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

<p>Abstract</p> <p>Background</p> <p>Sirodesmin PL is a secondary metabolite toxin made by the ascomycetous plant pathogen, <it>Leptosphaeria maculans</it>. The sirodesmin biosynthetic genes are clustered in the genome. The key genes are a non-ribosomal peptide synthetase, <it>sirP</it>, and a pathway-specific transcription factor, <it>sirZ</it>. Little is known about regulation of sirodesmin production.</p> <p>Results</p> <p>Genes involved in regulation of sirodesmin PL in <it>L. maculans </it>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 <it>sirZ </it>at very low levels. One of the affected genes had high sequence similarity to <it>Aspergillus fumigatus cpcA</it>, which regulates the cross-pathway control system in response to amino acid availability. This gene was silenced in <it>L. maculans </it>and the resultant mutant characterised. When amino acid starvation was artificially-induced by addition of 3-aminotriazole for 5 h, transcript levels of <it>sirP </it>and <it>sirZ </it>did not change in the wild type. In contrast, levels of <it>sirP </it>and <it>sirZ </it>transcripts increased in the silenced <it>cpcA </it>mutant. After prolonged amino acid starvation the silenced <it>cpcA </it>mutant produced much higher amounts of sirodesmin PL than the wild type.</p> <p>Conclusions</p> <p>Production of sirodesmin PL in <it>L. maculans </it>is regulated by the cross pathway control gene, <it>cpcA</it>, either directly or indirectly via the pathway-specific transcription factor, <it>sirZ</it>.</p

Digitalna procjena lisne površine krošnje stijenke vinove loze (Vitis vinifera cv. Sauvignon) korištenjem LIDAR mjerne tehnologije

A dosage rate reduction of plant protection products mixed with water, i.e. spray mixture, in a prescribed concentration in the vineyard will only be possible in the future, if the natural characteristics of vine canopy structures (leaf wall area) and canopy management are taken into account. In a practical experiment in the vineyard we evaluated the leaf wall area of the vine cv. Sauvignon on different segments on the left and right side of the vine canopy. We compared the results of manual measurements and laser measuring technology (LIDAR) with the corresponding algorithm, with which we enabled the digital reconstruction of the leaf wall area of the vine. The manual measurement of the leaf wall area was carried out using an automated image analyser. The digital system for measuring the leaf wall area on different segments consisted of a LIDAR sensor and a Differential Global Positioning System (hereinafter DGPS). To determine the exact DGPS position of the LIDAR sensor during the measurement, we set up a DGPS base station. Using the Excel software (CORREL function), we estimated the relationship between the dependent variable (digital number of points in the cloud) and an independent variable (leaf wall area, manually measured). An analysis of six randomly selected vines in the vineyard revealed the maximum value of the correlation coefficient r = 0.80 for the left side and r = 0.90 for the right side of the leaf wall area of the vine, respectively. In the near future the virtual three-dimensional space will provide more even control of spray mixture over the entire structure of the leaf wall area in the vineyard based on autonomous decision-making models.Smanjenje količine utroška sredstava za zaštitu bilja i same smjese za prskanje u budućnosti će biti moguće samo ako se uzmu u obzir prirodne karakteristike krošnje vinove loze tj. lisne površine krošnje trsa. U praktičnom pokusu u vinogradu procijenjena je lisna površinu krošnje vinove loze cv. Sauvignon na različitim segmentima s lijeve i desne strane krošnje uz pomoć ručnih mjerenja i laserske mjerne tehnologije (LIDAR). Dobiveni rezultati uspoređeni su s pripadajućim algoritmom čime je dobivena digitalna rekonstrukcija lisne površine vinove loze. Ručno mjerenje površine listova provedeno je u laboratoriju pomoću digitalnog lisnog skenera nakon što je lišće ručno pobrano s trsova i dopremljeno u sam laboratorij. Digitalni sustav za mjerenje lisne površine na različitim segmentima krošnje sastojao se od LIDAR senzora i DGPS navigacijskog sustava. Da bi se odredio točan DGPS položaj LIDAR senzora tijekom mjerenja, postavljena je DGPS bazna stanica. Pomoću regresijske metode utvrđen je odnos između zavisne varijable (digitalni broj točaka u oblaku) i nezavisne varijable (površina listova izmjerena skenerom). Rezultati analize imeđu dvije uspoređivane metode na šest slučajno odabranih trsova vinove loze otkrivaju vrijednost koeficijenta korelacije r = 0,80 za lijevu i r = 0,90 za desnu stranu krošnje. U bliskoj budućnosti virtualni trodimenzionalni prostor pružit će ravnomjerniju kontrolu smjese raspršivača preko cijele strukture područja stijenke lišča u vinogradu na temelju autonomnih modela odlučivanja

