THE ROLE OF TRANSPOSONS AND ANTISENSE TRANSCRIPT IN DROUGHT RESPONSE IN MAIZE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010579
• Project No.: IND011213
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2021

Project Director
Lisch, DA, R.

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Botany & Plant Pathology

Non Technical Summary
All of agriculture depends on exploitation of variation within population of plants and animals. Historically, farmers have selected for variants that optimized such qualities as yield, resistance to disease and ability to grow under various environmental stresses. Currently, seed companies have dramatically enhanced our ability to select for these variants and, at least in some cases, to genetically engineer favorable traits into crop species. In general, breeding strategies involve using variations in DNA sequence that are genetically linked to traits of interest to rapidly move traits from one genetic background to another (so called marker assisted breeding). However, in most cases, the genes that are required for conferring these traits is not known. We do know, however, that variation in the function of these genes is at the heart of crop improvement. Recent work in the field of genomics has revealed that an important source of such variation comes from transposable elements. These are short stretches of DNA that move from place to place within plant and animal genomes. As far as we can tell, they do so simply because they can. Like viruses, they use us to make extra copies of themselves. Unlike viruses, they never leave our cells. Instead, from generation to generation, they replicate themselves and are passed on to the next generation. In doing so, they can cause a wide variety of changes, from simply inactivating genes, to reprograming gene expression. Notable examples include Chardonnay wine grapes, in which a gene required for red color has been inactivated due to a transposon insertion and blood oranges, in which a gene required for color expression has been reprogrammed so that it expresses in the flesh of the orange. Interestingly, this only occurs if the oranges are subject to cold stress, which is likely due to the fact that this transposon, like many, is naturally activated under stress conditions. Similarly, dozens of examples of genes in rice have been identified that have been reprogrammed by transposons such that they are responsive to stress. Simply put, long before humans thought to genetically engineer crops, these natural genetic engineers have been reprogramming gene expression in an astonishing variety of ways, and that reprogramming is often related to stress response. This project seeks to explore some of the ways that genes in maize have been reprogramed due to transposon insertion in order to better understand the source of genetic variation that all of us depend on for continued crop improvement.

Animal Health Component

0%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1510	1040	20%
201	1510	1080	40%
202	1510	1060	10%
203	1510	1080	15%
203	1510	1020	15%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms; 202 - Plant Genetic Resources;

Subject Of Investigation
1510 - Corn;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1080 - Genetics; 1020 - Physiology;

Keywords

abiotic stress

transposable elements

maize

epigentic

drought

antisense transcript

rna expression

corn

genomes

Goals / Objectives
1) Identify instances in which adjacent TEs appear to induce down regulation of gene expression in two different maize inbred lines under drought stress.2) When available, compare these identified genes with Gene-wide association study (GWAS) mapped loci important in drought and/or stress response.3) Perform molecular analysis of changes in gene expression of a subset of differentially regulated genes that appear to map to GWAS mapped loci in families segregating for insertion polymorphisms.

