Progress 10/15/15 to 09/30/20
Outputs
Target Audience:The target audience for this research project are academic and industrial researchers working in the fields of mycotoxin research, plant biology and agricultural biotechnology (for the work on approaches to reduce mycotoxin contamination of crops through biotechnological approaches) as well as cell biologists and toxicologists (for the work on identifying cellular targets and mechanisms of mycotoxin action), growers, processors and end users of grain products, regulatory agencies concered with agriculture and food safety. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project involved a Ph. D graduate student, now graduated, who was funded by the teaching assistantship from the Plant Biology Graduate Program at Rutgers. During the course of the project, several undergraduate students in the majors of plant biology and plant biotechnology have also benefitted from the hands-on training in the areas of plant biology, plant tissue culture and engineering, plant biotechnology and molecular biology. Our results have also been incorporated into our teaching materials for courses in Plant Biology and Biotechnology. How have the results been disseminated to communities of interest?The results of these projects have been disseminated to the wider communities of interest through presentations made at scientific and professional meetings, such as the annual meeting ot he US Wheat and Barley Scab Initiative and through publications in the scientific literature. Results from the work on C. elegans exposed to DON were published in 2018. Results from work on gene-editing in Arabidopsis and the identification of barley orthologs of disease susceptibility genes were published in 2020.Our preliminary data has been shared with other participants of this project through email communications and through our annual meeting. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1 Impact: We successfully employed CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated endonuclease 9) gene editing technology to introduce mutations into genes associated with disease susceptibility to Fusarium Head Blight (FHB). These plants have been validated structurally, as being edited at the target genes, while lacking any transgenic sequences. This validates the approach of using CRISPR to generate genetically altered plants that do not carry any transgenes. These plants have been used to identify the corresponding susceptibility genes in barley, which are now the focus of gene editing in crop plants. Objective 2 Impact: Using the animal model C. elegans. for DON toxicity studies, we have shown that the mycotoxin DON acts on a number of distinct cellular and molecular targets, including pathways that control programmed cell death, toxin transport, and protein aggregation. By examining genome-wide gene expression, we have identified potentially important new targets for the toxin. The functional role of these genes in conditioning susceptibility to DON has been confirmed by RNAi functional studies. These approaches provide new insight into the mechanism of DON toxicity and provide new targets for amelioration of its effects in animals and humans. Accomplishments: Objective 1 CRISPR Editing of FHB Susceptibility Genes: We used CRISPR/Cas9 gene editing technology to mutate genes involved in conditioning FHB susceptibility. We employed Arabidopsis as the model plant to study the feasibility of knocking-out FHB susceptibility genes to achieve FHB resistance and, having generated such Arabidopsis knockout plants, have used them plants in complementation screens to identify barley functional orthologues. These orthologs are the focus of ongoing efforts to similarly knockout these loci in barley so that their contribution to FHB disease susceptibility can be determined. For the Arabidopsis studies, we focused on three candidate FHB susceptibility genes: ethylene insensitive 2 (EIN2), homoserine kinase (HSK) and 2-oxoglutarate Fe(II)-dependent oxygenase (2OGO). We used the Arabidopsis (At) CRISPR vector pAt-sgRNA-Cas9 and produced AtEIN2-, AtHSK- and At2OGO-edited Arabidopsis plants with vectors, pRD182, pRD212 and pRD207 respectively. Agrobacterium-mediated transformants (T1 and T2 generations) were validated for editing of target sites by restriction fragment length polymorphism (RFLP), T7 endonuclease 1 assay and sequencing of PCR-amplified gDNA fragment spanning the target sites. To-date, we have validated: 15 AtEIN2-edited plants each with different mutations at their respective target sites; 14 At2OGO-edited Arabidopsis plants, each with different mutations at their respective target sites. We have also produced AtHSK-KO Arabidopsis plants whose characterization at the DNA sequence level continues to be determined. For both AtEIN2-edited plants and At2OGO-edited plants, we selected individuals from the T2 generation and confirmed by RFLP analysis that these represent transgene-free homozygous plants. Both At2OGO and AtEIN2-KO plants displayed a markedly slower FHB disease development with a consequent reduction in the levels of Fusarium graminearum-GFP (determined by RT-qPCR), compared to WT plants. Objective 1. RNA-SEQ of Cultivar Conlon. To identify which barley homologs of 2OGO, EIN2 and HSK are involved in FHB susceptibility, we wished to complement the corresponding gene-edited Arabidopsis plants (At2OGO-KO and AtEIN2-KO) with barley cDNA isolated from the U.S. cultivar Conlon, which is more susceptible to Fusarium graminearum than Morex, whose genome has been sequenced. We used the Illumina HiSeq platform to perform