Biology subjects

Gardiner, D. M.

Publications and source records attributed to Gardiner, D. M..

9 recordsLinked to original sources

bioRxiv2024

dsRNAmax: a Multi-Target Chimeric dsRNA Designer for Safe And Effective Crop Protection

Crop protection is undergoing significant evolution, transitioning toward sustainable approaches that minimise impacts on the environment and human health. Exogenous application of double-stranded RNA (dsRNA) that silences pest or pathogen genes via RNAi (RNA interference) has promise as a safe and effective next generation crop protection platform without the need for genetic modification. However, exogenous dsRNA application at scale presents challenges. Specifically, a single dsRNA sequence needs to balance targeting the standing variation in a target pest or pathogen group against the potential for adverse impacts in a vast array of non-target and beneficial organisms at the application site and broader environment. To address these competing demands, we present dsRNAmax (https://github.com/sfletc/dsRNAmax), a software package that employs k-mer based assembly of chimeric dsRNA sequences to target multiple related RNA sequences, to broaden the target spectrum. The package ensures designed dsRNAs have no defined contiguous sequence homology with any off-target sequences, which can range from single transcriptomes through to metagenome sequence data and beyond. The efficacy of this package is demonstrated by a dsRNAmax-designed dsRNA that inhibits multiple root-knot nematode species but not a non-target nematode species, despite its susceptibility to environmental RNAi and high homology of the target gene. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/628581v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@c776c5org.highwire.dtl.DTLVardef@1468e45org.highwire.dtl.DTLVardef@1268b08org.highwire.dtl.DTLVardef@171ee19_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

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 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 transfer of Starships, implying that they frequency 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 defence, thereby having implications on the evolutionary stability of such elements.

microbiology↗

bioRxiv2022

A natural fungal gene drive enacts killing through targeting DNA

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 two evolutionary distant organisms, Escherichia coli (bacterium) and Saccharomyces cerevisiae (yeast). Using these systems, we show that the Podospora anserina spore killer protein Spok1 enacts killing through targeting DNA. Significance StatementNatural 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.

genetics↗

bioRxiv2021

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, performs well even without replication and can even account for differences in inherent growth properties of strains.

microbiology↗

bioRxiv2021

Unlocking inaccessible historical genomes preserved in formalin

BackgroundMuseum specimens represent an unparalleled record of historical genomic data. However, the wide-spread 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 1936 and 2015 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. ResultsWe recovered complete mitochondrial genomes and up to 3X nuclear genome coverage from formalin-fixed 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. ConclusionsWe 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 non-destructive assessment criteria. Sequencing of formalin-fixed 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.

genomics↗

bioRxiv2020

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

The fungal pathogen Fusarium graminearum infect 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 F. graminearum (Fg) during disease development is also an important health concern. Although how F. graminearum 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 F. graminearum during root infection of the model cereal Brachypodium distachyon. We also compared our Fg transcriptome data during root infection with those reported during wheat head infection. These analyses suggested that both shared and unique infection strategies employed by the pathogen during colonization of different host tissues. Several metabolite biosynthesis genes induced in F. graminearum during root infection could be linked to phytohormone production, implying that the pathogen likely interferes 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 virulence factor during root infection. Our results reveal new mechanistic insights into the below-ground strategies employed by F. graminearum that may benefit the development of new genetic tools to combat this important cereal pathogen.

microbiology↗

bioRxiv2020

Natural gene drives offer potential pathogen control strategies in plants

O_LIGlobally, 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. C_LIO_LIA 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 populations in only three generations. C_LIO_LIRepeat induce point mutation, a natural genome defence mechanism in fungi, may be useful to recall the gene drive following release, should a failsafe mechanism be required. C_LIO_LIWe propose that gene drive technology is a potential tool to control plant pathogens. C_LI

genetics↗

bioRxiv2019

Novel regulators of nitric oxide signaling triggered by host perception in a plant pathogen

The rhizosphere interaction between plant roots or pathogenic microbes is initiated by mutual exchange of signals. However, how soil pathogens sense host signals is largely unknown. Here, we studied early molecular events associated with host recognition in Fusarium graminearum, an economically important fungal pathogen that can infect both roots and heads of cereal crops. We found that host-sensing prior to physical contact with plant roots radically alters the transcriptome and triggers nitric oxide (NO) production in F. graminearum. We identified an ankyrin-repeat domain containing protein (FgANK1) required for host-mediated NO production and virulence in F. graminearum. In the absence of host plant, FgANK1 resides in the cytoplasm. In response to host signals, FgANK1 translocates to the nucleus and interacts with a zinc finger transcription factor (FgZC1), also required for NO production and virulence in F. graminearum. Our results reveal new mechanistic insights into host-recognition strategies employed by soil pathogens.

pathology↗

bioRxiv2018

Improved prediction of fungal effector proteins from secretomes with EffectorP 2.0

Plant-pathogenic fungi secrete effector proteins to facilitate infection. We describe extensive improvements to EffectorP, the first machine learning classifier for fungal effector prediction. EffectorP 2.0 is now trained on a larger set of effectors and utilizes a different approach based on an ensemble of classifiers trained on different subsets of negative data, offering different views on classification. EffectorP 2.0 achieves accuracy of 89%, compared to 82% for EffectorP 1.0 and 59.8% for a small size classifier. Important features for effector prediction appear to be protein size, protein net charge as well as the amino acids serine and cysteine. EffectorP 2.0 decreases the number of predicted effectors in secretomes of fungal plant symbionts and saprophytes by 40% when compared to EffectorP 1.0. However, EffectorP 1.0 retains value and combining EffectorP 1.0 and 2.0 results in a stringent classifier with low false positive rate of 9%. EffectorP 2.0 predicts significant enrichments of effectors in 12 out of 13 sets of infection-induced proteins from diverse fungal pathogens, whereas a small cysteine-rich classifier detects enrichment only in 7 out of 13. EffectorP 2.0 will fast-track prioritization of high-confidence effector candidates for functional validation and aid in improving our understanding of effector biology. EffectorP 2.0 is available at http://effectorp.csiro.au.

bioinformatics↗