Biology subjects

Goodwin, S. B.

Publications and source records attributed to Goodwin, S. B..

12 recordsLinked to original sources

bioRxiv2026

Development of a CRISPR/Cas9-mediated transformation procedure for the wheat pathogen Zymoseptoria tritici

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), one of the most destructive diseases of wheat worldwide. Although the Z. tritici genome encodes hundreds of predicted effector proteins, functional characterization through the use of genome-editing techniques has been limited due to low homologous recombination efficiency and extensive effector redundancy. In this study, we established and evaluated a CRISPR/Cas9-based genome editing procedure for targeted effector gene disruption in Z. tritici using in vitro-assembled Cas9-sgRNA ribonucleoprotein (RNP) complexes combined with short (60 bp) homologous donor DNA flanks. Using this approach, we successfully generated knockout mutants for a selected candidate effector gene, the Hce2 domain-containing effector Mycgr3107904. Virulence assays on the susceptible wheat cultivar Taichung 29 revealed that two independent{Delta} Mycgr3107904 mutants exhibited a pronounced delay in symptom development compared to the wild-type strain IPO323, with disease onset and progression delayed by approximately 4-5 days. While mutant strains ultimately followed a similar disease trajectory, wild-type-infected leaves displayed extensive necrosis and pycnidia formation at earlier time points, indicating a significant reduction in virulence upon loss of Mycgr3107904. Together, our results demonstrate the feasibility of CRISPR/Cas9-mediated effector gene knockout in Z. tritici and provide functional evidence that Mycgr3107904 contributes to timely disease progression. This work advances genome editing tools for Z. tritici and facilitates systematic dissection of effector functions underlying fungal virulence.

genetics↗

bioRxiv2026

Mapping and Genetic Dissection of a Novel Tar Spot Resistance QTL on Maize Chromosome 1

Tar spot, caused by the obligately biotrophic fungus Phyllachora maydis, significantly threatens maize (Zea mays L.) production across the Americas. Host genetic resistance offers the most viable long-term management strategy. Building on observed differential tar spot susceptibility, we utilized 92 recombinant-inbred lines (RILs) from the Intermated B73 x Mo17 (IBM-94) population to characterize the genetic architecture of resistance. Phenotypic analysis of 92 RILs plus the highly susceptible parent Mo17 and the moderately resistant parent B73 confirmed stable differences in susceptibility, with B73 consistently demonstrating moderate resistance compared to Mo17. Analysis of variance revealed highly significant genetic variation within the population (F = 12.96; p < 0.001). A high Pearson correlation (r = 0.8706, p < 0.0001) and coefficient of determination (R2 = 0.7579) across environments indicated that 76% of the phenotypic variance is attributable to genetic factors. Linkage mapping identified a novel, consistent major QTL cluster on chromosome 1. This cluster comprises five regions (qTAR_1.1 through qTAR_1.5) exceeding the significance threshold (LOD 3.8) in both years. We identified 74 candidate genes including bZIP and RING/U-box proteins at significant SNP peaks. Additionally, gene annotations revealed a high concentration of wall-associated kinases and S-locus lectin protein kinases within the qTAR_1.4 and qTAR_1.5 regions, alongside potential defense-related transcription factors (MYB, bZIP, and C2H2 zinc fingers). These results provide a framework for high-resolution mapping and functional validation to accelerate the development of tar spot-resistant maize cultivars.

plant biology↗

bioRxiv2025

Genome-Wide Association study in a US soft winter wheat population reveals novel and known sources of resistance to the Septoria tritici blotch pathogen Zymoseptoria tritici

