publications

2024

1. Overexpression of Vitis GRF4-GIF1 improves regeneration efficiency in diploid Fragaria vesca Hawaii 4

   Esther Rosales Sanchez , R. Jordan Price, Federico Marangelli , Kirsty McLeary , Richard J. Harrison , and 1 more author

   Research Square, 2024

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   Plant breeding played a very important role in transforming strawberries from being a niche crop with a small geographical footprint into an economically important crop grown across the planet. But even modern marker assisted breeding takes a considerable amount of time, over multiple plant generations, to produce a plant with desirable traits. As a quicker alternative, plants with desirable traits can be raised through tissue culture by doing precise genetic manipulations. Overexpression of morphogenic regulators previously known for meristem development provides an efficient strategy for easier regeneration and transformation in multiple crops. In this study, we show the results for overexpression of chimeric GRF4-GIF1 in diploid strawberry Fragaria vesca Hawaii 4 (strawberry) where Vitis GRF4-GIF1 chimera provides significantly higher regeneration efficiency. We present here a comprehensive protocol for strawberry regeneration and transformation under control condition as compared to ectopic expression of GRF4-GIF1 chimeras from different plants. We report that ectopic expression of Vitis vinifera VvGRF4-GIF1 provide significantly higher regeneration efficiency during retransformation over wild-type plants. On the other hand, deregulated expression of miRNA resistant version of Vitis GRF4-GIF1 or TaGRF4-GIF (wheat) resulted in abnormalities. Transcriptomic analysis between the different chimeric GRF4-GIF1 lines indicate that differential expression of FvExpansin might be responsible for the pleiotropic effects. Similarly, cytokinin dehydrogenase/oxygenase and cytokinin responsive response regulators also showed differential expression indicating GRF4-GIF1 pathway playing important role in controlling cytokinin homeostasis. Our data indicate that ectopic expression of Vitis vinifera VvGRF4-GIF1 chimera can provide significant advantage over wild-type plants during strawberry regeneration without producing any pleiotropic effects seen for the miRNA resistant VvGRF4-GIF1.

2. Comparative genomics and transcriptomics reveal differences in effector complement and expression between races of Fusarium oxysporum f.sp. lactucae

   Helen J. Bates , Jamie Pike , R. Jordan Price, Sascha Jenkins , John Connell , and 4 more authors

   bioRxiv, 2024

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   This study presents the first genome and transcriptome analyses for Fusarium oxysporum f.sp. lactucae (Fola) which causes Fusarium wilt disease of lettuce. Long-read genome sequencing of three race 1 (Fola1) and three race 4 (Fola4) isolates revealed key differences in putative effector complement between races and with other F. oxysporum f.spp. following mimp-based bioinformatic analyses. Notably, homologues of Secreted in Xylem (SIX) genes, also present in many other F. oxysporum f.spp, were identified in Fola, with both SIX9 and SIX14 (multiple copies with sequence variants) present in both Fola1 and Fola4. All Fola4 isolates also contained an additional single copy of SIX8. RNAseq of lettuce following infection with Fola1 and Fola4 isolates identified highly expressed effectors, some of which were homologues of those reported in other F. oxysporum f.spp. including several in F. oxysporum f.sp. apii. Although SIX8, SIX9 and SIX14 were all highly expressed in Fola4, of the two SIX genes present in Fola1, only SIX9 was expressed as further analysis revealed that copies of SIX14 gene copies were disrupted by insertion of a transposable element. Two variants of Fola4 were also identified based on different genome and effector-based analyses. This included two different SIX8 sequence variants which were divergently transcribed from a shared promoter with either PSE1 or PSL1 respectively. In addition there was evidence of two independent instances of HCT in the different Fola4 variants. The involvement of helitrons in Fola genome rearrangement and gene expression is discussed.Competing Interest StatementThe authors have declared no competing interest.The datasets generated for this study can be found in the article and supplementary material. All raw sequencing data and new genome assemblies presented have been submitted to the NCBI under the BioProject ID PRJNA1092066. Further enquiries can be directed to the corresponding author.

