Progress 11/01/23 to 10/31/24
Outputs
Target Audience:The target audience is professional scientists in academia, government agencies, and not-for-profit agricultural organizations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two postdoctoral scholars and one graduate student have received training on biochemistry, transcriptome-wide analyses (RNA-seq and CLIP), and phenotyping on this project. One of the postdoctorates additionally received training on mass spectrometric identification of nucleoside isoforms. How have the results been disseminated to communities of interest?The results were presented by Prof. Sarah Assmann at three conferences, with seminar titles and conference information indicated below: International Plant Resilience Summit, Michigan State University, East Lansing, MI (May, 2024) "Biotechnological and computational approaches toward improved climate resilience in rice" USDA/NIFA Awardee's Meeting, Honolulu, HI (June, 2024) "Cold shock proteins and multi-stress protection in rice: mechanisms and applications" RNA Canada, Ottowa, Canada (October, 2024) "RNA structure-function relationships in plant abiotic stress tolerance" ? What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, we plan to finish and publish the project results. Aim 1. Quantify Stress-Resistant Physiological and Morphological Phenotypes in CspB transgenic rice. Aim 1a. Quantitation of seedling phenotypes under optimal vs. stress conditions This aim has been completed. Aim 1b. Quantitation of reproductive stage phenotypes under optimal vs. stress conditions For the next reporting period we will quantify these phenotypes when abiotic stress is imposed during flowering. Aim 2. Identify the Molecular Basis of Multi-Stress Resistance in CspB transgenic rice Aim 2a. Stress protection through CspB alteration of the global mRNA transcriptome This aim has been completed. Aim 2b. Stress protection through CspB structural chaperoning of specific transcripts We are now applying eCLIP-seq (enhanced UV crosslinking and immunoprecipitation followed by sequencing) to identify the CspB-bound RNA. In this method, we use UV to crosslink RNA and protein followed by immunoprecipitation of the protein-RNA complex. After adding an adaptor to 3' end of RNA, reverse transcription is performed. Afterward, a second adaptor is ligated to the 3' end of the cDNA to facilitate amplification. The cDNA is then PCR amplified and sequenced, allowing for the identification of RNA sequences bound by CspB. To minimize the background, we will use the IgG epitope to preclear the sample before IP. We have done this eCLIP-seq once. We found that CspB could bind mRNA, but the sequencing reads we obtained were insufficient. Now we are optimizing aspects of the method including the UV condition and RNase inhibitor levels. We are also now developing CLAP-seq (doi: 10.1016/j.molcel.2024.01.026) to identify CspB-bound RNA. In this approach, Halo-tagged CspB (Halo-CspB) is expressed in rice, and the tagged protein is then covalently linked to a resin. The RNA-binding complexes formed by the interaction between Halo-CspB and its RNA targets are themselves covalent due to prior in vivo UV crosslinking. Stringent washing conditions are applied to this entirely covalent system to remove any non-specifically bound proteins and RNA, ensuring that only CspB-bound RNA remains in the final complex. This purified RNA can then be analyzed by next-generation sequencing, allowing for the identification of the specific RNA sequences bound by CspB. Additionally, in the next reporting period, we plan to conclude RNA structurome analyses in our transgenic lines. Aim 2c. Stress protection through CspB-based sequestration of RNAs into subcellular compartments. In the next year, our stress granule marker AtUBP1b-mTurq construct will be used to assess whether YFP-tagged CspB, CspB, and the plant Csps relocalize to stress granules following heat shock and cold shock. Aim 2d. Stress protection through CspB protection against RNA oxidation In the next reporting period, we will definitively determine whether CspB and/or CspM preferentially bind to 8-oxo-G using EMSA and related binding assays. We already have an 8-oxo-G-containing transcript for these tests in hand, with a single 8-oxo-G, and will prepare transcripts with multiple G-to-8-oxo-G substitutions either by transcription or by solid-phase synthesis in collaboration with Professor Marino Resendiz at U of Colorado, Denver. We will also apply mass spec approaches to rigorously quantify total 8-oxo-G, and any related isomers, in RNA from our transgenics with and without stress, and will compare these results to the readout of 8-oxoG recorded by G to T transversions. In addition, we will continue to pursue the identity of the unknown second peak which does not co-migrate with the 8-oxoG standard but has the same MS1 and MS2 peaks as 8-oxoG, meaning it is an isomer, analyzing additional plant and in vitro RNA samples and generating authentic standards of the oxidized nucleosides. Aim 3. Identify Functional Similarities and Differences Between Transgenic Lines Overexpressing Bacteria CspB vs. Oryza Csps. Aim 3a. Production and characterization of transgenic rice overexpressing full-length O. sativa Csp1 and Csp2 or truncated O. sativa Csp1 and Csp2 containing only the cold shock domain. Aim 3b. Production and characterization of transgenic rice expressing naturally-truncated wild rice Csp1 and Csp2 variants.? In the next reporting period, we will finalize rice Csp candidate selection and transform Kitaake rice with the selected rice Csps. Given the observations from our RNA-seq data (see previous section), our primary candidates are rice Csp1 and Csp2. The resultant transgenics will be phenotyped for improved abiotic stress tolerance.