RNAseq analysis of barley cv. Conlon, an important North American two-rowed cultivar and obtained high quality cDNA reads of 44170060, over 74% of which mapped to the Morex reference genome. The results have been uploaded to NCBI GenBank (Project #PRJNA563590). Complementation assay for Barley susceptibility genes: We identified and sequenced cDNAs of Conlon HvEIN2, HvHSK and Hv2OGO. Introduction of either gene into the corresponding Arabidopsis gene edited plants provided a complementation assay for ability to revert the phenotype of these plants back to fully-susceptible. Both HvEIN2 and Hv2OGO cDNAs were cloned into plant expression vectors with kanamycin resistance selectable marker and complement-transformed into the transgene-free, homozygous lines for the corresponding gene edited mutant plants. Our results showed that both AtEIN2- KO/HvEIN2 plants and At2OGO-KO/Hv2OGO plants recovered susceptibility to near-WT levels FHB and marks them as rationale targets for gene editing in barley and other grains. Development of a Barley tissue culture and regeneration system for barley cv. Conlon: We have used CRISPR/Cas9 gene editing to produce two Hv2OGO mutants in the Conlon cultivar. We have modified the barley tissue culture protocol to regenerate Conlon barley more efficiently and constructed several CRISPR-editing vectors using barley (Hv), rice (Os) and wheat (Ta) U3 or U6 promoter. Using immature or mature embryos with either gene gun or Agrobacterium transformation methods, we have regenerated four plantlets from pRD383 (targeting Hv2OGO). RFLP analysis indicates that three of the regenerated plants produced NdeIuncut fragments of the PCR-amplified, target site-spanning gDNA. Sequence analysis of the target site from these putatively edited barley plants confirm the presence of mutations near the NdeI target site, all resulting in amino acid changes in these two plants. These plants, together with gene-edited plants in progress for HvHSK and HvEIN2, provide the basis for determining the contribution of these genes to FHB susceptibility and to generating GMO-free, gene-edited plants with an enhanced disease performance in the field. OBJECTIVE 2: Identifying Novel Cellular and Molecular Targets for DON in Animals. We performed transcriptomic analysis of DON-treated C. elegans and identified several novel DON targets, including genes that condition programmed cell death, endocytotic transport and protein aggregation. The functional contribution of several genes has been confirmed by RNAi analysis. Our studies reveal that DON affects the expression of many genes outside of those involved in innate immunity, suggesting that this toxin may affect diverse molecular and cellular processes as well as development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Low, Y, Lawton, MA and Di, R. 2020. Validation of barley 2OGO gene as a functional orthologue of Arabidopsis DMR6 gene in Fusarium head blight susceptibility. Sci. Reports. 10:9935. https://doi.org/10.1038/s41598-020-67006-5
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience for this research project are academic and industrial researchers working in the fields of mycotoxin research, plant biology and agricultural biotechnology (for the work on approaches to reduce mycotoxin contamination of crops through biotechnological approaches) as well as cell biologists and toxicologists (for the work on identifying cellular targets and mechanisms of mycotoxin action), growers, processors and end users of grain products, regulatory agencies concered with agriculture and food safety. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has involved a Ph. D graduate student who is funded by the teaching assistantship from the Plant Biology Graduate Program at Rutgers. During the course of the project, several undergraduate students in the majors of plant biology and plant biotechnology have also benefitted from the hands-on training in the areas of plant biology, plant tissue culture and engineering, plant biotechnology and molecular biology. Our results have also been incorporated into our teaching materials forcourses in Plant Biology and Biotechnology. How have the results been disseminated to communities of interest?The results of these projects have been disseminatedto the wider communities of interest through presentations made at scientific and professional meetings, such as the annual meeting ot he US Wheat and Barley Scab Initiative and throughpublications in the scientific literature. Results from the work on C. elegans exposed to DON were published in 2018. A manuscript describing results from work on gene-editing in Arabidopsis and the identification of barley orthologs of disease susceptibility genes has been submitted and is under review. Our preliminary data has been shared with other participants of this project through email communications and through our annual meeting. What do you plan to do during the next reporting period to accomplish the goals?The focus in the remaining period of the project will be touse our CRISPR vectors to editbarley susceptibility genes that we have identified in these studies as being able to condition susceptibility to FHB. This goal is already underway and will be extended to the other gene targets described. Candidate mutant plants, edited intarget genes will be characterized at the molecular level and any effects (i.e. enhancement) on disease susceptibility determined. An associated goal, will be to continue to improve the efficiency of barley transformation and regeneration in the Conlon cultivar.
Impacts
What was accomplished under these goals?
