference. Characteristics of stress response were chosen, as they are relatively stable in their behavioural and physiological expression. Two consecutive experiments were done on isosexually kept males. In the first experiment, behavioural performance in a forced swim-test was noted, after basal endocrine status had been documented with three samples per individual. In a second experiment, the same males were presented to females in a round-robin choice paradigm. A discriminant analysis confirmed a categorization of males into groups that were preferred by different females either two times [more preferred (MP), n = 6] or less than that [less preferred (LP), n = 12]. Mean basal cortisol levels were comparable, but mean basal testosterone (T) was significantly higher in LP. This points at a scenario where higher T might be advantageous in male–male competition but perhaps less important for the formation of male–female bonds. Behaviourally, MP completed the swim-test significantly faster than LP, which may indicate greater goal directedness and motivation. We conclude that traits associated with stress responses may be components of male attractiveness, their stability perhaps reflecting adaptive qualities.     

X-ray crystal structure of Saccharomyces cerevisiae Pdx1 provides insights into the oligomeric nature of PLP synthases

The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis.

Structured summary

MINT-7147859: PDX1 (uniprotkb:P16451) and PDX1 (uniprotkb:P16451) bind (MI:0407) by cosedimentation in solution (MI:0028)MINT-7147899: PDX1 (uniprotkb:P37528) and PDX1 (uniprotkb:P37528) bind (MI:0407) by cosedimentation in solution (MI:0028)     

Secreted Fungal Effector Lipase Releases Free Fatty Acids to Inhibit Innate Immunity-Related Callose Formation during Wheat Head Infection

