First detection and complete genome sequence of a new potexvirus naturally infecting Adenium obesum.

Here, we report the detection and complete genome sequence of a novel potexvirus, tentatively named "Adenium obesum virus X" (AobVX), isolated from Adenium obesum, that was sent for virus screening at Australian Government post-entry quarantine (PEQ) facilities after being imported into Australia from China. The AobVX genome is 6781 nucleotides in length excluding the poly(A) tail and is predicted to encode conserved potexvirus proteins and sequence motifs across five open reading frames. The RNA-dependent RNA polymerase of this virus shares the highest amino acid sequence similarity with that of nerine potexvirus 1 (58.7% identity) and nerine virus X (58.58% identity). This is the first report of a positive-sense single-stranded RNA virus in A. obesum related to members of the genus Potexvirus in the family Alphaflexiviridae.

New Virus Diagnostic Approaches to Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand.

To protect New Zealand's unique ecosystems and primary industries, imported plant materials must be constantly monitored at the border for high-threat pathogens. Techniques adopted for this purpose must be robust, accurate, rapid, and sufficiently agile to respond to new and emerging threats. Polymerase chain reaction (PCR), especially real-time PCR, remains an essential diagnostic tool but it is now being complemented by high-throughput sequencing using both Oxford Nanopore and Illumina technologies, allowing unbiased screening of whole populations. The demand for and value of Point-of-Use (PoU) technologies, which allow for in situ screening, are also increasing. Isothermal PoU molecular diagnostics based on recombinase polymerase amplification (RPA) and loop-mediated amplification (LAMP) do not require expensive equipment and can reach PCR-comparable levels of sensitivity. Recent advances in PoU technologies offer opportunities for increased specificity, accuracy, and sensitivities which makes them suitable for wider utilization by frontline or border staff. National and international activities and initiatives are adopted to improve both the plant virus biosecurity infrastructure and the integration, development, and harmonization of new virus diagnostic technologies.

Implementation of GA-VirReport, a Web-Based Bioinformatics Toolkit for Post-Entry Quarantine Screening of Virus and Viroids in Plants.

High-throughput sequencing (HTS) of host plant small RNA (sRNA) is a popular approach for plant virus and viroid detection. The major bottlenecks for implementing this approach in routine virus screening of plants in quarantine include lack of computational resources and/or expertise in command-line environments and limited availability of curated plant virus and viroid databases. We developed: (1) virus and viroid report web-based bioinformatics workflows on Galaxy Australia called GA-VirReport and GA-VirReport-Stats for detecting viruses and viroids from host plant sRNA extracts and (2) a curated higher plant virus and viroid database (PVirDB). We implemented sRNA sequencing with unique dual indexing on a set of plants with known viruses. Sequencing data were analyzed using GA-VirReport and PVirDB to validate these resources. We detected all known viruses in this pilot study with no cross-sample contamination. We then conducted a large-scale diagnosis of 105 imported plants processed at the post-entry quarantine facility (PEQ), Australia. We detected various pathogens in 14 imported plants and discovered that de novo assembly using 21-22 nt sRNA fraction and the megablast algorithm yielded better sensitivity and specificity. This study reports the successful, large-scale implementation of HTS and a user-friendly bioinformatics workflow for virus and viroid screening of imported plants at the PEQ.

Complete genome sequence of a novel potyvirus infecting Miscanthus sinensis (silver grass).

Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named "Miscanthus sinensis mosaic virus" (MsiMV), isolated from Miscanthus sinensis (silver grass) held in a post-entry quarantine facility after being imported into Western Australia, Australia. The MsiMV genome is 9604 nucleotides (nt) in length, encoding a 3071-amino-acid (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to that of sorghum mosaic virus (SrMV), with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV), and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.

Side-by-Side Comparison of Post-Entry Quarantine and High Throughput Sequencing Methods for Virus and Viroid Diagnosis.

