REVIEW | doi:10.20944/preprints202002.0458.v1
Subject: Life Sciences, Biophysics Keywords: LacI; Lac repressor; TetR; TAL-Effector; transcription activator-like effector; Cas9d; dissociation rate constant
Online: 29 February 2020 (09:13:12 CET)
The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, TALE and CRISPR-Cas9 revealed common rules, which can help the optimization of activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression, and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor systematically optimized designer transcription factors to the needs of specific applications.
ARTICLE | doi:10.20944/preprints202205.0407.v1
Subject: Biology, Plant Sciences Keywords: Fusarium; tomato; novel effector candidates; cell death; Nicotiana benthamiana
Online: 31 May 2022 (03:20:05 CEST)
Fusarium oxysporum f. sp. lycopersici (Fol) causes vascular wilt disease in tomato. Upon colonization of the host, Fol secretes many small effector proteins into the xylem sap to facilitate infection. Besides known SIX (Secreted In Xylem) proteins, the identity of additional effectors that contribute to Fol pathogenicity remains largely unexplored. We have performed a deep RNA-sequencing analysis of Fol race 2-infected tomato, used the sequence data to annotate a published genome assembly generated via PacBio SMRT sequencing of the Fol race 2 reference strain Fol4287, and analysed the resulting transcriptome to identify Fol effector candidates among the newly annotated genes. We examined the Fol-infection expression profiles of all 13 SIX genes present in Fol race 2 and identified 27 new candidate effector genes that were likewise significantly upregulated upon Fol infection. Using Agrobacterium-mediated transformation, we tested the ability of 22 of the new candidate effector genes to suppress or induce cell death in leaves of Nicotiana benthamiana. One effector candidate designated Fol-EC19, encoding a secreted guanyl-specific ribonuclease, was found to trigger cell death and two effector candidates designated Fol-EC14 and Fol-EC20, encoding a glucanase and a secreted trypsin, respectively, were identified that can suppress Bax-mediated cell death. Remarkably, Fol-EC14 and Fol-EC20 were also found to suppress I-2/Avr2- and I/Avr1-mediated cell death. Using the yeast secretion-trap screening system, we showed that these three biologically-active effector candidates each contain a functional signal peptide for protein secretion. Our findings provide a basis for further understanding the virulence functions of Fol effectors.
ARTICLE | doi:10.20944/preprints202206.0376.v1
Subject: Biology, Other Keywords: effector proteins; genome-wide analysis; Ganoderma boninense; basal stem rot; genome architecture
Online: 28 June 2022 (04:59:14 CEST)
Ganoderma boninense is the major causal agent for the basal stem rot (BSR) disease in oil palm, causing the progressive rot of the basal part of the stem. Despite its prominence, key pathogenicity determinants for the aggressive nature of hemibiotrophic infection remain unknown. In this study, genome sequencing and annotation of G. boninense T10 were carried out using the Illumina sequencing platform and comparative genome analysis was performed with previously reported G. boninense strains (NJ3 and G3). The pan-secretome of G. boninense was constructed and comprised of 937 core orthogroups, 243 accessory orthogroups, and 84 strain-specific orthogroups. A set of core candidate effector proteins (CEPs) were found to be enriched with catalytic protein classified as the carbohydrate-active enzymes, hydrolases as well as non-catalytic proteins. Differential expression analysis revealed an upregulation of CEP genes which was linked to the suppression of PTI signaling cascade while the downregulation of CEP genes was linked to the inhibition of PTI by preventing host defense elicitation. Genome architecture analysis revealed the one-speed architecture of the G. boninense genome and the lack of preferential association of CEP genes to the transposable elements. The findings obtained from this study would aid in the characterization of pathogenicity determinants and molecular biomarkers of BSR disease.