Key message GWAS of 337 soft winter wheat genotypes from a breeding program in Indiana, USA identified marker-trait associations that likely correspond with existing and novel resistances to Septoria tritici blotch disease. Abstract Septoria tritici blotch (STB), caused by the ascomycete fungus Zymoseptoria tritici, is a major disease of wheat worldwide. To find additional sources of resistance, Genome-Wide Association (GWA) was used to analyze 337 soft winter wheat genotypes from Indiana, USA, by inoculating seedlings with two isolates of Z. tritici in a complete randomized design. Necrosis and pycnidia development were assessed at 14, 18 and 22 days post inoculation, enabling the calculation of area under the disease progress curve (AUDPC) for each parameter. Adjusted necrosis and pycnidia AUDPC scores were compared with 14,341 high-quality SNPs in a GWA analysis using the FarmCPU and GAPIT CMLM models to identify markers associated with the resistance. Significant (p < 0.05) isolate {xi} genotype interactions were identified, confirming that the phenotypic variation was caused by isolate-specific resistance genes. Overall, 12 marker-trait associations (MTAs) were identified with Z. tritici necrosis and pycnidia resistance. All mapped MTAs were isolate and necrosis/pycnidia specific. Distinct MTAs were mapped for necrosis and pycnidia on chromosome 6A, and for pycnidia on chromosomes 1A, 1D, 3B, 4A, 5B, 6A and 7A. MTAs on chromosomes 5B, 4A, 6A, and 1D likely corresponded with the known genes Stb1, Stb7, Stb15 and Stb19, respectively. MTAs on chromosomes 1A, 3B, 4A, 6A and 7A were not associated with previously known Stb genes and may be novel. Candidate genes near the marker locations have been identified for further investigations. The GWAS showed that Indiana soft winter wheat germplasm segregates for novel and known Stb genes and constitutes a valuable breeding resource for Z. tritici resistance.

plant biology↗

bioRxiv2025

Functional Characterization of Zymoseptoria tritici Candidate Effectors Reveals Their Role in Modulating Immunity in Nicotiana benthamiana

Zymoseptoria tritici is a significant wheat pathogen responsible for Septoria tritici blotch (STB) disease. Despite its economic impact, understanding of the molecular interactions between Z. tritici and its host remains limited, particularly the functions of many uncharacterized candidate effectors. Here, we assessed seven Z. tritici candidate effectors with elevated expression during the early biotrophic phase and transition to necrotrophy by transiently expressing them in the heterologous, non-host system Nicotiana benthamiana, with and without their predicted signal peptides. AlphaFold structural predictions revealed that two candidates share similarity with proteins of known function: a sterol-binding protein from Saccharomyces cerevisiae and a necrosis-inducing effector from the apple canker pathogen Valsa mali. Effector-associated phenotypes did not always correlate with expression timing, and signal peptide presence significantly influenced observed activities. Because N. benthamiana is not a natural host of Z. tritici, observed responses may reflect conserved, host-independent activities of effectors or immune recognition by N. benthamiana, rather than their native functions in wheat. Consistent with this distinction, two candidate effectors, Mycgr3107904 and Mycgr394290, induced cell death in N. benthamiana while also modulating ROS burst, likely indicative of immune recognition. Some candidates attenuate ROS production, suggesting potential targeting of conserved cellular processes, whereas none suppress PBR1-mediated cell death. Overall, our results indicate that candidate effector activities cannot be inferred from expression profiles alone, and that responses in the non-host N. benthamiana system reflect a combination of immune recognition and potential conserved activities, and primarily serve to prioritize candidates for validation in the native wheat-Z. tritici interaction.

molecular biology↗

bioRxiv2025

Real-Time Quantitative PCR Method for Assessing Wheat Cultivars for Resistance to Zymoseptoria tritici