OBJECTIVE 1: Gene Editing of FHB Susceptibility Loci. The Di and Lawton Labs have adopted CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated endonuclease 9) gene editing technology to mutate genes involved in conditioning FHB susceptibility. We have employed Arabidopsis as the model plant to study the feasibility of knocking-out FHB susceptibility genes to achieve FHB resistance. After generating and assaying the effects of gene editing of these loci in Arabidopsis, we have used these plants to identify their functional orthologues from barley via a complementation assay. Once confirmed, we then employ CRISPR/Cas9 to edit these loci in barley and determine their contribution to disease susceptibility We identified three FHB susceptibility genes that are likely involved in FHB susceptibility: ethylene insensitive 2 (EIN2), homoserine kinase (HSK) and 2-oxoglutarate Fe(II)-dependent oxygenase (2OGO). We used the Arabidopsis (At) CRISPR vector pAt-sgRNA-Cas9 and produced AtEIN2-, AtHSK- and At2OGO-edited Arabidopsis plants with vectors, pRD182, pRD212 and pRD207 respectively. Our vectors contain PAtU6::AtEIN2/AtHSK/At2OGO-gRNA//PAtUbi::Cas9/TAtUbi in the plant expression vector pCAMBIA1300, with the hygromycin resistance gene as the selectable marker. Agrobacterium-mediated transformants (T1 and T2 generations) were validated for editing of target sites by restriction fragment length polymorphism (RFLP), T7 endonuclease 1 assay and sequencing of PCR-amplified gDNA fragment spanning the target sites. To-date, we have validated: 15 AtEIN2-edited plants each with different mutations at their respective target sites. 14 At2OGO-edited Arabidopsis plants, each with different mutations at their respective target sites. We have also produced AtHSK-KO Arabidopsis plants whose characterization at the DNA sequence level is in progress. For both AtEIN2-edited plants and At2OGO-edited plants, we have selected from the T2 generation and confirmed by RFLP analysis transgene-free homozygous plants. This confirms the validity of the approach of using CRISPR/Cas9 gene editing to alter endogenous gene sequences in plants that are no longer considered to be GMOs. Inoculation of staged florets from WT and edited plants with Fg tagged with green fluorescent protein (GFP) allowed us to the progression of infection, monitor symptom development, and quantitate defense responses. We determined the levels of Fg-GFP by real-time PCR using GFP-specific primers and Arabidopsis actin gene as an endogenous control for relative quantification. Our results show that both At2OGO and AtEIN2-KO plants displayed a markedly slower FHB disease development with a consequent reduction in the levels of Fg-GFP, compared to WT plants. RNA-SEQ of Cultivar Conlon. To identify which barley homologs of 2OGO, EIN2 and HSK are involved in FHB susceptibility, we wished to complement the corresponding gene-edited Arabidopsis plants (At2OGO-KO and AtEIN2-KO) with barley cDNA isolated from the U.S. cultivar Conlon. The genome of Morex barley, a US spring six-row malting cultivar, has been published. We focused, however, on Conlon, because this cultivar is more susceptible to Fg than is Morex, and provides a better baseline for determining the effects of inactivating genes that condition susceptibility in barley. We used the Illumina HiSeq platform to perform RNAseq analysis of barley cv. Conlon, an important North American two-rowed cultivar and obtained high quality cDNA reads of 44170060, over 74% of which mapped to the Morex reference genome. The results have been uploaded to NCBI GenBank (Project #PRJNA563590). Complementation assay for Barley susceptibility genes. We identified and sequenced cDNAs of Conlon HvEIN2, HvHSK and Hv2OGO. Introduction of either gene into the corresponding Arabidopsis gene edited plants provided a complementation assay for ability to revert the phenotype of these plants back to fully-susceptible. Both HvEIN2 and Hv2OGO cDNAs were cloned into plant expression vectors with kanamycin resistance selectable marker and complement-transformed into the transgene-free, homozygous lines for the corresponding gene edited mutant plants. Our results showed that both AtEIN2-KO/HvEIN2 plants and At2OGO-KO/Hv2OGO plants recovered susceptibility to near-WT levels FHB. These results indicate that both the barley HvEIN2 Hv2OGO genes condition FHB susceptibility and marks them as rationale targets for gene editing in barley and other grains. These findings have been incorporated into a manuscript that will be submitted for publication in the period just following this reporting period. Development of a Barley tissue culture and regeneration system for barley cv. Conlon. We have used CRISPR/Cas9 gene editing to produce two Hv2OGO mutants in the Conlon cultivar. We have modified the barley tissue culture protocol to regenerate Conlon barley more efficiently, as this is a bottleneck for our studies and others in the field. Therefore, we constructed several CRISPR-editing vectors using barley (Hv), rice (Os) and wheat (Ta) U3 or U6 promoter, which have been used effectively in other cultivars, such as Golden Promise. Using immature or mature embryos with either gene gun or Agrobacterium transformation methods, we have regenerated four plantlets from pRD383 (targeting Hv2OGO). RFLP analysis indicates that three of the regenerated plants produced NdeI-uncut fragments of the PCR-amplified, target site-spanning gDNA. Sequence analysis of the target site from these putatively edited barley plants confirm the presence of mutations near the NdeI target site, all resulting in amino acid changes in these two plants. These plants, together with gene-edited