Impacts
What was accomplished under these goals?
Aim 1a. Quantitation of seedling phenotypes under optimal vs. stress conditions These experiments were completed in a previous reporting period for our abiotic stressors. Last year we established a collaboration with the lab of Dr. Jan Leach (Colorado State University) to assess the impact of B. subtilis CspB expression on biotic stress resistance. They performed trials assaying two Xanthomans oryzae pv. oryzae strains (PXO99A and PXO86) on the four genotypes (wild-type, empty vector, CspB and Cspm) at normal and high temperatures. In two of the three trials performed, they found no statistically significant differences in the size of pathogen-induced lesions across the different transgenics, indicating that CspB may not involved in regulating resistance to these pathogens. Aim 1b. Quantitation of reproductive stage phenotypes under optimal vs. stress conditions. In the past year, we had intended to assess the effect on yield of stress imposed during the reproductive state itself, but we discovered that we first needed to identify some new transgenic lines, as some of the lines that we had previously utilized were exhibiting transgene suppression. Aim 2a. Stress protection through CspB alteration of the global mRNA transcriptome We analyzed our RNA-seq data (3 biological replicates) from CspB, Cspm, and empty vector transgenic lines after heat and cold stress. In CspB transgenic rice, significant transcriptomic changes were observed compared to the wild type under different temperature conditions. The heatmap of RNA-Seq data revealed distinct clustering patterns for samples and genes across different conditions. Samples grouped tightly by treatment, with clear separation between heat-stressed, cold-stressed, and ambient conditions. Genes are clustered into expression modules, with one cluster showing significant up-regulation in CspB transgenic rice under heat stress, particularly involving genes related to stress tolerance pathways. Conversely, a cluster of up-regulated genes under cold stress included genes associated with RNA metabolic processing. These patterns suggest a strong transcriptional response to heat stress and cold stress mediated by CspB. Another interesting finding is that among the five OsCsps identified in rice, only OsCsp1 and OsCsp2 are expressed genes. Aim 2b. Stress protection through CspB structural chaperoning of specific transcripts We previously utilized a standard RNA immunoprecipitation (RIP-seq) methodology with a UV cross-linking step to enforce specific protein-to-RNA covalent bonding affinity for the identification of CspB interacting RNA partners. With this method, transcripts of ROS- and stress-responsive genes, spliceosomal RNAs, transcription factors, and RNAs encoding chaperone proteins were found to be associated with both CspB and Cspm complexes post-stress. However, further data analysis identified high background in the empty vector lines; most of the background is ribosomal RNA or spliceosome RNA, both of which are abundant in cells. We are currently pursuing improved RIP-seq methods (eCLIP) as well as a new method, termed CLAP-seq, to more rigorously identify the transcripts that selectively interact with CspB and Cspm. We also pursued experiments to assess how CspB and Cspm globally alter the RNA "structurome". We have conducted a complete pilot of the method and have verified that the entire experimental pipeline is working correctly. Aim 2c. Stress protection through CspB-based sequestration of RNAs into subcellular compartments. We found that CspB-YFP and Cspm-YFP transiently expressed in Nicotiana benthamiana are observed in both the nucleus and the cytoplasm. Additionally, we found that YFP-tagged O. sativa OsCsp1, OsCsp2, or their respective truncated versions showlocalization of each protein in both the nucleus and the cytoplasm. The removal of the glycine-rich and zinc finger domains from the