Project Methods
Data will be provided by our colleagues at Sichuan Agricultural University including a number of variables concerning drought stress and its effects on gene expression. This will include methylome data, expression data, small RNA profiling and GWAS association mapping data, much of which is already available. Extensive data has been obtained from two maize parental lines, AC7643 and AC7729/TZSRW, which differ in their response to drought stress, as well as two derivative Recombinant Inbred Lines (RILs), RIL208 and RIL64, which were selected based on drought tolerance or sensitivity, as was previously characterized (Lu et al. 2010). The initial characterization of these data will be described in a manuscript that is being currently revised for publication. Going forward, our goal will be to examine in more detail the specific mechanisms involved in regulation of antisense expression of these genes, the potential impact of variation in these genes on drought response and the degree to which transposable elements may have contributed to drought responsiveness. This will involve confirming the stress responsiveness of a subset of transcripts using RT-PCR and genetic analysis of segregating families, confirming a potential role for TEs in triggering antisense transcription by examining presence/absence polymorphism, and investigating the role of epigenetic regulation of these genes using available mutants.Objective 1:Using strand specific RNAseq, our collaborators have identified all antisense transcripts associated with drought stress in AC7643 and AC7729/TZSRW and the two derivative Recombinant Inbred Lines (RILs), RIL208 and RIL64. To assess the impact of transposable elements on antisense transcription, the maize reference genome B73 will be analyzed using bedtools (Quinlan and Hall 2010) according to the MIPS transposon annotation database (ftp://ftp.gramene.org/pub/gramene/maizesequence.org/release-5a/repeats/). This data will be augmented by our laboratory's in-house protocol for identification of maize transposable elements. The objective is to identify all instances in which transposons of any kind may be responsible for driving antisense transcription in the reference inbred and in the drought tolerant and susceptible lines. However, these lines may be polymorphic with respect to TE insertions relative to the reference genome, and whole genome sequence data for them is currently unavailable. Therefore, for each accession, the presence or absence of a given insertion associated with a given antisense transcript will be validated using PCR, or, when available, low pass high throughput sequencing data. This should give us a set of candidate genes with associated transposons in these lines. When available, genome sequence data from other maize accessions will also be examined for evidence of presence or absence of transposon insertions at the affected loci (Makarevitch et al. 2015), and expression of these genes in these accessions under drought stress can also be examined. Ultimately, the goal is to identify polymorphic insertions that are causally related to the production of antisense transcript.Objective 2:Loci producing antisense transcripts that are drought responsive have been compared with data obtained from a panel of 368 maize inbred lines (Li et al. 2013) to determine association with drought survival (Liu et al. 2013). Those that are significantly associated with drought survival will be subject to more detailed molecular analysis in AC7643 and AC7729/TZSRW and the two derivative Recombinant Inbred Lines (RILs), RIL208 and RIL64, as well as F2 backcross populations derived from AC7643 AC7729/TZSRW hybrids that will segregate for polymorphic antisense loci. This analysis will initially involve validation of high throughput results using strand-specific qRT-PCR under drought stress conditions. In these experiments, the expectation is that drought responsiveness of a limited number of TE associated alleles will segregate as expected in F2 back cross families. This will provide strong genetic evidence for a role for TEs in drought responsiveness of a subset of antisense genes in these accessions. Ideally, at least a subset of these insertion polymorphisms will be associated with drought resistance, a hypothesis that can be directly tested in these segregating families.Objective 3:The reference genome for B73 is available now, and that of the W22 genome will be available soon, which will allow us to identify polymorphisms for TE insertions in these backgrounds that are known to be associated with drought response in AC7643, AC7729/TZSRW, RIL208 and RIL64. An important part of our analysis will involve examination of the effects of mutants on antisense transcription. This is based on observations that antisense regulation involves the production of small RNAs that are dependent on a number of components of the trans-acting small RNA pathway. Mutants in this pathway are available in both B73 and W22. These lines will be subject to drought stress and control conditions and two week old seedlings will be harvested for analysis. This analysis will include strand-specific RT-PCR (to validate antisense expression in these genetic backgrounds under these conditions). The mutant leaf bladeless1 (lbl1), which is known to be required for repression of at least some sense/antisense transcripts under abiotic stress will be used to determine the extent to which down-regulation in response is due to the tasiRNA pathway, which is known to be involved in sense natural antisense gene regulation. The protein encoded by Lbl1 is essential for production of amplification of small RNAs resulting from the interaction of cognate sense and antisense transcripts, and also plays an important role in the trans-acting small RNA pathway, as well as system responses to viral infection and transposon silencing (Borges and Martienssen 2015). Similarly, the mutant mediatior of paramutation1 (mop1) (and similar mutants in the same pathway) will be used to determine the extent to which epigenetic regulation of transposons is responsible for drought responsive transcription, a phenomenon that has been already been observed at the NAC gene in maize (Mao et al. 2015). In this case, the hypothesis is that TEs that are normally repressed by small RNAs that require Mop1 might generate antisense transcript if that repression is relieved. If we observe that levels of sense or antisense transcription is significantly altered in the mutants the hypothesis that these transcripts are epigenetically regulated would be supported. By making the appropriate crosses and back-crosses, with both insertion polymorphisms and the mutants segregating, it should be possible to perform high quality genetic analysis of candidate loci. Fortunately, a large number of mutations are now available in W22, so if there is a polymorphic insertion between W22 and B73 it should be relatively easy to determine whether or on a mutant alters the effect of that insertion on expression of antisense transcript. Finally, chromatin Immunoprecipitation (ChiP) will be used to determine the degree to which expression of antisense transcript contributes to changes in chromatin, and (at least potentially) somatically heritable changes in gene expression. Ultimately, our expectation is that we will identify and characterize a relatively small number of loci that we can determine with high confidence that are 1) regulated due to the presence of a transposon insertion 2) related to drought stress and 3) subject to epigenetically regulation.

