Biology subjects

Shim, W. B.

Publications and source records attributed to Shim, W. B..

12 recordsLinked to original sources

bioRxiv2025

Machine Learning-Guided Synthetic Microbial Communities Enable Functional and Sustainable Degradation of Persistent Environmental Pollutants

Persistent environmental pollutants demand the use of diverse microbial metabolic capabilities for effective degradation. While naturally occurring consortia or single strains often fall short in efficiency, synthetic microbial communities (SynComs) hold greater promise for enhanced degradation. To address this challenge, we developed GENIA (Genomically and Environmentally Networked Intelligent Assemblies), a genome-informed and machine learning-guided framework for the rational design of SynComs capable of multi-pollutant degradation under simulated environmental conditions. Using a microfluidic high-throughput cultivation platform, 2,155 bacterial strains were isolated from xenobiotic-enriched environments and screened for pollutant-specific growth. Whole-genome sequencing and functional annotation of 45 prioritized strains revealed metabolic traits associated with the potential degradation of challenging persistent environmental pollutants as proof of concept, i.e., lignin oxidation, atrazine dechlorination, and PFAS defluorination. These genomic profiles were encoded into spline-based graph representations and integrated within the GENIA pipeline, which combines graph neural networks, pathway complementarity modeling, and functional redundancy minimization to predict optimal community assemblies. The resulting nine-member community--comprising Pantoea dispersa, Atlantibacter hermannii, Pseudomonas fulva, Paenibacillus polymyxa, Bacillus cabrialesii, Micrococcus luteus, Bacillus pseudomycoides, Bacillus licheniformis, and Pseudomonas pergaminensis--was predicted to exhibit broad catabolic capacity and minimal intra-community competition. Kinetic experiments in minimal medium demonstrated simultaneous multi-pollutant degradation: lignin (91.6% removal by day 5), atrazine (91.4% removal by day 3), and PFOS (93.1% removal within seven days), representing a 2-4-fold improvement over existing approaches. GENIA establishes a scalable and generalizable framework that integrates systems-level genomics, phenotypic screening, and predictive modeling to engineer ecologically coherent microbial consortia with application to complex environmental bioremediation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/677392v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@1beee39org.highwire.dtl.DTLVardef@a0a1corg.highwire.dtl.DTLVardef@11ddc4aorg.highwire.dtl.DTLVardef@169ba57_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Fusarium oxysporum f. sp. vasinfectum race 4 (Fov4) FNP1, a unique non-ribosomal peptide synthetase gene, plays an important role in fungal development and cotton Fusarium wilt pathogenesis

Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum (Fov), is one of the most destructive early-season cotton diseases worldwide. The recent emergence of the highly virulent Fov race 4 (Fov4) and its aggressiveness have raised significant concerns for the U.S. cotton industry. Unlike predominant Fov races in US cotton production, which require root-knot nematodes to cause damage, Fov4 is known to infect cotton independent of nematodes. However, molecular mechanisms of Fov4 virulence in cotton are not clearly understood. Secondary metabolites are often identified as the culprits in pathogen virulence toward plant hosts. To investigate these factors in Fov4, we analyzed the genomes of Fov1 and Fov4 using Fungal antiSMASH and identified a Fov4-specific nonribosomal peptide synthetase (NRPS) gene FNP1. To investigate its function, we generated FNP1 knock-out mutant using CRISPR-Cas9 approach. Growth assays revealed that the mutants exhibit significantly attenuated hyphal production on media containing cotton roots as the sole carbon source, increased sensitivity to cell stress agents, as well as lagged spore germination. Furthermore, the mutant exhibited defect in cotton root rot virulence and significant decrease in Fusaric acid production. Microscopic observation of GFP-labeled FNP1 deletion mutant showed impeded infection progression in cotton roots compared to the wild type (WT), which further explained the impeded virulence in FNP1 mutant. Gene complementation restored the observed defects, confirming that FNP1 is critical for Fov4 virulence, hyphal development, Fusaric acid production, and stress responses. HighlightsComparative genomic analysis between Fov1 and Fov4 identified FNP1 as a gene specific to Fov4. CRISPR/Cas9 system was employed in Fov4 to generate gene deletion mutants and GFP labeling. FNP1 plays a critical role in Fov4 hyphal development, virulence, fusaric acid production, and stress responses. This study is the first report to identify and functionally characterize a virulence gene in Fusarium oxysporum f. sp. vasinfectum (Fov) race 4 (Fov4) in cotton wilt pathogenesis.

