12 research outputs found
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Male gametophyte specific expression helps identify a conserved gene associated with increased pollen fitness
GRMZM2G372877 was identified as a gene with potential function in the male gametophyte based on its strong expression in mature pollen relative to other maize tissues (Chettoor et al. 2014). Identification of a Ds insertion mutation in this gene from the Brutnell/Vollbrecht collection provided further support for this hypothesis, as initial data indicated the insertion was associated with a male-specific transmission defect. In this study, we confirmed the location of GRMZM2G372877 on chromosome 9, approximately 25 map units away from wx1. We used linkage of the Ds insertion to Wx1+, as well as PCR genotyping, to follow up on the initial results, confirming a male-specific transmission defect from mutant heterozygotes. Because the severity of the transmission defect varied with different crosses (2% to 13%), we tested the idea that the defect decreased pollen fitness when in competition with wild-type pollen. Consistent with this idea, we found that male transmission of the mutation increases in frequency when less pollen is applied to the silk (12% to 43%). Based on DNA sequence, we found GRMZM2G372877 was orthologous to a gene (delegen14) included in a 65-kb deletion associated with the rice no-pollen mutant (Osnop) (Jiang et al 2005), suggesting a conserved function for this gene in pollen. We have tentatively named the gene nop1*, and it encodes a protein with C2 and GRAM domains that are predicted to interact with calcium and phosphoinositides, respectively. Results from microscopy experiments, to visualize specific cellular defects, and to help better determine the function for this gene in pollen or pollen tube development, will also be presente
Heritable site-specific mutagenesis using TALENs in maize
Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize g lossy2 ( gl2 ) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement
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Discovery of novel transcripts and gametophytic functions via RNA-seq analysis of maize gametophytic transcriptomes
BACKGROUND:
Plant gametophytes play central roles in sexual reproduction. A hallmark of the plant life
cycle is that gene expression is required in the haploid gametophytes. Consequently, many
mutant phenotypes are expressed in this phase.
RESULTS:
We perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the
embryo sacs removed, mature pollen, and seedlings to assist the identification of
gametophyte functions in maize. Expression levels were determined for annotated genes in
both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq
reads. Transposon-related transcripts are present in high levels in both gametophytes,
suggesting a connection between gamete production and transposon expression in maize not
previously identified in any female gametophytes. Two classes of small signaling proteins
and several transcription factor gene families are enriched in gametophyte transcriptomes.
Expression patterns of maize genes with duplicates in subgenome 1 and subgenome 2
indicate that pollen-expressed genes in subgenome 2 are retained at a higher rate than
subgenome 2 genes with other expression patterns. Analysis of available insertion mutant
collections shows a statistically significant deficit in insertions in gametophyte-expressed
genes.
CONCLUSIONS:
This analysis, the first RNA-seq study to compare both gametophytes in a monocot, identifies
maize gametophyte functions, gametophyte expression of transposon-related sequences, and
unannotated, novel transcripts. Reduced recovery of mutations in gametophyte-expressed
genes is supporting evidence for their function in the gametophytes. Expression patterns of
extant, duplicated maize genes reveals that selective pressures based on male gametophytic
function have likely had a disproportionate effect on plant genomes
Rapid “Open-Source” Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification
Summary—Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zincfinger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multi-finger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1 to 50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally-integrated EGFP reporter gene. In summary, OPEN provides an “opensource” method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy
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ColeRexBotanyPlantPathologyDiscoveryNovelTranscripts_AdditionalFiles.zip
BACKGROUND:
Plant gametophytes play central roles in sexual reproduction. A hallmark of the plant life
cycle is that gene expression is required in the haploid gametophytes. Consequently, many
mutant phenotypes are expressed in this phase.
RESULTS:
We perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the
embryo sacs removed, mature pollen, and seedlings to assist the identification of
gametophyte functions in maize. Expression levels were determined for annotated genes in
both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq
reads. Transposon-related transcripts are present in high levels in both gametophytes,
suggesting a connection between gamete production and transposon expression in maize not
previously identified in any female gametophytes. Two classes of small signaling proteins
and several transcription factor gene families are enriched in gametophyte transcriptomes.
Expression patterns of maize genes with duplicates in subgenome 1 and subgenome 2
indicate that pollen-expressed genes in subgenome 2 are retained at a higher rate than
subgenome 2 genes with other expression patterns. Analysis of available insertion mutant
collections shows a statistically significant deficit in insertions in gametophyte-expressed
genes.
