Proteomic analysis of the Mycocentrospora acerina-carrot interaction during storage

During post harvest storage, a large proportion of carrots (more than 50%) may have to be discarded due to the development of liquorice rot caused by Mycocentrospora acerina. This fungus is soil borne and brought into the store in to soil adhering to the root. Liquorice rot development is mainly related to physiological or structural resistance of carrot, therefore the control of this storage disease is based on cultural practices and storage conditions. It is believed that carrots at the beginning of storage can resist disease developments due to chemical defence mechanisms involving some proteins, peptides and secondary metabolites. The hypothesis is that proteome changes during storage of carrots are related to the susceptibility to M. acerina. During root-pathogen interactions, several genes have been reported to provide resistance against pathogens but only few proteins have been identified using proteomic approaches. Little is known about proteins involved during M. acerina - carrot interaction. The carrots used in this study are grown under two different agricultural practices (one conventional, one organic) in order to investigate the effect of the cropping system on the susceptibility to liquorice rot. We developed a bioassay for infection studies of M. acerina on conventional and organic carrots in order to determine the important time points of the infection process. Then the proteome is investigated at these different time points. The protocol for extraction of proteins has been improved so that it can be used to obtain an optimal recovery of proteins from both plant and pathogen on their own as well as from infected carrot roots. Proteomes of carrot and of M. acerina are characterized by two dimensional gel electrophoreses and the proteins whose synthesis varies significantly in the course of pathogen infection are identified by mass spectrometry (MALDI TOF-TOF)

Gulerodens farefulde vej fra marken til forbrugeren

Kølelagring af Lammefjords-gulerødder muligør, at der kan leveres danskproducerede gulerødder i perioden fra november til april. Sidst på lagringssæsonen kan mere ed 50% af rødderne dog være kassable på grund af lagersygdomme. I marken angribes rødderne af forskellige jordboende svampe, der allerede ved høst kan resultere i kassable rødder. I lagerperioden kan tilsyneladende raske gulerøder dog også udvikle sygdomme forårsaget af de mikrosvampe der forkommer naturligt på rødderne. Sår på rodoverfladen fremmer angreb under lagring og desuden reduceres gulerøddernes modstandsdygtighed overfor sygdomme i takt med røddernes aldring. I artiklen beskrives de mest betydende sygdomsfremkaldende organismer (patogener), faktorer der er af betydning for udvikling af lagersygdomme samt muligheder for forbedret lagerkvalitet ved hjælp af biologiske forebyggelsesmetoder

Mobilization of Pollutant-Degrading Bacteria by Eukaryotic Zoospores

This study was supported by the Spanish Ministry of Science and Innovation (CGL2010-22068-C02-01 and CGL2013- 44554-R), the Andalusian Government (RNM 2337), and the CSIC JAE Program (RS). PvW has funding support from the BBSRC and NERC. Thanks are also given to Sara Hosseini of the Uppsala BioCenter, SLU, Uppsala, Sweden for a useful discussion on oomycete zoospores.Peer reviewedPostprin

Deciphering common and specific transcriptional immune responses in pea towards the oomycete pathogens Aphanomyces euteiches and Phytophthora pisi

Primers used in this study for RT-qPCR analysis. (DOCX 20 kb

Insights into the ecological generalist lifestyle of Clonostachys fungi through analysis of their predicted secretomes

IntroductionThe fungal secretome comprise diverse proteins that are involved in various aspects of fungal lifestyles, including adaptation to ecological niches and environmental interactions. The aim of this study was to investigate the composition and activity of fungal secretomes in mycoparasitic and beneficial fungal-plant interactions. MethodsWe used six Clonostachys spp. that exhibit saprotrophic, mycotrophic and plant endophytic lifestyles. Genome-wide analyses was performed to investigate the composition, diversity, evolution and gene expression of Clonostachys secretomes in relation to their potential role in mycoparasitic and endophytic lifestyles. Results and discussionOur analyses showed that the predicted secretomes of the analyzed species comprised between 7 and 8% of the respective proteomes. Mining of transcriptome data collected during previous studies showed that 18% of the genes encoding predicted secreted proteins were upregulated during the interactions with the mycohosts Fusarium graminearum and Helminthosporium solani. Functional annotation of the predicted secretomes revealed that the most represented protease family was subclass S8A (11-14% of the total), which include members that are shown to be involved in the response to nematodes and mycohosts. Conversely, the most numerous lipases and carbohydrate-active enzyme (CAZyme) groups appeared to be potentially involved in eliciting defense responses in the plants. For example, analysis of gene family evolution identified nine CAZyme orthogroups evolving for gene gains (p <= 0.05), predicted to be involved in hemicellulose degradation, potentially producing plant defense-inducing oligomers. Moreover, 8-10% of the secretomes was composed of cysteine-enriched proteins, including hydrophobins, important for root colonization. Effectors were more numerous, comprising 35-37% of the secretomes, where certain members belonged to seven orthogroups evolving for gene gains and were induced during the C. rosea response to F. graminearum or H. solani. Furthermore, the considered Clonostachys spp. possessed high numbers of proteins containing Common in Fungal Extracellular Membranes (CFEM) modules, known for their role in fungal virulence. Overall, this study improves our understanding of Clonostachys spp. adaptation to diverse ecological niches and establishes a basis for future investigation aiming at sustainable biocontrol of plant diseases

