Fluorescently Tagged Verticillium dahliae to Understand the Infection Process on Cotton (Gossypium hirsutum) and Weed Plant Species.

Verticillium wilt is a soil-borne disease caused by distinct vegetative compatibility groups (VCG) of the fungus Verticillium dahliae. Defoliating (VCG 1A) and non-defoliating (VCG 2A) pathotypes of V. dahliae have contributed to yield losses of cotton production in Australia. To study the virulence and the infection process of V. dahliae on cotton, two isolates, one representing each VCG, have been transformed with fluorescent protein genes. The transformants maintained their ability to infect the host, and both strains were observed to move through the plant vasculature to induce wilt symptoms. Furthermore, virulence testing suggests that the cotton V. dahliae strains can endophytically colonise common weed plant species found in the Australian landscape, and that is contrasted by their ability to infect and colonise native tobacco plants. The fluorescently labelled strains of V. dahliae not only allowed us to gain a thorough understanding of the infection process but also provided a method to rapidly identify recovered isolates from host colonisation studies.

A comparison between the role of enniatins and deoxynivalenol in Fusarium virulence on different tissues of common wheat.

BACKGROUND: Fusarium graminearum and Fusarium avenaceum are two of the most important causal agents of Fusarium head blight (FHB) of wheat. They can produce mycotoxins that accumulate in infected wheat heads, including deoxynivalenol (DON) and enniatins (ENNs), produced by F. graminearum and F. avenaceum, respectively. While the role of DON as a virulence factor in F. graminearum toward wheat is well known, ENNs in F. avenaceum has been poorly explored. Results obtained to-date indicate that ENNs may confer an advantage to F. avenaceum only on particular hosts. RESULTS: In this study, with the use of ENN-producing and ENN non-producing F. avenaceum strains, the role of ENNs on F. avenaceum virulence was investigated on the root, stem base and head of common wheat, and compared with the role of DON, using DON-producing and DON non-producing F. graminearum strains. The DON-producing F. graminearum strain showed a significantly higher ability to cause symptoms and colonise each of the tested tissues than the non-producing strain. On the other hand, the ability to produce ENNs increased initial symptoms of the disease and fungal biomass accumulation, measured by qPCR, only in wheat heads, and not in roots or stem bases. LC-MS/MS analysis was used to confirm the presence of ENNs and DON in the different strains, and results, both in vitro and in wheat heads, were consistent with the genetics of each strain. CONCLUSION: While the key role of DON on F. graminearum virulence towards three different wheat tissues was noticeable, ENNs seemed to have a role only in influencing F. avenaceum virulence on common wheat heads probably due to an initial delay in the appearance of symptoms.

Double-stranded RNA prevents and cures infection by rust fungi.

Fungal pathogens that impact perennial plants or natural ecosystems require management strategies beyond fungicides and breeding for resistance. Rust fungi, some of the most economically and environmentally important plant pathogens, have shown amenability to double-stranded RNA (dsRNA) mediated control. To date, dsRNA treatments have been applied prior to infection or together with the inoculum. Here we show that a dsRNA spray can effectively prevent and cure infection by Austropuccinia psidii (cause of myrtle rust) at different stages of the disease cycle. Significant reductions in disease coverage were observed in plants treated with dsRNA targeting essential fungal genes 48 h pre-infection through to 14 days post-infection. For curative treatments, improvements in plant health and photosynthetic capacity were seen 2-6 weeks post-infection. Two-photon microscopy suggests inhibitory activity of dsRNA on intercellular hyphae or haustoria. Our results show that dsRNA acts both preventively and curatively against myrtle rust disease, with treated plants recovering from severe infection. These findings have immediate potential in the management of the more than 10-year epidemic of myrtle rust in Australia.

RNA-Based Control of Fungal Pathogens in Plants.

Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20-40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

Starships are active eukaryotic transposable elements mobilized by a new family of tyrosine recombinases.

Transposable elements in eukaryotic organisms have historically been considered "selfish," at best conferring indirect benefits to their host organisms. The Starships are a recently discovered feature in fungal genomes that are, in some cases, predicted to confer beneficial traits to their hosts and also have hallmarks of being transposable elements. Here, we provide experimental evidence that Starships are indeed autonomous transposons, using the model Paecilomyces variotii, and identify the HhpA "Captain" tyrosine recombinase as essential for their mobilization into genomic sites with a specific target site consensus sequence. Furthermore, we identify multiple recent horizontal gene transfers of Starships, implying that they jump between species. Fungal genomes have mechanisms to defend against mobile elements, which are frequently detrimental to the host. We discover that Starships are also vulnerable to repeat-induced point mutation defense, thereby having implications on the evolutionary stability of such elements.

