Unearthing the plant-microbe quid pro quo in root associations with beneficial fungi.

Mutualistic symbiotic associations between multicellular eukaryotes and their microbiota are driven by the exchange of nutrients in a quid pro quo manner. In the widespread arbuscular mycorrhizal (AM) symbiosis involving plant roots and Glomeromycotina fungi, the mycobiont is supplied with carbon through photosynthesis, which in return supplies the host plant with essential minerals such as phosphorus (P). Most terrestrial plants are largely dependent on AM fungi for nutrients, which raises the question of how plants that are unable to form a functional AM sustain their P nutrition. AM nonhost plants can form alternative, evolutionarily younger, mycorrhizal associations such as the ectomycorrhiza, ericoid and orchid mycorrhiza. However, it is unclear how plants such as the Brassicaceae species Arabidopsis thaliana, which do not form known mycorrhizal symbioses, have adapted to the loss of these essential mycorrhizal traits. Isotope tracing experiments with root-colonizing fungi have revealed the existence of new 'mycorrhizal-like' fungi capable of transferring nutrients such as nitrogen (N) and P to plants, including Brassicaceae. Here, we provide an overview of the biology of trophic relationships between roots and fungi and how these associations might support plant adaptation to climate change.

Dysfunction in the arbuscular mycorrhizal symbiosis has consistent but small effects on the establishment of the fungal microbiota in Lotus japonicus.

Most land plants establish mutualistic interactions with arbuscular mycorrhizal (AM) fungi. Intracellular accommodation of AM fungal symbionts remodels important host traits like root morphology and nutrient acquisition. How mycorrhizal colonization impacts plant microbiota is unclear. To understand the impact of AM symbiosis on fungal microbiota, ten Lotus japonicus mutants impaired at different stages of AM formation were grown in non-sterile natural soil and their root-associated fungal communities were studied. Plant mutants lacking the capacity to form mature arbuscules (arb- ) exhibited limited growth performance associated with altered phosphorus (P) acquisition and reduction-oxidation (redox) processes. Furthermore, arb- plants assembled moderately but consistently different root-associated fungal microbiota, characterized by the depletion of Glomeromycota and the concomitant enrichment of Ascomycota, including Dactylonectria torresensis. Single and co-inoculation experiments showed a strong reduction of root colonization by D. torresensis in the presence of AM fungus Rhizophagus irregularis, particularly in arbuscule-forming plants. Our results suggest that impairment of central symbiotic functions in AM host plants leads to specific changes in root microbiomes and in tripartite interactions between the host plant, AM and non-AM fungi. This lays the foundation for mechanistic studies on microbe-microbe and microbe-host interactions in AM symbiosis of the model L. japonicus.

Plant-mediated effects of soil phosphorus on the root-associated fungal microbiota in Arabidopsis thaliana.

Plants respond to phosphorus (P) limitation through an array of morphological, physiological and metabolic changes which are part of the phosphate (Pi) starvation response (PSR). This response influences the establishment of the arbuscular mycorrhizal (AM) symbiosis in most land plants. It is, however, unknown to what extent available P and the PSR redefine plant interactions with the fungal microbiota in soil. Using amplicon sequencing of the fungal taxonomic marker ITS2, we examined the changes in root-associated fungal communities in the AM nonhost species Arabidopsis thaliana in response to soil amendment with P and to genetic perturbations in the plant PSR. We observed robust shifts in root-associated fungal communities of P-replete plants in comparison with their P-deprived counterparts, while bulk soil communities remained unaltered. Moreover, plants carrying mutations in the phosphate signaling network genes, phr1, phl1 and pho2, exhibited similarly altered root fungal communities characterized by the depletion of the chytridiomycete taxon Olpidium brassicae specifically under P-replete conditions. This study highlights the nutritional status and the underlying nutrient signaling network of an AM nonhost plant as previously unrecognized factors influencing the assembly of the plant fungal microbiota in response to P in nonsterile soil.

Oral Rabies Vaccine Strain SPBN GASGAS: Genetic Stability after Serial In Vitro and In Vivo Passaging.

Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered rabies virus vaccine strain SPBN GASGAS (Rabitec), are based on replication-competent viruses. To evaluate the genetic stability of this vaccine strain, five serial passages of the Master Seed Virus (MSV) in the production cell line BHK21 Cl13 were performed. Furthermore, to test possible reversion to virulence, a back-passage study in suckling mouse brain (SMB) was performed. Subsequently, the pooled 5th SMB passage was inoculated intracerebrally (i.c.) in adult and suckling mice. The full genome sequences of the isolated 5th passage, in vivo and in vitro, were compared at both the consensus and the quasispecies level with the MSV. Additionally, the full genome sequence of the 6th SMB passage from the individual animals was determined and compared. Full-length integration of the double glycoprotein and modified base substitutions at amino acid position 194 and 333 of the glycoprotein could be verified in all 5th and 6th passage samples. Overall, 11 single nucleotide polymorphisms (SNPs) were detected in the 5th pooled SMB passage, 4 with frequency between 10 and 20%, and 7 with between 2.5 and 10%. SNPs that resulted in amino acid exchange were found in genes: N (one SNP), G (four SNPs), and L (three SNPs). However, none of these SNPs were associated with reversion to virulence since all adult mice inoculated i.c. with this material survived. In the individual samples of the 6th SMB passage 24 additional SNPs (>2.5%) were found, of which only 1 SNP (L-gene, position 6969) had a prevalence of >50% in 3 of 17 samples. The obtained results confirmed the stable expression of genetic modifications and the genetic stability of the consensus strain after serial in vivo and in vitro passaging.

LABRADOR-A Computational Workflow for Virus Detection in High-Throughput Sequencing Data.

High-throughput sequencing (HTS) allows detection of known and unknown viruses in samples of broad origin. This makes HTS a perfect technology to determine whether or not the biological products, such as vaccines are free from the adventitious agents, which could support or replace extensive testing using various in vitro and in vivo assays. Due to bioinformatics complexities, there is a need for standardized and reliable methods to manage HTS generated data in this field. Thus, we developed LABRADOR-an analysis pipeline for adventitious virus detection. The pipeline consists of several third-party programs and is divided into two major parts: (i) direct reads classification based on the comparison of characteristic profiles between reads and sequences deposited in the database supported with alignment of to the best matching reference sequence and (ii) de novo assembly of contigs and their classification on nucleotide and amino acid levels. To meet the requirements published in guidelines for biologicals' safety we generated a custom nucleotide database with viral sequences. We tested our pipeline on publicly available HTS datasets and showed that LABRADOR can reliably detect viruses in mixtures of model viruses, vaccines and clinical samples.

Neighboring plants divergently modulate effects of loss-of-function in maize mycorrhizal phosphate uptake on host physiology and root fungal microbiota.

Maize, a main crop worldwide, establishes a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi providing nutrients to the roots from soil volumes which are normally not in reach of the non-colonized root. The mycorrhizal phosphate uptake pathway (MPU) spans from extraradical hyphae to root cortex cells housing fungal arbuscules and promotes the supply of phosphate to the mycorrhizal host in exchange for photosynthetic carbon. This symbiotic association with the mycobiont has been shown to affect plant host nutritional status and growth performance. However, whether and how the MPU affects the root microbial community associated with mycorrhizal hosts in association with neighboring plants, remains to be demonstrated. Here the maize germinal Mu transposon insertion mutant pht1;6, defective in mycorrhiza-specific Pi transporter PHT1;6 gene, and wild type B73 (wt) plants were grown in mono- and mixed culture and examined under greenhouse and field conditions. Disruption of the MPU in pht1;6 resulted in strongly diminished growth performance, in reduced P allocation to photosynthetic source leaves, and in imbalances in leaf elemental composition beyond P. At the microbial community level a loss of MPU activity had a minor effect on the root-associated fungal microbiome which was almost fully restricted to AM fungi of the Glomeromycotina. Moreover, while wt grew better in presence of pht1;6, pht1;6 accumulated little biomass irrespective of whether it was grown in mono- or mixed culture and despite of an enhanced fungal colonization of its roots in co-culture with wt. This suggested that a functional MPU is prerequisite to maintain maize growth and that neighboring plants competed for AM fungal Pi in low P soil. Thus future strategies towards improving yield in maize populations on soils with low inputs of P fertilizer could be realized by enhancing MPU at the individual plant level while leaving the root-associated fungal community largely unaffected.