LysM Proteins Regulate Fungal Development and Contribute to Hyphal Protection and Biocontrol Traits in Clonostachys rosea

Lysin motif (LysM) modules are approximately 50 amino acids long and bind to peptidoglycan, chitin and its derivatives. Certain LysM proteins in plant pathogenic and entomopathogenic fungi are shown to scavenge chitin oligosaccharides and thereby dampen host defense reactions. Other LysM proteins can protect the fungal cell wall against hydrolytic enzymes. In this study, we investigated the biological function of LysM proteins in the mycoparasitic fungus Clonostachys rosea. The C. rosea genome contained three genes coding for LysM-containing proteins and gene expression analysis revealed that lysm1 and lysm2 were induced during mycoparasitic interaction with Fusarium graminearum and during colonization of wheat roots. Lysm1 was suppressed in germinating conidia, while lysm2 was induced during growth in chitin or peptidoglycan-containing medium. Deletion of lysm1 and lysm2 resulted in mutants with increased levels of conidiation and conidial germination, but reduced ability to control plant diseases caused by F. graminearum and Botrytis cinerea. The Delta lysm2 strain showed a distinct, accelerated mycelial disintegration phenotype accompanied by reduced biomass production and hyphal protection against hydrolytic enzymes including chitinases, suggesting a role of LYSM2 in hyphal protection against chitinases. The Delta lysm2 and Delta lysm1 Delta lysm2 strains displayed reduced ability to colonize wheat roots, while only Delta lysm1 Delta lysm2 failed to suppress expression of the wheat defense response genes PR1 and PR4. Based on our data, we propose a role of LYSM1 as a regulator of fungal development and of LYSM2 in cell wall protection against endogenous hydrolytic enzymes, while both are required to suppress plant defense responses. Our findings expand the understanding of the role of LysM proteins in fungal-fungal interactions and biocontrol

Comparative genomics highlights the importance of drug efflux transporters during evolution of mycoparasitism in Clonostachys subgenus Bionectria (Fungi, Ascomycota, Hypocreales)

Various strains of the mycoparasitic fungal speciesClonostachys roseaare used commercially as biological control agents for the control of fungal plant diseases in agricultural crop production. Further improvements of the use and efficacy ofC. roseain biocontrol require a mechanistic understanding of the factors that determines the outcome of the interaction betweenC. roseaand plant pathogenic fungi. Here, we determined the genome sequences of 11Clonostachysstrains, representing five species inClonostachyssubgenusBionectria, and performed a comparative genomic analysis with the aim to identify gene families evolving under selection for gene gains or losses. Several gene families predicted to encode proteins involved in biosynthesis of secondary metabolites, including polyketide synthases, nonribosomal peptide syntethases and cytochrome P450s, evolved under selection for gene gains (p <= .05) in theBionectriasubgenus lineage. This was accompanied with gene copy number increases (p <= .05) in ATP-binding cassette (ABC) transporters and major facilitator superfamily (MFS) transporters predicted to contribute to drug efflux. MostClonostachysspecies were also characterized by high numbers of auxiliary activity (AA) family 9 lytic polysaccharide monooxygenases, AA3 glucose-methanol-choline oxidoreductases and additional carbohydrate-active enzyme gene families with putative activity (or binding) towards xylan and rhamnose/pectin substrates. Particular features of theC. roseagenome included expansions (p <= .05) of the ABC-B4 multidrug resistance transporters, the ABC-C5 multidrug resistance-related transporters and the 2.A.1.3 drug:H + antiporter-2 MFS drug resistance transporters. The ABC-G1 pleiotropic drug resistance transporter geneabcG6inC. roseawas induced (p <= .009) by exposure to the antifungalFusariummycotoxin zearalenone (1121-fold) and various fungicides. Deletion ofabcG6resulted in mutants with reduced (p < .001) growth rates on media containing the fungicides boscalid, fenhexamid and iprodione. Our results emphasize the role of biosynthesis of, and protection against, secondary metabolites inClonostachyssubgenusBionectria