pathology↗

bioRxiv2021

Streptomyces and Bacillus species utilize volatile organic compounds to impact Fusarium oxysporum f.sp. vasinfectum race 4 (Fov4) virulence and suppress Fusarium wilt in Pima cotton

Emergence of a highly virulent Fusarium oxysporum f.sp. vasinfectum race 4 (Fov4) with aggressiveness towards Pima cotton (Gossypium barbadense) has raised significant concern for cotton producers while revealing challenges in soil-borne cotton disease management strategies which rely heavily on crop resistance and chemical controls. An alternative management approach uses antagonistic bacteria as biocontrol agents against Fov4. Initial studies showed a unique combination of bacteria Bacillus Rz141 and Streptomyces HC658 isolates displayed a mutualistic relationship capable of altering Fov4 growth. Notably, experimental design placed Fov4 between each isolate preventing direct physical contact of bacterial colonies. These observations led us to hypothesize that bacterial volatile organic compounds (VOCs) impact the growth and virulence of Fov4. Ensuring physical separation, I-plate cultures showed Rz141 had a VOC inhibition of 24%. Similarly, physically separated cultures of Rz141 and HC658 showed slight increase in VOC inhibition, 26% with some loss of Fov4 pigmentation. Pathogenicity assays where Fov4-infected Pima cotton was exposed to VOCs from physically separated Rz141 and HC658 showed VOCs can suppress Fov4 infection and reduce tissue darkening. Our results provide evidence that rhizosphere bacteria can use VOCs as a communication tool impacting fungal physiology and virulence, and ultimately Fov4-cotton interactions without direct physical contact.

microbiology↗

bioRxiv2021

Soil-free pathogenicity assay of Fusarium oxysporum f.sp. vasinfectum race 4 on Pima cotton (Gossypium barbadense) seedlings

One of the devastating early season diseases of cotton is Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum (Fov). Recent emergence of highly virulent Fov race 4 (Fov4) and its aggressiveness toward Gossypium barbadense (pima) cultivars are raising significant concerns for the US cotton industry. One of the key challenges in studying Fov4 virulence and cotton Fusarium wilt pathogenesis is establishing a disease assay strategy that can help researcher overcome several technical challenges, including efficient infection and highly reproducible and consistent symptom development. Here, we have developed a small-scale, soil-free Fusarium wilt disease assay that can complement conventional assays with faster symptom development and high reproducibility in infected pima cotton seedlings. Our data showed statistically significant differences (p<0.0001) between Fov4-infected and non-infected pima cotton at 4 and 6 days post inoculation (dpi) when compared to control experiments. At 6 dpi, longitudinal observations under magnification showed Fov4 colonization in primary xylem of infected plants, which is a common symptom observed in Fov4 triggered Fusarium wilt in pima cotton. While this is an artificial assay system, this soil-free disease testing strategy can offer another strategy to supplement current assays when studying pathogen-host interaction in soil-borne diseases.

plant biology↗

bioRxiv2021

Distinct function of Mediator subunits in fungal development, stress response and secondary metabolism in fungal pathogen Fusarium verticillioides

