Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall–degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application

Presentation_1_Profiling of the Transcriptomic Responses of Clonostachys rosea Upon Treatment With Fusarium graminearum Secretome.PDF

<p>Clonostachys rosea strain ACM941 is a fungal bio-control agent patented against the causative agent of Fusarium Head Blight, Fusarium graminearum. Although the molecular details remain enigmatic, previous studies have suggested that C. rosea may secrete F. graminearum growth inhibitors. Further toward this, experiments described herein show that induction of C. rosea cultures by the addition of an aliquot of F. graminearum(Fg)-spent media (including macroconidia), yield C. rosea (Cr)-spent media that elicited higher anti-F. graminearum activity than either control or deoxynivalenol (DON)-induced Cr-spent media. To gain additional insight into the genetic and metabolic factors modulating this interaction, transcriptomic (RNAseq) profiles of C. rosea in response to DON and Fg-spent media treatment, were developed. This analysis revealed 24,112 C. rosea unigenes, of which 5,605 and 6,285 were differentially regulated by DON and F-spent media, respectively. More than half of these unigenes were up-regulated, with annotations, most notably in the Fg-spent media treatment data, suggesting enhancement of polyketide (PK) and non-ribosomal peptide (NRP) secondary metabolite precursor synthesis, and PK/NRP-like synthases. Four ABC transporters were also up-regulated in response to Fg-spent media. Further analysis showed that the PK and NRP-like synthases belong to three gene clusters that also include ABC transporters, and other genes known to tailor secondary metabolite biosynthesis. The RNAseq data was further validated using quantitative RT-qPCR. Taken together, these results show that C. rosea responds to the presence of Fg-spent media (and to a lesser extent, DON-alone) by up-regulating unique aspects of its secondary metabolism-related genetic repertoire. The identities and roles of C. rosea secondary metabolites produced by the targeted gene clusters are now under investigation.</p

Characterization of the ATP-translocating properties of the predicted Arabidopsis thaliana mitochondrial adenine nucleotide translocator 2

In this study we demonstrate that the predicted Arabidopsis thaliana (L.) Heynh. mitochondrial adenine nucleotide translocator 2 (ANT2) mediates ATP translocation. The demonstration that recombinantly produced ANT2 mediates ATP uptake into proteoliposomes, confirms previous sequence-based identification of the protein as a member of the adenine nucleotide translocator family. Expressed in Saccharomyces cerevisiae with a C-terminal His6 tag, localization was confirmed in yeast membrane extracts. The recombinant protein was solubilized with Triton X-100, enriched by immobilized metal affinity chromatography, and reconstituted into liposomes. Functionality of the reconstituted protein was confirmed by demonstration of pyridoxal 5\ue2\u20ac\ub2-phosphate-sensitive [3H]ATP uptake. Transport assays showed first order kinetic uptake of ATP with an approximate Km value of 15 \uc2\ub5mol\uc2\ub7L\ue2\u20ac\u201c1. Competition assays indicated that the reconstituted protein had highest specificity for ATP. Overall, these results indicate that Arabidopsis ANT2 is an adenine nucleotide translocator.Dans cette \uc3\ua9tude, les auteurs d\uc3\ua9montrent leur pr\uc3\ua9diction \uc3 l'effet que le transporteur du nucl\uc3\ua9otide ad\uc3\ua9nine (ANT2) mitochondrial intervient dans le transport de l'ATP. Que l'ANT2 produit par recombinaison intervienne dans l'absorption de l'ATP dans des prot\uc3\ua9oliposomes confirme l'identification bas\uc3\ua9e sur les s\uc3\ua9quences de la prot\uc3\ua9ine comme membre de la famille des transporteurs du nucl\uc3\ua9otide ad\uc3\ua9nine. Les auteurs confirment sa pr\uc3\ua9sence dans des extraits de membranes de levure, lorsqu'on l'exprime chez le Saccharomyces cerevisiae, avec un marqueur C-terminal His6. On a solubiliz\uc3\ua9 la prot\uc3\ua9ine recombinante avec du Triton X-100 et on l'a enrichie par chromatographie d'affinit\uc3\ua9 sur m\uc3\ua9tal immobilis\uc3\ua9, avant de la reconstituer dans des liposomes. Les auteurs ont ainsi confirm\uc3\ua9 la fonctionnalit\uc3\ua9 de la prot\uc3\ua9ine reconstitu\uc3\ua9e en d\uc3\ua9montrant l'absorption du pyridoxal 5\ue2\u20ac\ub2 [3H]ATP sensible au phosphate. Des essais de transport montrent une absorption cin\uc3\ua9tique de premier ordre de l'ATP, avec une valeur Km approximative de 15 \uc2\ub5mol\uc2\ub7L\ue2\u20ac\u201c1. Les essais de comp\uc3\ua9tition indiquent que la prot\uc3\ua9ine reconstitu\uc3\ua9e poss\uc3\ua8de une plus grande affinit\uc3\ua9 pour l'ATP. En g\uc3\ua9n\uc3\ua9ral, ces r\uc3\ua9sultats montrent que l'ANT2 de l'Arabidopsis constitue un transporteur du nucl\uc3\ua9otide ad\uc3\ua9nine.Peer reviewed: YesNRC publication: Ye