Septoria tritici blotch, caused by Zymoseptoria tritici (formerly Mycosphaerella graminicola), is an economically significant disease of wheat (Triticum aestivum) worldwide. However, there is little understanding of the fungus growth dynamics during the 14- to 18-day latent period between penetration and symptom expression, making it challenging to develop wheat cultivars resistant to Z. tritici Furthermore, environmental factors and variations in disease-scoring systems among evaluators add to the complexity. To address these issues and quantify the fungus growth during the initial stages of infection, we developed a real-time quantitative polymerase chain reaction (real-time PCR) method to monitor the T. aestivum - Z. tritici pathosystem. The assay used specific primers designed from {beta}-tubulin gene sequences of Z. tritici to quantify fungal DNA in susceptible and resistant wheat cultivars and segregating recombinant-inbred lines (RILs) that were inoculated at seedling and adult-plant stages with low or high concentrations of inoculum. The real-time PCR method was compared with visual disease assessment for 0 to 27 days after inoculation (DAI). The results showed that fungal DNA increased more quickly in two susceptible cultivars than in resistant cultivars with the Stb4 or Stb8 genes for resistance. In the susceptible cultivars, the amount of fungal DNA remained low until symptoms became visible at around 18 DAI. Disease severity and fungal DNA in the two resistant cultivars were less than in either susceptible cultivar, starting at 12 DAI. The differences in fungal DNA between resistant and susceptible cultivars were more significant in adult plant tests that used a higher concentration of inoculum. The data analyses showed that the fungus was not eliminated during resistant interactions but could persist throughout the 27 days. Our results suggest that the real-time PCR method can distinguish between resistant and susceptible cultivars starting at 12 DAI and can be used to evaluate early-stage breeding materials for both quantitative and qualitative resistance to Z. tritici.

plant biology↗

bioRxiv2025

Differences Among Fungal Microbiomes Associated with Tar Spot of Corn in Ecuador, Guatemala, and Indiana, USA

Tar spot of corn, caused by the obligate fungal pathogen Phyllachora maydis, was previously found to be associated with Monographella maydis (syn. Microdochium maydis) and Coniothyrium phyllachorae in Mexico. While M. maydis was once believed to cause fisheye lesions and C. phyllachorae was described as a mycoparasite of P. maydis, their presence and roles in tar spot remain uncertain. This study hypothesizes that the tar spot-associated fungal microbiome varies geographically and includes fungal species with inconsistent associations with the disease. To test this, tar spot-infected leaves were collected in Guatemala, Ecuador, and the USA. This study shows that the distribution and abundance of these fungi exhibit a geographical pattern and are not consistently associated with tar spot lesions in all locations. We confirmed the presence of Microdochium in some parts of Ecuador and Guatemala but not in the USA. The role of Microdochium remains unclear and whether it can be a primary pathogen needs to be tested by additional research, as its occurrence and abundance depend more on geographical locations rather than the presence of fisheye lesions. Coniothyrium was detected at low abundance in samples from Ecuador and Guatemala but was absent in the USA. Additionally, we identified the likely mycoparasite Paraphaeosphaeria as highly prevalent across samples from all three countries. Microbiome diversity analysis grouped tar spot samples into four distinct clusters: two dominated by P. maydis, one primarily associated with Microdochium, and a fourth characterized by a combination of P. maydis and Paraphaeosphaeria.

plant biology↗

bioRxiv2024

Comparative genomics of the extremophile Cryomyces antarcticus and other psychrophilic Dothideomycetes

Cryomyces antarcticus is an endolithic fungus that inhabits rock outcrops in Antarctica. It survives extremes of cold, humidity and solar radiation in one of the least habitable environments on Earth. This fungus is unusual because it produces heavily melanized, meristematic growth and is thought to be haploid and asexual. Due to its growth in the most extreme environment, it has been suggested as an organism that could survive on Mars. However, the mechanisms it uses to achieve its extremophilic nature are not known. Over a billion years of fungal evolution has enabled representatives of this kingdom to populate almost all parts of planet Earth and to adapt to some of its most uninhabitable environments including extremes of temperature, salinity, pH, water, light, or other sources of radiation. Comparative genomics can provide clues to the processes underlying biological diversity, evolution, and adaptation. This effort has been greatly facilitated by the 1000 Fungal Genomes project and the JGI MycoCosm portal where sequenced genomes have been assembled into phylogenetic and ecological groups representing different projects, lifestyles, ecologies, and evolutionary histories. Comparative genomics within and between these groups provides insights into fungal adaptations, for example to extreme environmental conditions. Here, we analyze two Cryomyces genomes in the context of additional psychrophilic fungi, as well as non-psychrophilic fungi with diverse lifestyles selected from the MycoCosm database. This analysis identifies families of genes that are expanded and contracted in Cryomyces and other psychrophiles and may explain their extremophilic lifestyle. Higher GC contents of genes and of bases in the third positions of codons may help to stabilize DNA under extreme conditions. Numerous smaller contigs in C. antarcticus suggest the presence of an alternative haplotype that could indicate that the sequenced isolate is diploid or dikaryotic. These analyses provide a first step to unraveling the secrets of the extreme lifestyle of C. antarcticus.

