Progress 09/30/22 to 09/29/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:With the delay in release of funds and also changes in post docs working on this project, we will be extending the work for another four months to accomplish remaining goals of the project and write publications based on these results. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Over the next few months, we will finalizing the results and writing publications. We anticipate at least two publications that will be drafted for submission in peer-reviewed journals.
Impacts
What was accomplished under these goals?
?DNA-based real-time quantitative PCR (Polymerase Chain Reaction) assay for the quantification of A. japonica growth and the assessment of resistance/susceptibility of pennycress germplasm lines Over time, many techniques including the counting of infection site numbers per leaf area, image-scanning methodologies, molecular biological techniques such as RNA- or DNA-based quantitative real-time polymerase chain reaction (qRT-PCR) assay, development of transgenic pathogens expressing reporter genes and enzyme-linked immunosorbent assays have been developed for the quantification of the fungal growth progression in planta. Of these, the RNA- or DNA-based quantitative PCR method has been proven as the most feasible rapid sensitive economical approach. A DNA-based qRT-PCR assay was developed for the quantification of A. japonica growth on pennycress plant and to assess the resistance/susceptibility of the two pennycress germplasm lines (MN106 and 2032) in response to A. japonica infection. To initiate with, the qRT-PCR forward and reverse primer pairs were designed, and the assay was optimized for the efficient amplification of the target sequences from the target genes, respectively (Table 1). Elaborately, a primer-pair was designed from the ITS1 (internal transcribed sequence1) region of the 5.8S rRNA gene of A. japonica whereas the two primer-pairs were from pennycress plant for amplifying the intron-exon sequence region of the respective two housekeeping genes (TaActin and TaUbiquitin) as a proxy for measuring the biomass of pathogen and plant, respectively. Further, given that a reliable PCR assay requires the robust sensitive primers that can efficiently amplify the specific target nucleotide sequences, a PCR-based gel assay using the genomic DNA (gDNA) from mock and infected pods and 8th leaves at 3dpi as well as the A. japonica field isolate (as positive control) of pennycress was initially performed to validate the amplification potential of the designed primers. No template samples were used as the negative control. The results obtained were as expected. The ITS-based primers from A. japonica 5.8S rRNA gene did not amplify in the mock and no template samples compared to the infected and A. japonica field isolate samples whereas, the TaActin and TaUbiquitin gene primers amplified in all the samples except the positive (showed non-specific amplification) and negative controls (Figure 2a). To determine the efficiency of these primer-pairs, a 10-fold dilution series of gDNA from A. japonica, uninfected pennycress plant and A. japonica-infected pennycress plant were used as template for qRT-PCR assay. All the primer sets exhibited and yielded an efficient linear amplification over a 1000-fold range of template inputs (0.05 ng/µl to 50ng/µl) for all the DNA samples with a significant correlation coefficient (R2) ranging from >0.95 to >0.99. (Figure 2c-e). The percentage qPCR efficiency of all the primers for different gDNA templates are given in Table 1. Additionally, to assess the primer specificity, the dissociation curve analysis was performed on the amplicons from all the tested DNA samples. As expected, all the primers AjITS, TaUbiquitin and TaActin produced single well-defined peaks distinctively at Tm = 81.00°C, 75.50°C and 77.50°C from the gDNA of A. japonica, uninfected pennycress plant, A. japonica-infected pennycress plant. The PCR-based gel assay also confirms that these primers provide single bands of expected size from infected and positive control samples indicating that primer sets can specifically amplify the target regions from target genes (Figure 2a). Overall, these suggest that all the primers are suitable for quantification of the target genes even at low concentrations of input template DNA. Development of detached leaf and pod infection assays for visual screening of S. sclerotiorum susceptibility Similar to the detached leaf and pod assays described