truncated proteins does not alter their subcellular localization. The stress granule marker, oligouridylate-binding protein 1b (AtUBP1b, At1g17370), has been cloned and fused with the fluorescence marker mTurquoise. The plasmid construction has been completed, and the gene sequence has been confirmed. The cloned vector has been successfully transformed into Agrobacterium tumefaciens strain C58 for agroinfiltration into Nicotiana benthamiana. Aim 2d. Stress protection through CspB protection against RNA oxidation (1) We have shown that CspB can bind RNA using an electromobility shift assay (EMSA). (2) We have pursued mass spec quantification of 8-oxoG in plant RNA by comparison to in vitro transcripts with and without 8-oxoG introduced during T7 transcription via 8-oxoGTP pairing to dA. We observed a significant peak corresponding to 8-oxo-G in the plant RNA and in the +8-oxoG in vitro transcript in LC-MS (as expected), as determined by comparison to an authentic 8-oxoG nucleoside standard. This peak was much smaller in the LC-MS of the -8-oxoG in vitro transcript, although not entirely gone suggesting possible oxidation this transcript, which is G-rich. In only the plant sample, we also saw a second peak, which does not co-migrate with the 8-oxoG standard on HPLC but has the same MS1 and MS2 peaks as 8-oxoG, meaning it is an isomer and likely has the same base MW and sugar MW as 8-oxoG. (3) Considering that 8-oxoG can assume the syn conformation and then base pair with A, we analyzed the G-to-T transversion as a means to quantify the content of 8-oxoG in transcriptomes. By examining our previously published RNA sequencing data from wild-type rice under heat stress (doi: 10.1073/pnas.1807988115), we observed an increase in the amount of 8-oxoG compared to ambient condition, as indicated by the quantification of G to T transversions (p-value of 0.0038, two-way ANOVA). However, we did not see a similar increase in 8-oxoG in wild type under heat stress compared to ambient environment while analyzing our newtranscriptome data from Aim 2A. There is also no difference in the amount of 8-oxoG between CspB-ambient and CspB-heat-stressed samples. It will be important to assess the reliability of these G-to-T transversions as a readout of 8-oxoG through comparison with quantification of 8-oxoG in these samples by mass spectrometric analysis. Aim 3a. Production and characterization of transgenic rice overexpressing full-length O. sativa Csp1 and Csp2 or truncated O. sativa Csp1 and Csp2 containing only the cold shock domain. Aim 3b. Production and characterization of transgenic rice expressing naturally-truncated wild rice Csp1 and Csp2 variants. We utilize a hairpin nucleic acid molecular beacon tagged with a quencher and a fluorophorethatemits fluorescence when the structure melts out. In the past year, we have discovered variations in quality between fluorescent beacon probes with different quencher molecules (Black Hole Quencher vs. Iowa Black FQ). The Iowa Black FQ produces a large fluorescent signal following a nuclease treatment, as expected, whereas the Black Hole Quencher does not; this indicates that the Black Hole Quencher may not be a valid reporter of nucleic acid refolding for these assays. In the past year, we further optimized the His-tagged protein purification protocol for recombinant Csps. We observed that O. sativa Csp1 and Csp2 show little melting activity at our lowest protein concentration (70 pmol), but their melting activities increase markedly when the protein concentration is higher (210 pmol). This may suggest that cooperative binding promotes activity. With regard to transgenic rice production, five genes (O. sativa Csp1 and Csp2, truncated O. sativa Csp1 (OsT1) and Csp2 (OsT2) (lacking zinc-finger and glycine-rich domains), and an mVenus empty vector control) were cloned into the same rice transformation vector as was used to create stable transgenic CspB/Cspm rice lines.