genomics↗

bioRxiv2024

Tar Spot Disease Severity Influences Phyllosphere-Associated Bacterial and Fungal Microbiomes

Tar spot, caused by the obligate fungal pathogen Phyllachora maydis, is a foliar disease of corn that has become a major economic concern in the United States. To test the hypothesis that P. maydis can interact with other foliar microorganisms, we investigated phyllosphere microbiomes in relation to corn inbreds with differential tar spot symptoms under natural infestation in the field. Leaf samples from sixteen inbred lines were assessed for tar spot symptoms, and bacterial and fungal microbiomes were characterized by paired-end sequencing on the Illumina MiSeq platform. Comparison of the phyllosphere microbiomes revealed distinct bacterial and fungal communities between resistant and susceptible lines. Bacterial and fungal species richness was significantly higher in resistant compared to susceptible inbred lines in a sample-specific manner. In contrast, there were no clear differences in diversity when including evenness of bacterial communities between the resistant and susceptible lines. Diversity of fungal communities differed significantly, particularly between twelve of the fourteen susceptible lines versus resistant lines. Plant-beneficial bacterial genera such as Methylorubrum and Quadrisphaera were associated with resistant lines, while Pantoea, Deinococcus and Pseudomonas were the least abundant. The second-most commonly detected fungus likely was a Coniothyrium, but whether it is the same species that was identified as a possible mycoparasite of P. maydis in Central and South America is not known. Fungal genera Cladosporium, Papiliotrema, Cryptococcus, Tilletiopsis and Alternaria were associated with resistant lines while Sphaerellopsis was the least-abundant genus. In contrast, Puccinia, Sphaerellopsis and Phyllachora were the dominant fungal genera in susceptible lines. Our findings imply that P. maydis infection may result in a distinct microbiota with lower diversity. Further analyses of these distinct microbiota between resistant and susceptible lines could lead to a better understanding of the potential role of foliar microbiomes in causing or resisting P. maydis infection.

plant biology↗

bioRxiv2023

Mechanisms of infection and response of the fungal wheat pathogen Zymoseptoria tritici during compatible, incompatible and non-host interactions

Zymoseptoria tritici is responsible for Septoria tritici blotch, a disease causing significant annual yield losses in wheat. To investigate infection phase-specific gene expression in the pathogen, we analyzed gene expression during infection of susceptible (Taichung 29) and resistant (Veranopolis and Israel 493) wheat cultivars, plus the non-host species barley at 1, 3, 6, 10, 17 and 23 days post inoculation (DPI). There were dramatic differences in pathogen gene expression at 10 DPI in the susceptible compared to both resistant interactions. The most pronounced differences in pathogen gene expression were observed at 3 DPI in both the susceptible and resistant host interactions compared to the non-host interaction. Thirty-one putative effectors showed early expression during the susceptible interaction compared to the non-host interaction, and six effectors were selected for subcellular localization studies. Using Agrobacterium-mediated transient expression in Nicotiana benthamiana, subcellular localization assays revealed that two candidate effectors, Mycgr3109710 and Mycgr394290, localized to putative mobile cytosolic bodies when expressed without their signal peptides, suggesting potential roles in intracellular signaling or host gene regulation. When expressed with their native signal peptides, four candidate effectors localized to the cytosol, while one effector, Mycgr3107904, did not accumulate to detectable levels, as shown by immunoblot analysis, indicating degradation. Comparison of pathogen gene expression in the susceptible host to expression in the resistant hosts, allowed us to identify genes that are expressed during the transition from biotrophic to necrotrophic growth at 10 DPI. Comparison of pathogen gene expression in resistant and susceptible hosts, versus in the non-host barley, allowed us to identify genes involved in initial colonization and host recognition. In addition, our study contributes to understanding candidate effectors that are activated early during infection and may play a role in the initial suppression of plant immunity, making them strong candidates for functional characterization.

plant biology↗

bioRxiv2023

Small but significant genetic differentiation among populations of Phyllachora maydis in the midwestern United States revealed by microsatellite (SSR) markers.

