scholarly journals Improving disease resistance in plants by editing the epigenome

2022 ◽  
Author(s):  
Kira M Veley ◽  
Kiona Elliott ◽  
Greg Jensen ◽  
Zhenhui Zhong ◽  
Suhua Feng ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Disease Resistance ◽  
Transcriptional Activation ◽  
Epigenetic Modification ◽  
Crop Improvement ◽  
Host Susceptibility ◽  
Transcription Activator ◽  
Cassava Bacterial Blight ◽  
S Genes

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas axonopodis pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. We directed methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display increased resistance to CBB while maintaining normal growth and development. This work establishes epigenome editing as a new strategy for crop improvement.

scholarly journals Functional analysis of the heterotrimeric NF-Y transcription factor complex in cassava disease resistance

2019 ◽  
Vol 124 (7) ◽  
pp. 1185-1197 ◽  
Author(s):  
Xinyi He ◽  
Guoyin Liu ◽  
Bing Li ◽  
Yanwei Xie ◽  
Yunxie Wei ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Disease Resistance ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Plant Disease Resistance ◽  
Plant Defence ◽  
Virus Induced Gene Silencing ◽  
Cassava Bacterial Blight ◽  
Transcription Factor Complex

Abstract Background and Aims The nuclear factor Y (NF-Y) transcription factor complex is important in plant growth, development and stress response. Information regarding this transcription factor complex is limited in cassava (Manihot esculenta). In this study, 15 MeNF-YAs, 21 MeNF-YBs and 15 MeNF-YCs were comprehensively characterized during plant defence. Methods Gene expression in MeNF-Ys was examined during interaction with the bacterial pathogen Xanthomonas axonopodis pv. manihotis (Xam). The yeast two-hybrid system was employed to investigate protein–protein interactions in the heterotrimeric NF-Y transcription factor complex. The in vivo roles of MeNF-Ys were revealed by virus-induced gene silencing (VIGS) in cassava. Key Results The regulation of MeNF-Ys in response to Xam indicated their possible roles in response to cassava bacterial blight. Protein–protein interaction assays identified the heterotrimeric NF-Y transcription factor complex (MeNF-YA1/3, MeNF-YB11/16 and MeNF-YC11/12). Moreover, the members of the heterotrimeric NF-Y transcription factor complex were located in the cell nucleus and conferred transcriptional activation activity to the CCAAT motif. Notably, the heterotrimeric NF-Y transcription factor complex positively regulated plant disease resistance to Xam, confirmed by a disease phenotype in overexpressing plants in Nicotiana benthamiana and VIGS in cassava. Consistently, the heterotrimeric NF-Y transcription factor complex positively regulated the expression of pathogenesis-related genes (MePRs). Conclusions The NF-Y transcription factor complex (MeNF-YA1/3, MeNF-YB11/16 and MeNF-YC11/12) characterized here was shown to play a role in transcriptional activation of MePR promoters, contributing to the plant defence response in cassava.

scholarly journals Gene tagging via CRISPR-mediated homology-directed repair in cassava

2021 ◽  
Vol 11 (4) ◽  
Author(s):  
Kira M Veley ◽  
Ihuoma Okwuonu ◽  
Greg Jensen ◽  
Marisa Yoder ◽  
Nigel J Taylor ◽  
...  
Keyword(s):  
Bacterial Blight ◽  
Gene Editing ◽  
Crop Improvement ◽  
Transcription Activator ◽  
Manihot Esculenta Crantz ◽  
Cassava Bacterial Blight ◽  
Tal Effector ◽  
Homology Directed Repair ◽  
Important Disease

Abstract Research on a few model plant–pathogen systems has benefitted from years of tool and resource development. This is not the case for the vast majority of economically and nutritionally important plants, creating a crop improvement bottleneck. Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is an important disease in all regions where cassava (Manihot esculenta Crantz) is grown. Here, we describe the development of cassava that can be used to visualize one of the initial steps of CBB infection in vivo. Using CRISPR-mediated homology-directed repair (HDR), we generated plants containing scarless insertion of GFP at the 3’ end of CBB susceptibility (S) gene MeSWEET10a. Activation of MeSWEET10a-GFP by the transcription activator-like (TAL) effector TAL20 was subsequently visualized at transcriptional and translational levels. To our knowledge, this is the first such demonstration of HDR via gene editing in cassava.

scholarly journals DNA methylation presents distinct binding sites for human transcription factors

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Shaohui Hu ◽  
Jun Wan ◽  
Yijing Su ◽  
Qifeng Song ◽  
Yaxue Zeng ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Epigenetic Modification ◽  
Specific Binding ◽  
Protein Microarray ◽  
Embryonic Stem ◽  
Underlying Mechanism ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Promoter Regions

DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription.