Progress 10/01/16 to 09/30/21

Outputs
Target Audience: Our target audience are national and international scientists working on stress response in plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Our collaborator, Jie Xu, has been able to secure additional funds based entirely on our work together. In addition, a graduate student, Wei Guo, who examined the effects of heat stress on maize gene expression, has now graduated and is a postdoctoral fellow at Rutgers University. In addition, a graduat e student in our collaborator, Demeke Bayable in Tesfe Mengiste'slaboratory gained extensive experience in molecular characterization of regulation of gene expression in response to disease pressure that will complement his understanding of mapping and breeding. He is now employed by the USDA. How have the results been disseminated to communities of interest?Our results have been published in high impact journals and our raw data has been deposited in the NCBI repository. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact statement: Given anticipated and current changes in the climate, drought stress is likely to be a growing issue for agriculture. An essential tool to address that issue will be genetic variants that are better able to cope with drought. To fully understand these genetic variants, it is necessary to understand how variation in specific genes helps plants deal with drought. Although some variation is in the proteins encoded by genes, it has become clear that a great deal of genetic variation responsible for advantageous alleles (versions) of genes occurs not in the coding sequence, but in regulatory sequences responsible for when and where a gene turns on. One kind of regulatory change that has only recently been discovered involves the production of anti-sense transcripts that may interfere with the normal transcripts and thus modulate levels of expression of key genes under conditions of drought stress. With our collaborators, we have found strong evidence that a subset of antisense transcripts is indeed important for drought stress in maize, and we have identified genes that are directly implicated in this process. This is likely to have important implications for breeding programs that seek to optimize drought response in this essential commodity crop. In addition, using a very similar set of tools, PI Lisch in collaboration with another professor at Purdue university, has found strong evidence that antisense transcription driven by the insertion of a mobile genetic element plays a role in resistance to Anthracnose, a major disease of sorghum worldwide. Because this resistance gene confers strong resistance to several pathogens, this discovery is likely to have broad implications, particularly for farmers in Africa, who depend on sorghum as a staple crop. 1) Although we found ample evidence of natural antisense transcripts in maize lines that are associated with drought stress in maize, we did not find evidence that transposable elements (TEs) are associated with production of these transcripts. Indeed, we found that TEs tend to be excludedantisense transcripts and their associated promoters. Although somewhat surprising, this is actually and important negative result. Because TE insertion is random with respect to effects on function (the TEs can't "know what effects they will have), one would expect that most TE polymorphisms would be neutral or mildly deleterious. And instances in which any particular insertion of a TE is causally related to a new adaptive function (drought response) would be a tiny minority of insertions. Similarly, any anti-sense transcript causally associated wiith drought stress would be a tiny minority of antisense transcripts in general. What we found is that TEs appear not to be a primary cause of antisense. However, the purging of TEs associated with antisense provides good evidence for selection against TEs in antisense genes, and thus supports a functionaly role for these transcripts. This is important because it provides evidence against the null hypothesis, that antisense transcription is simply "noise" that does not provide any benefit. 2) We did find compelling evidence from GWAS analysis that a subset of antisense transcripts are indeed genetically linked to drought stress in maize. This important result was published in the journal Nucleic Acids research, with PI Lisch included as a co-corresponding author. To our knowledge,this was the first strong evidence for an involvement of cis-natural antisense regulation of genes being directly and causally linked to stress response in plants. 3) Our collaborators recently published a paper that directly links a specific locus producing antisense transcript with drought response. This takes our global analysis and provides additional strong evidence to support our hypothesis. 4) PI Lisch's work with antisense regulation ended up having an unexpected benefit in analysis of disease response in a second plant species, sorghum. In collaboration with Tesfaye Mengiste, a professor here at Purdue, we found that a gene linked to broad spectrum resistance to fungal infection is regulated via antisense transcription driven by TE insertions. It is unlikely that PI Lisch would have recognized the importance of this antisense transcript and the role that TEs play in producing them if he had not been engaged in exactly this kind of analysis in maize. This represents an excellent example of the synergistic effects of providing support for basic research. This work has recently been accepted for publication in The Plant Cell and is likely to have significant real world implications given the importance of fungal disease in sorghum in the U.S. and the rest of the world.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Sanghun Lee, Fuyou Fu, Chao-Jan Liao, Demeke B. Mewa, Adedayo Adeyanju, Gebisa Ejeta, Damon Lisch and Tesfaye Mengiste, 2022. Broad spectrum and complete fungal resistance in sorghum is conferred through a complex regulation of an immune receptor embedded in a natural antisense transcript. The Plant Cell, in press.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Yan Mao, Jie Xu, Qi Wang, Guobang Li, Xin Tang, Tianhong Liu,Xuanjun Feng, Fengkai Wu, Menglu Li, Wubing Xie, Yanli Lu, 2021. A natural antisense transcript acts as a negative regulator for the maize drought stress response gene ZmNAC48 Journal of Experimental Botany, Volume 72, Issue 7, 29 March 2021, Pages 27902806