The biochemical characterization of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound inhibits lateral branching

Peer reviewed: YesNRC publication: Ye

A Universally Primed-Polymerase Chain Reaction (UP-PCR) Marker to Discriminate <i>Clonostachys rosea</i> ACM941 from Related Strains

Clonostachys rosea strain ACM941 is an effective biocontrol agent against several crop diseases including Fusarium head blight. In anticipation of its increased relevance going forward, the development of a reliable DNA-based molecular marker to track it is essential. Universally primed-PCR (UP-PCR) has been used successfully to differentiate other C. rosea strains. Herein, the development of a UP-PCR marker for ACM941 is described. A combination of two primers (AS15 and L45) produced a ~450 bp fragment that was unique to ACM941 compared to other commercial biocontrol agents. Primers subsequently designed based on the obtained fragment also produced a similarly unique band from ACM941 alone. BLAST analysis of the amplified sequence did not yield any homologous sequence in available online databases or within the closely related C. rosea IK726 and CBS125111 strains&#8217; genomes. The specificity of this marker for ACM941 was validated against ten additional C. rosea strains isolated from Canada, with ACM941 producing the brightest band. Taken together, these results imply that the UP-PCR primers AS15 and L45 and the amplified fragment can be used to detect and monitor the ACM941 strain after its release into the environment

Identification of an attenuated barley stripe mosaic virus for the virus-induced gene silencing of pathogenesis-related wheat genes

Background: Virus-induced gene silencing (VIGS) has become an emerging technology for the rapid, efficient functional genomic screening of monocot and dicot species. The barley stripe mosaic virus (BSMV) has been described as an effective VIGS vehicle for the evaluation of genes involved in wheat and barley phytopathogenesis; however, these studies have been obscured by BSMV-induced phenotypes and defense responses. The utility of BSMV VIGS may be improved using a BSMV genetic background which is more tolerable to the host plant especially upon secondary infection of highly aggressive, necrotrophic pathogens such as Fusarium graminearum. Results: BSMV-induced VIGS in Triticum aestivum (bread wheat) cv. 'Fielder' was assessed for the study of wheat genes putatively related to Fusarium Head Blight (FHB), the necrotrophism of wheat and other cereals by F. graminearum. Due to the lack of 'Fielder' spike viability and increased accumulation of Fusarium-derived deoxynivalenol contamination upon co-infection of BSMV and FHB, an attenuated BSMV construct was generated by the addition of a glycine-rich, C-terminal peptide to the BSMV \u3b3 b protein. This attenuated BSMV effectively silenced target wheat genes while limiting disease severity, deoxynivalenol contamination, and yield loss upon Fusarium co-infection compared to the original BSMV construct. The attenuated BSMV-infected tissue exhibited reduced abscisic, jasmonic, and salicylic acid defense phytohormone accumulation upon secondary Fusarium infection. Finally, the attenuated BSMV was used to investigate the role of the salicylic acid-responsive pathogenesis-related 1 in response to FHB. Conclusions: The use of an attenuated BSMV may be advantageous in characterizing wheat genes involved in phytopathogenesis, including Fusarium necrotrophism, where minimal viral background effects on defense are required. Additionally, the attenuated BSMV elicits reduced defense hormone accumulation, suggesting that this genotype may have applications for the investigation of phytohormone-related signaling, developmental responses, and pathogen defense.Peer reviewed: YesNRC publication: Ye

Molecular mechanisms in the selective basal activation of pyrabactin receptor 1: Comparative analysis of mutants

Pyrabactin receptors (PYR) play a central role in abscisic acid (ABA) signal transduction; they are ABA receptors that inhibit type 2C protein phosphatases (PP2C). Molecular aspects contributing to increased basal activity of PYR against PP2C are studied by molecular dynamics (MD) simulations. An extensive series of MD simulations of the apo-form of mutagenized PYR1 as a homodimer and in complex with homology to ABA-insensitive 1 (HAB1) phosphatase are reported. In order to investigate the detailed molecular mechanisms mediating PYR1 activity, the MD data was analyzed by essential collective dynamics (ECD), a novel approach that allows the identification, with atomic resolution, of persistent dynamic correlations based on relatively short MD trajectories. Employing the ECD method, the effects of select mutations on the structure and dynamics of the PYR1 complexes were investigated and considered in the context of experimentally determined constitutive activities against HAB1. Approaches to rationally design constitutively active PYR1 constructs to increase PP2C inhibition are discussed.Peer reviewed: YesNRC publication: Ye

Structure of non-activated Pisum sativum Rubisco in complex with its substrate ribulose-1,5-bisphosphate

The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20 \uc5 resolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the `closed' conformation observed in other substrate/inhibitor-bound Rubisco structures.Peer reviewed: YesNRC publication: Ye