Exogenous double-stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta.

Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems.

A Natural Fungal Gene Drive Enacts Killing via DNA Disruption.

Fungal spore killers are a class of selfish genetic elements that positively bias their own inheritance by killing non-inheriting gametes following meiosis. As killing takes place specifically within the developing fungal ascus, a tissue which is experimentally difficult to isolate, our understanding of the mechanisms underlying spore killers are limited. In particular, how these loci kill other spores within the fungal ascus is largely unknown. Here, we overcome these experimental barriers by developing model systems in 2 evolutionary distant organisms, Escherichia coli (bacterium) and Saccharomyces cerevisiae (yeast), similar to previous approaches taken to examine the wtf spore killers. Using these systems, we show that the Podospora anserina spore killer protein SPOK1 enacts killing through targeting DNA. IMPORTANCE Natural gene drives have shaped the genomes of many eukaryotes and recently have been considered for applications to control undesirable species. In fungi, these loci are called spore killers. Despite their importance in evolutionary processes and possible applications, our understanding of how they enact killing is limited. We show that the spore killer protein Spok1, which has homologues throughout the fungal tree of life, acts via DNA disruption. Spok1 is only the second spore killer locus in which the cellular target of killing has been identified and is the first known to target DNA. We also show that the DNA disrupting activity of Spok1 is functional in both bacteria and yeast suggesting a highly conserved mode of action.

BioClay™ prolongs RNA interference-mediated crop protection against Botrytis cinerea.

One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double-stranded RNA (dsRNA) targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea, a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides.

Early discontinuation of adjuvant chemotherapy in patients with early-stage pancreatic cancer correlates with inferior survival: A multicenter population-based cohort study.

BACKGROUND: The current study aimed to determine the association between timing and completion of adjuvant chemotherapy and outcomes in real-world patients with early-stage pancreatic cancer. METHODS: In this multi-center cohort study patients with early-stage pancreatic cancer who were diagnosed from 2007-2017 and underwent complete resection in the province of Saskatchewan were examined. Cox proportional multivariate analyses were performed for correlation with recurrence and survival. RESULTS: Of 168 patients, 71 eligible patients with median age of 69 years and M:F of 37:34 were identified. Median time to the start of adjuvant therapy from surgery was 73 days. Of all patients, 49 (69%) patients completed adjuvant chemotherapy and 22 (31%) required early treatment discontinuation. Median recurrence-free survival of patients who completed treatment was 22 months (95%CI:15.8-28.2) vs. 9 months (3.3-14.7) if treatment was discontinued early (P<0.001). Median overall survival of those who completed treatment was 33 (17.5-48.5) vs. 16 months (17.5-48.5) with early treatment discontinuation (P<0.001). In the multivariate analysis, treatment discontinuation was significantly correlated with recurrent disease, hazard ratio (HR), 2.57 (1.41-4.68), P = 0.002 and inferior survival, HR, 2.55 (1.39-4.68), P = 0.003. No correlation between treatment timing and survival was noted. CONCLUSIONS: Early discontinuation but not the timing of adjuvant chemotherapy correlates with inferior outcomes.

Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase.

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS's, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively.

Transcriptome analysis reveals infection strategies employed by Fusarium graminearum as a root pathogen.

The fungal pathogen Fusarium graminearum (Fg) infects both heads and roots of cereal crops causing several economically important diseases such as head blight, seedling blight, crown rot and root rot. Trichothecene mycotoxins such as deoxynivalenol (DON), a well-known virulence factor, produced by Fg during disease development is also an important health concern. Although how Fg infects above-ground tissues is relatively well studied, very little is known about molecular processes employed by the pathogen during below-ground infection. Also unknown is the role of DON during root infection. In the present study, we analyzed the transcriptome of Fg during root infection of the model cereal Brachypodium distachyon (Bd). We also compared our Fg transcriptome data obtained during Bd root infection with those reported during wheat head infection. These analyses suggested that both shared and unique infection strategies were employed by the pathogen during colonization of different host tissues. Several metabolite biosynthesis genes induced in Fg during root infection could be linked to phytohormone production, implying that the pathogen likely interferes with root specific defenses. In addition, to understand the role of DON in Fg root infection, we analyzed the transcriptome of the DON deficient Tri5 mutant. These analyses showed that the absence of DON had a significant effect on fungal transcriptional responses. Although DON was produced in infected roots, this mycotoxin did not act as a Fg virulence factor during root infection. Our results reveal new mechanistic insights into the below-ground strategies employed by Fg that may benefit the development of new genetic tools to combat this important cereal pathogen.