plants in progress for HvHSK and HvEIN2, provide the basis for determining the contribution of these genes to FHB susceptibility and to generating GMO-free, gene-edited plants with an enhanced disease performance in the field. OBJECTIVE 2: Identifying Novel Cellular and Molecular Targets for DON in Animals. We have addressed the second goal using the model multicellular animal C. elegans. DON appears to act in humans a number of different levels, and many of these features can also be addressed in the C. elegans model. These include the roles of genes that condition programmed cell death, the role of endocytotic pathways in toxin cellular transport, and the role of toxin-induced protein aggregation in neurological degeneration. With the previous multistate fund, we have established the worm system to study the mode of DON intoxication. We have mapped out the gene expression profile throughout the entire genome of C. elegans upon DON intoxication by genome-wide RNAseq analysis. The interaction of some of these genes has been confirmed by RNAi (RNA interference) analysis. Our studies reveal that DON affects the expression of many genes outside of those involved in innate immunity, suggesting that this toxin may affect diverse molecular and cellular processes as well as development. Deoxynivalenol (DON) is a mycotoxin produced by Fusarium spp. that causes Fusarium head blight (FHB) disease in cereal crops. Ingestion of food contaminated with DON poses serious human health complications. However, the DON cytotoxicity has been mostly deduced from animal studies. In this study, we used the nematode Caenorhabditis elegans (C. elegans) as a tractable animal model to dissect the toxic effect of DON. Our results indicate that DON reduces the fecundity and lifespan of C. elegans. The results of this ongoing study provide insights to the targets of DON cytotoxicity and potential mitigation measures.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience for this research project comprises academic and industrial researchers working in the fields of mycotoxin research, plant biology and agricultural biotechnology (for work on approaches to reduce mycotoxin contamination of crops through biotechnological approaches) as well as cell biologists, toxicologists and the medical community (for work on identifying cellular targets and mechanisms of mycotoxin action). Additional targets include growers, processors, those involved in testing and certifying grain crop contamination, malters and brewers, and other downstream processors and distributors in the food industry. Changes/Problems:The advent of RNP based CRISPR methods provides an opportuity to speed up the editing of susceptibility genes while also avoiding problems of public acceptance associated with the use of transgenics. Consequently, we will make use of this technology where it serves the interest of the project and the needs of end users. What opportunities for training and professional development has the project provided?This project has involved a Ph. D graduate student who is funded by a teaching assistantship from the Plant Biology Graduate Program at Rutgers. During the course of the project, several undergraduate students in the majors of plant biology and plant biotechnology have also benefitted from the hands-on training in the areas of plant biology, plant tissue culture and engineering, plant biotechnology and molecular biology. Our results have also been incorporated into our teaching materials for the courses in Plant Biology and Biotechnology. How have the results been disseminated to communities of interest?The results of these projects are not yet ready for dissemination to the wider communities of interest as we are still waiting for extant transgenic plants to be tested and for additional plants to be constructed and tested. The results of the C. elegans work have been published this year. This reporting period we hosted the project annual meeting at Rutgers. We shared our reliminary data and where project members presented the results of their research. The new project sub-committee structure put in place since the previous meeting was discussed and project members were assigned to coordinate project collaboration (both research and funding). The project web site (https://www.mycotoxins-nc1183.com/) has been completely redesigned to better coordinate research between members of the project, to facilitate collaboration and to serve as an educational and outreach tool for the project's stakeholders and the public. The Project web site is now established and has been linked to other resources of use to the community of mycotoxin researchers and other stakeholders. What do you plan to do during the next reporting period to accomplish the goals?In the coming year, we expect to make significant progress on the construction of CRISPR knockout lines for designated susceptibility genes in barley. We are in the process of constructing CRISPR constructs that target these gene in barley, using the transformation and regeneration protocols we have recently established in our lab. We will also make use of the RNP approach to directly deliver CRISPR/gRNAs into cells. The effects of mutating these genes on FHB disease susceptibility in barley will be determined in the subsequent reporting period. We will create additional knock-down lines in C. elegans using RNAi and examine the effects on DON susceptibility. If successful, we, along with our collaborators in this project, will explore ways to translate the beneficial effects of inhibiting these genes and their encoded proteins in other animal systems.
Impacts
What was accomplished under these goals?