Rapid and safe access to new plant genetic stocks is crucial for primary plant industries to remain profitable, sustainable, and internationally competitive. Imported plant species may spend several years in Post Entry Quarantine (PEQ) facilities, undergoing pathogen testing which can impact the ability of plant industries to quickly adapt to new global market opportunities by accessing new varieties. Advances in high throughput sequencing (HTS) technologies provide new opportunities for a broad range of fields, including phytosanitary diagnostics. In this study, we compare the performance of two HTS methods (RNA-Seq and sRNA-Seq) with that of existing PEQ molecular assays in detecting and identifying viruses and viroids from various plant commodities. To analyze the data, we tested several bioinformatics tools which rely on different approaches, including direct-read, de novo, and reference-guided assembly. We implemented VirusReport, a new portable, scalable, and reproducible nextflow pipeline that analyses sRNA datasets to detect and identify viruses and viroids. We raise awareness of the need to evaluate cross-sample contamination when analyzing HTS data routinely and of using methods to mitigate index cross-talk. Overall, our results suggest that sRNA analyzed using VirReport provides opportunities to improve quarantine testing at PEQ by detecting all regulated exotic viruses from imported plants in a single assay.

Evolution of Plant Virus Diagnostics Used in Australian Post Entry Quarantine.

As part of a special edition for MDPI on plant virology in Australia, this review provides a brief high-level overview on the evolution of diagnostic techniques used in Australian government Post-Entry Quarantine (PEQ) facilities for testing imported plants for viruses. A comprehensive range of traditional and modern diagnostic approaches have historically been employed in PEQ facilities using bioassays, serological, and molecular techniques. Whilst these techniques have been effective, they are time consuming, resource intensive and expensive. The review highlights the importance of ensuring the best available science and diagnostic developments are constantly tested, evaluated, and implemented by regulators to ensure primary producers have rapid and safe access to new genetics to remain productive, sustainable and competitive.

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.

Constitutive expression of transcription factor SirZ blocks pathogenicity in Leptosphaeria maculans independently of sirodesmin production.

Sirodesmin, the major secondary metabolite produced by the plant pathogenic fungus Leptosphaeria maculans in vitro, has been linked to disease on Brassica species since the 1970s, and yet its role has remained ambiguous. Re-examination of gene expression data revealed that all previously described genes and two newly identified genes within the sir gene cluster in the genome are down-regulated during the crucial early establishment stages of blackleg disease on Brassica napus. To test if this is a strategy employed by the fungus to avoid damage to and then detection by the host plant during the L. maculans asymptomatic biotrophic phase, sirodesmin was produced constitutively by overexpressing the sirZ gene encoding the transcription factor that coordinates the regulation of the other genes in the sir cluster. The sirZ over-expression strains had a major reduction in pathogenicity. Mutation of the over-expression construct restored pathogenicity. However, mutation of two genes, sirP and sirG, required for specific steps in the sirodesmin biosynthesis pathway, in the sirZ over-expression background resulted in strains that were unable to synthesize sirodesmin, yet were still non-pathogenic. Elucidating the basis for this pathogenicity defect or finding ways to overexpress sirZ during disease may provide new strategies for the control of blackleg disease.

Identification of a gene cluster for the synthesis of the plant hormone abscisic acid in the plant pathogen Leptosphaeria maculans.

Leptosphaeria maculans is an ascomycetous fungus that causes the disease blackleg on Brassica napus (canola). In spite of the importance of the disease worldwide, the mechanisms of disease development are poorly understood. Secondary metabolites, which are one of the common virulence factors of pathogenic fungi, have not been extensively explored from this fungus. An RNA-seq dataset was examined to find genes responsible for secondary metabolite synthesis by this fungus during infection. One polyketide synthase gene, pks5, was found to be upregulated during the early biotrophic stage of development. In addition to pks5, six other genes adjacent to the pks5 gene, including one encoding a Zn(II)2Cys6 transcription factor abscisic acid-like 7 gene (abl7), were also upregulated during that time. A striking feature of the L. maculans genome is that it contains large AT-rich regions that are gene-poor and large GC-rich regions that are gene rich. This set of seven co-regulated genes is embedded within and separated by two such AT-rich regions. Three of the genes in the cluster have similarities to those known to be involved in the synthesis of abscisic acid (ABA) in other fungi. When L. maculans is grown in axenic culture the genes in this cluster are not expressed and ABA is not produced. Overexpressing abl7, encoding the putative transcription factor, resulted in the transcription of the six adjacent genes in axenic culture and in the production of ABA, as detected by liquid chromatography quadrupole-time-of-flight mass spectrometry analysis. Mutation of two genes of the cluster using CRISPR/Cas9 did not affect pathogenicity on canola cotyledons. The characterization of the ABA gene cluster has led to the discovery of the co-regulation of genes within an AT-rich region by a transcription factor, and the first report of the plant hormone abscisic acid being produced by L. maculans.