264 ANALYSIS OF DNA METHYLATION PATTERN IN PRE-IMPLANTATION-STAGE EMBRYOS DERIVED FROM NUCLEAR TRANSFER USING PORCINE EMBRYONIC GERM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 248
Author(s):  
D.-H. Choi ◽  
C.-H. Park ◽  
S.-G. Lee ◽  
H.-S. Kim ◽  
H.-Y. Son ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Perinatal Death ◽  
Epigenetic Modification ◽  
Methylation Pattern ◽  
Paternal Allele ◽  
High Birth Weight ◽  
Aberrant Methylation

Somatic cell nuclear transfer (SCNT) has been successfully used to produce live cloned offspring in various mammals. However, some studies had reported that cloned embryos by SCNT had many problems in reprogramming or epigenetic modification, such as DNA methylation. DNA methylation is an essential process in epigenetic modification for development, and aberrant methylation in cloned embryos gives rise to abortion, high birth weight, and perinatal death. In this study, embryonic germ (EG) cells were used as donor cells for nuclear transfer. EG cells may have less reprogramming or demethylation than SCNT because these are already in erased status. However, little is known about methylation state or developmental capacity of the EG cell as a donor. The objective of this study was to analyze the methylation pattern of pre-implantation embryos cloned from porcine EG cells. Two regions, PRE-1 and microsatellite (MS), were analyzed for methylation patterns of cloned embryos from porcine EG cells and compared with the pattern of mature oocytes and in vitro-fertilized (IVF) embryos as a control. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro for 44 h, followed by use for IVF and NT with porcine EG cells. The porcine EG cells were prepared from 28-day-old fetuses after mating; genital ridges were isolated from fetuses, and then transferred into a culture medium on a feeder layer. The number of embryos for analysis was 300 for matured oocytes, 50–80 for 4–8 cell embryos, 30–40 for morulae, and 20–30 for blastocysts. The genomic DNA was isolated from the embryos and treated with bisulfite solution. PCR was performed for the amplification of PRE-1 and MS regions. The PCR products were sequenced by using an automatic DNA sequencer. The methylation rates of the PRE-1 and MS regions in IVF embryos showed that the demethylation process had occurred during the pre-implantation stage, which is a typical phenomenon of in vivo counterparts (Kang et al. 2001 J. Biol. Chem. 276, 39 980). However, compared to IVF embryos, embryos derived from NT using EG cells showed differences at the morula (PRE-1) and blastocyst (MS) stage. These results indicate that porcine EG cells also have problems in reprogramming during NT. For detailed and reliable results, the methylation pattern analysis of the imprinting region, for example, H19 in maternal allele and Igf2 in paternal allele, must be examined. Table 1.Methylation of PRE-1 and MS regions in embryos derived from IVF and NT using porcine EG cells

scholarly journals A strain of an emerging Indian pathotype of Xanthomonas oryzae pv. oryzae defeats the rice bacterial blight resistance gene xa13 without inducing a clade III SWEET gene and is nearly identical to a recent Thai isolate

2018 ◽  
Author(s):  
Sara C. D. Carpenter ◽  
Prashant Mishra ◽  
Chandrika Ghoshal ◽  
Prasanta Dash ◽  
Li Wang ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Bacterial Blight ◽  
Xanthomonas Oryzae ◽  
Host Susceptibility ◽  
Transcription Activator ◽  
Rice Bacterial Blight ◽  
Indian Strain ◽  
Bacterial Blight Pathogen ◽  
Insight Into ◽  
S Genes

