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Wood-degrading brown rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation (LOX) by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and nonselective, these two steps are likely segregated. A common hypothesis has been that brown rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that LOX components involved in ROS production are differentially expressed by brown rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole-transcriptome shotgun sequencing (RNA-seq) and assayed relevant enzyme activities. We found a marked pattern of LOX up-regulation in a narrow (5-mm, 48-h) zone at the hyphal front, which included many genes likely involved in ROS generation. Up-regulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with the notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for brown rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin-and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization.B rown rot wood-degrading fungi release sequestered carbon from lignocellulose in forests (1) and have the unique ability to accomplish this without significantly removing the recalcitrant lignin that encases the structural polysaccharides. Accordingly, their decay mechanisms may provide a model for new biomass conversion technologies that not only function despite the presence of lignin but also yield lignin as a potentially useful coproduct (1-3). Deviating from their white rot ancestors, brown rot fungi have evolved mechanisms that are generally faster (4, 5) and more polysaccharide-specific because they circumvent lignin (4,(6)(7)(8). This enhanced efficiency is coupled with losses, not expansions, of key white rot genes, including many linked to lignin degradation and processive cellulose hydrolysis. For example, few brown rot fungi produce the cellobiohydrolases that are included in commercial synergistic glycoside hydrolase (GH) mixtures (9-12). These observations imply that brown rot fungi harbor novel pathways to improve saccharification yields.To explain why brown rot fungi are so efficient, despite their minimal toolkit of biodegradative enzymes, low-molecular-weight (LMW) oxidative agents have been proposed to operate in tandem with the enzymes. ...

Several species of the filamentous fungus Fusarium colonize plants and produce toxic small molecules that contaminate agricultural products, rendering them unsuitable for consumption. Among the most destructive of these species is F. graminearum, which causes disease in wheat and barley and often infests the grain with harmful trichothecene mycotoxins. Synthesis of these secondary metabolites is induced during plant infection or in culture in response to chemical signals. Our results show that trichothecene biosynthesis involves a complex developmental process that includes dynamic changes in cell morphology and the biogenesis of novel subcellular structures. Two cytochrome P-450 oxygenases (Tri4p and Tri1p) involved in early and late steps in trichothecene biosynthesis were tagged with fluorescent proteins and shown to co-localize to vesicles we provisionally call “toxisomes.” Toxisomes, the inferred site of trichothecene biosynthesis, dynamically interact with motile vesicles containing a predicted major facilitator superfamily protein (Tri12p) previously implicated in trichothecene export and tolerance. The immediate isoprenoid precursor of trichothecenes is the primary metabolite farnesyl pyrophosphate. Changes occur in the cellular localization of the isoprenoid biosynthetic enzyme HMG CoA reductase when cultures non-induced for trichothecene biosynthesis are transferred to trichothecene biosynthesis inducing medium. Initially localized in the cellular endomembrane system, HMG CoA reductase, upon induction of trichothecene biosynthesis, increasingly is targeted to toxisomes. Metabolic pathways of primary and secondary metabolism thus may be coordinated and co-localized under conditions when trichothecene biosynthesis occurs.

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.

A previously undescribed caulimo-like virus was identified in the hybrid tobacco species Nicotiana edwardsonii, and was named tobacco vein clearing virus (TVCV) after the symptoms associated with its occurrence in this plant. The virions of TVCV are 50 nm in diameter and are composed of a 45 kDa capsid protein and a 7767 bp dsDNA genome. Each strand of the genome is interrupted by a site-specific discontinuity. In genome sequence and arrangement of ORFs TVCV was most similar to cassava vein mosaic virus, indicating that TVCV is a pararetrovirus. No serological relationship was detected between TVCV and any other caulimoviruses, including petunia vein clearing virus, which has similar biological properties. In N. edwardsonii TVCV was seed-transmitted to 100 % of progeny plants, but was not transmitted by mechanical inoculation, grafting or Myzus persicae to any of seven other Nicotiana spp. Genomic DNA of TVCV hybridized to genomic DNA of N. edwardsonii and of N. glutinosa, its male parent, but not to genomic DNA of N. clevelandii, the female parent. TVCV has 78 % sequence identity with pararetrovirus-like sequences that are present in high copy number in the N. tabacum genome, and TVCV genomic DNA hybridized to genomic DNA of N. tabacum and N. rustica. These observations suggest that the episomal form of TVCV may arise from integrated pararetroviral elements present in N. edwardsonii, that these integrants were inherited from the male parent N. glutinosa, and that these elements are related but not identical to pararetroviral elements occurring in other Nicotiana spp.