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

<p>Abstract</p> <p>Background</p> <p>Genes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.</p> <p>Results</p> <p>Putative ETP gene clusters are present in 14 ascomycete taxa, but absent in numerous other ascomycetes examined. These clusters are discontinuously distributed in ascomycete lineages. Gene content is not absolutely fixed, however, common genes are identified and phylogenies of six of these are separately inferred. In each phylogeny almost all cluster genes form monophyletic clades with non-cluster fungal paralogues being the nearest outgroups. This relatedness of cluster genes suggests that a progenitor ETP gene cluster assembled within an ancestral taxon. Within each of the cluster clades, the cluster genes group together in consistent subclades, however, these relationships do not always reflect the phylogeny of ascomycetes. Micro-synteny of several of the genes within the clusters provides further support for these subclades.</p> <p>Conclusion</p> <p>ETP gene clusters appear to have a single origin and have been inherited relatively intact rather than assembling independently in the different ascomycete lineages. This progenitor cluster has given rise to a small number of distinct phylogenetic classes of clusters that are represented in a discontinuous pattern throughout ascomycetes. The disjunct heredity of these clusters is discussed with consideration to multiple instances of independent cluster loss and lateral transfer of gene clusters between lineages.</p

Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1) and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis

Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products

A new set of international Leptosphaeria maculans isolates as a resource for elucidation of the basis and evolution of blackleg disease on Brassica napus

© 2023 The Authors. Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology. This is an open access article under the terms of the Creative Commons Attribution-Non Commercial-No Derivs License. https://creativecommons.org/licenses/by-nc-nd/4.0/A collection of isolates of the fungi Leptosphaeria maculans and L. biglobosa, which cause blackleg disease on Brassica napus (canola/oilseed rape) and other Brassicaceae species, was assembled to represent the global diversity of these pathogens and a resource for international research. The collection consists of 226 isolates (205 L. maculans and 21 L. biglobosa) from 11 countries. The genomes of all 205 L. maculans isolates were sequenced, and the distribution and identity of avirulence gene alleles were determined based on genotypic information and phenotypic reactions on B. napus lines that hosted specific resistance genes. Whilst the frequencies of some avirulence alleles were consistent across each of the regions, others differed dramatically, potentially reflecting the canola/oilseed rape cultivars grown in those countries. Analyses of the single-nucleotide polymorphism (SNP) diversity within these L. maculans isolates revealed geographical separation of the populations. This "open access" resource provides a standardized set of isolates that can be used to define the basis for how these fungal pathogens cause disease, and as a tool for discovery of new resistance traits in Brassica species.Peer reviewe

A global-scale expert assessment of drivers and risks associated with pollinator decline.