The role of nutrient balance in shaping plant root-fungal interactions: facts and speculation.

Microbiota colonizing plant roots and their vicinity were shown not to be just random associations, but compose, at least to some extent, host-selected microbial consortia. The plant physiological status, especially the nutrient status, prompts changes in plant morphology and metabolism, which successively imposes a selective pressure on microbial communities. It is well established that a low phosphate status of the host plant activates the molecular machinery underlying the development of mutualistic associations in the host root with arbuscular mycorrhizal fungi (AMF). We hypothesize that the plant´s response to changing nutrient stoichiometry affects processes at the root-mycosphere interface which promote or repress also root interactions with microbial taxa other than AMF. As a consequence, fundamental mechanisms underlying these interactions would be shared in AM host and non-host plants. A detailed understanding of the processes involved in maintenance of plant nutrient homeostasis could contribute to novel strategies in tailoring predominantly parasitic or commensalistic plant-microbe interactions towards beneficial associations.

Intracellular phosphate homeostasis - A short way from metabolism to signaling.

Phosphorus in plant cells occurs in inorganic form as both ortho- and pyrophosphate or bound to organic compounds, like e.g., nucleotides, phosphorylated metabolites, phospholipids, phosphorylated proteins, or phytate as P storage in the vacuoles of seeds. Individual compartments of the cell are surrounded by membranes that are selective barriers to avoid uncontrolled solute exchange. A controlled exchange of phosphate or phosphorylated metabolites is accomplished by specific phosphate transporters (PHTs) and the plastidial phosphate translocator family (PTs) of the inner envelope membrane. Plastids, in particular chloroplasts, are the site of various anabolic sequences of enzyme-catalyzed reactions. Apart from their role in metabolism PHTs and PTs are presumed to be also involved in communication between organelles and plant organs. Here we will focus on the integration of phosphate transport and homeostasis in signaling processes. Recent developments in this field will be critically assessed and potential future developments discussed. In particular, the occurrence of various plastid types in one organ (i.e. the leaf) with different functions with respect to metabolism or sensing, as has been documented recently following a tissue-specific proteomics approach (Beltran et al., 2018), will shed new light on functional aspects of phosphate homeostasis.

Long-Sought Vacuolar Phosphate Transporters Identified.

The vacuole is an important subcellular compartment that serves as main phosphate storage in plants among other functions. Three recent studies shed light on the underlying molecular mechanisms for vacuolar phosphate transport that had long remained unknown.

Plant-pathogenic fungi in seeds of different pea cultivars in poland.

Legume crops are exposed to infection by fungal pathogens, which often results in contamination with mycotoxins. The aim of this study was to evaluate the level of field resistance/susceptibility of edible and fodder pea cultivars to the colonization of seeds by fungal pathogens in two subsequent seasons, as well as to identify the pathogens present in the seeds of the tested cultivars. Alternaria spp. were the most common fungi isolated from pea seeds in both seasons, followed by Fusarium spp., Stemphylium spp., Ulocladium spp., Botrytis cinerea Pers., Epicoccum nigrum Link., and Phomapinodella L. K. Jones. The highest percentage of infected seeds (55 %) was recorded for cultivar Ezop. The presence of a large number of fungi was found in 2012 for cultivars Santana, Tarchalska, Medal, Cysterski, Mentor, Lasso, and Ezop. Fodder cultivars displayed a lower infection level than edible cultivars. We can conclude that Alternaria spp. were the most frequent fungi present in pea seeds in Poland and Fusarium spp. were likely the most dangerous, having in mind their established mycotoxigenic abilities.