2023

1. Chromosome-scale genome sequence assemblies of the ‘Autumn Bliss’ and ‘Malling Jewel’ cultivars of the highly heterozygous red raspberry (Rubus idaeus L.) derived from long-read Oxford Nanopore sequence data

   R. Jordan Price, Jahn Davik , Felicidad Fernandéz Fernandéz , Helen J. Bates , Samantha Lynn , and 7 more authors

   PLOS ONE, May 2023

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   Red raspberry (Rubus idaeus L.) is an economically valuable soft-fruit species with a relatively small ( 300 Mb) but highly heterozygous diploid (2n = 2x = 14) genome. Chromosome-scale genome sequences are a vital tool in unravelling the genetic complexity controlling traits of interest in crop plants such as red raspberry, as well as for functional genomics, evolutionary studies, and pan-genomics diversity studies. In this study, we developed genome sequences of a primocane fruiting variety (‘Autumn Bliss’) and a floricane variety (‘Malling Jewel’). The use of long-read Oxford Nanopore Technologies sequencing data yielded long read lengths that permitted well resolved genome sequences for the two cultivars to be assembled. The de novo assemblies of ‘Malling Jewel’ and ‘Autumn Bliss’ contained 79 and 136 contigs respectively, and 263.0 Mb of the ‘Autumn Bliss’ and 265.5 Mb of the ‘Malling Jewel’ assembly could be anchored unambiguously to a previously published red raspberry genome sequence of the cultivar ‘Anitra’. Single copy ortholog analysis (BUSCO) revealed high levels of completeness in both genomes sequenced, with 97.4% of sequences identified in ‘Autumn Bliss’ and 97.7% in ‘Malling Jewel’. The density of repetitive sequence contained in the ‘Autumn Bliss’ and ‘Malling Jewel’ assemblies was significantly higher than in the previously published assembly and centromeric and telomeric regions were identified in both assemblies. A total of 42,823 protein coding regions were identified in the ‘Autumn Bliss’ assembly, whilst 43,027 were identified in the ‘Malling Jewel’ assembly. These chromosome-scale genome sequences represent an excellent genomics resource for red raspberry, particularly around the highly repetitive centromeric and telomeric regions of the genome that are less complete in the previously published ‘Anitra’ genome sequence.

2022

1. Stress combined with loss of the Candida albicans SUMO protease Ulp2 triggers selection of aneuploidy via a two-step process

   Marzia Rizzo , Natthapon Soisangwan , Samuel Vega-Estevez , R. Jordan Price, Chloe Uyl , and 5 more authors

   PLOS Genetics, Dec 2022

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   A delicate balance between genome stability and instability ensures genome integrity while generating genetic diversity, a critical step for evolution. Indeed, while excessive genome instability is harmful, moderated genome instability can drive adaptation to novel environments by maximising genetic variation. Candida albicans, a human fungal pathogen that colonises different parts of the human body, adapts rapidly and frequently to different hostile host microenvironments. In this organism, the ability to generate large-scale genomic variation is a key adaptative mechanism triggering dangerous infections even in the presence of antifungal drugs. Understanding how fitter novel karyotypes are selected is key to determining how C. albicans and other microbial pathogens establish infections. Here, we identified the SUMO protease Ulp2 as a regulator of C. albicans genome integrity through genetic screening. Deletion of ULP2 leads to increased genome instability, enhanced genome variation and reduced fitness in the absence of additional stress. The combined stress caused by the lack of ULP2 and antifungal drug treatment leads to the selection of adaptive segmental aneuploidies that partially rescue the fitness defects of ulp2Δ/Δ cells. Short and long-read genomic sequencing demonstrates that these novel genotypes are selected via a two-step process leading to the formation of novel chromosomal fragments with breakpoints at microhomology regions and DNA repeats.