Analysis of Repeat Induced Point (RIP) Mutations in Leptosphaeria maculans Indicates Variability in the RIP Process Between Fungal Species.

Gene duplication contributes to evolutionary potential, yet many duplications in a genome arise from the activity of "selfish" genetic elements such as transposable elements. Fungi have a number of mechanisms by which they limit the expansion of transposons, including Repeat Induced Point mutation (RIP). RIP has been best characterized in the Sordariomycete Neurospora crassa, wherein duplicated DNA regions are recognized after cell fusion, but before nuclear fusion during the sexual cycle, and then mutated. While "signatures" of RIP appear in the genome sequences of many fungi, the species most distant from N. crassa in which the process has been experimentally demonstrated to occur is the Dothideomycete Leptosphaeria maculans In the current study, we show that similar to N. crassa, nonlinked duplications can trigger RIP; however, the frequency of the generated RIP mutations is extremely low in L maculans (< 0.1%) and requires a large duplication to initiate RIP, and that multiple premeiotic mitoses are involved in the RIP process. However, a single sexual cycle leads to the generation of progeny with unique haplotypes, despite progeny pairs being generated from mitosis. We hypothesize that these different haplotypes may be the result of the deamination process occurring post karyogamy, leading to unique mutations within each of the progeny pairs. These findings indicate that the RIP process, while common to many fungi, differs between fungi and that this impacts on the fate of duplicated DNA.

Clues to an Evolutionary Mystery: The Genes for T-Toxin, Enabler of the Devastating 1970 Southern Corn Leaf Blight Epidemic, Are Present in Ancestral Species, Suggesting an Ancient Origin.

The Southern corn leaf blight (SCLB) epidemic of 1970 devastated fields of T-cytoplasm corn planted in monoculture throughout the eastern United States. The epidemic was driven by race T, a previously unseen race of Cochliobolus heterostrophus. A second fungus, Phyllosticta zeae-maydis, with the same biological specificity, appeared coincidentally. Race T produces T-toxin, while Phyllosticta zeae-maydis produces PM-toxin, both host-selective polyketide toxins necessary for supervirulence. The present abundance of genome sequences offers an opportunity to tackle the evolutionary origins of T- and PM- toxin biosynthetic genes, previously thought unique to these species. Using the C. heterostrophus genes as probes, we identified orthologs in six additional Dothideomycete and three Eurotiomycete species. In stark contrast to the genetically fragmented race T Tox1 locus that encodes these genes, all newly found Tox1-like genes in other species reside at a single collinear locus. This compact arrangement, phylogenetic analyses, comparisons of Tox1 protein tree topology to a species tree, and Tox1 gene characteristics suggest that the locus is ancient and that some species, including C. heterostrophus, gained Tox1 by horizontal gene transfer. C. heterostrophus and Phyllosticta zeae-maydis did not exchange Tox1 DNA at the time of the SCLB epidemic, but how they acquired Tox1 remains uncertain. The presence of additional genes in Tox1-like clusters of other species, although not in C. heterostrophus and Phyllosticta zeae-maydis, suggests that the metabolites produced differ from T- and PM-toxin.

A silver bullet in a golden age of functional genomics: the impact of Agrobacterium-mediated transformation of fungi.

The implementation of Agrobacterium tumefaciens as a transformation tool revolutionized approaches to discover and understand gene functions in a large number of fungal species. A. tumefaciens mediated transformation (AtMT) is one of the most transformative technologies for research on fungi developed in the last 20 years, a development arguably only surpassed by the impact of genomics. AtMT has been widely applied in forward genetics, whereby generation of strain libraries using random T-DNA insertional mutagenesis, combined with phenotypic screening, has enabled the genetic basis of many processes to be elucidated. Alternatively, AtMT has been fundamental for reverse genetics, where mutant isolates are generated with targeted gene deletions or disruptions, enabling gene functional roles to be determined. When combined with concomitant advances in genomics, both forward and reverse approaches using AtMT have enabled complex fungal phenotypes to be dissected at the molecular and genetic level. Additionally, in several cases AtMT has paved the way for the development of new species to act as models for specific areas of fungal biology, particularly in plant pathogenic ascomycetes and in a number of basidiomycete species. Despite its impact, the implementation of AtMT has been uneven in the fungi. This review provides insight into the dynamics of expansion of new research tools into a large research community and across multiple organisms. As such, AtMT in the fungi, beyond the demonstrated and continuing power for gene discovery and as a facile transformation tool, provides a model to understand how other technologies that are just being pioneered, e.g. CRISPR/Cas, may play roles in fungi and other eukaryotic species.