Unlocking inaccessible historical genomes preserved in formalin.

Museum specimens represent an unparalleled record of historical genomic data. However, the widespread practice of formalin preservation has thus far impeded genomic analysis of a large proportion of specimens. Limited DNA sequencing from formalin-preserved specimens has yielded low genomic coverage with unpredictable success. We set out to refine sample processing methods and to identify specimen characteristics predictive of sequencing success. With a set of taxonomically diverse specimens collected between 1962 and 2006 and ranging in preservation quality, we compared the efficacy of several end-to-end whole genome sequencing workflows alongside a k-mer-based trimming-free read alignment approach to maximize mapping of endogenous sequence. We recovered complete mitochondrial genomes and up to 3× nuclear genome coverage from formalin-preserved tissues. Hot alkaline lysis coupled with phenol-chloroform extraction out-performed proteinase K digestion in recovering DNA, while library preparation method had little impact on sequencing success. The strongest predictor of DNA yield was overall specimen condition, which additively interacts with preservation conditions to accelerate DNA degradation. Here, we demonstrate a significant advance in capability beyond limited recovery of a small number of loci via PCR or target-capture sequencing. To facilitate strategic selection of suitable specimens for genomic sequencing, we present a decision-making framework that utilizes independent and nondestructive assessment criteria. Sequencing of formalin-preserved specimens will contribute to a greater understanding of temporal trends in genetic adaptation, including those associated with a changing climate. Our work enhances the value of museum collections worldwide by unlocking genomes of specimens that have been disregarded as a valid molecular resource.

A method for high-throughput image-based antifungal screening.

Robust antifungal screening is technically challenging particularly for filamentous fungi. We present a method for undertaking antifungal screening assays that builds upon existing broth dilution protocols and incorporates time resolved image-based assessment of fungal growth. We show that the method performs with different fungi, particularly those for which spores can be used as inoculum, and with different compound classes, can accurately assess susceptibility or otherwise in only few hours and can even account for differences in inherent growth properties of strains.

Adaptive defence and sensing responses of host plant roots to fungal pathogen attack revealed by transcriptome and metabolome analyses.

Plant root-produced constitutive and inducible defences inhibit pathogenic microorganisms within roots and in the rhizosphere. However, regulatory mechanisms underlying host responses during root-pathogen interactions are largely unexplored. Using the model species Brachypodium distachyon (Bd), we studied transcriptional and metabolic responses altered in Bd roots following challenge with Fusarium graminearum (Fg), a fungal pathogen that causes diseases in diverse organs of cereal crops. Shared gene expression patterns were found between Bd roots and spikes during Fg infection associated with the mycotoxin deoxynivalenol (DON). Overexpression of BdMYB78, an up-regulated transcription factor, significantly increased root resistance during Fg infection. We show that Bd roots recognize encroaching Fg prior to physical contact by altering transcription of genes associated with multiple cellular processes such as reactive oxygen species and cell development. These changes coincide with altered levels of secreted host metabolites detected by an untargeted metabolomic approach. The secretion of Bd metabolites was suppressed by Fg as enhanced levels of defence-associated metabolites were found in roots during pre-contact with a Fg mutant defective in host perception and the ability to cause disease. Our results help to understand root defence strategies employed by plants, with potential implications for improving the resistance of cereal crops to soil pathogens.

Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum.

DNA methylation mediates organisms' adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

A conserved Zn2Cys6 transcription factor, identified in a spontaneous mutant from in vitro passaging, is involved in pathogenicity of the blackleg fungus Leptosphaeria maculans.