Mycotoxigenic fungi contamination of grain and forage crops is a significant and ongoing problem agriculture and the food and brewing industries and for animal and human health. Improved, more sensitive, rapid and precise methods are needed to monitor, treat or prevent grain contamination. Effective monitoring or elimination of mycotoxins from grain crops will benefit US consumers, and allow US commodities to compete more effectively in world markets. The goal of this project is to identify novel targets for mycotoxins in plant and animal cells and develop strategies for eliminating or ameliorating the effects of mycotoxins. Identification of novel targets in plants will inform strategies to enhance plant disease resistance and reduce both pre- and post-harvest infection in grain crops. Identification of novel cellular targets in the C. elegans model will guide amelioration and preventative strategies for animals and humans exposed to these toxins. By reducing the both the incidence and impact of mycotoxins in grain crops, we expect to enhance animal and human health, assure food security and protect the integrity of US and international grain production and markets. Objective 1. Develop data for use in risk assessment of mycotoxins in human and animal health. The Caenorhabditis elegans model system has been developed to study mechanisms of toxicity of DON produced by Fusarium spp.. This model has allowed us to explore the mechanisms of DON toxicity in animals, identify cellular targets, and develop strategies for reducing the effects of exposure to DON. These studies have shown that worm lifespan is significantly reduced following exposure to DON. RNA seq analysis has uncovered many genes, involved in a variety of cellular and molecular functions, that are either up- or down-regulated upon DON intoxication. This analysis has identified genes that are most highly induced in response to DON, including genes that encode proteins likely to be involved in DON detoxification as well as proteins involved in the innate immune response. Expression of a number of genes is down-regulated by exposure to DON. These represent targets whose expression may help protect against DON and other mycotoxins. Genes whose expression was altered following DON treatment were analyzed by RT-qPCR. The functional significance of these induced genes was explored by RNAi suppression of expression. C. elegans was fed E. coli expressing RNAi constructs that target the three most highly DON-induced genes. After 24 hr, L2 worms fed with E. coli expressing RNAi constructs displayed a reduced viability, compared to controls, when exposed to DON. These results show that the RNAi suppression of DON-inducible genes could further reduce C. elegans viability when worms are exposed to DON. one possible explanation is that these genes are involved in DONA detoxification. A manuscript reporting these findings has recently been published. In collaboration with Multi-State Project Member Dr. Chris Schardl, University of Kentucky, we confirmed that the C. elegans system could also be used to analyze the cytotoxicity of N-formylloline (NFL) from endophyte Epichloe. It should now be possible to similarly examine the transcriptome of C. elegans exposed to these mycotoxins and ask if a similar or distinct set of genes is up- or down-regulated in NFL and DON-treated worms. Objective 2: Establish integrated strategies to monitor and reduce mycotoxin contamination in cereal grains and distiller grains. In addition to developing a regeneration and transformation system in Barley, we are also using Brachypodium (Bd21 variety) and Arabidopsis as model systems for assessing infection by F. graminearum and the responses to application of DON. We have developed our own CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 nuclease) gene-editing platforms to disrupt genes that condition susceptibility to Fusarium graminearum (Fg) infection to engineer FHB resistance in Brachypodium, Arabidopsis and barley. In addition to the use of DNA-based systems, we have also had success in the use of Ribonucleoprotein (RNP)-based CRISPR methods that avoid the need for cloning and transgenic plants. F. graminearum exploits ethylene signaling to colonize both dicotyledonous and monocotyledonous plants and that the EIN2-RNAi (ethylene insensitive 2-RNA interference)-silenced wheat plants are more resistant to FHB with reduced DON accumulation. The EIN2 gene has also been implicated in Brachypodium susceptibility to FHB. It was reported in 2009 that an ethyl methanesulfonate (EMS)-induced mutation in Arabidopsis DMR1 gene encoding homoserine kinase (HSK) resulted in homoserine accumulation and resistance to downy mildew (DM) caused by Hyaloperonospora arabidopsidis. Later, it was shown that dmr1 Arabidopsis mutant plants were also resistant to F. culmorum and F. graminearum, and that exogenous application of L-homoserine reduced bud infection in both dmr1 and wild type (WT) plants. Another Arabidopsis EMS mutant dmr6 in which the gene encoding a 2-oxoglutarate Fe(II)-dependent oxygenase (2OGO) was mutated was found to be resistant to H. arabidopsidis, and the resistance was a result of enhanced plant immunit. The dmr6 Arabidopsis mutant plants were highly resistant to F. culmorum and F. graminearum. We have now made CRISPR constructs that target the EIN2, HSK and 2OGO genes in Arabidopsis, Brachypodium and barley. We have produced AtHSK- and At2OGO-knock out (KO) mutant Arabidopsis plants. Detached leaves from mutant Arabidopsis plants were inoculated with F. graminearum-GFP. Our results have shown that the AtHSK and At2OGO Arabidopsis mutant plants are more resistant to F. graminearum-GFP infection. The GFP gene copy of F. graminearum in the inoculated leaves of AtHSK and At2OGO Arabidopsis mutant plants was much lower compared to that in the WT plants, as measured by real-time quantitative PCR analysis. We have now developed our own barley (Hordeum vulgare, Hv; cv. Conlon) tissue culture protocol, which paves the way for future transgenic studies. We have performed RNAseq analysis of Conlon barley, cloned the cDNAs of HvHSK and Hv2OGO and the partial genomic DNAs of HvHSK, Hv2OGO and HvEIN2, in preparation for complementation studies in Arabidopsis. We have also constructed CRISPR gene-editing vectors for HvHSK, Hv2OGO and HvEIN2, in preparation for transformation of barley calli derived from immature seeds by gene gun bombardment.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Di, R.; Zhang, H.; Lawton, M.A. Transcriptome Analysis of C. elegans Reveals Novel Targets for DON Cytotoxicity. Toxins 2018, 10, 262. doi:10.3390/toxins10070262
