Identification of Multiple Phytotoxins Produced by Fusarium virguliforme Including a Phytotoxic Effector (FvNIS1) Associated With Sudden Death Syndrome Foliar Symptoms.

Sudden death syndrome (SDS) of soybean is caused by a soilborne pathogen, Fusarium virguliforme. Phytotoxins produced by F. virguliforme are translocated from infected roots to leaves, in which they cause SDS foliar symptoms. In this study, additional putative phytotoxins of F. virguliforme were identified, including three secondary metabolites and 11 effectors. While citrinin, fusaric acid, and radicicol induced foliar chlorosis and wilting, Soybean mosaic virus (SMV)-mediated overexpression of F. virguliforme necrosis-inducing secreted protein 1 (FvNIS1) induced SDS foliar symptoms that mimicked the development of foliar symptoms in the field. The expression level of fvnis1 remained steady over time, although foliar symptoms were delayed compared with the expression levels. SMV::FvNIS1 also displayed genotype-specific toxicity to which 75 of 80 soybean cultivars were susceptible. Genome-wide association mapping further identified three single nucleotide polymorphisms at two loci, where three leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes were found. Culture filtrates of fvnis1 knockout mutants displayed a mild reduction in phytotoxicity, indicating that FvNIS1 is one of the phytotoxins responsible for SDS foliar symptoms and may contribute to the quantitative susceptibility of soybean by interacting with the LRR-RLK genes.

14-3-3 proteins SGF14c and SGF14l play critical roles during soybean nodulation.

The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules.

Electrochemical biosensor for rapid detection of fungal contamination in fuel systems.

There is an increased demand for real-time monitoring of biological and biochemical processes. While most sensor research focuses on physiological conditions, less has been done towards developing real-time biosensors that can operate in and survive exposure to extreme environments and harsh chemicals such as fuel. One interesting application is monitoring microbial load in fuel tanks to prevent both fuel spoilage and biocorrosion. We developed a comprehensive method to enable the first reagentless, real-time, microbial sensor platform that is also fuel resistant. We first identified an extracellular protein epitope conserved in fuel-degrading fungi then used this epitope to develop a suitable biorecognition element (BRE) through biopanning of a 7-mer phage displayed peptide library. After demonstrating the BRE's affinity to fungi using molecular and fluorescence assays, we incorporated the BRE into a reagentless, real-time electrochemical sensing platform based on a self-assembled monolayer of peptide BREs and redox reporters. Finally, we incorporated this real-time electrochemical sensing platform into a microfluidic device. We demonstrated detection of Yarrowia lipolytica as low as 1 × 104 CFU/mL in a bath cell, and demonstrate a microfluidic cell that functions even after exposure to jet fuel. In summary, this work describes development of a fuel-resistant biosensor for monitoring microbial growth in extreme environments.

Transcriptional analysis of soybean root response to Fusarium virguliforme, the causal agent of sudden death syndrome.

Sudden death syndrome (SDS) of soybean can be caused by any of four distinct Fusarium species, with F. virguliforme and F. tucumaniae being the main casual agents in North and South America, respectively. Although the fungal tissue is largely confined to the roots, the fungus releases a toxin that is translocated to leaf tissues, in which it causes interveinal chlorosis and necrosis leading to scorching symptoms and possible defoliation. In this study, we report on an Affymetrix analysis measuring transcript abundances in resistant (PI 567.374) and susceptible (Essex) roots upon infection by F. virguliforme, 5 and 7 days postinoculation. Many of the genes with increased expression were common between resistant and susceptible plants (including genes related to programmed cell death, the phenylpropanoid pathway, defense, signal transduction, and transcription factors), but some genotype-specific expression was noted. Changes in small (sm)RNA levels between inoculated and mock-treated samples were also studied and implicate a role for these molecules in this interaction. In total, 2,467 genes were significantly changing in the experiment, with 1,694 changing in response to the pathogen; 93 smRNA and 42 microRNA that have putative soybean gene targets were identified from infected tissue. Comparing genotypes, 247 genes were uniquely modulating in the resistant host, whereas 378 genes were uniquely modulating in the susceptible host. Comparing locations of differentially expressed genes to known resistant quantitative trait loci as well as identifying smRNA that increased while their putative targets decreased (or vice versa) allowed for the narrowing of candidate SDS defense-associated genes.

Complete transcriptome of the soybean root hair cell, a single-cell model, and its alteration in response to Bradyrhizobium japonicum infection.