AbstractThe rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) injects transcription activator-like effectors (TALEs) that bind and activate host ‘susceptibility’ (S) genes important for disease. Clade III SWEET genes are major S genes for bacterial blight. The resistance genes xa5, which reduces TALE activity generally, and xa13, a SWEET11 allele not recognized by the cognate TALE, have been effectively deployed. However, strains that defeat both resistance genes individually were recently reported in India and Thailand. To gain insight into the mechanism(s), we completely sequenced the genome of one such strain from each country and examined the encoded TALEs. Strikingly, the two strains are clones, sharing nearly identical TALE repertoires, including a TALE known to activate SWEET11 strongly enough to be effective even when diminished by xa5. We next investigated SWEET gene induction by the Indian strain. The Indian strain induced no clade III SWEET in plants harbouring xa13, indicating a pathogen adaptation that relieves dependence on these genes for susceptibility. The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective disease resistance.

scholarly journals Zbtb24 binding protects promoter activity by antagonizing DNA methylation in mESCs

2019 ◽  
Author(s):  
Haoyu Wu ◽  
David San Leon Granado ◽  
Maja Vukic ◽  
Kelly K.D. Vonk ◽  
Cor Breukel ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcriptional Activation ◽  
Zinc Finger Protein ◽  
Epigenetic Modification ◽  
Embryonic Stem ◽  
Dna Hypomethylation ◽  
Finger Protein ◽  
Genome Bisulfite Sequencing ◽  
Methylation Patterns ◽  
Unmethylated State

ABSTRACTDNA methylation is a key epigenetic modification essential for normal development. How particular factors control DNA methylation patterns and activity of a given locus is incompletely understood. The zinc finger protein Zbtb24 has been implicated in transcriptional activation/repression and the DNA methylation maintenance pathway. Here, using whole genome bisulfite sequencing in mouse embryonic stem cells, we report that besides a general trend towards DNA hypomethylation, many genomic sites gain methylation in the absence of Zbtb24 and they include promoters of actively transcribed genes. DNA hypomethylation is not generally associated with gene expression changes, suggesting that additional epigenetic safeguards are in place that ensure silencing of the affected loci. Remarkably, we identify a set of genes that is particularly susceptible to Zbtb24 occupancy. At these sites, Zbtb24 binding is not only required for gene activity but also required for maintaining the unmethylated state of the promoter.

scholarly journals Rational design of a compact CRISPR-Cas9 activator for AAV-mediated delivery

2018 ◽  
Author(s):  
Suhani Vora ◽  
Jenny Cheng ◽  
Ru Xiao ◽  
Nathan J. VanDusen ◽  
Luis Quintino ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Rational Design ◽  
Somatic Tissue ◽  
Transcription Activator ◽  
Adeno Associated Virus ◽  
Guide Rna ◽  
Delivery Vector ◽  
Transcriptional Modulators ◽  
Rna Complex

AbstractAkin to Zinc Finger and Transcription Activator Like Effector based transcriptional modulators, nuclease-null CRISPR-Cas9 provides a groundbreaking programmable DNA binding platform, begetting an arsenal of targetable regulators for transcriptional and epigenetic perturbation, by either directly tethering, or recruiting, transcription enhancing effectors to either component of the Cas9/guide RNA complex. Application of these programmable regulators is now gaining traction for the modulation of disease-causing genes or activation of therapeutic genes, in vivo. Adeno-Associated Virus (AAV) is an optimal delivery vehicle for in vivo delivery of such regulators to adult somatic tissue, due to the efficacy of viral delivery with minimal concerns about immunogenicity or integration. However, present Cas9 activator systems are notably beyond the packaging capacity of a single AAV delivery vector capsid. Here, we engineer a compact CRISPR-Cas9 activator for convenient AAV-mediated delivery. We validate efficacy of the CRISPR-Cas9 transcriptional activation using AAV delivery in several cell lines.

scholarly journals Active DNA Demethylation in Plants

2019 ◽  
Vol 20 (19) ◽  
pp. 4683 ◽  
Author(s):  
Jara Teresa Parrilla-Doblas ◽  
Teresa Roldán-Arjona ◽  
Rafael R. Ariza ◽  
Dolores Córdoba-Cañero
Keyword(s):  
Dna Methylation ◽  
Transcriptional Activation ◽  
Excision Repair ◽  
Epigenetic Modification ◽  
Dna Demethylation ◽  
Plant Responses ◽  
Dna Glycosylases ◽  
Active Dna Demethylation ◽  
Physiological Processes ◽  
Base Excision

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.

scholarly journals Characterization of Neurospora CPC1, a bZIP DNA-binding protein that does not require aligned heptad leucines for dimerization.