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Our target audience are national and international scientists working on stress response in plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Our graduate student, Wei Guo, is spending a significant amount of time learning how do do whole genome analysis of gene expression and DNA methylation in maize. Given the current urgent need for training in this field, this will proved to be an invaluable skill set for Wei Guo, who we anticipate will be graduating next summer. How have the results been disseminated to communities of interest?A series of talks and poster presentations. What do you plan to do during the next reporting period to accomplish the goals?We anticipate that our work with heat stress, as well as our collaboration on drought stress induced circular RNAs will result in publications in major journals in the next year.

Impacts
What was accomplished under these goals? In addition to drought stress, we have also been investigating the effects of heat on transposable element activity. To our surprise, we have found that heat stress has a major effect on heritable activity of a particular transposable element in maize. Briefly, we have discovered that a brief period of heat strest can cause rapid and heritable changes in the activity of a previously silenced transposable element. We are now extending that observation to whole genome analysis in the inbred lines B73 and Mo17, with the expectation that our analysis of a single transposable element will be generalizable to large numbers of silenced transposons. Given that silenced transposons can have a significant effect on nearby genes, we will be curious to see how heat affects heritable gene expression. In addition we are continuing our long term collaboration with Jie Xu, who is extending her analysis of drought stress to include drought-inducedcircular RNAs (circRNAs), many of which are composed of sequences including transposable elements.

Publications

• Type: Book Chapters Status: Published Year Published: 2020 Citation: Ziang, X. and D. Lisch. Cost-effective profiling of Mutator transposon insertions in maize by next-generation sequencing, 2020. Methods Mol Biol. PMID: 31541437.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhang, X., Zhao, M., McCarty, D. and D. Lisch , 2020. Transposable elements employ distinct integration strategies with respect to transcriptional landscapes in eukaryotic genomes. Nucleic Acids Research, PMID 32442316.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bayer Public Seminar Series, Online. Heat Stress Rapidly and Heritably Reactivates a Silenced Maize Transposable element. Damon Lisch.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Invited Talk. 62nd Annual Maize Genetics Meeting. 62nd Annual Maize Genetics Meeting. Rapid, Heat-Induced Transgenerational Reactivation of a Silenced Transposable Element in Maize. Wei Guo (Lisch Lab Graduate student).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Invited Talk. Plant and Animal Genome XXVIII. Rapid, Heat-Induced Transgenerational Reactivation of a Silenced Transposable Element in Maize. Wei Guo (Lisch Lab Graduate student).