The deposition of the (1,3)-β-glucan cell wall polymer callose at sites of attempted penetration is a common plant defense response to intruding pathogens and part of the plant’s innate immunity. Infection of the Fusarium graminearum disruption mutant Δfgl1, which lacks the effector lipase FGL1, is restricted to inoculated wheat (Triticum aestivum) spikelets, whereas the wild-type strain colonized the whole wheat spike. Our studies here were aimed at analyzing the role of FGL1 in establishing full F. graminearum virulence. Confocal laser-scanning microscopy revealed that the Δfgl1 mutant strongly induced the deposition of spot-like callose patches in vascular bundles of directly inoculated spikelets, while these callose deposits were not observed in infections by the wild type. Elevated concentrations of the polyunsaturated free fatty acids (FFAs) linoleic and α-linolenic acid, which we detected in F. graminearum wild type-infected wheat spike tissue compared with Δfgl1-infected tissue, provided clear evidence for a suggested function of FGL1 in suppressing callose biosynthesis. These FFAs not only inhibited plant callose biosynthesis in vitro and in planta but also partially restored virulence to the Δfgl1 mutant when applied during infection of wheat spikelets. Additional FFA analysis confirmed that the purified effector lipase FGL1 was sufficient to release linoleic and α-linolenic acids from wheat spike tissue. We concluded that these two FFAs have a major function in the suppression of the innate immunity-related callose biosynthesis and, hence, the progress of F. graminearum wheat infection.The molecular and physiological regulation of the biosynthesis of callose, which is a (1,3)-β-glucan polymer with some (1,6)-branches (Aspinall and Kessler, 1957), and its importance for plant development as well as plant defense are still under examination. Regarding the involvement of callose in plant defense responses, particular attention has been focused on the formation of cell wall thickenings in plants, so-called papillae, at sites of microbial attack. They were already described 150 years ago (deBary, 1863) and reported to commonly contain callose (Mangin, 1895). Since then, examinations have identified callose as the most abundant chemical constituent in papillae, which may also include proteins (e.g. peroxidases and antimicrobial thionins), phenolics, and other constituents (Aist and Williams, 1971; Sherwood and Vance, 1976; Mims et al., 2000). Papillae have been regarded as an early defense reaction that may not completely stop the pathogen; rather, they have been considered to act as a physical barrier to slow pathogen invasion (Stone and Clarke, 1992; Voigt and Somerville, 2009) and to contribute to the plant’s innate immunity (Jones and Dangl, 2006; Schwessinger and Ronald, 2012). The host plant can gain time to initiate defense reactions that require gene activation and expression, such as the hypersensitive reactions, phytoalexin production, and pathogenesis-related protein synthesis (Lamb and Dixon, 1997; Brown et al., 1998). However, our recent study revealed that callose can also act as a barrier that completely prevents fungal penetration. The overexpression of POWDERY MILDEW RESISTANT4 (PMR4), a gene encoding a stress-induced callose synthase, resulted in early elevated callose deposition at sites of attempted powdery mildew penetration in Arabidopsis (Arabidopsis thaliana; Ellinger et al., 2013). Interestingly, the pmr4 deletion mutant also showed an increased resistance to powdery mildew that, however, was induced at later stages of powdery mildew infection because an initial fungal penetration still occurred. In fact, the absence of the functional callose synthase PMR4 in the pmr4 mutant resulted in papillae that were free from callose but also induced a hyperactivation of the salicylic acid defense pathway, which was shown to be the basis of resistance in double mutant and microarray analyses (Jacobs et al., 2003; Nishimura et al., 2003). The callose synthase gene PMR4 from Arabidopsis belongs to the GLUCAN SYNTHASE-LIKE (GSL) family, genes that have been identified in higher plants including wheat (Triticum aestivum; Cui et al., 2001; Doblin et al., 2001; Hong et al., 2001; Østergaard et al., 2002; Voigt et al., 2006). The predicted function of these genes as callose synthases is generally supported by homology with the yeast FK506 SENSITIVITY (FKS) genes, which are believed to be subunits of (1,3)-β-glucan synthase complexes (Douglas et al., 1994; Dijkgraaf et al., 2002). Additionally, the predicted proteins encoded by the GSL genes correlate with the approximately 200-kD catalytic subunit of putative callose synthases. Li et al. (2003) showed that the amino acid sequence predicted from a GSL gene in barley (Hordeum vulgare; HvGSL1) correlates with the amino acid sequence of an active (1,3)-β-glucan synthase fraction.In this study, we aimed to examine the involvement of callose synthesis and callose deposition in plant defense against intruding fungal pathogens in the pathosystem wheat-Fusarium graminearum. We focused on the ability of wheat to inhibit a further spread of fungal pathogens after an initial, successful infection. This resistance to fungal spread within the host has been referred to as type II resistance and is part of a widely accepted two-component system of resistance, which includes type I resistance operating against initial infection (Schroeder and Christensen, 1963). For our analyses, we used the direct interaction between wheat as host and F. graminearum as a pathogen. On the one hand, Fusarium head blight (FHB) of wheat, caused by F. graminearum, is one of the most destructive crop diseases worldwide (McMullen et al., 1997; del Blanco et al., 2003; Madgwick et al., 2011) and classifies this fungus as a top 10 plant pathogen based on its importance in science and agriculture (Dean et al., 2012). On the other hand, only a limited number of wheat cultivars were identified that revealed FHB resistance. However, these cultivars did not qualify for commercial cultivation or breeding approaches due to inappropriate agronomic traits (Buerstmayr et al., 2009). Further elucidation of the mechanisms of spreading resistance could support the generation of FHB-resistant wheat cultivars.In this regard, we demonstrated that the secreted lipase FGL1 of F. graminearum is a virulence factor required for wheat infection (Voigt et al., 2005). A strong resistance to fungal spread was observed in a susceptible wheat cultivar after infection with the lipase-deficient F. graminearum strain Δfgl1. Light microscopy indicated barrier formation in the transition zone of rachilla and rachis of directly inoculated spikelets. In contrast, neither spreading resistance nor barrier formation was observed during F. graminearum wild type infection. An active role of lipases in establishing full virulence was also recently proposed for the plant pathogen Fusarium oxysporum f. sp. lycopersici, where reduced lipolytic activity due to the deletion of lipase regulatory genes resulted in reduced colonization of tomato (Solanum lycopersicum) plants (Bravo-Ruiz et al., 2013). Because the expression of the lipase-encoding gene LIP1 was induced in the biotrophic fungus Blumeria graminis during early stages of infection (Feng et al., 2009) and disruption of the putative secreted lipase gene lipA resulted in reduced virulence of the bacterial plant pathogen Xanthomonas campestris (Tamir-Ariel et al., 2012), a general importance of extracellular lipolytic activity during plant colonization is indicated.We evaluated a possible role of callose in plant defense by infecting wheat spikes with the virulent fungal pathogen F. graminearum wild type, the virulence-deficient F. graminearum deletion mutant Δfgl1, and the barley leaf pathogen Pyrenophora teres, the latter intended to induce strong plant defense responses as known from incompatible, nonhost interactions. The formation of callose plugs within the vascular bundles of inoculated spikelets and the callose synthase activity of infected spikelet tissue correlated directly with increased plant resistance. Subsequent analyses of free fatty acid (FFA) concentrations revealed that those polyunsaturated FFAs were enriched during wheat infection with the F. graminearum wild-type strain that could inhibit callose synthase activity in vitro as well as in planta and partially restored the virulence of the lipase-deficient F. graminearum strain Δfgl1. On the basis of these results, we propose a model for FHB where defense-related callose synthase is inhibited by specific FFAs whose accumulation is caused by the fungus during fungal infection; this inhibition is required for full infection of the wheat head.     

Computer-Guided Trypanocidal Activity of Natural Lactones Produced by Endophytic Fungus of Euphorbia umbellata

Hundreds of millions of people worldwide are affected by Chagas’ disease caused by Trypanosoma cruzi. Since the current treatment lack efficacy, specificity, and suffers from several side-effects, novel therapeutics are mandatory. Natural products from endophytic fungi have been useful sources of lead compounds. In this study, three lactones isolated from an endophytic strain culture were in silico evaluated for rational guidance of their bioassay screening. All lactones displayed in vitro activity against T. cruzi epimastigote and trypomastigote forms. Notably, the IC₅₀ values of (+)-phomolactone were lower than benznidazole (0.86 vs. 30.78 μM against epimastigotes and 0.41 vs. 4.88 μM against trypomastigotes). Target-based studies suggested that lactones displayed their trypanocidal activities due to T. cruzi glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH) inhibition, and the binding free energy for all three TcGAPDH-lactone complexes suggested that (+)-phomolactone has a lower score value (−3.38), corroborating with IC₅₀ assays. These results highlight the potential of these lactones for further anti-T. cruzi drug development.