Continuous passaging in vitro can lead to the accumulation of changes in DNA sequence that potentially affect the properties of microbes, making them different from the original isolates. The identification of such genetic alterations is rare in fungi. A set of insertional mutants in the plant pathogenic fungus Leptosphaeria maculans, all derived from the same transformation experiment, had independent Agrobacterium T-DNA insertions and reduced pathogenicity on canola (Brassica napus). None of the insertions co-segregated in progeny from crosses with the reduction in pathogenicity. Genome sequences of three strains were analysed, and a mutation identified in a gene (ptf1, for pathogenicity-associated transcription factor 1) encoding a putative Zn2(II)Cys6 transcription factor. Homologs are found in other ascomycetes, and are required for pathogenicity by Fusarium graminearum, Fusarium oxysporum and Magnaporthe oryzae. The mutation in the L. maculans ptf1 gene co-segregates in progeny from crosses with the reduction in pathogenicity, a strain with an independent mutant allele isolated using CRISPR-Cas9 editing has reduced pathogenicity, and addition of wild type copies of the gene restores pathogenicity. Thus, this work defines a base pair substitution that occurred during in vitro passaging of a fungus that contributed to an attenuation of pathogenicity.

Comparison of Perioperative Chemotherapy versus Postoperative Chemoradiotherapy for Operable Stomach Cancer: A Western Canadian Province Experience.

BACKGROUND: The standard approaches for resectable stomach cancer are postoperative chemoradiotherapy (PCR) or perioperative chemotherapy (PC). Limited evidence is available regarding the superiority of one of the two approaches. We aimed to compare the survival of patients with operable stomach cancer who were treated with PC or PCR. METHODS: In this retrospective cohort study, patients with operable stomach cancer diagnosed between 2005-2015 in the province of Saskatchewan were identified and, based on type of treatment, were placed into PCR and PC groups. A Cox proportional multivariate analysis was performed to assess independent prognostic variables, including survival advantage of PC over PCR. RESULTS: A total of 88 eligible patients with a median age of 66 (56-71) and a male to female ratio of 1:0.44 were identified. Seventy-three (83%) patients had pathologically node positive disease. Sixty-seven (76%) patients received PCR, while 21 (24%) patients received PC. The median overall survival of the whole group was 34 months, with 38 months (95% CI 24.6-51.3) in the PCR group vs. 30 months (14.3-45.7) in the PC group (p = 0.29). Median relapse-free survival was 34 months (20.7-47.3) in the PCR group vs. 23 months (6.7-39.3) in the PC group (p = 0.20). Toxicities were comparable. On multivariate analysis, T ≥ 3 tumor (HR, 3.57 (1.39-8.56)), neutrophil to lymphocyte ratio (LNR) > 2.8 (HR, 1.85 (1.05-3.25)), and positive resection margins (HR, 1.89 (1.06-3.37)) were independently correlated with inferior survival. CONCLUSIONS: This well-designed population based cohort study suggests a lack of survival benefit of PC over PCR. Both treatment options remain viable approaches for resectable stomach cancer.

Map-based cloning identifies velvet A as a critical component of virulence in Fusarium pseudograminearum during infection of wheat heads.

Although better known as a pathogen of wheat stem bases, Fusarium pseudograminearum also causes Fusarium head blight. A natural isolate of F. pseudograminearum was identified that showed severely reduced virulence towards wheat heads and a map-based cloning approach was undertaken to identify the genetic basis of this phenotype. Using a population of 95 individuals, a single locus on chromosome 1 was shown to be responsible for the low virulence. Fine mapping narrowed the region to just five possible SNPs of which one was in the F. pseudograminearum homologue of velvet A. Knockout mutants of velvet A, which were non-pathogenic towards wheat, confirmed that velvet A regulates virulence in this pathogen. The mutation in velvet A was only found in a single field isolate and the origin of the mutation is unknown.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Can natural gene drives be part of future fungal pathogen control strategies in plants?

Globally, fungal pathogens cause enormous crop losses and current control practices are not always effective, economical or environmentally sustainable. Tools enabling genetic management of wild pathogen populations could potentially solve many problems associated with plant diseases. A natural gene drive from a heterologous species can be used in the globally important cereal pathogen Fusarium graminearum to remove pathogenic traits from contained populations of the fungus. The gene drive element became fixed in a freely crossing population in only three generations. Repeat-induced point mutation (RIP), a natural genome defence mechanism in fungi that causes C to T mutations during meiosis in highly similar sequences, may be useful to recall the gene drive following release, should a failsafe mechanism be required. We propose that gene drive technology is a potential tool to control plant pathogens once its efficacy is demonstrated under natural settings.