Pollinator decline has attracted global attention and substantial efforts are underway to respond through national pollinator strategies and action plans. These policy responses require clarity on what is driving pollinator decline and what risks it generates for society in different parts of the world. Using a formal expert elicitation process, we evaluated the relative regional and global importance of eight drivers of pollinator decline and ten consequent risks to human well-being. Our results indicate that global policy responses should focus on reducing pressure from changes in land cover and configuration, land management and pesticides, as these were considered very important drivers in most regions. We quantify how the importance of drivers and risks from pollinator decline, differ among regions. For example, losing access to managed pollinators was considered a serious risk only for people in North America, whereas yield instability in pollinator-dependent crops was classed as a serious or high risk in four regions but only a moderate risk in Europe and North America. Overall, perceived risks were substantially higher in the Global South. Despite extensive research on pollinator decline, our analysis reveals considerable scientific uncertainty about what this means for human society.University of Reading’s Building Outstanding Impact Support Programm

Use of DNA–Damaging Agents and RNA Pooling to Assess Expression Profiles Associated with BRCA1 and BRCA2 Mutation Status in Familial Breast Cancer Patients

A large number of rare sequence variants of unknown clinical significance have been identified in the breast cancer susceptibility genes, BRCA1 and BRCA2. Laboratory-based methods that can distinguish between carriers of pathogenic mutations and non-carriers are likely to have utility for the classification of these sequence variants. To identify predictors of pathogenic mutation status in familial breast cancer patients, we explored the use of gene expression arrays to assess the effect of two DNA–damaging agents (irradiation and mitomycin C) on cellular response in relation to BRCA1 and BRCA2 mutation status. A range of regimes was used to treat 27 lymphoblastoid cell-lines (LCLs) derived from affected women in high-risk breast cancer families (nine BRCA1, nine BRCA2, and nine non-BRCA1/2 or BRCAX individuals) and nine LCLs from healthy individuals. Using an RNA–pooling strategy, we found that treating LCLs with 1.2 µM mitomycin C and measuring the gene expression profiles 1 hour post-treatment had the greatest potential to discriminate BRCA1, BRCA2, and BRCAX mutation status. A classifier was built using the expression profile of nine QRT–PCR validated genes that were associated with BRCA1, BRCA2, and BRCAX status in RNA pools. These nine genes could distinguish BRCA1 from BRCA2 carriers with 83% accuracy in individual samples, but three-way analysis for BRCA1, BRCA2, and BRCAX had a maximum of 59% prediction accuracy. Our results suggest that, compared to BRCA1 and BRCA2 mutation carriers, non-BRCA1/2 (BRCAX) individuals are genetically heterogeneous. This study also demonstrates the effectiveness of RNA pools to compare the expression profiles of cell-lines from BRCA1, BRCA2, and BRCAX cases after treatment with irradiation and mitomycin C as a method to prioritize treatment regimes for detailed downstream expression analysis

Insertion of an Esterase Gene into a Specific Locust Pathogen (Metarhizium acridum) Enables It to Infect Caterpillars

An enduring theme in pathogenic microbiology is poor understanding of the mechanisms of host specificity. Metarhizium is a cosmopolitan genus of invertebrate pathogens that contains generalist species with broad host ranges such as M. robertsii (formerly known as M. anisopliae var. anisopliae) as well as specialists such as the acridid-specific grasshopper pathogen M. acridum. During growth on caterpillar (Manduca sexta) cuticle, M. robertsii up-regulates a gene (Mest1) that is absent in M. acridum and most other fungi. Disrupting M. robertsii Mest1 reduced virulence and overexpression increased virulence to caterpillars (Galleria mellonella and M. sexta), while virulence to grasshoppers (Melanoplus femurrubrum) was unaffected. When Mest1 was transferred to M. acridum under control of its native M. robertsii promoter, the transformants killed and colonized caterpillars in a similar fashion to M. robertsii. MEST1 localized exclusively to lipid droplets in M. robertsii conidia and infection structures was up-regulated during nutrient deprivation and had esterase activity against lipids with short chain fatty acids. The mobilization of stored lipids was delayed in the Mest1 disruptant mutant. Overall, our results suggest that expression of Mest1 allows rapid hydrolysis of stored lipids, and promotes germination and infection structure formation by M. robertsii during nutrient deprivation and invasion, while Mest1 expression in M. acridum broadens its host range by bypassing the regulatory signals found on natural hosts that trigger the mobilization of endogenous nutrient reserves. This study suggests that speciation in an insect pathogen could potentially be driven by host shifts resulting from changes in a single gene