An avirulence gene, AvrLmJ1, from the blackleg fungus, Leptosphaeria maculans, confers avirulence to Brassica juncea cultivars.

The fungus Leptosphaeria maculans causes blackleg of Brassica species. Here, we report the mapping and subsequent cloning of an avirulence gene from L. maculans. This gene, termed AvrLmJ1, confers avirulence towards all three Brassica juncea cultivars tested. Analysis of RNA-seq data showed that AvrLmJ1 is housed in a region of the L. maculans genome which contains only one gene that is highly expressed in planta. The closest genes are 57 and 33 kb away and, like other avirulence genes of L. maculans, AvrLmJ1 is located within an AT-rich, gene-poor region of the genome. The encoded protein is 141 amino acids, has a predicted signal peptide and is cysteine rich. Two virulent isolates contain a premature stop codon in AvrLmJ1. Complementation of an isolate that forms cotyledonary lesions on B. juncea with the wild-type allele of AvrLmJ1 confers avirulence towards all three B. juncea cultivars tested, suggesting that the gene may confer species-specific avirulence activity.

Next-generation genome sequencing can be used to rapidly characterise sequences flanking T-DNA insertions in random insertional mutants of Leptosphaeria maculans.

BACKGROUND: Banks of mutants with random insertions of T-DNA from Agrobacterium tumefaciens are often used in forward genetics approaches to identify phenotypes of interest. Upon identification of mutants of interest, the flanking sequences of the inserted T-DNA must be identified so that the mutated gene can be characterised. However, for many fungi, this task is not trivial as widely used PCR-based methods such as thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) are not successful. FINDINGS: Next-generation Illumina sequencing was used to locate T-DNA insertion sites in four mutants of Leptosphaeria maculans, a fungal plant pathogen. Sequence reads of up to 150 bp and coverage ranging from 6 to 24 times, were sufficient for identification of insertion sites in all mutants. All T-DNA border sequences were truncated to different extents. Additionally, next-generation sequencing revealed chromosomal rearrangements associated with the insertion in one of the mutants. CONCLUSIONS: Next-generation sequencing is a cost-effective and rapid method of identifying sites of T-DNA insertions, and associated genomic rearrangements in Leptosphaeria maculans and potentially in other fungal species.

A workflow from untargeted LC-MS profiling to targeted natural product isolation.

Liquid chromatography-mass spectrometry (LC-MS or HPLC-MS) is an extremely sensitive analytical technique that enables the detection of metabolites with a vast range of chemistries and molecular masses. Extracts from any biological starting material are first fractionated chromatographically on a stationary phase suitable for the retention of the target molecules. The eluent is then transferred directly to the ionization source for MS detection. There is a vast range of chromatographic separation methods and MS configurations. This chapter describes a method for the detection of a broad range of metabolites using reversed phase (C18) LC-MS as well as a method for the isolation of targeted metabolites of interest.

A gene cluster responsible for biosynthesis of phomenoic acid in the plant pathogenic fungus, Leptosphaeria maculans.

Phomenoic acid, a long chain aliphatic carboxylic acid is a major metabolite produced by Leptosphaeria maculans, a fungal pathogen of Brassica napus (canola). This fungus has 15 predicted polyketide synthases (PKS) and seven of them have the appropriate domains for the biosynthesis of phomenoic acid. The most highly expressed PKS gene after 7 days growth in 10% V8 juice, PKS2, was silenced and the resultant mutant produced very low levels of phomenoic acid, indicating that this PKS is involved in phomenoic acid biosynthesis. This gene is part of a co-regulated cluster of genes. Reduced expression of an adjacent gene encoding the transcriptional regulator C6TF, led to reduced expression of genes for PKS2, P450, a cytochrome P450 monoxygenase, YogA, an alcohol dehydrogenase/quinone reductase, RTA1, a lipid transport exporter superfamily member and MFS, a Major Facilitator Superfamily transporter, as well as a marked reduction in phomenoic acid production. Phomenoic acid is toxic towards another canola pathogen Leptosphaeria biglobosa 'canadensis', but not towards L. maculans and only moderately toxic towards the wheat pathogen Stagonospora nodorum. This molecule is detected in infected stems and stubble of B. napus, but biosynthesis of it does not appear to be essential for pathogenicity of L. maculans. Phomenoic acid may play a role in allowing L. maculans to outcompete other fungi in its environmental niche.

