The search for a new gene: RPS6

Abstract only availablePlants are exposed to a wide variety of pathogens including viruses, bacteria, fungi, nematodes and protozoa. In response, plants have developed a plethora of strategies aimed at blocking infection by potential pathogens. One form of induced response is the hypersensitive response (HR), during which cells immediately surrounding the site of infection rapidly die. This interaction between these pathogens and plants is governed by the genetics of both organisms. The genes responsible for deterring infection are called disease resistance genes. In fact, disease resistance genes are employed to specifically recognize pathogens expressing cognate genes (appropriately called the avirulence gene). Historically, this has been explained by the gene-for-gene hypothesis. This hypothesis predicts that if the pathogen carries an avirulence gene, which is “recognized” by a specific resistance gene in the plant, a plant resistance response is induced. If either the avirulence gene or the resistance gene is absent, then the pathogen causes disease on the plant. In many cases, control of disease resistance conforms to the gene-for-gene hypothesis. I have been pursuing the identification, and mapping of a new disease-resistance gene, RPS6 , which has been named by our lab as the “ hopPsyA project”. I have been screening Arabidopsis plants to isolate mutants, and I have thus far isolated two true mutants with the desired trait. I have also sequenced the eds1 gene in two of these mutants. Because the eds1 gene is already known to be required for RPS6 function, verifying that the mutation is not in the eds1 gene (which our results confirm) gets us one step closer to indicate that the mutation is most probably in the RPS6 gene. I am further focusing my project on narrowing down the location of the gene responsible for the desired trait and have crossed one of my mutants to a resistant line.MU Monsanto Undergraduate Research Fellowshi

Determining the function of SNC1 in Arabidopsis plants

Abstract only availableWhen Arabidopsis must defend itself from bacterial infections, the process can potentially be harmful to the host plant. The specific gene I am focusing on in my project is SRFR1 (suppressor of rps4-RLD), which is involved in the avrRps4 triggered disease resistance pathway. SRFR1 is located close to the bottom of Arabidopsis' fourth chromosome. It was found that if a T-DNA insertion was made into exon 3 of SRFR1 in the Arabidopsis accession Columbia-0 (Col-0), one fourth of the plants are stunted, indicating that stunting is a recessive trait. Stunting in plants is a phenotype that is related to plants that have their defense mechanisms activated nonstop. However, mutations in SRFR1 in the Arabidopsis accession RLD did not cause stunting, indicating that genetic differences exist between Col-0 and RLD that control the stunted phenotype. In order for my project to begin, srfr1-3 plants (T-DNA insertion in SRFR1, Col-0 background) were crossed to RLD, and it was determined that stunting was based on two recessive genes. One candidate gene to contribute to stunting caused by the srfr1-3 allele is SNC1, a Col-0 gene known to cause stunting if SNC1 expression is misregulated. The major goal of my project is to find out if SNC1 is the other gene that is required for stunting. The method I will use to find out if SNC1 is the second gene required for stunting is by crossing srfr1-3 plants with snc1-11 plants. If srfr1-3 causes stunting but needs SNC1 to do so, if the SNC1 gene is not functional theoretically plants should not be stunted. In the F2 generation, I will genotype stunted plants and determine if the SNC1 gene is activated or deactivated. If the SNC1 gene is activated in stunted plants then there is a possibility of SNC1 being the second responsible gene. If SNC1 is deactivated in stunted plants, then it cannot be the second gene responsible for stunting. The second part of my project takes an unbiased approach by mapping (or finding the location on the chromosome) the additional gene required for stunting. I will run several polymerase chain reaction (PCR) based genetic markers on a selected group of plants and compare phenotypes and genotypes to determine where the target gene is located.NSF Grant to W. Gassman