Publications
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Progress 11/01/22 to 10/31/23
Outputs
Target Audience:The target audience is professional scientists in academia, government agencies, and not-for-profit agricultural organizations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two postdoctoral scholars and one graduate student have received training on biochemistry, transcriptome-wide analyses, and phenotyping on this project. In addition, one rotating graduate student learned cloning, transient expression, and confocal imaging using a Csp construct. How have the results been disseminated to communities of interest?The results were discussed by Prof. Sarah Assmann at a one-day conference on Structured Nucleic Acids held at Penn State in April, 2023. Post-doctorate Dr. Hong-Li Chou presented results at an internal RNA mini-symposium at Penn State in December, 2023. What do you plan to do during the next reporting period to accomplish the goals? Aim 1. Quantify Stress-Resistant Physiological and Morphological Phenotypes in CspB transgenic rice. Aim 1a. Quantitation of seedling phenotypes under optimal vs. stress conditions This aim has been completed for our four abiotic stressors. We have established a new collaboration with the lab of Dr. Jan Leach (Colorado State University) to assess the impact of B. subtilis CspB expression on biotic stress resistance, and will have initial data from those experiments in the next year. Aim 1b. Quantitation of reproductive stage phenotypes under optimal vs. stress conditions We have completed these experiments for our four abiotic stresses imposed at the seedling stage. For the next reporting period we will quantify these phenotypes when abiotic stress is imposed during flowering. Aim 2. Identify the Molecular Basis of Multi-Stress Resistance in CspB transgenic rice Aim 2a. Stress protection through CspB alteration of the global mRNA transcriptome For the next reporting period, we will finish our analyses of the RNA-seq data from the heat- and cold-stressed plants. Aim 2b. Stress protection through CspB structural chaperoning of specific transcripts The RNA-seq experiments to determine the RNA binding partners of CspB and Cspm have been completed. In the next year, we plan to perform Structure-seq to assess how the mRNA structurome differs in wild-type vs. CspB and Cspm transgenics, and in mRNAs that CspB/Cspm bind vs. mRNAs that CspB/Cspm do not bind. Aim 2c. Stress protection through CspB-based sequestration of RNAs into subcellular compartments. Based on both cellular fractionation and on the identity of their protein partners, both CspB and Cspm localize to both the nucleus and the cytoplasm. In the next year, in an orthogonal approach, we will visualize the subcellular localization of CspB-YFP and Cspm-YFP transiently expressed in Nicotiana benthamiana. We will also use this methodology to determine whether acute heat shock or cold stress induce CspB or Cspm re-localization into stress granules. Aim 2d. Stress protection through CspB protection against RNA oxidation In the next year, we will use EMSA to determine whether CspB and/or CspM preferentially bind 8-oxo-guanine. We will quantify total 8-oxo-G in our transgenics with and without stress by mass spec approaches. We will bioinformatically analyze our RNA-seq data for signature mutations in cDNA known to be introduced opposite 8-oxo-G by reverse transcriptase. Aim 3. Identify Functional Similarities and Differences Between Transgenic Lines Overexpressing Bacteria CspB vs. Oryza Csps. Aim 3a. Production and characterization of transgenic rice overexpressing full-length O. sativa Csp1 and Csp2 or truncated O. sativa Csp1 and Csp2 containing only the cold shock domain. Aim 3b. Production and characterization of transgenic rice expressing naturally-truncated wild rice Csp1 and Csp2 variants. In the next year, we will finish the melting assays on all Csps. Plant candidates that are most active in these assays (currently O. barthii Csp and OsT2) will be over-expressed in Kitaake rice, along with the full-length O. sativa Csp1 and Csp2 for comparison, and the plants will be phenotyped for improved abiotic stress tolerance.
Impacts
What was accomplished under these goals?