Phyllachora maydis Maubl, the causal pathogen of tar spot of corn (Zea mays L.), has emerged recently in the United States and Canada. Studies related to its genetic diversity and population structure are limited and are necessary to improve our understanding of this pathogens biology, ecology, epidemiology, and evolutionary potential within this region. This study developed and used 13 microsatellites (SSR markers) to assess the genetic population structure, diversity, gene flow and reproductive mode of 181 P. maydis samples across five states in the Midwest U.S. The polymorphic information content (PIC) of loci ranged from 0.32 to 0.72 per locus, indicating their high utility for assessing the dynamics of P. maydis populations. Analysis of molecular variance (AMOVA) detected a significantly low, but statistically significant genetic differentiation (FST = 0.15) among populations, where 85% of the variance resided within populations. P. maydis populations were highly diverse (He = 0.55), with moderate gene flow (Nm = 2.80), and showed evidence of sexual recombination ([r]d; p = > 0.001). Structure analysis showed the samples were not geographically structured but rather grouped into two genetic clusters (k =2) of severe genetic admixture suggesting possible long-distance dispersal of aerial spores or infected corn materials among the five Midwest states. Both principal coordinate analysis (PCoA) and discriminate analysis of principal component (DAPC) supported the STRUCTURE analysis of the two clusters. These 13 highly polymorphic molecular markers could be used for future investigations of this pathogens population dynamics within the U.S., and possibly populations outside.

pathology↗

bioRxiv2023

Improved gene annotation of the fungal wheat pathogen Zymoseptoria tritici based on combined Iso-Seq and RNA-Seq evidence

Despite large omics datasets, the establishment of a reliable gene annotation is still challenging for eukaryotic genomes. Here, we used the reference genome of the major fungal wheat pathogen Zymoseptoria tritici (isolate IPO323) as a case study to develop methods to improve eukaryotic gene prediction. Four previous IPO323 annotations identified 10,933 to 13,260 gene models, but only one third of these coding sequences (CDS) have identical structures. To resolve these discrepancies and improve gene models, we generated full-length transcripts using long-read sequencing. This dataset was used together with other evidence (RNA-Seq transcripts and protein sequences) to generate novel ab initio gene models. The selection of the best structure among novel and existing gene models was performed according to transcript and protein evidence using InGenAnnot, a novel bioinformatics suite. Overall, 13,414 re-annotated gene models (RGMs) were predicted, including 671 new genes among which 53 encoded effector candidates. This process corrected many of the errors (15%) observed in previous gene models (coding sequence fusions, false introns, missing exons). While fungal genomes have poor annotations of untranslated regions (UTRs), our Iso-Seq long-read sequences outlined 5 and 3UTRs for 73% of the RGMs. Alternative transcripts were identified for 13% of RGMs, mostly due to intron retention (75%), likely corresponding to unprocessed pre-mRNAs. A total of 353 genes displayed alternative transcripts with combinations of previously predicted or novel exons. Long non-coding transcripts (lncRNAs) and double-stranded RNAs from two fungal viruses were also identified. Most lncRNAs corresponded to antisense transcripts of genes (52%). lncRNAs that were up or down regulated during infection were enriched in antisense transcripts (70%), suggesting their involvement in the control of gene expression. Our results showed that combining different ab initio gene predictions and evidence-driven curation using InGenAnnot improved the quality of gene annotations of a compact eukaryotic genome. Our analysis also provided new insights into the transcriptional landscape of Z. tritici, helping develop an increasingly complex picture of its biology.

genomics↗

bioRxiv2022

A thousand-genome panel retraces the global spread and climatic adaptation of a major crop pathogen

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.

genomics↗