Nodulation is the result of a mutualistic interaction between legumes and symbiotic soil bacteria (e.g. soybean [Glycine max] and Bradyrhizobium japonicum) initiated by the infection of plant root hair cells by the symbiont. Fewer than 20 plant genes involved in the nodulation process have been functionally characterized. Considering the complexity of the symbiosis, significantly more genes are likely involved. To identify genes involved in root hair cell infection, we performed a large-scale transcriptome analysis of B. japonicum-inoculated and mock-inoculated soybean root hairs using three different technologies: microarray hybridization, Illumina sequencing, and quantitative real-time reverse transcription-polymerase chain reaction. Together, a total of 1,973 soybean genes were differentially expressed with high significance during root hair infection, including orthologs of previously characterized root hair infection-related genes such as NFR5 and NIN. The regulation of 60 genes was confirmed by quantitative real-time reverse transcription-polymerase chain reaction. Our analysis also highlighted changes in the expression pattern of some homeologous and tandemly duplicated soybean genes, supporting their rapid specialization.

Downy mildew (Pl ( 8 ) and Pl ( 14 )) and rust (R ( Adv )) resistance genes reside in close proximity to tandemly duplicated clusters of non-TIR-like NBS-LRR-encoding genes on sunflower chromosomes 1 and 13.

Nucleotide binding site-leucine rich repeat (NBS-LRR) proteins are encoded by a ubiquitous gene family in sunflower and frequently harbor disease resistance genes. We investigated NBS-LRR-encoding resistance gene candidates (RGCs) flanking the downy mildew resistance genes Pl ( 8 ) and Pl ( 14 ) and the rust resistance gene R ( Adv ), which map on the NBS-LRR clusters of linkage groups 1 and 13 in sunflower genome. We shotgun sequenced bacterial artificial chromosome (BAC) clones proximal to Pl ( 8 ), Pl ( 14 ) , and R ( Adv ) and identified seven novel non-Toll/interleukin-1 receptor (TIR)-like NBS-LRR RGCs, which clustered with previously identified RGCs of linkage group 13 but were phylogenetically distant from the TIR- and non-TIR-NBS-LRR-encoding superfamilies of sunflower. Six of the seven predicted RGCs have intact open reading frames and reside in genomic segments with abundant transposable elements. The genomic localization and sequence similarity of the novel non-TIR-like predicted RGCs suggests that they originated from tandem duplications. RGCs in the proximity of Pl ( 8 ) and R ( Adv ) were likely introgressed from silverleaf sunflower genome, where the RGC cluster of linkage group 13 is duplicated in two independent chromosomes that have different architecture and level of recombination from the respective common sunflower chromosomes.

Molecular characterization of two types of resistance in sunflower to Plasmopara halstedii, the causal agent of downy mildew.

Depending on host-pathotype combination, two types of sunflower-Plasmopara halstedii incompatibility reactions have previously been identified. Type I resistance can restrict the growth of the pathogen in the basal region of the hypocotyls, whereas type II cannot, thus allowing the pathogen to reach the cotyledons. In type II resistance, a large portion of the hypocotyls is invaded by the pathogen and, subsequently, a hypersensitive reaction (HR) is activated over a long portion of the hypocotyls. Thus, the HR in type II resistance coincides with a higher induction of hsr203j sunflower homologue in comparison with type I resistance, where the HR is activated only in the basal part of hypocotyls. Although the pathogen was not detected in cotyledons of type I resistant plants, semiquantitative polymerase chain reaction confirmed the early abundant growth of the pathogen in cotyledons of susceptible plants by 6 days postinfection (dpi). This was in contrast to scarce growth of the pathogen in cotyledons of type II-resistant plants at a later time point (12 dpi). This suggests that pathogen growth differs according to the host-pathogen combination. To get more information about sunflower downy mildew resistance genes, the full-length cDNAs of RGC151 and RGC203, which segregated with the PlARG gene (resistance type I) and Pl14 gene (resistance type II), were cloned and sequenced. Sequence analyses revealed that RGC151 belongs to the Toll/interleukin-1 receptor (TIR) nucleotide-binding site leucine-rich repeat (NBS-LRR) class whereas RGC203 belongs to class coiled-coil (CC)-NBS-LRR. This study suggests that type II resistance may be controlled by CC-NBS-LRR gene transcripts which are enhanced upon infection by P. halstedii, rather than by the TIR-NBS-LRR genes that might control type I resistance.

Metagenome-Assembled Genomes of 12 Bacterial Species from Biofouled Plastic Fabrics Harbor Multiple Genes for Degradation of Hydrocarbons.

We report the metagenome-assembled genomes (MAGs) of 12 different bacterial species recovered from environmental microbiomes associated with biofouled plastic fabrics. The MAGs have estimated sizes of 2.53 to 7.66 Mb with 3,229 to 9,289 proteins, 26.20% to 99.1% genome completeness, 48.9% to 72.6% G+C content, and multiple genes for hydrocarbon degradation.

Black Yeast Genomes Assembled from Plastic Fabric Metagenomes Reveal an Abundance of Hydrocarbon Degradation Genes.