Aim 1. Quantify Stress-Resistant Physiological and Morphological Phenotypes in CspB transgenic rice. Aim 1a. Quantitation of seedling phenotypes under optimal vs. stress conditions Twelve-day-old rice seedlings of wildtype (WT), and transgenic plants with the genetic transformation events of empty vector (EV), functional bacterial (Bacillus subtilis) cold shock protein B (CspB) and the cold shock protein B mutant (Cspm), which contains a point mutation of F30R that eliminates the RNA unfolding activity, were subjected to abiotic stress treatments (heat, cold, salinity, and drought) respectively for phenotype quantification. Observations from 15 biological replicates per genotype indicate enhanced stress tolerance in CspB transgenic plants, while Cspm transgenic plants showed hypersensitivity to stress. Aim 1b. Quantitation of reproductive stage phenotypes under optimal vs. stress conditions. We quantified 1000-grain weight and panicle number to assess rice yield across genotypes (WT, EV, CspB, and Cspm), with 15 biological replicates for each rice genotype. While these phenotypes did not differ under normal growth conditions, a significant increase in panicle numbers (~12%) was noted in CspB riceafter a 2-day heat stress (38°C-6hr) or a 3-day cold stress (10°C-constant) treatment of twelve-day-old rice seedlings. No significant differences were observed among 1000-grain weight comparisons in these four genotypes post-temperature stress treatments. Aim 2. Identify the Molecular Basis of Multi-Stress Resistance in CspB transgenic rice Aim 2a. Stress protection through CspB alteration of the global mRNA transcriptome To assess the role of CspB in transcriptome-wide stress protection, we conducted an mRNA transcriptome study and gene ontology (GO) enrichment analysis across the four genotypes under optimal growth conditions. In the class of upregulated RNA transcripts (log2Fold change >= 1), GO enrichment analysis indicates a significant apoptotic process in the Cspm plants' exclusive transcripts while a significant GO class of oxygen reductase activities was found enriched in CspB plants' exclusive transcripts. Given that reactive oxygen species (ROS) are commonly overproduced in abiotic stress stimuli, and that supra-optimal ROS concentration can cause cell damage, our preliminary findings suggest a possible ROS scavenging pathway activated in CspB transgenics for multi-stress protection, while the enriched apoptotic processes in Cspm plants might explain its stress hypersensitivity. In the class of downregulated RNA transcripts (log2Fold change <= -1), a significant GO enrichment of DNA binding was found in both CspB and Cspm plants, indicating possible transcriptional regulation events in both of these transgenic plants, while the exclusively downregulated GOs in Cspm (electron carrier activity, ROS related) and CspB (apoptotic processes) plants consistently suggesting increased ROS-stress in Cspm plants and decreased apoptotic activities in Csp plants, consistent and opposite to the trends seen in upregulated RNA transcripts in these genotypes. We have also acquired RNA-seq data (3 biological replicates) from these genotypes after heat and cold stress, and are currently analyzing these data. Aim 2b. Stress protection through CspB structural chaperoning of specific transcripts To assess CspB structural chaperoning transcripts, we enhanced a standard RNA immunoprecipitation (RIP-seq) methodology by including a UV cross-linking step to enforce specific protein-to-RNA covalent bonding affinity for the identification of CspB interacting RNA partners. With this improved method, transcripts of ROS- and stress-responsive genes, spliceosomal RNAs, transcription factors, and RNAs encoding chaperone proteins were found to be associated with both CspB and Cspm complexes post-stress. Aim 2c. Stress protection through CspB-based sequestration of RNAs into subcellular compartments. To assess the subcellular location of FLAG epitope fused CspB/Cspm, a column gradient separation method was adopted to isolate the nucleic and cytoplasmic fractions from these 4 genotypes. Proteins then were purified from each subcellular fraction and subjected immunoblotting to confirm Csp/Cspm-FLAG localization by FLAG monoclonal antibody. Anti-histone-H3 and anti-Rubisco were adopted as well as subcellular specific protein markers for the indication of nucleic and cytoplasmic fractions respectively. Interestingly, we found that both CspB/Cspm were localized in both the nucleoplasm and cytoplasm. To provide insights into the protein network of CspB for its roles in stress protection, we conducted Immunoprecipitation-Mass Spectrometry (IP-MS) study to identify the interacting protein partners with biological replicates. In parallel to the RIP-Seq study, the growth stage of 12-day-old rice seedlings of the 4 genotypes was also used in the IP-MS study. These