ARTICLE | doi:10.20944/preprints201910.0147.v1
Subject: Life Sciences, Biotechnology Keywords: blackleg; brassica; diagnosis; effector; genome alignment; leptosphaeria biglobosa; leptosphaeria maculans; marker; PCR
Online: 13 October 2019 (16:08:55 CEST)
Background: Accurate diagnosis of the differentially aggressive fungus Leptosphaeria maculans and Leptosphaeria biglobosa causing Blackleg in crucifers is crucial. Available markers were designed decades ago which may become ineffective due to the ever evolving nature of the fungus, requiring the development of more precise molecular markers. Methods: The whole genomes of available isolates belonging to this two species were aligned using progressive MAUVE tool, species specific genomic regions were extracted and species specific primers were designed from the sequences that encode for effector proteins. Results: Three (Lm1, Lm2 and Lm5) and two (Lb3 and Lb3’) primer sets specifically detected the isolates of target species in PCR based assay, of which the primers Lm5 and Lb3’ were multiplexed for detection of Leptosphaeria maculans and Leptosphaeria biglobosa, generating PCR amplicons of 230 and 834 bp, respectively from a single PCR reaction. The markers were highly sensitive and were able to amplify target species from crude ‘pseudothecia and ascospores suspension’ without requiring DNA extraction. Conclusions: These markers, solitarily or in combination, designed from species specific genomic segments will serve as precise, sensitive and rapid detection of Leptosphaeria maculans and Leptosphaeria biglobosa species and will be helpful for surveillance, management and transboundary quarantine of the devastating disease.
ARTICLE | doi:10.20944/preprints202203.0275.v1
Subject: Materials Science, General Materials Science Keywords: alumina; Additive Manufacturing (AM); CerAMfacturing; vat photopolymerization (VPP); digi-tal light processing (DLP); Lithography-based Ceramic Manufacturing (LCM); cold-gas nozzle; aerospike nozzle
Online: 21 March 2022 (07:31:31 CET)
Advanced ceramics are recognized as key enabling materials possessing combinations of properties not achievable in other material classes. They are characterized by very high thermal, chemical and mechanical resistance and also usually have a lower density than metals. These properties predestine ceramics for many different applications, especially space applications.In the aerospace sector aerospike nozzles promise performance and application advantages compared to classic bell nozzles but are also inherently more complex to manufacture due to their shape. AM methods drastically simplify or even enable the fabrication of those complex structures while minimising the number of individual parts. The applicability of ceramic AM (“CerAMfacturing”) on rocket engines and especially nozzles is consequently investigated in the frame of the “MACARONIS” project, a cooperation of the Institute of Aerospace Engineering at Technische Universität Dresden and the Fraunhofer Institute for Ceramic Technologies and Systems (IKTS) in Dresden. The goal is to develop novel large size aerospike thrust nozzles including areas of highest resolution and fineness. Finding a suitable AM process that enables the realisation of both aspects is extremely challenging. One possibility could be the hybridization of shaping methods, in that case CerAM VPP (ceramic additive manufacturing via vat photopolymerization) and CerAM FFF (ceramic additive manufacturing via fused filament fabrication) in combination with sinter joining. This contribution focuses on the high resolution CerAM VPP process, in particular the development, characterization and testing of a new photoreactive Al2O3 suspension validated by AM of novel aerospike nozzles.
ARTICLE | doi:10.20944/preprints202012.0697.v1
Subject: Life Sciences, Biochemistry Keywords: Repeat-Induced Point Mutations; RIP; Accessory Chromosome; Genome Compartmentalization; GC Content; Genetic Variation; Fusarium circinatum; Transposable Elements; Interstitial Telomeric Repeats; Effector Genes.
Online: 28 December 2020 (12:18:11 CET)
Repeat-Induced Point mutations (RIP) serves as a genome defence mechanism that impedes the deleterious consequences of repeated motifs such as transposable elements in fungi. Genomic regions with RIP are biased for adenosine and thymine transitions and the cumulative influence of RIP is thought to have a considerable impact on genome composition. We investigated the impact of RIP on localized genomic regions and whole-genome sequences for representatives of the pine pathogen, Fusarium circinatum. We set out to determine the intraspecific variation in acquired RIP and the role of RIP in the development of diverse F. circinatum sub-genomic compartments. The results of the study show that the AT-enriched sub-genomic compartment accounts for ca. 97% of the calculated RIP and was further prominent in both core and accessory genomic regions. However, more extensive RIP was observed in the accessory sub-compartment and more variable regions of the genome. Regions with RIP indicated increased intrinsic curvature of the DNA which may influence DNA-protein interactions and may promote constitutive heterochromatin formation. The results show that RIP is an important source of functional novelty and genome variation. RIP contributes to the evolution of the genetic landscape and differentiation of diverse sub-genomic compartments of this important fungal pathogen.