We report the assembly and annotation of 10 different black yeast genomes from microbiome metagenomic data derived from biofouled plastic fabrics. The draft genomes are estimated to be 9 to 33.2 Mb, with 357 to 5,108 contigs and G+C contents of 43.9% to 57.4%, and they harbor multiple genes for hydrocarbon adaptation and degradation.

GC-MS hydrocarbon degradation profile data of Pseudomonas frederiksbergensis SI8, a bacterium capable of degrading aromatics at low temperatures.

The ability of the psychrotrophic bacterium Pseudomonas frederiksbergensis SI8 to grow and degrade aromatic hydrocarbons efficiently at low temperature is shown in this study. The robust growth of P. frederiksbergensis SI8 was demonstrated in jet fuel and an aromatic blend. The bacterium showed 2.5 to 3-fold faster growth in the aromatic blend than in jet fuel. The hydrocarbons degradation profile of P. frederiksbergensis SI8 at ambient temperature (i.e., 28 °C) and low temperature (i.e., 4 °C) was characterized by Gas Chromatography-Mass Spectrometry (GC-MS) analysis. GC-MS data demonstrated that P. frederiksbergensis SI8 is a novel psychrotrophic bacterium with the ability to degrade aromatic hydrocarbons at temperatures as low as 4 °C. Specifically, P. frederiksbergensis SI8 consumed toluene, ethylbenzene, n-propylbenzene and methyl ethyl benzene efficiently. The data presented here serves to characterize the hydrocarbon degradation profile of P. frederiksbergensis SI8 and corroborates the capacity of this bacterium to degrade aromatic hydrocarbons at low temperatures. The raw GC-MS data for the degradation of hydrocarbons by P. frederiksbergensis SI8 grown at 4 °C and 28 °C for 14 days have been deposited in Mendeley Data and can be retrieved from https://dx.doi.org/10.17632/z9292bvdmh.1 and https://dx.doi.org/10.17632/dp3sgwpj23.1. The datasets and raw data presented here were associated with the main research work "Metagenomic characterization reveals complex association of soil hydrocarbon-degrading bacteria" [1].

Shotgun metagenomic data of microbiomes on plastic fabrics exposed to harsh tropical environments.

The development of more affordable high-throughput DNA sequencing technologies and powerful bioinformatics is making of shotgun metagenomics a common tool for effective characterization of microbiomes and robust functional genomics. A shotgun metagenomic approach was applied in the characterization of microbial communities associated with plasticized fabric materials exposed to a harsh tropical environment for 14 months. High-throughput sequencing of TruSeq paired-end libraries was conducted using a whole-genome shotgun (WGS) approach on an Illumina HiSeq2000 platform generating 100 bp reads. A multifaceted bioinformatics pipeline was developed and applied to conduct quality control and trimming of raw reads, microbial classification, assembly of multi-microbial genomes, binning of assembled contigs to individual genomes, and prediction of microbial genes and proteins. The bioinformatic analysis of the large 161 Gb sequence dataset generated 3,314,688 contigs and 120 microbial genomes. The raw metagenomic data and the detailed description of the bioinformatics pipeline applied in data analysis provide an important resource for the genomic characterization of microbial communities associated with biodegraded plastic fabric materials. The raw shotgun metagenomics sequence data of microbial communities on plastic fabric materials have been deposited in MG-RAST (https://www.mg-rast.org/) under accession numbers: mgm4794685.3-mgm4794690.3. The datasets and raw data presented here were associated with the main research work "Metagenomic characterization of microbial communities on plasticized fabric materials exposed to harsh tropical environments" (Radwan et al., 2020).

An AMA1/MSP119 Adjuvanted Malaria Transplastomic Plant-Based Vaccine Induces Immune Responses in Test Animals.

Malaria is a tropical human disease, caused by protozoan parasites, wherein a significant number of the world's population is at risk. Annually, more than 219 million new cases are reported. Although there are prevention treatments, there are no highly and widely effective licensed anti-malarial vaccines available for use. Opportunities for utilization of plant-based vaccines as novel platforms for developing safe, reliable, and affordable treatments offer promise for developing such a vaccine against malaria. In this study, a Malchloroplast candidate vaccine was designed, composed of segments of AMA1 and MSP1 proteins, two epitopes of Plasmodium falciparum, along with a GK1 peptide from Taenia solium as adjuvant, and this was expressed in tobacco chloroplasts. Transplastomic tobacco lines were generated using biolistic transformation, and these were confirmed to carry the synthetic gene construct. Expression of the synthetic GK1 peptide was confirmed using RT-PCR and Western blots. Furthermore, the GK1 peptide was detected by HPLC at levels of up to 6 µg g-1 dry weight of tobacco leaf tissue. The plant-derived Malchloroplast candidate vaccine was subsequently tested in BALB/c female mice following subcutaneous administration, and was found to elicit specific humoral responses. Furthermore, components of this candidate vaccine were recognized by antibodies in Plasmodium falciparum malaria patients and were immunogenic in test mice. Thus, this study provided a 'proof of concept' for a promising plant-based candidate subunit vaccine against malaria.