The cross-pathway control system regulates production of the secondary metabolite toxin, sirodesmin PL, in the ascomycete, Leptosphaeria maculans.

BACKGROUND: Sirodesmin PL is a secondary metabolite toxin made by the ascomycetous plant pathogen, Leptosphaeria maculans. The sirodesmin biosynthetic genes are clustered in the genome. The key genes are a non-ribosomal peptide synthetase, sirP, and a pathway-specific transcription factor, sirZ. Little is known about regulation of sirodesmin production. RESULTS: Genes involved in regulation of sirodesmin PL in L. maculans have been identified. Two hundred random insertional T-DNA mutants were screened with an antibacterial assay for ones producing low levels of sirodesmin PL. Three such mutants were isolated and each transcribed sirZ at very low levels. One of the affected genes had high sequence similarity to Aspergillus fumigatus cpcA, which regulates the cross-pathway control system in response to amino acid availability. This gene was silenced in L. maculans and the resultant mutant characterised. When amino acid starvation was artificially-induced by addition of 3-aminotriazole for 5 h, transcript levels of sirP and sirZ did not change in the wild type. In contrast, levels of sirP and sirZ transcripts increased in the silenced cpcA mutant. After prolonged amino acid starvation the silenced cpcA mutant produced much higher amounts of sirodesmin PL than the wild type. CONCLUSIONS: Production of sirodesmin PL in L. maculans is regulated by the cross pathway control gene, cpcA, either directly or indirectly via the pathway-specific transcription factor, sirZ.

Mutations to LmIFRD affect cell wall integrity, development and pathogenicity of the ascomycete Leptosphaeria maculans.

Maintaining cell wall integrity is essential for fungal growth and development. We describe two mutants with altered expression of a gene, LmIFRD, from the ascomycete Leptosphaeria maculans. Truncation of the LmIFRD transcript in a T-DNA insertional mutant led to slower germination, less sporulation and loss-of-pathogenicity towards Brassica napus, whereas silencing of the LmIFRD transcript led to increased germination, sporulation and earlier infection. The increased tolerance to cell wall lysing enzymes and cell wall-disrupting compounds of the T-DNA mutant contrasts with decreased tolerance of the silenced mutant and suggests altered cell wall integrity and accessibility to 1,3-linked glucan and chitin. Lectin binding experiments and monosaccharide analysis revealed altered polysaccharide content and structure within the cell wall of the LmIFRD mutants, notably increased 1,3-linked galactose and chitin within the cell wall of the T-DNA mutant. This is the first analysis of monosaccharide linkage composition of cell walls of spores and mycelia for any dothideomycete.

Mutation of a gene in the fungus Leptosphaeria maculans allows increased frequency of penetration of stomatal apertures of Arabidopsis thaliana.

Leptosphaeria maculans, a pathogen of Brassica napus, is unable to invade most wild-type accessions of Arabidopsis thaliana, although several mutants are susceptible. The infection pathway of L. maculans via a non-invasive inoculation method on A. thaliana lms1 (undefined), pmr4-1 (defective in callose deposition), and pen1-1 and pen2-1 (defective in non-host responses to several pathogens) mutants is described. On wild types Col-0 and Ler-0, hyphae are generally arrested at stomatal apertures. A T-DNA insertional mutant of L. maculans (A22) that penetrates stomatal apertures of Col-0 and Ler-0 five to seven times more often than the wild-type isolate is described. The higher penetration frequency of isolate A22 is associated with an increased hypersensitive response, which includes callose deposition. Complementation analysis showed that the phenotype of this isolate is due to T-DNA insertion in an intronless gene denoted as ipa (increased penetration on Arabidopsis). This gene is predicted to encode a protein of 702 amino acids with best matches to hypothetical proteins in other filamentous ascomycetes. The ipa gene is expressed in the wild-type isolate at low levels in culture and during infection of A. thaliana and B. napus.