Identifying plant resistance pathways in Arabidopsis thaliana

Abstract only availableThe destruction of plants by a pathogen results in millions of dollars lost in crop yield annually. Identifying the plant response pathway to the presence of a pathogen is key to combating plant disease. The gene-for-gene hypothesis suggests that for every pathogen avirulence gene (avr), there is a corresponding specific plant resistance (R) gene that recognizes it and elicits a defense response. One method for discovering resistance pathways is through the use of a suppressor screen in which the deletion or mutation of a negative regulator can reactivate a signaling pathway. Our srfr (suppressor of rps4-RLD) mutants were discovered to provide resistance to the Pseudomonas syringae pv. tomato DC3000 expressing avrRps4 and thus is possibly a regulatory gene. The srfr mutants reactivated resistance to avrRps4 in plants that have a non-functional RPS4 gene. We are studying to see if the srfr gene signaling pathway reactivates avr responses dependent on R genes other than RPS4. We chose the well-studied RPM1 gene, which functions in detecting bacteria expressing avrRpm1. After crossing srfr3 and rpm1-3 mutants and harvesting F1 seeds, we grow these F1 seeds to get the F2 generation population. We isolated genomic DNA from F2 plants and then applied PCR based markers to find homozygous double mutants using 2 genetic markers linked to the srfr3 and rpm1-3 mutation, respectively. The F3 generation plants will be available to test disease symptoms in these double mutants. Based on the results of disease symptoms of the F3 population against bacteria expressing avrRpm1, we will propose whether or not the srfr3 gene is involved in the RPM1 defense signaling pathway. If the F3 generation is susceptible to avrRpm1, then srfr3 is not involved in the RPM1 defense signaling pathway.Plant Genomics Internship @ M

Identifying the interaction between AtSRFR1 with AtTPL and AtTLR3 in planta

Abstract only availablePlant disease caused by pathogens results in large economic losses in crop yield annually. Our previous study found a negative regulator of effector triggered immunity in Arabidopsis thaliana. Mutations in SRFR1 (Suppressor of rps4-RLD) enhance resistance to the pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4. We hypothesize that SRFR1 functions similar to Ssn6, a possible ortholog of SRFR1. Ssn6 - Tup1 is a well- known conserved system of transcriptional repression in eukaryotes. We are investigating whether SRFR1 interacts with Tup1-orthologs of Arabidopsis. We chose TOPLESS (TPL), which functions as a transcriptional repressor regulating shoot development, and TOPLESS RELATED (TLR). We first amplified cDNAs of TPL and TLR from Arabidopsis and cloned them into the entry vector of the Gateway compatible system. We then subcloned TPL and TLR into various vectors using Gateway system. These vectors introduced sequences of various tags such as Myc or HA at the 5' end. Using Agrobacterium -mediated transient expression in Nicotiana benthamiana, we will test for interactions between SRFR1 with TPL and TLR. Protein samples will be extracted for co-immunoprecipitation assay to reveal the presence or absence of interactions between these proteins. We will perform western blots to look for these interactions. Cloning TPL and TLR transcripts into plasmid vectors for Agrobacterium transformation required the majority of time and effort for this project. If the two proteins interact, it will be evidence of the importance for SRFR1 in transcriptional repression.Gyeongsang National Universit

Tissue-specific expression of a FMR1/β-galactosidase fusion gene in transgenic mice

Fragile X syndrome is one of the most common genetic causes of mental retardation, yet the mechanisms controlling expression of the fragile X mental retardation gene FMR1 are poorly understood. To identify sequences regulating FMR1 transcription, transgenic mouse lines were established using a fusion gene consisting of an E.coli β-galactosidase reporter gene (lacZ) linked to a 2.8 kb fragment spanning the 5′-region of FMR1. Five transgenic mouse lines showed lacZ expression in brain, in particular in neurons of the hippocampus and the granular layer of the cerebellum. Expression of the reporter gene was also detected in Leydig cells and spermatogonia in the testis, in many epithelia of adult mice, and in the two other steroidogenic cell types, adrenal cortex cells and ovarian follicle cells. Embryonic tissues which showed strong activity of the reporter gene included the telencephalon, the genital ridge, and the notochord. This expression pattern closely resembles the endogenous one, indicating that the 5′ FMR1 gene promoter region used in this study contains most cis-acting elements regulating FMR1 transcriptio

Alternative transcripts of the RPS4 resistance gene in Arabidopsis: Do they produce truncated proteins?