Draft Genome Sequence of Lecanicillium sp. Isolate LEC01, a Fungus Capable of Hydrocarbon Degradation.

Lecanicillium sp. isolate LEC01 is adapted to grow in the presence of jet fuel, employing genes involved in the degradation of alkanes and aromatic hydrocarbons. The draft genome is estimated at 31,407,988 bp and has 9,737 proteins, 50.0% G+C content, and high similarity to Lecanicillium sp. strain CCF 5233.

Draft Genome Sequence of Byssochlamys sp. Isolate BYSS01, a Filamentous Fungus Adapted to the Fuel Environment.

Byssochlamys sp. isolate BYSS01 (anamorph, Paecilomyces sp.), which was isolated from jet fuel, is highly adapted to grow in hydrocarbons, having predicted genes involved in degradation of n-alkanes, branched alkanes, and aromatic compounds. The draft genome size is estimated at 29 Mb, containing 8,509 proteins.

Draft Genome Sequence of Fusarium fujikuroi, a Fungus Adapted to the Fuel Environment.

Fusarium fujikuroi isolate FUS01 is highly adapted to grow in jet fuel with predicted genes involved in hydrocarbon catabolism and carbon assimilation. The draft genome size is estimated at 49 Mb containing 18,578 proteins with high similarity to that of F. fujikuroi isolate B14.

Draft Genome Sequence of Achromobacter spanius Strain 6, a Soil Bacterium Isolated from a Hydrocarbon-Degrading Microcosm.

Achromobacter spanius strain 6 is a Gram-negative soil bacterium isolated from a hydrocarbon-degrading microcosm. The draft genome sequence of A. spanius strain 6 is 6.57 Mb with a G+C content of 64.7% and 5,855 protein coding genes. Multiple genes involved in degradation of aromatics are present in this strain.

IMA Genome-F 4: Draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata.

The genomes of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygami, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata are presented in this genome announcement. These seven genomes are from endophytes, plant pathogens and economically important fungal species. The genome sizes range from 26.6 Mb in the case of Leptographium lundbergii to 44 Mb for Chrysoporthe austroafricana. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera, and may contribute to our understanding of the lifestyles through comparative studies with closely related organisms.

Effect of Fusarium virguliforme phytotoxin on soybean gene expression suggests a role in multidimensional defence.

Sudden death syndrome (SDS), caused by Fusarium virguliforme, is an important yield-limiting disease of soybean. This soil-borne fungus colonizes soybean roots causing root rot, and also releases a phytotoxin that is translocated to leaves causing interveinal chlorosis and necrosis leading to defoliation and early maturation. The objective of our study was to compare gene expression profiles during the early response of soybean leaves exposed to sterile culture filtrates of F. virguliforme in soybean genotypes with different levels of resistance to SDS. The analysis identified SDS-related defence genes that were induced in the most resistant genotype, but not in the other genotypes. Further functional annotations based on sequence homology suggested that some of the induced genes probably encode proteins involved in cell wall modification, detoxification, defence responses, primary metabolism and membrane transport. Quantitative real-time reverse-transcribed polymerase chain reaction confirmed the differential transcript accumulation of a subset of these genes. In addition, in silico mapping of differentially expressed genes to SDS-resistant quantitative trait loci allowed for the identification of new potential defence genes that could be genetically mapped to the soybean genome, and could be used further in a marker-assisted selection programme. A comparison of the response of soybean to F. virguliforme phytotoxin (Fv toxin) relative to other biotic and abiotic stresses revealed that the resistance response to Fv toxin is quite similar to the response to inoculation with an incompatible Pseudomonas syringae pv. glycinea strain, suggesting that Fv toxin might induce hypersensitive response pathways in soybean leaf tissues in the absence of pathogen in these tissues.

Phylogenetic analyses of peanut resistance gene candidates and screening of different genotypes for polymorphic markers.

The nucleotide-binding-site-leucine-rich-repeat (NBS-LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. Here, we describe a collection of peanut NBS-LRR resistance gene candidates (RGCs) isolated from peanut (Arachis) species by mining Gene Bank data base. NBS-LRR sequences assembled into TIR-NBS-LRR (75.4%) and non-TIR-NBS-LRR (24.6%) subfamilies. Total of 20 distinct clades were identified and showed a high level of sequence divergence within TIR-NBS and non-TIR-NBS subfamilies. Thirty-four primer pairs were designed from these RGC sequences and used for screening different genotypes belonging to wild and cultivated peanuts. Therefore, peanut RGC identified in this study will provide useful tools for developing DNA markers and cloning the genes for resistance to different pathogens in peanut.