plants were then subjected to cold (10oC for 3 days) or heat (38oC for 6hr for 2 days) stress treatments, followed by FLAG-tagged CspB/Cspm complex isolation immediately after the end of the stresses. After heat stress treatment, several stress tolerance and ROS-responsive proteins were exclusively identified in CspB plants while several nucleic acid binding proteins were exclusively identified in the Cspm IP-MS. This finding suggests that CspB and Cspm might be involved in different protein regulatory networks in response to stress stimuli. Interestingly, this phenomenon is enhanced under cold stress, where we obtained almost twice the number of protein candidates identified under cold relative to those under heat stress conditions. Aim 2d. Stress protection through CspB protection against RNA oxidation We observed decreased RNA oxidation in our CspB transgenics based on a competitive ELISA assay for 8-oxo-guanine. In the past year, we have developed methodology for incorporation of 8-oxo-guanine into in vitro transcribed transcripts. This will allow us to perform electrophoretic mobility shift assays (EMSA) to determine whether CspB and/or Cspm preferentially bind 8-oxo-guanine. We have also worked on methodology to quantify 8-oxo-G by LC-MS/MS. Aim 3. Identify Functional Similarities and Differences Between Transgenic Lines Overexpressing Bacteria CspB vs. Oryza Csps. Aim 3a. Production and characterization of transgenic rice overexpressing full-length O. sativa Csp1 and Csp2 or truncated O. sativa Csp1 and Csp2 containing only the cold shock domain. Aim 3b. Production and characterization of transgenic rice expressing naturally-truncated wild rice Csp1 and Csp2 variants. Five genes (O. sativa Csp1 and Csp2, truncated O. sativa Csp1 (OsT1) and Csp2 (OsT2), and mVenus control) have been cloned into the rice transformation vector used for CspB/Cspm lines. Analysis of predicted proteins from genome sequence has identified three other O. sativa Csps: Os01g0546250 (OsCsp3), Os03g0277800 (OsCsp4), and Os05g0524666 (OsCsp5). In addition, we have cloned a Csp from the rice wild relative, O. barthii (OBART05G07870). Recombinant O. sativa Csp1, Csp2, T1, T2, Csp3 and OBART05G07870 have been successfully expressed to date. In preliminary molecular beacon assays of nucleic acid melting, OsCsp3 had only minor melting activity. Bacillus subtilis CspB was the most effective, but O. barthii Csp was also highly active in the melting assays. OsT1 and OsT2 were more active than full-length O. sativa Csp1 or O. sativa Csp2, respectively. These results are particularly exciting because O. barthii Csp and the truncated proteins OsT1 and OsT2 are similar to B. subtilis CspB in lacking the glycine-rich zinc finger domain characteristic of plant Csps. These results therefore offer a possible way forward to improve rice stress tolerance by non-transgenic approaches based on introgression of O. barthii Csp or CRISPR editing to truncate O. sativa Csp1 or Csp2.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Assmann SM, Chou HL, Bevilacqua PC. Rock, scissors, paper: How RNA structure informs function. Plant Cell. 2023 May 29;35(6):1671-1707. doi: 10.1093/plcell/koad026. PMID: 36747354; PMCID: PMC10226581.
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Progress 11/01/21 to 10/31/22
Outputs
Target Audience:The target audience is professional scientists in academia, government agencies, and not-for-profit agricultural organizations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral scholar and one graduate student have received scientific training on this project.Postdoctorate Hong-Li Chou received training in professional development and networking through presenting a posteron his results at two international meetings in 2022 (see next section). How have the results been disseminated to communities of interest?Our research findings were presented by post-doctorate Dr. Hong-Li Chou as a poster at two conferences: 1) the 2022 Gordon Research Conference on Post-Transcriptional Gene Regulation; 2) the 23rd Penn State Plant Biology Symposium: RNA Biology. Our research findings were also presented by PI Sarah M. Assmann in an invited talk at the 23rd Penn State Plant Biology Symposium: RNA Biology, and by PI Sarah M. Assmann in an invited plenary talk for the President's Symposium at the annual meeting of the American Society of Plant Biologists in Portland, OR in July, 2022. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Quantify Stress-Resistant Physiological and Morphological Phenotypes in CspB transgenic rice. Aim 1a. Quantitation of seedling phenotypes under optimal vs. stress conditions This aim has essentially been completed for heat and cold stress. For salinity and drought stress, we will repeat the experiments in order to obtain quantitative data. Aim 1b. Quantitation of reproductive stage phenotypes under optimal vs. stress conditions We will obtain measurements of yield in our CspB transgenics vs wild-type under optimal vs. stress conditions. Aim 2. Identify the Molecular Basis of Multi-Stress Resistance in CspB transgenic rice Aim 2a. Stress protection through CspB alteration of the global mRNA transcriptome This aim has been completed, although we may obtain transcriptomic data under additional stresses. Aim 2b. Stress protection through CspB structural chaperoning of specific transcripts In the next reporting period, we will improve our RIP-seq methodology by including a UV cross-linking step, and will obtain replicate datasets of the RNA partners of CspB and mCspB in rice. Aim 2c. Stress protection through CspB-based sequestration of RNAs into subcellular compartments. In the next reporting period, we will obtain more replicates of our CspB immunoprecipitates to definitively identify interacting protein partners of CspB. Aim 3. Identify Functional Similarities and Differences Between Transgenic Lines Overexpressing Bacteria CspB vs. Oryza Csps. Aim 3a. Production and characterization of transgenic rice overexpressing full-length O. sativa Csp1 and Csp2 or truncated O. sativa Csp1 and Csp2 containing only the cold shock domain. In the next reporting period, all CspB genes will be cloned into vectors for expression of the recombinant protein and the CspBs' RNA unfolding activity assayed. CspB genes encoding proteins with efficacious RNA unfolding activity will be transformed into rice. Aim 3b. Production and characterization of transgenic rice expressing naturally-truncated wild rice Csp1 and Csp2 variants. In the next reporting period, we will finish the cloning of wild rice CspB genes and will assay the RNA unfolding activity of the resultant recombinant proteins. CspB genes encoding proteins with efficacious RNA unfolding activity will be transformed into Oryza sativa (cultivated rice).
Impacts
What was accomplished under these goals?
Aim 1. Quantify Stress-Resistant Physiological and Morphological Phenotypes in CspB transgenic rice. Aim 1a. Quantitation of seedling phenotypes under optimal vs. stress conditions Twelve day-old rice seedlings of wildtype (WT), and transgenic plants with the genetic transformation events of empty vector (EV), functional bacterial (Bacillus subtilis) cold shock protein B (CspB) and the cold shock protein B mutant (Cspm, which contains a point mutation of F30R that eliminates the RNA unfolding activity) were subjected to abiotic stress treatments (heat, cold, salinity, and drought) respectively for phenotype quantifications. For temperature stress assays, either a 2-day heat stress (38oC-6hr) or a 3-day cold stress (10oC- constant) was conducted, followed by a 2-week recovery period for plant height measurement. CspB transgenic plants consistently displayed vigorous plant growth where the plant height was significantly taller than the wild-type plants after heat or cold stress. Regarding drought tolerance, we performed a stress treatment by terminating watering plants for one week and then measuring plant height and root length immediately after the end of the stress. Observations suggest improved drought stress tolerance in CspB transgenic plants and hyper-sensitivity to drought in Cspm transgenic plants. We performed salinity stress by hydroponically growing seedlings of these rice genotypes (WT, EV, CspB, and Cspm) for 12-days, followed by one day of 200 mM NaCl treatment. CspB transgenic plants displayed less leaf browning immediately after this salinity stress. Aim 2. Identify the Molecular Basis of Multi-Stress Resistance in CspB transgenic rice Aim 2a. Stress protection through CspB alteration of the global mRNA transcriptome To assess the role of CspB in transcriptome-wide stress protection, we conducted an mRNA transcriptome study and gene ontology (GO) enrichment analysis across the four genotypes under ambient growth conditions. 120 million reads were collected by the NextSeq 150 nt Mid output single-end read sequencing method on a total of 12 samples among these 4 genotypes with 3 biological replicates per each genotype. The transcript per million (TPM) abundance was normalized by the dataset of empty vector transgenic plants and compared to the wild-type plants by analyzing the representative log2 fold changes. These data were then processed to Venn diagrams and GO enrichment analyses. In the class of upregulated RNA transcripts (log2Fold change >= 1), GO enrichment analysis indicates a significant apoptotic process in the Cspm plants-exclusive transcripts while a significant GO class of oxygen reductase activities was found enriched in CspB plants-exclusive transcripts. Given that reactive oxygen species (ROS) are commonly overproduced in abiotic stress stimuli, and that supra-optimal ROS concentration can cause cell damage, our preliminary findings suggest a possible ROS scavenging pathway