Biology subjects

White, F. F.

Publications and source records attributed to White, F. F..

9 recordsLinked to original sources

bioRxiv2025

From lab to field: analyses of genome-edited bacterial blight resistant rice

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the major drivers of yield losses in Africa and Asia. Xoo secretes TAL-effectors (TALe) that induce host SWEET sucrose uniporter by binding to the effector binding element (EBE) of SWEET promoters, likely required for Xoo reproduction and virulence. We had multiplex edited the EBEs of three SWEET genes to prevent TALe binding, producing genome-edited (GEd) rice mega-varieties (IR64, Ciherang-Sub1 for Asia, and Komboka for Africa) that were resistant to a wide spectrum of Xoo strains. Here, we report comprehensive analyses of the GEd lines, including evaluation of agronomic performance in multi-location multi-season experimental field plots under different fertilization regimes, and tests for the presence/absence of foreign DNA/transgene in the offspring of GEd lines (IR64-BC1T6, Ciherang-Sub1-BC1T5, Komboka-T3). Various strategies were evaluated, including herbicide tolerance, PCR, DNA gel blotting, whole genome sequencing (WGS), and specific tests stipulated by country-specific biosafety guidelines. Different WGS technologies were evaluated and also used to identify heritability of the edits, single nucleotide polymorphisms (SNPs), and insertions/deletions (indels) that might have resulted from somaclonal variation and potential GE-induced off-target mutations. Complete genome reference sequences for the parental lines IR64, Ciherang-Sub1, and Komboka are provided. In the field experiments, the GEd lines did not show performance defects. Together, the results indicate that select GE lines do not contain foreign DNA or transgene fragments and fulfill the requirements for treatment equivalent to classical breeding lines in countries such as India and Kenya.

plant biology↗

bioRxiv2023

Genome analysis and hyphal movement characterization of the hitchhiker endohyphal Enterobacter sp. from Rhizoctonia solani

Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria (EHB) have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. (En-Cren) isolated from the generalist fungal pathogen Rhizoctonia solani. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved. ImportanceThe genome characterization and comparative genomics analysis of En-Cren provided the foundation and resources for a better understanding of the ecology and evolution of this EHB in the rhizosphere. The ability to produce IAA and PAA may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction yet to be explored.

microbiology↗

bioRxiv2021

Bacterium-Enabled Transient Gene Activation by Artificial Transcription Factor for Resolving Gene Regulation in Maize

Cellular functions are diversified through intricate transcription regulations, and an understanding gene regulation networks is essential to elucidating many developmental processes and environmental responses. Here, we employed the Transcriptional-Activator Like effectors (TALes), which represent a family of transcription factors that are synthesized by members of the {gamma}-proteobacterium genus Xanthomonas and secreted to host cells for activation of targeted host genes. Through delivery by the maize pathogen, Xanthomonas vasicola pv. vasculorum, designer TALes (dTALes), which are synthetic TALes, were used to induce the expression of the maize gene glossy3 (gl3), a MYB transcription factor gene involved in the cuticular wax biosynthesis. RNA-Seq analysis of leaf samples identified 146 gl3 downstream genes. Eight of the nine known genes known to be involved in the cuticular wax biosynthesis were up-regulated by at least one dTALe. A top-down Gaussian graphical model predicted that 68 gl3 downstream genes were directly regulated by GL3. A chemically induced mutant of the gene Zm00001d017418 from the gl3 downstream gene, encoding aldehyde dehydrogenase, exhibited a typical glossy leaf phenotype and reduced epicuticular waxes. The bacterial protein delivery of artificial transcription factors, dTALes, proved to be a straightforward and powerful approach for the revelation of gene regulation in plants.

plant biology↗

bioRxiv2020

Chromosome-level Genome Assembly of a Regenerable Maize Inbred Line A188

The highly embryogenic and transformable maize inbred line A188 is an attractive model for analyzing maize gene function. Here we constructed a chromosome-level genome assembly of A188 using long reads and optical maps. Genome comparison of A188 with the reference line B73 identified pervasive structural variation, including a 1.8 Mb duplication on the Gametophyte factor1 locus for unilateral cross-incompatibility and six inversions of 0.7 Mb or greater. Increased copy number of the gene, carotenoid cleavage dioxygenase 1 (ccd1) in A188 is associated with elevated expression during seed development. High ccd1 expression together with low expression of yellow endosperm 1 (y1) condition reduced carotenoid accumulation, which accounts for the white seed phenotype of A188 that contrasts with the yellow seed of B73 that has high expression of y1 and low expression of the single-copy ccd1. Further, transcriptome and epigenome analyses with the A188 reference genome revealed enhanced expression of defense pathways and altered DNA methylation patterns of embryonic callus.

genomics↗

bioRxiv2020

Estimating Assembly Base Errors Using K-mer Abundance Difference (KAD) Between Short Reads and Genome Assembled Sequences