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Target audiences have included scientists in genetics and related fields both nationally and internationally. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The most important outcome with respect to training and development has been the successful elevation of a former trainee of Dr. Lisch, Jie Xu, who is now an associate professor at theMaize Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China. This would have not been possible without publication of our work on natural antisense transcripts in maize. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to continue follow up experiments on individual genes implicated in sense-natural antisense regulation of response to water stress. One such gene pair, which involves a WRKY transcription factor appears to be involved in shoot branching. Overexpression of both sense and antisense members of the pair are expected to provide clues as to the function of this and other gene pairs.

Impacts
What was accomplished under these goals? In large part, the goals of this project have been achieved. The primary hypothesis was that transposable elements (TEs) can drive expression of antisense transcript, which in turn could, at least in principle, downregulate gene expression. Because TEs are remarkably polymorphic, and because TEs are often stress responsive, one could imagine a scenario in which polymorphism in TE insertions drive polymorphisms in stress response. This hypothesis was rigorously tested in two inbred lines of maize, along with derivived recominant inbred lines, that vary with respect to responsiveness to drought. The data obtained suggest that the overall hypothesis, as appealing as it might be, is not supported. Although there is evidence for a truely remarkable number of sense natural antisense (SNAS) gene pairs, few of them appear to be the result of TE activity. Indeed, SNAS gene pairs have an over reduction in enrichment of TEs, likely because the vast majority of insertions are deleterious to normal gene function. However, and importantly, we did find that SNAS gene pairs are in fact significantly associated with drought responsively. This conclusion is based on analysis of NIL and GWAS studies that show linkage between SNAS gene pairs and drought responsiveness in these inbred lines. The results of these analysis were published in Nucleic Acids research in 2017. Since then, Dr. Lisch has continued to communicate with Dr. Jei on follow up experiments designed to alter expression of specific SNAS pairs.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lisch D, Burns KH. Editorial overview: Genome architecture and expression: Mobile elements at work. Curr Opin Genet Dev. 2018 Apr;49:iv-v. doi: 10.1016/j.gde.2018.05.003.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: The long and short of doubling down: polyploidy, epigenetics, and the temporal dynamics of genome fractionation. Wendel JF, Lisch D, Hu G, Mason AS. Curr Opin Genet Dev. 2018 Apr;49:1-7. doi: 10.1016/j.gde.2018.01.004. Epub 2018 Feb 10. Review.PMID: 29438956
• Type: Other Status: Published Year Published: 2019 Citation: Invited Seminar Speaker, URPP Evolution Seminar, University of Zurich, Zurich, Switzerland "The Long and Short of Doubling Down: Transposable elements as both causes and consequences of subgenome differentiation in polyploid"
• Type: Other Status: Published Year Published: 2019 Citation: Invited Seminar Speaker, Biological Science Colloquium, Florida State University, Florida. "Death and Resurrection of a Maize Transposable Element"
• Type: Other Status: Published Year Published: 2019 Citation: Invited Speaker, Mobile Genetic Elements 2019, Woods Hole Oceanographic Institute, Woods Hole, Massachusetts. "Heat Stress Rapidily and Heritably Reactivates a Silenced Maize Transposable element"
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Meixia Zhao, Biao Zhang, Jianxin Ma, Damon Lisch. Genome-wide Estimation of Evolutionary Distance and Phylogenetic Analysis of Homologous Genes BIO-PROTOCOLVol 8, Issue 23, Dec 05, 2018,DOI:10.21769/BioProtoc.3097

Progress 10/01/17 to 09/30/18

Outputs
Target Audience: PI Lischpresented the results of our anaysis of the relationship between heat stress and transposon activation in a regional conference on chromatin and epigenetics. Graduate student Wei Guo has also presented these results in two student poster presentations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Ongoing training of a graduate student, who is looking at the relationship between stress and transposon activity. The student has presented the results of his work at the annual national Maize Meeting as well as local poster presentations. This graduate student has also taken a bioinformatics course, which has signficantly enhanced his ability to deal with high throughput data.One postdoc associated with the project has obtained a tenure-track position. How have the results been disseminated to communities of interest?The results of our major analysis concerning drought was published in 2017. More recently, results relating to the relationship between heat stress and transposon activiation have been presented at a regional conference on chromatin and epigenetics. What do you plan to do during the next reporting period to accomplish the goals?We plan on examining in more detail specific loci that produce antisense RNA and that are genetically linked to drought stress in maize. In addition, we plan on determine the extent to which stress can produce heritable changes in gene expression using our model transposon system in maize. In addiiton, we are expanding our analysis of the relationship between stress and epigenetic regulation by determining the extent to which salt and heat stress affects heritable reactivation of a transposable element. We will also use a particular mutant, leaf bladeless1, which interferes with antisense regulation to test hypotheses concerning the mechanism by which stress responsive genes are regulated via antisense.