CONCLUSIONS:
This analysis, the first RNA-seq study to compare both gametophytes in a monocot, identifies
maize gametophyte functions, gametophyte expression of transposon-related sequences, and
unannotated, novel transcripts. Reduced recovery of mutations in gametophyte-expressed
genes is supporting evidence for their function in the gametophytes. Expression patterns of
extant, duplicated maize genes reveals that selective pressures based on male gametophytic
function have likely had a disproportionate effect on plant genomes
Recommended from our members
ColeRexBotanyPlantPathologyDiscoveryNovelTranscripts.pdf
BACKGROUND:
Plant gametophytes play central roles in sexual reproduction. A hallmark of the plant life
cycle is that gene expression is required in the haploid gametophytes. Consequently, many
mutant phenotypes are expressed in this phase.
RESULTS:
We perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the
embryo sacs removed, mature pollen, and seedlings to assist the identification of
gametophyte functions in maize. Expression levels were determined for annotated genes in
both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq
reads. Transposon-related transcripts are present in high levels in both gametophytes,
suggesting a connection between gamete production and transposon expression in maize not
previously identified in any female gametophytes. Two classes of small signaling proteins
and several transcription factor gene families are enriched in gametophyte transcriptomes.
Expression patterns of maize genes with duplicates in subgenome 1 and subgenome 2
indicate that pollen-expressed genes in subgenome 2 are retained at a higher rate than
subgenome 2 genes with other expression patterns. Analysis of available insertion mutant
collections shows a statistically significant deficit in insertions in gametophyte-expressed
genes.
CONCLUSIONS:
This analysis, the first RNA-seq study to compare both gametophytes in a monocot, identifies
maize gametophyte functions, gametophyte expression of transposon-related sequences, and
unannotated, novel transcripts. Reduced recovery of mutations in gametophyte-expressed
genes is supporting evidence for their function in the gametophytes. Expression patterns of
extant, duplicated maize genes reveals that selective pressures based on male gametophytic
function have likely had a disproportionate effect on plant genomes
Heritable site-specific mutagenesis using TALENs in maize
Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize g lossy2 ( gl2 ) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.This article is published as Char, Si Nian, Erica Unger‐Wallace, Bronwyn Frame, Sarah A. Briggs, Marcy Main, Martin H. Spalding, Erik Vollbrecht, Kan Wang, and Bing Yang. "Heritable site‐specific mutagenesis using TALENs in maize." Plant biotechnology journal 13, no. 7 (2015): 1002-1010. 10.1111/pbi.12344</p
High expression in maize pollen correlates with genetic contributions to pollen fitness as well as with coordinated transcription from neighboring transposable elements.
In flowering plants, gene expression in the haploid male gametophyte (pollen) is essential for sperm delivery and double fertilization. Pollen also undergoes dynamic epigenetic regulation of expression from transposable elements (TEs), but how this process interacts with gene expression is not clearly understood. To explore relationships among these processes, we quantified transcript levels in four male reproductive stages of maize (tassel primordia, microspores, mature pollen, and sperm cells) via RNA-seq. We found that, in contrast with vegetative cell-limited TE expression in Arabidopsis pollen, TE transcripts in maize accumulate as early as the microspore stage and are also present in sperm cells. Intriguingly, coordinate expression was observed between highly expressed protein-coding genes and their neighboring TEs, specifically in mature pollen and sperm cells. To investigate a potential relationship between elevated gene transcript level and pollen function, we measured the fitness cost (male-specific transmission defect) of GFP-tagged coding sequence insertion mutations in over 50 genes identified as highly expressed in the pollen vegetative cell, sperm cell, or seedling (as a sporophytic control). Insertions in seedling genes or sperm cell genes (with one exception) exhibited no difference from the expected 1:1 transmission ratio. In contrast, insertions in over 20% of vegetative cell genes were associated with significant reductions in fitness, showing a positive correlation of transcript level with non-Mendelian segregation when mutant. Insertions in maize gamete expressed2 (Zm gex2), the sole sperm cell gene with measured contributions to fitness, also triggered seed defects when crossed as a male, indicating a conserved role in double fertilization, given the similar phenotype previously demonstrated for the Arabidopsis ortholog GEX2. Overall, our study demonstrates a developmentally programmed and coordinated transcriptional activation of TEs and genes in pollen, and further identifies maize pollen as a model in which transcriptomic data have predictive value for quantitative phenotypes
Genome-Wide Distribution of Transposed Dissociation Elements in Maize[W][OA]
Inherited transpositions of the endogenous Ds create stable insertion lines, a resource for targeting gene knockouts and examining mechanisms of transposition. Ds preferentially inserts into genes, at target sites within 16-bpair segments of DNA with specific structural properties. These results suggest approaches to predict insertion sites in transposon mutagenesis experiments