2019

1. Chromatin Profiling of the Repetitive and Nonrepetitive Genomes of the Human Fungal Pathogen Candida albicans

   R. Jordan Price, Esther Weindling , Judith Berman , and Alessia Buscaino

   mBio, Dec 2019

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   The fungus Candida albicans is an opportunistic pathogen that normally lives on the human body without causing any harm. However, C. albicans is also a dangerous pathogen responsible for millions of infections annually. C. albicans is such a successful pathogen because it can adapt to and thrive in different environments. Chemical modifications of chromatin, the structure that packages DNA into cells, can allow environmental adaptation by regulating gene expression and genome organization. Surprisingly, the contribution of chromatin modification to C. albicans biology is still largely unknown. For the first time, we analyzed C. albicans chromatin modifications on a genome-wide basis. We demonstrate that specific chromatin states are associated with distinct regions of the C. albicans genome and identify the roles of the chromatin modifiers Sir2 and Set1 in shaping C. albicans chromatin and gene expression. Eukaryotic genomes are packaged into chromatin structures that play pivotal roles in regulating all DNA-associated processes. Histone posttranslational modifications modulate chromatin structure and function, leading to rapid regulation of gene expression and genome stability, key steps in environmental adaptation. Candida albicans, a prevalent fungal pathogen in humans, can rapidly adapt and thrive in diverse host niches. The contribution of chromatin to C. albicans biology is largely unexplored. Here, we generated the first comprehensive chromatin profile of histone modifications (histone H3 trimethylated on lysine 4 [H3K4me3], histone H3 acetylated on lysine 9 [H3K9Ac], acetylated lysine 16 on histone H4 [H4K16Ac], and γH2A) across the C. albicans genome and investigated its relationship to gene expression by harnessing genome-wide sequencing approaches. We demonstrated that gene-rich nonrepetitive regions are packaged into canonical euchromatin in association with histone modifications that mirror their transcriptional activity. In contrast, repetitive regions are assembled into distinct chromatin states; subtelomeric regions and the ribosomal DNA (rDNA) locus are assembled into heterochromatin, while major repeat sequences and transposons are packaged in chromatin that bears features of euchromatin and heterochromatin. Genome-wide mapping of γH2A, a marker of genome instability, identified potential recombination-prone genomic loci. Finally, we present the first quantitative chromatin profiling in C. albicans to delineate the role of the chromatin modifiers Sir2 and Set1 in controlling chromatin structure and gene expression. This report presents the first genome-wide chromatin profiling of histone modifications associated with the C. albicans genome. These epigenomic maps provide an invaluable resource to understand the contribution of chromatin to C. albicans biology and identify aspects of C. albicans chromatin organization that differ from that of other yeasts. IMPORTANCE The fungus Candida albicans is an opportunistic pathogen that normally lives on the human body without causing any harm. However, C. albicans is also a dangerous pathogen responsible for millions of infections annually. C. albicans is such a successful pathogen because it can adapt to and thrive in different environments. Chemical modifications of chromatin, the structure that packages DNA into cells, can allow environmental adaptation by regulating gene expression and genome organization. Surprisingly, the contribution of chromatin modification to C. albicans biology is still largely unknown. For the first time, we analyzed C. albicans chromatin modifications on a genome-wide basis. We demonstrate that specific chromatin states are associated with distinct regions of the C. albicans genome and identify the roles of the chromatin modifiers Sir2 and Set1 in shaping C. albicans chromatin and gene expression.

2018

1. The fungal-specific Hda2 and Hda3 proteins regulate morphological switches in the human fungal pathogen Candida albicans

   Misty R. Peterson , R. Jordan Price, Sarah Gourlay , Alisha May , Jennifer Tullet , and 1 more author

   bioRxiv, Dec 2018

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   The human fungal pathogen Candida albicans is responsible for millions of infections annually. Due to the few available anti-fungal drugs and the increasing incidence of drug resistance, the number of C. albicans infections is dramatically increasing. Morphological switches, such as the white-opaque switch and the yeast-hyphae switch, are key for the development of C. albicans pathogenic traits. Lysine deacetylases are emerging as important regulators of morphological switches. Yet, targeting lysine deacetylases for drug development is problematic due to the high homology between the fungal and human proteins that could result in toxicity. Here we provide evidence that the fungal specific proteins Hda2 and Hda3 interact with the lysine deacetylase Hda1. By combining phenotypic analyses with genome-wide transcriptome analyses, we demonstrate that Hda2 and Hda3 control C. albicans morphological switches. Under nutrient-rich conditions, deletion of HDA2 or HDA3 leads to moderate overexpression of the master regulator of white-opaque switching WOR1 and increase switching frequency. Under hyphae inducing conditions, deletion of HDA2 and HDA3 block hyphae development. However, deletion of HDA2 and HDA3 does not affect hyphae-formation and virulence in vivo. We propose that Hda2 and Hda3 are good targets for the development of anti-fungal drugs to be used in combination therapy.

2016

1. The Chromatin of Candida albicans Pericentromeres Bears Features of Both Euchromatin and Heterochromatin

   Verónica Freire-Benéitez , R. Jordan Price, and Alessia Buscaino

   Frontiers in Microbiology, Dec 2016

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   Centromeres, sites of kinetochore assembly, are important for chromosome stability and integrity. Most eukaryotes have regional centromeres epigenetically specified by the presence of the histone H3 variant CENP-A. CENP-A chromatin is often surrounded by pericentromeric regions packaged into transcriptionally silent heterochromatin. Candida albicans, the most common human fungal pathogen, possesses small regional centromeres assembled into CENP-A chromatin. The chromatin state of C. albicans pericentromeric regions is unknown. Here, for the first time, we address this question. We find that C. albicans pericentromeres are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Pericentromeric chromatin is associated with nucleosomes that are highly acetylated, as found in euchromatic regions of the genome; and hypomethylated on H3K4, as found in heterochromatin. This intermediate chromatin state is inhibitory to transcription and partially represses expression of proximal genes and inserted marker genes. Our analysis identifies a new chromatin state associated with pericentromeric regions.