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience for this research project continue to be academic and industrial researchers working in the fields of mycotoxin research, plant biology and agricultural biotechnology (for the work on approaches to reduce mycotoxin contamination of crops through biotechnological approaches) as well as cell biologists and toxicologists (for the work on identifying cellular targets and mechanisms of mycotoxin action). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has involved a Ph. D graduate student who is funded by a teaching assistantship from the Plant Biology Graduate Program at Rutgers. During the course of the project, several undergraduate students in the majors of plant biology and plant biotechnology have also benefitted from the hands-on training in the areas of plant biology, plant tissue culture and engineering, plant biotechnology and molecular biology. Our results have also been incorporated into our teaching materials for the courses in Plant Biology and Biotechnology. How have the results been disseminated to communities of interest?The results of these projects are not yet ready for dissemination to the wider communities of interest as we are still waiting for extant transgenic plants to be tested and for additional plants to be constructed and tested. The results of the C. elegans work have been summarized in a manuscript that willbe submitted for publication shortly. This reporting period we hosted the project annual meeting at which we shared our preliminary data and where project members presented the results of their research. Outcomes of this meeting included the establishment of a new project committee structure designed to better coordinate the objectives of the project and to secure outside funding. The meeting also led to a decision to revamp the project web site to better coordinate research between members of the project, to facilitate collaboration and to serve as an educational an outreach tool for the project's stakeholders and the public. The web site is now established and has been linked to other resources of use to the community of mycotoxin researchers and other stakeholders. What do you plan to do during the next reporting period to accomplish the goals?The CRISPR knockout lines constructed in Arabidopsis for disease susceptibility genes will be used in complementation assays to identify Barley gene homologs that are functionally equivalent. Once these Barley genes are identified we will construct CRISPR constructs that target these gene in barley, using the transformation and regeneration protocols we have recently established in our lab.The effects of mutating these genes on FHB disease susceptibility in barley will be determined. We will create additional knock-down lines in C. elegans using RNAi and examine the effects on DON susceptibility. If successful, we, along withour collaborators in this project, will exploreways to translate the beneficial effects of inhibiting these genes and their encoded proteins in other animal systems.
Impacts
What was accomplished under these goals?
Mycotoxigenic fungi contamination of grain and forage crops presents an ongoing problem and challenge for agriculture, for human and animal health and for industrial applications, fermentation of grains by microorganisms. Better, faster and more accurate methods to monitor, treat or prevent the grain contamination will benefit US consumers, and allow US commodities to compete more effectively in foreign markets. This project aims to identify novel targets for mycotoxins in plant and animal cells. Identification of such targets in plants will guide strategies to enhance disease resistance to infection in grain crops, both pre- and post-harvest. Identification of cellular targets in C. elegans will help in the development of amelioration and preventative strategies for animals and humans exposed to these toxins. Reducing the incidence and impact of mycotoxins in grain crops will directly enhance animal and human health, assure food security and protect the integrity of US and international grain production and markets. Objective 1. Develop data for use in risk assessment of mycotoxins in human and animal health. We have developed the Caenorhabditis elegans system to study the toxicity mechanisms of DON produced by Fusarium spp. This model allows us to understand the mechanisms of DON toxicity in animals, identify cellular targets, and explore strategies for amelioration of DON toxicity. This system can, and has been used to study other mycotoxins. Our results have shown that the lifespan of the DON-treated worms is significantly reduced compared to worms treated with DMSO alone. RNAseq analysis of the 250 mg/ml DON-treated worms indicates that many genes involved in diverse worm biological processes and molecular functions are either up- or down-regulated upon DON intoxication. This analysis has allowed us to identify those genes that are most highly induced in response to DON. These include genes that encode proteins likely to be involved in DON detoxification as well as proteins involved in the expression of innate immunity. Just as significant are a number of genes whose expression is severely down-regulated by DON. These represent targets whose expression may provide a natural defense or protection against DON and other mycotoxins. Changes of expression for selected genes of interest were validated by real-time RT-qPCR analysis of different samples of DON-treated worms. The significance of these induced genes was further explored using RNAi suppression. C. elegans was fed E. coli expressing