activated in CspB transgenics for multi-stress protection, while the enriched apoptotic processes in Cspm plants might explain its stress hypersensitivity. In the class of downregulated RNA transcripts (log2Fold change <= -1), a significant GO enrichment of DNA binding was found in both CspB and Cspm plants, indicating possible transcriptional regulation events in both of these transgenic plants, while the exclusively downregulated GOs in Cspm (electron carrier activity, ROS related) and CspB (apoptotic processes) plants consistently suggesting increased ROS-stress in Cspm plants and decreased apoptotic activities in Csp plants, consistent and opposite to the trends seen in upregulated RNA transcripts in these genotypes. Aim 2b. Stress protection through CspB structural chaperoning of specific transcripts To assess CspB structural chaperoning transcripts, we first conducted RNA immunoprecipitation followed by regular RNA sequencing (RIP-Seq) for the identification of CspB interacting RNA partners. Spliceosomal RNAs and RNAs encoding chaperone proteins were found to be associated with both CspB and Cspm complexes post-stress. Aim 2c. Stress protection through CspB-based sequestration of RNAs into subcellular compartments. To assess the subcellular location of FLAG epitope fused CspB/Cspm, a column gradient separation method was adopted to isolate the nucleic and cytoplasmic fractions from these 4 genotypes. Proteins then were purified from each subcellular fraction and went through immunoblotting to confirm Csp/Cspm-FLAG localization by FLAG monoclonal antibody. Anti-histone-H3 and anti-Rubisco were adopted as well as subcellular specific protein markers for the indication of nucleic and cytoplasmic fractions respectively. Interestingly, we found that both CspB/Cspm were localized in both the nucleoplasm and cytoplasm. To provide insights into the protein network of CspB for its roles in stress protection, we conducted Immunoprecipitation-Mass Spectrometry (IP-MS) study to identify the interacting protein partners. In parallel to the RIP-Seq study, the growth stage of 12-day-old rice seedlings of the 4 genotypes was also used in the IP-MS study. These plants were then subjected to cold (10oC for 3 days) or heat (38oC for 6hr) stress treatments, followed by Csp-containing RBP (RNA binding protein) complex isolation immediately after the end of the stresses. After heat stress treatment, several stress tolerance and ROS-responsive proteins were exclusively identified in CspB plants while several nucleic acid binding proteins were exclusively identified in the Cspm IP-MS. This finding suggests that CspB and Cspm might be involved in different protein regulatory networks in response to stress stimuli. Interestingly, this phenomenon is enhanced under cold stress stimuli where we obtained almost twice the number of protein candidates identified under cold relative to those under heat stress conditions. Aim 2d. Stress protection through CspB protection against RNA oxidation We have observed decreased RNA oxidation in our CspB transgenics based on a competitive ELISA assay for 8-oxo-guanine. Aim 3. Identify Functional Similarities and Differences Between Transgenic Lines Overexpressing Bacteria CspB vs. Oryza Csps. Aim 3a. Production and characterization of transgenic rice overexpressing full-length O. sativa Csp1 and Csp2 or truncated O. sativa Csp1 and Csp2 containing only the cold shock domain. Five genes (O. sativa Csp1 and Csp2, truncated O. sativa Csp1 and Csp2, and mVenus control) have been cloned into the same vector used for CspB/mCspB lines. Analysis of predicted proteins from genomic sequences have identified three other Csps belonging to O. sativa (Os01g0546250, Os03g0277800, and Os05g0524666) which have been successfully cloned into a holding vector. In order to narrow down transformation candidates, all proteins will be cloned into recombinant protein vectors to measure melting activity (correlated to CspB lines' multi-stress tolerant phenotypes). O. sativa Csp1 and Csp2 as well as their truncated counterparts have been cloned the recombinant protein vectors thus far. Aim 3b. Production and characterization of transgenic rice expressing naturally-truncated wild rice Csp1 and Csp2 variants. Initial sequence alignments found divergent sequences in O. rufipogon (ORUFI08G02050 and ORUFI03G13150), O. barthii (OBART05G07870) and O. brachyantha (OB02G23180 and OB08G12040) in comparison to O. sativa Csps. However, cloning and sequencing of ORUFI08G02050 and ORUFI03G13150 revealed misannotations in the genomic sequence as these genes had high nucleotide and protein similarity to O. sativa Csps. Cloning of OBART05G07870 revealed five nucleotide discrepancies between the annotation and two independent amplifications, but the gene remains a divergent sequence nonetheless.
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