Mediator is a conserved, nucleus-localized, multi-subunit complex highly conserved across eukaryotes. It interacts with RNA polymerase II transcription machinery as well as various transcription factors to regulate gene expression. However, systematic characterization of Mediator complex has not be thoroughly performed in filamentous fungi. In current study, our aim was to investigate key biological functions of Mediator subunits in a mycotoxigentic plant pathogen Fusarium verticillioides. While we recognized some level of divergence in subunits constituent, overall structure remained conserved between Saccharomyces cerevisiae and F. verticillioides. We were able to generate 11 Mediator subunit deletion mutants and characterize serveal important phenotypes associated with fungal development, environmental stress, nutrient utilization, virulence as well as secondary metabolism. While each Mediator subunit deletion mutant showed deficiencies in at least three of the phenotypes tested, it is important to note that different modules or subunits showed unique regulatory role in different cellular processes. Significantly, the deletion of FvMed1 led to increased FB1 production, and we were able to confirm that FvMed1 is transported from the nucleus to the cytoplasm under fumonisin-producing conditions. Taken together, our study characterized the different biological functions of Mediator subunits in plant pathogen F. verticillioides and the possibility of select subunits unique cytoplasmic function independent of the core Mediator. IMPORTANCEMediator is a highly conserved component of RNA polymerase II pre-initiation complex (PIC) in eukaryotes. Studies in yeast have demonstrated the important roles of Mediator subunits, but we lack deeper understanding in filamentous fungi. Here, we selected Fusarium verticillioides, a major fungal pathogen of maize worldwide, to study transcriptional profile followed by mutational analyses of all Mediator genes. Each Mediator subunit deletion mutant showed some shared deficiencies but it is also important that different modules or subunits showed unique regulatory role in different cellular processes. Significantly, FvMed1 deletion mutant showed elevated levels of FB1 production, and we were able to confirm that FvMed1 translocates from the nucleus to cytoplasmic organelles under fumonisin-producing conditions. This is a first study demonstrating that a Mediator subunit regulate cellular functions while not directly associating with PIC in filamentous fungi.

microbiology↗

bioRxiv2020

Two Regulators of G-protein signaling (RGS) proteins FlbA1 and FlbA2 differentially regulate fumonisin B1 biosynthesis in Fusarium verticillioides

Fumonisins are a group of mycotoxins produced by maize pathogen Fusarium verticillioides that pose health concerns to humans and animals. Yet we still lack a clear understanding of the mechanism of fumonisins regulation during pathogenesis. The heterotrimeric G protein complex, which consists of G, G{beta}, and G{gamma} subunits, plays an important role in transducing signals under environmental stress. Furthermore, regulators of G-protein signaling (RGS) proteins act as negative regulators in heterotrimeric G protein signaling. Earlier studies demonstrated that G and G{beta} subunits are positive regulators of fumonisin B1 (FB1) biosynthesis and that two RGS genes, FvFlbA1 and FvFlbA2, were highly upregulated in G{beta} deletion mutant {Delta}Fvgbb1. Saccharomyces cerevisiae and Aspergillus nidulans contain a single copy of FlbA, but F. verticillioides has two putative FvFlbA paralogs, FvFlbA1 and FvFlbA2. Importantly, FvFlbA2 has a negative role in FB1 regulation. In this study, we further characterized functional roles of FvFlbA1 and FvFlbA2. While {Delta}FvflbA1 deletion mutant exhibited no significant defects, {Delta}FvflbA2 and {Delta}FvflbA2/A1 mutants showed thinner aerial hyphal growth while promoting FB1 production. FvFlbA2 is required for proper expression of key conidia regulation genes, including putative FvBRLA, FvWETA, and FvABAA, while suppressing FUM21, FUM1, and FUM8 expression. Split luciferase assays suggest that FvFlbA paralogs interact with key heterotrimeric G protein components to impact F. verticillioides FB1 production and asexual development.

microbiology↗

bioRxiv2020

Fusarium verticillioides peroxins associated with peroxisomal docking/translocation module serve important roles in fumonisin biosynthesis and virulence

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfill various important metabolic functions. In this study, we investigated the role of Docking/Translocation Module (DTM) peroxins, mainly FvPex8, FvPex13, FvPex14, and FvPex33, in Fusarium verticillioides virulence and fumonisin B1 (FB1) biosynthesis. Protein interaction experiments suggested that FvPex13 serves as the core subunit of F. verticillioides DTM. When we generated gene deletion mutants ({Delta}Fvpex8, {Delta}Fvpex13, {Delta}Fvpex14, {Delta}Fvpex33, {Delta}Fvpex33/14) and examined whether the expression of other peroxin genes were affected in the DTM mutants, {Delta}Fvpex8 strain showed most drastic changes to PEX gene expression profiles. Deletion mutants exhibited disparity in carbon source utilization and defect in cell wall integrity when stress agents were applied. Under nutrient starvation, mutants also showed higher levels of lipid droplet accumulation. Notably, {Delta}Fvpex8 mutant showed significant FB1 reduction and altered expression of FUM1 and FUM19 genes. However, FvPex13 was primarily responsible for virulence, while {Delta}Fvpex33/14 double mutant also showed virulence defect. In summary, our study suggests that FvPex13 is the core component of DTM, regulating peroxisome membrane biogenesis as well as PTS1- and PTS2-mediated transmembrane cargo transportation. Importantly, we predict FvPex8 as a key component in DTM that affects peroxisome function in FB1 biosynthesis in F. verticillioides.