Impacts
What was accomplished under these goals? As discussed in the previous report, we have rigorously tested the hypothesis that TEs are a ubiquitous source of sense-natural antisense transcripts triggered by stress. They do not appear to be, at least with respect to drought stress in maize. However, we have found evidence that salt stress can result in the production of anti-sense dependent down regulation of a maize gene, aparently driven by a transposable element. We are now following up on this result.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Invited Seminar Speaker, University of Missouris Interdisciplinary Plant Group (IPG) Seminar Series, Columbia Missouri. Mutant analysis reveals a surprising connection between RNA-Directed DNA methylation, microRNA function and plant morphology in maize
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Invited Speaker, EMBL Symposium: The Mobile Genome: Genetic and Physiological Impacts of Transposable Elements, Heidelberg Germany. Epimutants reveal a surprising connection between RNA directed DNA methylation and microRNA function in maize
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Invited Speaker, Midwest Chromatin and Epigenetics Meeting. Epigenetic changes associated with transgenerational silencing of a maize transposon"
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Invited Speaker, Midwest Chromatin and Epigenetics Meeting. Epigenetic changes associated with transgenerational silencing of a maize transposon 

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The research resulted in a manuscript that has been published in the highly regarded journal, Nucleic Acids Research. The audience for this journal are basic research scientists who study the nature and function of DNA and RNA. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student has been trained in the analysis of differential gene expression in plants under stress. Further, a postdoctoral fellow atSichuan Agricultural University has recieved extensive training in analysis of high throughput data analysis. How have the results been disseminated to communities of interest?The results of these analysis have been disseminated in a well respectedpeer reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?Selected sense-antisense gene pairs will be subject to more detailed molecular analysis under a variety of stress conditions and in mutant backgrounds in order to determine cause effect relationships between phenotypes and genotypes.

Impacts
What was accomplished under these goals? 1) Our intial goal was to identify transposable elements that may be involved in response to stress, particularly with respect to the generation of antisense transcripts. This was based on some preliminary data suggesting that transposons can drive antisense expression under salt stress, which, in turn, can down-regulate expression of an adjacent gene. Drought stress is an important variable in maize yeild. Through a collaboration with the Lu group at Sichuan Agricultural Universitywe had available two inbred lines that were more and less drought tolerant along with recombinant inbred lines that made it possible to map drought resistance. We performed analysis of small RNAs, DNA methylation, gene expression, histone modification and antisense transcript abundance under normal and drought stress conditions for each maize line. We found evidence for changes in each of these variables. However, we did not find evidence for an involvement of transposable elements with these changes. Indeed, we found that transposable elements were underrepresented among sense-antisense gene pairs, presumably because they would interfere with 2) We found a clear and signficant association between sense-antisense gene pairs under drought conditions and survival and yield using both Recombinant Inbred Lines and GWAS analysis. This represents an important step forward in our understanding of the connection between regulation of sense-antisense gene pairs and drought stress, which is likely to be an increasingly important challenge for farmers. 3) Follow up analysis of candidate sense natural antisense gene pairs is ongoing.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Xu, J., Wang, Q., Freeling, M., Zhang, X., Xu, Y., Mao, Y., Tang, X., Wu, F., Lan, H., Cao, M., Rong T, Lisch, D, and Lu, Y. Natural antisense transcripts are significantly involved in regulation of drought stress in maize. Nucleic Acids Res, 2017 PMID: 28175341. Dr.s Lisch and Lu were co-corresponding authors