Genome sequences provide genomic maps with a single-base resolution for exploring genetic contents. Sequencing technologies, particularly long reads, have revolutionized genome assemblies for producing highly continuous genome sequences. However, current long-read sequencing technologies generate inaccurate reads that contain many errors. Some errors are retained in assembled sequences, which are typically not completely corrected by using either long reads or more accurate short reads. The issue commonly exists but few tools are dedicated for computing error rates or determining error locations. In this study, we developed a novel approach, referred to as K-mer Abundance Difference (KAD), to compare the inferred copy number of each k-mer indicated by short reads and the observed copy number in the assembly. Simple KAD metrics enable to classify k-mers into categories that reflect the quality of the assembly. Specifically, the KAD method can be used to identify base errors and estimate the overall error rate. In addition, sequence insertion and deletion as well as sequence redundancy can also be detected. Therefore, KAD is valuable for quality evaluation of genome assemblies and, potentially, provides a diagnostic tool to aid in precise error correction. KAD software has been developed to facilitate public uses.

bioinformatics↗

bioRxiv2019

An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly

TALes (Transcription Activator-Like effectors) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single strand annealing and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately one month compared to three months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

microbiology↗

bioRxiv2019

Genomic acquisitions in emerging populations of Xanthomonas vasicola pv. vasculorum infecting corn in the U.S. and Argentina

Xanthomonas vasicola pv. vasculorum (Xvv) is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this bacteria have greatly increased in the past years in South America and in the U.S., where it is now present in most of the corn producing states. Phenotypic characterization showed that the emerging U.S. and South American Xvv populations may have increased virulence in corn compared to older strains. To understand the genetic mechanisms behind the increased virulence in this group, we used comparative genomics to identify gene acquisitions in Xvv genomes from the U.S. and Argentina. We sequenced 41 genomes of Xvv and the related sorghum-infecting X. vasicola pv. holcicola (Xvh). A comparison of all available X. vasicola genomes showed the phylogenetic relationships in the group and identified clusters of genes associated with the emerging Xvv populations. The newly acquired gene clusters showed evidence of horizontal transfer to Xvv and included candidate virulence factors. One cluster, in particular, corresponded to a prophage transferred from Xvh to all Xvv from Argentina and the U.S. The prophage contains putative secreted proteins, which represent candidates for virulence determinants in these populations and await further molecular characterization.

microbiology↗

bioRxiv2018

Temporal Small RNA Expression Profiling Under Drought Reveals a Potential Regulatory Role of snoRNAs in Drought Responses of Maize

Small RNAs (sRNAs) are short noncoding RNAs that play roles in many biological processes, including drought responses in plants. However, how the expression of sRNAs dynamically changes with the gradual imposition of drought stress in plants is largely unknown. We generated time-series sRNA sequence data from maize seedlings under drought stress and under well-watered conditions at the same time points. Analyses of length, functional annotation, and abundance of 736,372 non-redundant sRNAs from both drought and well-watered data, as well as genome copy number and chromatin modifications at the corresponding genomic regions, revealed distinct patterns of abundance, genome organization, and chromatin modifications for different sRNA classes of sRNAs. The analysis identified 6,646 sRNAs whose regulation was altered in response to drought stress. Among drought-responsive sRNAs, 1,325 showed transient down-regulation by the seventh day, coinciding with visible symptoms of drought stress. The profiles revealed drought-responsive microRNAs, as well as other sRNAs that originated from ribosomal RNAs (rRNAs), splicing small nuclear RNAs, and small nucleolar RNAs (snoRNA). Expression profiles of their sRNA derivers indicated that snoRNAs might play a regulatory role through regulating stability of rRNAs and splicing small nuclear RNAs under drought condition.

genomics↗

bioRxiv2018

Effector Gene Reshuffling Involves Dispensable Mini-chromosomes in the Wheat Blast Fungus

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. To understand genetic diversity in wheat-infecting strains, we report a near-finished reference genome of a recent field isolate generated using long read sequencing and a novel scaffolding approach with long-distance paired genomic sequences. The genome assemblage includes seven core chromosomes and sequences from a dispensable mini-chromosome that harbors effector genes normally found on the ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and two mini-chromosomes from another field isolate each contain different effector homologous genes and core chromosome end sequences. The mini-chromosome is highly repetitive and is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and non-dispensable core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.\n\nSignificance statementThe emerging blast disease on wheat is proving even harder to control than the ancient, still-problematic rice blast disease. Potential wheat resistance identified using strains isolated soon after disease emergence are no longer effective in controlling recent aggressive field isolates from wheat in South America and South Asia. We report that recent wheat pathogens can contain one or two highly-variable conditionally-dispensable mini-chromosomes, each with an amalgamation of effector sequences that are duplicated or absent from pathogen core chromosome ends. Well-studied effectors found on different core chromosomes in rice pathogens appear side-by-side in wheat pathogen mini-chromosomes. The rice pathogen often overcomes deployed resistance genes by deleting triggering effector genes. Localization of effectors on mini-chromosomes, which are unstably transmitted during growth, would accelerate pathogen adaptation in the field.

genomics↗