1991 ◽  
Vol 11 (2) ◽  
pp. 935-944 ◽  
Author(s):  
J L Paluh ◽  
C Yanofsky
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Activator ◽  
Dimerization Domain ◽  
Activation Domain ◽  
Band Shift ◽  
Transcriptional Activation Domain ◽  
Chimeric Polypeptide

CPC1 is the transcriptional activator of amino acid biosynthetic genes of Neurospora crassa. CPC1 function in vivo was abolished upon deletion of segments of cpc-1 corresponding to the presumed transcription activation domain, the DNA-binding and dimerization domains, or a 52-residue connector segment of CPC1. A truncated CPC1 polypeptide containing only the carboxy-terminal 57-residue segment of CPC1 was sufficient to form homodimers that bound DNA. However, deletion of the segment of cpc-1 corresponding to the connector segment in the full-length CPC1 polypeptide abolished DNA binding. Removal of a segment of cpc-1 corresponding to the GIn-rich region of CPC1 reduced in vivo function only slightly. The homologous transcription activator of Saccharomyces cerevisiae, GCN4, did not substitute for CPC1 in N. crassa. Chimeric CPC1-GCN4 polypeptides that contained the GCN4 transcriptional activation domain or the domain of GCN4 that corresponds to the essential 52-residue connector segment of CPC1, functioned with reduced efficiency. However, a chimeric polypeptide containing the GCN4 DNA-binding and dimerization domains in place of those of CPC1 functioned essentially as well as wild-type CPC1. The basic and dimerization domains of CPC1 were characterized by introducing deletions or site-directed amino acid replacements. The basic region was required for DNA binding but not for dimerization. CPC1 has a short dimerization domain containing heptad residues Leu-1, Leu-2, Trp-3, and His-4. When Val was substituted for Leu-1 or Leu-2, CPC1 was fully active, but when Val replaced Trp-3, dimerization and DNA binding were prevented. DNA band shift analyses with CPC1 heterodimers demonstrated that CPC1 does not require aligned heptad leucine residues for dimerization. Replacement of two charged residues located between Leu-1 and Leu-2 of CPC1 abolished dimerization and DNA binding.

scholarly journals Induced Methylation in Plants as a Crop Improvement Tool: Progress and Perspectives

Agronomy ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1484
Author(s):  
Clémentine Mercé ◽  
Philipp E. Bayer ◽  
Cassandria Tay Fernandez ◽  
Jacqueline Batley ◽  
David Edwards
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Epigenetic Modification ◽  
Crop Improvement ◽  
Regulation Of Gene Expression ◽  
Gene Promoters ◽  
Control Of Gene Expression ◽  
Underlying Mechanisms ◽  
Successive Generations ◽  
Methylation Patterns

The methylation of gene promoters is an epigenetic process that can have a major impact on plant phenotypes through its control of gene expression. This phenomenon can be observed as a response to stress, such as drought, cold/heat stress or pathogen infection. The transgenerational heritability of DNA methylation marks could enable breeders to fix beneficial methylation patterns in crops over successive generations. These properties of DNA methylation, its impact on the phenotype and its heritability, could be used to support the accelerated breeding of improved crop varieties. Induced DNA methylation has the potential to complement the existing plant breeding process, supporting the introduction of desirable characteristics in crops within a single generation that persist in its progeny. Therefore, it is important to understand the underlying mechanisms involved in the regulation of gene expression through DNA methylation and to develop methods for precisely modulating methylation patterns for crop improvement. Here we describe the currently available epigenetic editing tools and their advantages and limitations in the domain of crop breeding. Finally, we discuss the biological and legislative limitations currently restricting the development of epigenetic modification as a crop improvement tool.

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