Abstract only availablePlant disease due to pathogenic infection causes large economic losses worldwide. The study of plant disease resistance will potentially lead to crop improvements in an effort to feed the growing world population. This study focuses on the RPS4 resistance gene in Arabidopsis thaliana, which confers resistance to the pathogenic bacteria Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 in a gene-for-gene specific interaction. Like many related resistance genes, RPS4 produces multiple alternative transcripts whose functions are not yet fully understood. The dominant RPS4 alternative transcripts are produced via alternative splicing causing the retention of either intron 3 or introns 2 and 3. Previous research revealed the combined presence of both the full and alternative transcripts was required for proper RPS4 function. These alternative transcripts have the potential to be translated into truncated proteins due to the presence of premature in-frame stop codons within the retained introns. The goal of this study was to detect and analyze the function of these putative truncated proteins. To detect truncated proteins, N- and C-terminal epitope tags were added to both the genomic RPS4 sequence and cDNAs of different lengths. These constructs will be transiently expressed in tobacco leaves using Agrobacterium. Protein samples will be extracted for an immunoblot assay to reveal the presence or absence of truncated RPS4 proteins. Subcloning RPS4 transcripts into plasmid vectors for Agrobacterium transformation required the majority of time and effort for this project. To determine the biological function of the putative truncated proteins, an Agrobacterium-mediated transient assay on tobacco will be performed using the same RPS4 constructs. The plants will be observed for the presence of a ubiquitous defense response called the hypersensitive response (HR). If a certain RPS4 construct or combination of constructs causes HR, it will lend evidence toward the functional importance of those constructs in generating defense responses. It has been hypothesized that in resting cells, R-proteins are self-inhibited by intramolecular interactions. When the plant recognizes an invading pathogen, these intramolecular interactions are disrupted, leading to defense responses. To test this hypothesis on the RPS4 gene, another transient assay will be performed using RPS4 constructs with N- and C-terminal serial deletions in various combinations. If a certain combination of constructs causes HR, it will provide evidence that these sequences interact intramolecularly. The serial deletion constructs are in the initial stages of development and will be used in the future to perform this experiment. The sum of this research will improve our understanding of alternative transcripts and their function in plant resistance.Plant Genomics Internship @ M

NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor family of cytokines that control vascular and lymphatic endothelium development. It has been implicated in promoting angiogenesis in pathological conditions via signaling to vascular endothelial growth factor receptor-1. PlGF expression is induced by hypoxia and proinflammatory stimuli. Metal responsive transcription factor 1 (MTF-1) was shown to take part in the hypoxic induction of PlGF in Ras-transformed mouse embryonic fibroblasts. Here we report that PlGF expression is also controlled by NF-κB. We identified several putative binding sites for NF-κB in the PlGF promoter/enhancer region by sequence analyses, and show binding and transcriptional activity of NF-κB p65 at these sites. Expression of NF-κB p65 from a plasmid vector in HEK293 cells caused a substantial increase of PlGF transcript levels. Furthermore, we found that hypoxic conditions induce nuclear translocation and interaction of MTF-1 and NF-κB p65 proteins, suggesting a role for this complex in hypoxia-induced transcription of PlG

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean

High-Throughput RNA Sequencing of Pseudomonas-Infected Arabidopsis Reveals Hidden Transcriptome Complexity and Novel Splice Variants

We report the results of a genome-wide analysis of transcription in Arabidopsis thaliana after treatment with Pseudomonas syringae pathovar tomato. Our time course RNA-Seq experiment uses over 500 million read pairs to provide a detailed characterization of the response to infection in both susceptible and resistant hosts. The set of observed differentially expressed genes is consistent with previous studies, confirming and extending existing findings about genes likely to play an important role in the defense response to Pseudomonas syringae. The high coverage of the Arabidopsis transcriptome resulted in the discovery of a surprisingly large number of alternative splicing (AS) events – more than 44% of multi-exon genes showed evidence for novel AS in at least one of the probed conditions. This demonstrates that the Arabidopsis transcriptome annotation is still highly incomplete, and that AS events are more abundant than expected. To further refine our predictions, we identified genes with statistically significant changes in the ratios of alternative isoforms between treatments. This set includes several genes previously known to be alternatively spliced or expressed during the defense response, and it may serve as a pool of candidate genes for regulated alternative splicing with possible biological relevance for the defense response against invasive pathogens