RNAi constructs that target the three most highly DON-induced genes. After 24 hr, L2 worms fed with E. coli expressing RNAi constructs displayed a reduced viability, compared to controls, when exposed to DON. These results demonstrate that the knocking-down through RNAi DON-inducible genes further reduced the C. elegans viability when fed with DON, indicating the possible involvement of these genes in the detoxification mechanism of C. elegans. A manuscript reporting these findings is in the final stages of preparation. In collaboration with Dr. C. Schardl at University of Kentucky, we used the C. elegans system to analyze the cytotoxicity of N-formylloline (NFL) from endophyte Epichloe. Our data showed that 250 mg/ml NFL killed all the L2 worms within 24 hr, whereas 250 mg/ml DON only reduced the worm's viability by 2-5%, indicating NFL is more toxic than DON. NFL also reduced the egg-laying capability of C. elegans dose-dependently as DON. This results shows that C. elegans can be used to study the cytotoxicity of fungal toxins other than DON. Objective 2: Establish integrated strategies to monitor and reduce mycotoxin contamination in cereal grains and distiller grains We have adopted Brachypodium (Bd21 variety) and Arabidopsis as model systems for assessing infection by F. graminearum and the responses to application of DON. We have developed our own CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 nuclease) gene-editing platforms to disrupt genes that condition susceptibility to Fusarium graminearum (Fg) infection to engineer FHB resistance in Brachypodium, Arabidopsis and barley. It has been shown that F. graminearum exploits ethylene signaling to colonize both dicotyledonous and monocotyledonous plants and that the EIN2-RNAi (ethylene insensitive 2-RNA interference)-silenced wheat plants are more resistant to FHB with reduced DON accumulation. The EIN2 gene has also been implicated in Brachypodium susceptibility to FHB. It was reported in 2009 that an ethyl methanesulfonate (EMS)-induced mutation in Arabidopsis DMR1 gene encoding homoserine kinase (HSK) resulted in homoserine accumulation and resistance to downy mildew (DM) caused by Hyaloperonospora arabidopsidis. Later, it was shown that dmr1 Arabidopsis mutant plants were also resistant to F. culmorum and F. graminearum, and that exogenous application of L-homoserine reduced bud infection in both dmr1 and wild type (WT) plants. Another Arabidopsis EMS mutant dmr6 in which the gene encoding a 2-oxoglutarate Fe(II)-dependent oxygenase (2OGO) was mutated was found to be resistant to H. arabidopsidis, and the resistance was a result of enhanced plant immunit. The dmr6 Arabidopsis mutant plants were highly resistant to F. culmorum and F. graminearum. Accordingly, we have made CRISPR constructs that target the EIN2, HSK and 2OGO genes in Arabidopsis, Brachypodium and barley. We have produced AtHSK- and At2OGO-knock out (KO) mutant Arabidopsis plants. Detached leaves from mutant Arabidopsis plants were inoculated with F. graminearum-GFP. Our results showed that the AtHSK and At2OGO Arabidopsis mutant plants were more resistant to F. graminearum-GFP infection. The GFP gene copy of F. graminearum in the inoculated leaves of AtHSK and At2OGO Arabidopsis mutant plants was much lower compared to that in the WT plants, as measured by real-time quantitative PCR analysis. We have developed our own barley (Hordeum vulgare, Hv; cv. Conlon) tissue culture protocol, and have conducted RNAseq analysis of Conlon barley, cloned the cDNAs of HvHSK and Hv2OGO and the partial genomic DNAs of HvHSK, Hv2OGO and HvEIN2. We are in the process of constructing the CRISPR gene-editing vectors for HvHSK, Hv2OGO and HvEIN2, and will transform barley calli derived from immature seeds by gene gun bombardment.
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Progress 10/15/15 to 09/30/16
Outputs
Target Audience:The target audience for this research project are academic and industrial researchers working in the fields of mycotoxin research, plant biology and agricultural biotechnology (for the work on approaches to reduce mycotoxin contamination of crops through biotechnological approaches) as well as cell biologists and toxicologists (for the work on identifying cellular targets and mechanisms of mycotoxin action). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has involved a Ph. D graduate student who is funded by the teaching assistantship from the Plant Biology Graduate Program at Rutgers. During the course of the project, several undergraduate students in the majors of plant biology and plant biotechnology have also benefitted from the hands-on training in the areas of plant biology, plant tissue culture and engineering, plant biotechnology and molecular biology. Our results have also been incorporated into our teaching materials for the courses in Plant Biology and Biotechnology. How have the results been disseminated to communities of interest?The results of these projects are not yet ready for dissemination to the wider communities of interest as we are still waiting for extant transgenic plants to be tested and for additional platns to be constructed and tested. The results of the C. elegans work are being prepared for a manuscript that will be submitted for publication. Our prelimnary data will be shared with the other participants of thisproject through email and through our annual meeting. What do you plan to do during the next reporting period to accomplish the goals? We will continue to produce Arabidopsis AtDFR- mutant with pRD271, and Brachypodium mutants in BdEIN2, BdHSK and BdDFR. We will clone the gDNAs of barley HvHSK and HvDFR genes, identify the gene targeting sequences and construct CRISPR vectors. We will use these CRISPR vectors to mutant the genes in barley, leading to FHB resistant barley plants. The barley HvEIN2 cDNA has been cloned by RT-PCR. We will also clone the cDNAs for HvHSK and HvDFR and construct transformation vectors, which will be used to transform into the equivalent gene knocked-out Arabidopsis or Brachypodium mutant plants to study the interaction of these genes with host plants.