microbiology↗

bioRxiv2019

FvLcp1, a type-D fungal LysM protein with Chitin-binding domains, is a secreted protein involved in host recognition and fumonisin production in Fusarium verticillioides - maize kernel interaction

O_LIFusarium verticillioides is one of the key maize ear rot pathogens and produces fumonisins, a group of mycotoxins detrimental to humans and animals. Unfortunately, our understanding on how this fungus interacts with maize kernels to trigger mycotoxin biosynthesis is very limited.\nC_LIO_LIWe performed a systematic computational network-based analysis of large-scale F. verticillioides RNA-seq datasets to identify potential gene subnetwork modules that are associated with virulence and fumonisin regulation.\nC_LIO_LIAmong the highly discriminative subnetwork modules, we identified a putative hub gene FvLCP1, which encodes a putative a type-D fungal LysM protein with a signal peptide, three LysM domains, and two chitin binding domains. FvLcp1 is a unique protein that harbors these domains amongst five representative Fusarium species.\nC_LIO_LIFvLcp1 is a secreted protein important for fumonisin production with LysM domain playing acritical role. Chitin-binding domain was essential for in vitro chitin binding.\nC_LIO_LIUsing rice blast fungus, we learned that FvLcp1 accumulates in appressoria, a key infection structure, suggesting that FvLcp1 could be involved in host recognition and infection. Also, full length FvLcp1 was able to suppress the BAX triggered plant cell death in Nicotiana benthamiana.\nC_LIO_LIThis is the first report where a unique type-D LysM secreted protein with chitin-binding domain in mycotoxigenic fungus F. verticillioides was shown to be potentially involved in suppressing host cell death and promoting fumonisins biosynthesis while the pathogen colonizes maize kernels.\nC_LI

microbiology↗

bioRxiv2019

Characterization of non-canonical G beta-like protein FvGbb2 and its relationship with heterotrimeric G proteins in Fusarium verticillioides

Fusarium verticillioides is a fungal pathogen that is responsible for maize ear rot and stalk rot diseases worldwide. The fungus also produces carcinogenic mycotoxins, fumonisins, on infested maize. Unfortunately, we still lack clear understanding of how the pathogen responds to host and environmental stimuli to trigger fumonisin biosynthesis. The heterotrimeric G protein complex, consisting of canonical G, G{beta}, and G{gamma} subunits, is involved in transducing signals from external stimuli to regulate downstream signal transduction pathways. Previously, we demonstrated that G{beta} protein FvGbb1 has direct impact on fumonisin regulation but no other physiological aspects in F. verticillioides. In this study, we identified and characterized a putative receptor for activated C kinase 1 (RACK1) homolog FvGbb2 as a putative G{beta}-like protein in F. verticillioides. The mutant exhibited severe defects not only in fumonisin biosynthesis but also vegetative growth and conidiation. FvGbb2 was positively associated with carbon source utilization and stress agents but negatively regulated general amino acid control. While FvGbb2 does not interact with canonical G protein subunits, it may interact with diverse proteins in the cytoplasm to regulate vegetative growth, virulence, fumonisin biosynthesis, and stress response in F. verticillioides.

microbiology↗

bioRxiv2019

Fusarium verticillioides SNARE protein FvSyn1 harbors two key functional motifs that play selective roles in fungal development and virulence