2. Candida albicans repetitive elements display epigenetic diversity and plasticity

   Verónica Freire-Benéitez , R. Jordan Price, Daniel Tarrant , Judith Berman , and Alessia Buscaino

   Scientific Reports, Mar 2016

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   Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30\thinspace\textdegreeC, while robust heterochromatin is assembled over these regions at 39\thinspace\textdegreeC, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation.

3. Folic acid induces cell type-specific changes in the transcriptome of breast cancer cell lines: a proof-of-concept study

   R. Jordan Price, Karen A. Lillycrop , and Graham C. Burdge

   Journal of Nutritional Science, Mar 2016

2015

1. Folic acid supplementation in vitro induces cell type–specific changes in BRCA1 and BRCA 2 mRNA expression, but does not alter DNA methylation of their promoters or DNA repair

   R. Jordan Price, Karen A. Lillycrop , and Graham C. Burdge

   Nutrition Research, Mar 2015

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   Dietary supplementation with folic acid (FA) has been shown to induce opposing effects on cancer-related outcomes. The mechanism underlying such heterogeneity is unclear. We hypothesized that FA supplementation induces changes in breast cancer–associated (BRCA) genes 1 and 2 expression and function through altered epigenetic regulation in a cell type–dependent manner. We investigated the effect of treating normal and cancer cells with physiologically relevant FA concentrations on the mRNA and protein expression, capacity for DNA repair, and DNA methylation of BRCA1 and BRCA2. FA treatment induced dose-related increases in BRCA1 mRNA expression in HepG2, Huh-7D12, Hs578T, and JURKAT and in BRCA2 in HepG2, Hs578T, MCF7, and MDA-MB-157 cells. FA did not affect the corresponding normal cells or on any of the ovarian cell lines. Folic acid induced increased BRCA1 protein expression in Hs578T, but not HepG2 cells, whereas BRCA2 protein levels were undetectable. FA treatment did not alter DNA repair in liver-derived cells, whereas there were transient effects on breast-derived cells. There was no effect of FA treatment on BRCA1 or BRCA2 DNA methylation, although there was some variation in the methylation of specific CpG loci between some cell lines. Overall, these findings show that the effects of FA on BRCA-related outcomes differ between cells lines, but the biological consequences of induced changes in BRCA expression appear to be at most limited.

2. The Link Between Early Life Nutrition and Cancer Risk

   R. Jordan Price, Graham C. Burdge , and Karen A. Lillycrop

   Current Nutrition Reports, Mar 2015

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   Traditionally, cancer has been considered a disease caused by genetic alterations. However, there is growing evidence that the environment, particularly a person’s early life environment, can influence cancer risk. The mechanism by which the environment has been suggested to influence cancer risk is through the altered epigenetic regulation of genes. Epigenetic processes, which include DNA methylation, induce stable changes in gene expression without altering the gene sequence. A number of environmental factors, including nutrition, have been shown to alter the epigenome, leading to long term changes in gene expression and an altered susceptibility to disease. Using evidence from epidemiological and experimental studies, this review will discuss the hypothesis that changes in diet during early development can lead to an altered susceptibility to cancer as the result of modified epigenetic regulation of genes.

2013

1. Depletion of Uhrf1 inhibits chromosomal DNA replication in Xenopus egg extracts

   Elaine M. Taylor , Nicola M. Bonsu , R. Jordan Price, and Howard D. Lindsay

   Nucleic Acids Research, Jun 2013

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   UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) has a well-established role in epigenetic regulation through the recognition of various histone marks and interaction with chromatin-modifying proteins. However, its function in regulating cell cycle progression remains poorly understood and has been largely attributed to a role in transcriptional regulation. In this study we have used Xenopus laevis egg extracts to analyse Uhrf1 function in DNA replication in the absence of transcriptional influences. We demonstrate that removal of Uhrf1 inhibits chromosomal replication in this system. We further show that this requirement for Uhrf1, or an associated factor, occurs at an early stage of DNA replication and that the consequences of Uhrf1 depletion are not solely due to its role in loading Dnmt1 onto newly replicated DNA. We describe the pattern of Uhrf1 chromatin association before the initiation of DNA replication and show that this reflects functional requirements both before and after origin licensing. Our data demonstrate that the removal of Xenopus Uhrf1 influences the chromatin association of key replication proteins and reveal Uhrf1 as an important new factor required for metazoan DNA replication.