Abstract1. Despite the central role of saprotrophic fungi in wood decomposition and terrestrial carbon cycling, the diversity and functioning of wood endophytes (i.e. fungi that asymptomatically colonize living plant tissue) on decay remains poorly understood.2. In a 4-year field experiment in a boreal forest in the upper midwestern United States, we investigated whether endophytes influenced fungal community structure and subsequent wood decomposition via priority effects. We compared decay of sterilized and non-sterilized birch (Betula papyrifera) logs using both highthroughput sequencing and wood physiochemical analyses (i.e. density loss, dilute alkali solubility, ratio of lignin loss relative to density loss).3. Endophyte presence significantly altered initial fungal species composition during the first 2 years and enhanced mass loss over the experiment's duration. Results suggest that following tree death the immediate utilization of organic substrates by wood endophytes significantly alters establishment patterns of later arriving fungal saprotrophs.4. Independent of endophyte presence, white rot was the wood decay outcome at all sampling times, despite an initial presence of both brown and white rot fungi. 5.Collectively, these findings demonstrate that wood endophytes can affect early community assembly and subsequent decay rates, although environmental filtering leads to consistent selection for fungi with lignin-targeted decay strategies. K E Y W O R D Sendophyte, fungal community, historical contingency, priority effects, wood decomposition

BackgroundGenes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood.ResultsIn order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects.ConclusionsThis research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5399-x) contains supplementary material, which is available to authorized users.

This research characterized the genetics of resistance of wild barley accession PI 466423 to a widely virulent pathotype of Cochliobolus sativus . Breeding lines were identified that combine the Midwest Six-rowed Durable Resistance Haplotype and resistance to the virulent isolate ND4008. Spot blotch, caused by Cochliobolus sativus, is a historically important foliar disease of barley (Hordeum vulgare L.) in the Upper Midwest region of the USA. However, for the last 50 years this disease has been of little consequence due to the deployment of resistant six-rowed malting cultivars. These durably resistant cultivars carry the Midwest Six-rowed Durable Resistant Haplotype (MSDRH) comprised of three Quantitative Trait Loci (QTL) on chromosomes 1H, 3H and 7H, originally contributed by breeding line NDB112. Recent reports of C. sativus isolates (e.g. ND4008) with virulence on NDB112 indicate that widely grown cultivars of the region are vulnerable to spot blotch epidemics. Wild barley (H. vulgare ssp. spontaneum), the progenitor of cultivated barley, is a rich source of novel alleles, especially for disease resistance. Wild barley accession PI 466423 is highly resistant to C. sativus isolate ND4008. To determine the genetic architecture of resistance to isolate ND4008 in PI 466423, we phenotyped and genotyped an advanced backcross population (N = 244) derived from the wild accession and the recurrent parent 'Rasmusson', a Minnesota cultivar with the MSDRH. Disease phenotyping was done on BC2F4 seedlings in the greenhouse using isolate ND4008. The Rasmusson/PI 466423 population was genotyped with 7842 single nucleotide polymorphic markers. QTL analysis using composite interval mapping revealed four resistance loci on chromosomes 1H, 2H, 4H and 5H explaining 10.3, 7.4, 6.4 and 8.4 % of the variance, respectively. Resistance alleles on chromosomes 1H, 4H and 5H were contributed by PI 466423, whereas the one on chromosome 2H was contributed by Rasmusson. All four resistance QTL are likely coincident with previously identified QTL. Agronomically advanced two- and six-rowed lines combining the MSDRH and resistance alleles to isolate ND4008 have been identified and are being utilized in breeding. These results reaffirm the value of using wild relatives as a source of novel resistance alleles.