Fusarium verticillioides is one of the key fungal pathogens responsible for maize stalk rots. While stalk rot pathogens are prevalent worldwide, our understanding of stalk rot virulence mechanism in pathogenic fungi is still very limited. We previously identified F. verticillioides FvSYN1 gene, which was demonstrated to plays an important role in maize stalk rot virulence. FvSyn1 belongs to a family of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that play critical roles in a variety of developmental processes. In this study, we further characterized the cellular features of FvSyn1 protein motifs in F. verticillioides development and virulence. We generated FvSyn1 motif-specific deletion mutants to further investigate how different motifs contribute to development and virulence. Microscopic observation showed that {Delta}fvsyn1 mutant exhibits rough and hyper-branched hyphae when compared to the wild type progenitor. Moreover, {Delta}fvsyn1 mutant was sensitive to cell wall stress agents resulting in vegetative growth reduction. We showed that FvSyn1::GFP protein is associated with endomembrane but this outcome did not clarify why the deletion of this protein led to stress sensitivity and aberrant hyphal development. Characterization of FvSyn1 domains indicated that both Syntaxin N-terminus (SynN) domain and SNARE C-terminus domain play distinct roles in fungal development but collectively function in virulence. We also determined that two domains in FvSyn1 is not required for fumonisin production. Interestingly, these two domains were involved in carbon nutrient utilization including pectin, starch and sorbitol. This study further characterized the role of FvSyn1 in hyphal growth, localization, cell wall stress response and virulence in F. verticillioides.\n\nHighlights{blacktriangleright} F. verticillioides SNARE protein FvSYN1 is important for vegetative growth and virulence.\n\n {blacktriangleright}FvSYN1 deletion mutant performed better under cell wall stressors.\n\n {blacktriangleright}Dissecting of two domains and investigate the roles.

molecular biology↗

bioRxiv2019

A Rab GTPase protein FvSec4 is necessary for fumonisin B1 biosynthesis and virulence in Fusarium verticillioides

Rab GTPases are responsible for a variety of membrane trafficking and vesicular transportation in fungi. But the role of Rab GTPases in Fusarium verticillioides, one of the key corn pathogens worldwide, remains elusive. These Small GTPases in fungi, particularly those homologous to Saccharomyces cerevisiae Sec4, are known to be associated with protein secretion, vesicular trafficking, secondary metabolism and pathogenicity. Here, we characterized the molecular functions of FvSec4 by generating a null mutant and learned that it is important for vegetative growth, hyphal branching, and conidiation. Interestingly, the mutation did not impair the expression of key conidiation-related genes. Meanwhile, the mutant did not show any defect in sexual development, including perithecia production. GFP-FvSec4 localized to growing hyphal tips, and raised the possibility that FvSec4 is involved in protein trafficking and endocytosis. The mutant exhibited defect in corn stalk rot virulence and also significant alteration of fumonisn B1 production. The mutation led to more sensitivity to oxidative and cell wall stress agents, and defects in carbon utilization. Gene complementation fully restored the defects in the mutant demonstrating that FvSec4 plays important role in these functions. Taken together, our data indicate that FvSec4 plays important roles in F. verticillioides hyphal development, virulence, mycotoxin production and stresses response. Further study is needed to characterize whether the mutation in FvSec4 leads to altered vesicle trafficking and protein secretion, which ultimately impact F. verticillioides physiology and virulence.

microbiology↗

bioRxiv2017

Characterizing co-expression networks underpinning maize stalk rot virulence in Fusarium verticillioides through computational subnetwork module analyses

Fusarium verticillioides is recognized as an important stalk rot pathogen of maize worldwide, but our knowledge of genetic mechanisms underpinning this pathosystem is limited. Previously, we identified a striatin-like protein Fsr1 that plays an important role in stalk rot. To further characterize transcriptome networks downstream of Fsr1, we performed next-generation sequencing (NGS) to investigate relative read abundance and also to infer co-expression networks utilizing the preprocessed expression data through partial correlation. We used a probabilistic pathway activity inference strategy to identify functional subnetwork modules likely involved in virulence. Each subnetwork modules consisted of multiple correlated genes with coordinated expression patterns, but the collective activation levels were significantly different in F. verticillioides wild type versus the mutant. We also identified putative hub genes from predicted subnetworks for functional validation and network robustness studies through mutagenesis, virulence and qPCR studies. Our results suggest that these genes are important virulence genes that regulate the expression of closely correlated genes, demonstrating that these are important hubs of their respective subnetworks. Lastly, we used key F. verticillioides virulence genes to computationally predict a subnetwork of maize genes that potentially respond to fungal genes by applying cointegration-correlation-expression strategy.

microbiology↗