Impacts
What was accomplished under these goals?
Objective 1: Develop novel strategies to enhance the resistance of crop plants to infection with Fusarium graminearum, the causal agent of wheat and barley head blight (FHB) and the source of grain contamination with mytoxins such as deoxynivalenol (DON). For the first goal, we have adopted CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated endonuclease 9) gene editing technology. This technology is efficient, selective and reproducible and has been used to produce gene edited or mutagenized Arabidopsis, rice, wheat, tobacco and soybean plants. Our aim is to mutate genes involved in FHB susceptibility. We employed dicotyledonous Arabidopsis and monocotyledonous Brachypodium as model plants to study the feasibility of knocking-out FHB susceptibility genes to achieve FHB resistance. We then applied the same CRISPR-gene editing technology to mutate the equivalent FHB susceptibility genes in barley cv. Conlon to produce FHB resistant barley plants. Based on literature and published results in Arabidopsis, Brachypodium, wheat and barley, we have identified the following three genes involved in FHB susceptibility: EIN2 (ethylene insensitive 2), HSK (homoserine kinase) and DFR (dihydroflavonol-4-reductase). We have constructed CRISPR-editing vectors to target these three genes in Arabidopsis, pRD182 (AtEIN2), pRD212 (AtHSK) and pRD271 (AtDFR). In these vectors, the 20-bp target sequence for each Arabidopsis gene is under the control of the Arabidopsis U6 promoter followed by the scaffold RNA sequence, and the Cas9 endonuclease gene is driven by the Arabidopsis ubiquitin promoter and terminated by the nopoline synthase terminator. With Agrobacterium-mediated transformation, we have produced several Arabidopsis small nucleotide deletion mutants with pRD182 (AtEIN2) and pRD212 (AtHSK). We are in the process of segregating the T2 generation of these mutant plants to screen for transgene (gRNA+Cas9 cassettes)-free homozygous mutant plants. As the involvement of HvEIN2, HvHSK and HvDFR genes in barley FHB susceptibility, and the mechanisms of these gene mutations leading to FHB resistance are largely unknown, creating specific gene knocked-outs in Arabidopsis will provide a great platform to study the interaction between F. graminearum and plants. We have also constructed CRISPR-editing vectors for Brachypodium, pRD262 (BdEIN2), pRD273 (BdHSK) and pRD274 (BdDFR). In these vectors, the gDNAs are under the control of rice U3 promoter and the Cas9 cassette is driven by the rice ubiquitin promoter. By gene gun bombardment method, we have produced a few lines of putative gene knocked-out Brachypodium plants. We are in the process of characterizing these potential mutant Brachypodium plants. We have developed detached leaf inoculation method for F. graminearum infection and DON inoculation. Our preliminary data have shown that the T1 pRD182 (AtEIN2) Arabidopsis plants are resistant to F. graminearum-GFP (green fluorescent protein). To CRISPR-edit the FHB susceptibility genes in the crop barley plants, we have cloned and sequenced the complete gDNA of HvEIN2. We have identified the HvEIN2 gene targeting sequence and constructed the CRISPR vectors, pRD281 with hygromycin resistance gene as the selectable marker and pRD282 with kanamycin resistance gene as the selectable marker. We have used the gene gun bombardment method to transform barley calli generated from immature Conlon seeds with pRD281 and pRD282. The transformed calli are currently being selected on antibiotic-containing media. Objective 2: Use the C. elegans model system to define the molecular mechanisms and cellular targets of DON and to devise and test novel preventative and ameliorative treatments. We have addressed the second goal using the model multicellular animal C. elegans. DON appears to act in humans a number of different levels, and many of these features can also be addressed in the C. elegans model. These include the roles of genes that condition programmed cell death, the role of endocytotic pathways in toxin cellular transport, and the role of toxin-induced protein aggregation in neurological degeneration. With the previous multistate fund, we have established the worm system to study the mode of DON intoxication. We have mapped out the gene expression profile throughout the entire genome of C. elegans upon DON intoxication by genome-wide RNAseq analysis. The interaction of some of these genes has been confirmed by RNAi (RNA interference) analysis. Our studies reveal that DON affects the expression of many genes outside of those involved in innate immunity, suggesting that this toxin may affect diverse molecular and cellular processes as well as development. Our findings are currently being incorporated into a manuscript for publication.
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