Fungi have been used to better the lives of everyday people and unravel the mysteries of higher eukaryotic organisms for decades. However, comparing progress and development stemming from fungal research to that of human, plant, and bacterial research, fungi remain largely understudied and underutilized. Recent commercial ventures have begun to gain popularity in society, providing a new surge of interest in fungi, mycelia, and potential new applications of these organisms to various aspects of research. Biotechnological advancements in fungal research cannot occur without intensive amounts of time, investments, and research tool development. In this review, we highlight past breakthroughs in fungal biotechnology, discuss requirements to advance fungal biotechnology even further, and touch on the horizon of new breakthroughs with the highest potential to positively impact both research and society.

Deoxynivalenol (DON) contamination of cereal grains caused by Fusarium head blight may be addressed by future RNA interference (RNAi)-based gene silencing approaches. However, utilizing these approaches will require a greater understanding of the principles that govern RNAi effectiveness in the pathogen Fusarium graminearum. RNAi in higher eukaryotes, including fungi, involves processing double stranded RNA (dsRNA) into small interfering RNA (siRNA) that silence gene expression based on base pair complementarity. This study examined virulence, DON production, and the small RNA (sRNA) populations in response to RNAi-based silencing of TRI6, a transcription factor that positively regulates DON synthesis via control of TRI5 expression. Silencing was accomplished via the expression of transgenes encoding inverted repeats targeting various regions of TRI6 (RNAi vectors). Transgene expression was associated with novel, TRI6-specific siRNAs. For RNAi vectors targeting the majority of TRI6 sequence (~600 bp), a discontinuous, repeatable pattern was observed in which most siRNAs mapped to specific regions of TRI6. Targeting shorter regions (250–350 bp) did not alter the siRNA populations corresponding to that region of TRI6. No phased processing was observed. The 5' base of ~83% of siRNAs was uracil, consistent with DICER processing and ARGONAUTE binding preferences for siRNA. Mutant lines showed TRI6 siRNA-associated reductions of TRI5 expression on toxin inducing media and DON in infected wheat and barley spikes. Shorter RNAi vectors resulted in variable levels of silencing that were less than for the ~600 bp RNAi vector, with a 343 bp RNAi vector targeting the 5’ end of TRI6 having the best silencing efficiency. This work identifies efficient shorter region for silencing of TRI6 and describes the patterns of siRNA corresponding to those regions.

For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host–pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and genders to discuss the diverse roles of mycotoxins in the lifestyles of filamentous fungi of the families Clavicipitaceae, Trichocomaceae (Eurotiales), and Nectriaceae (Hypocreales).

Benzoxazinoid (Bx) metabolites produced by wheat and other members of the Poaceae have activity against Fusarium sp. that cause cereal diseases including Fusarium head blight (FHB) on wheat and barley. Certain Bx metabolites can be detoxified by Fusarium sp. with the arylamine N-acetyltransferase NAT1. Investigation of this pathway may reveal strategies for increasing FHB resistance, such as selection for higher levels of Bx metabolites within existing germplasm and/or engineering fungal susceptibility via host induced silencing of NAT1. We assessed the reactions of fifteen wheat cultivars or breeding lines adapted to the Northwestern United States to infection with F. graminearum Δnat1 mutants that should be sensitive to Bx metabolites. Significant differences were noted in disease severity and deoxynivalenol (DON) among the cultivars 21 d after inoculation with either mutant or wildtype (PH1) strains. Mutant vs. wildtype strains did not result in significant variation for infection severity (as measured by % infected florets), but inoculation with Δnat1 mutants vs. wildtype resulted in significantly lower DON concentrations in mature kernels (p < 0.0001). Of the cultivars tested, HRS3419 was the most resistant cultivar to PH1 (severity = 62%, DON = 45 ppm) and Δnat1 mutants (severity = 61%, DON = 30 ppm). The cultivar most susceptible to infection was Kelse with PH1 (severity = 100%, DON = 292 ppm) and Δnat1 mutants (severity = 100%, DON = 158 ppm). We hypothesized that sub-lethal Bx metabolite levels may suppress DON production in F. graminearum Δnat1 mutants. In vitro assays of Bx metabolites BOA, MBOA, and DIMBOA at 30 μM did not affect growth, but did reduce DON production by Δnat1 and PH1. Although the levels of Bx metabolites are likely too low in the wheat cultivars we tested to suppress FHB, higher levels of Bx metabolites may contribute towards reductions in DON and FHB.

Fusarium solani is a species complex encompassing a large phylogenetic clade with diverse members occupying varied habitats. We recently reported a unique opportunistic F. solani associated with unusual dark galls in sugarbeet. We assembled the chromosome-level genome of the F. solani sugarbeet isolate strain SB1 using Oxford Nanopore and Hi-C sequencing. The average size of F. solani genomes is 54 Mb, whereas SB1 has a larger genome of 59.38 Mb, organized into 15 chromosomes. The genome expansion of strain SB1 is due to the high repeats and segmental duplications within its three potentially accessory chromosomes. These chromosomes are absent in the closest reference genome with chromosome-level assembly, F. vanettenii 77-13-4. Segmental duplications were found in three chromosomes but are most extensive between two specific SB1 chromosomes, suggesting that this isolate may have doubled its accessory genes. Further comparison of the F. solani strain SB1 genome demonstrates inversions and syntenic regions to an accessory chromosome of F. vanettenii 77-13-4. The pan-genome of 12 publicly available F. solani isolates nearly reached gene saturation, with few new genes discovered after the addition of the last genome. Based on orthogroups and average nucleotide identity, F. solani is not grouped by lifestyle or origin. The pan-genome analysis further revealed the enrichment of several enzymes-coding genes within the dispensable (accessory + unique genes) genome, such as hydrolases, transferases, oxidoreductases, lyases, ligases, isomerase, and dehydrogenase. The evidence presented here suggests that genome plasticity, genetic diversity, and adaptive traits in Fusarium solani are driven by the dispensable genome with significant contributions from segmental duplications.

Key message: We transferred the Tri6 gene into the elite barley GemCraft via new transformation method through shoot organogenesis and identified the rearrangements of transgenes and phenotypic variations in the transgenic plants. Despite its agronomic and economic importance, barley transformation is still very challenging for many elite varieties. In this study, we used direct shoot organogenesis to transform the elite barley cultivar GemCraft with the RNAi constructs containing Tri6 gene of Fusarium graminearum, which causes fusarium head blight (FHB). We isolated 4432 shoot tips and co-cultured these explants with Agrobacterium tumefaciens. A total of 25 independent T0 transgenic plants were generated including 15 events for which transgene-specific PCR amplicons were observed. To further determine the presence of transgenes, the T1 progenies of all 15 T0 plants were analyzed, and the expected PCR products were obtained in 10 T1 lines. Droplet digital (dd) PCR analysis revealed various copy numbers of transgenes in the transgenic plants. We determined the insertion site of transgenes using long-read sequencing data and observed the rearrangements of transgenes. We found phenotypic variations in both T1 and T2 generation plants. FHB disease was evaluated under growth chamber conditions, but no significant differences in disease severity or deoxynivalenol accumulation were observed between two Tri6 transgenic lines and the wildtype. Our results demonstrate the feasibility of the shoot tip transformation and may open the door for applying this system for genetic improvement and gene function research in other barley genotypes.