above for A. japonica, S. sclerotiorum inoculations were developed using leaves from the vegetative stage (4.5 to 5 week-old non-vernalized plants). A pennycress isolate of S. sclerotiorum was grown on potato dextrose agar (PDA) plates. Three days after transfer to fresh PDA plates, 2mm agar plugs of S. sclerotiorum mycelia were placed onto the 8th, 9th, and 10th rosette leaves. Three days post inoculation (dpi), the necrotic lesions from S. sclerotiorium infection are visibly larger on 2032 leaves than MN106 leaves (Figure 4a). These lesions are easily quantifiable from pictures using FIJI software. The area of the lesions at 3dpi are significantly larger on 2032 leaves than MN106 leaves (Figure 4b). We also developed assays to screen for S. sclerotiorum resistance in the reproductive stage. For S. sclerotiorum, pods from vernalized plants were detached and spray-inoculated with fragmented mycelia (OD600 = 0.5). At this concentration, not every pod develops necrotic symptoms. Across multiple experiments, 33% of 2032 pods develop necrosis from S. sclerotiorum infection, compared to 13% of MN106 pods (Figure 4c). To screen for S. sclerotiorum resistance in the stems, agar plugs were placed onto cut stems. Although the necrotic lesions spread at an equal rate down the stem, more sclerotia (fungal resting structures) developed on 2032 stems than MN106 stems (Figure 4d). Taken together, these results indicate that in a variety of tissues and developmental stages, MN106 plants are more resistant to S. sclerotiorum than 2032 plants, as we observed with A. japonica. ?A multitude of genes are differentially regulated in MN106 compared to 2032 leaves infected with S. sclerotiorum We performed an RNAseq experiment to identify genes that are induced during early S. sclerotiorum infection. One purpose, described in the Phase II proposal, was to identify promoters that we could use to inducibly drive expression of antifungal peptides upon infection. We selected 3 and 6hpi as the time points for this experiment, as these occur prior to the development of necrotic symptoms which we first observe at 12 hpi. Another purpose of this experiment was to identify the key differences in gene expression between infected MN106 and 2032 plants, differences that might reveal what genes are driving the difference in immunity between these two varieties. We inoculated the 8th leaf of 5 week-old plants with 6mm agar disks of S. sclerotiorum grown on PDA plates. Control samples were inoculated with sterile PDA agar disks. After 3 and 6hr, the leaf tissue under and around the agar disks (1cm in diameter) was collected, and total RNA was isolated. mRNA enrichment, library preparation, and Illumina sequencing (151nt paired-end reads) was performed by Novogene. Differential gene expression analysis was performed in R using the DESeq2 package, using cutoffs of log2(fold change) >1 and p-adjusted values < 0.05. Using these cutoffs, 6hr of infection in MN106 leaves leads to the upregulation of 1814 genes and downregulation of 399 genes, as compared to expression in mock controls. Similarly, there are 2180 upregulated and 616 downregulated genes in 2032 leaves after 6hr of infection. KEGG pathway enrichment analysis reveals that many of the genes induced by infection in both genotypes are involved in secondary metabolite biosynthesis (Figure 6a). This KEGG pathway includes genes involved in phenylpropanoid and glucosinolate biosynthesis, compounds that contribute to S. sclerotiorum resistance (Zhang et al. 2015; Ranjan et al. 2019). Fewer genes are differentially expressed after 3hr of infection; there are only 12 upregulated and 33 downregulated genes in 2032 at 3hpi, and no differentially expressed genes (DEGs) in MN106 at 3hpi.
Publications
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Progress 09/30/21 to 09/29/23
Outputs
Target Audience:During the project, researchers from academia and industry organizations interested in pennycress and CoverCress domestication learnt about the progress of this research project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We were able to successful train two post-doctrates and one of them has joined CoverCress Inc. to pursue her professional career. How have the results been disseminated to communities of interest?We are currently preparing two manuscripts using the data obtained during the course of this grant funding. The first is a methods paper, describing the development of the assays used to screen for fungal resistance in pennycress. We anticipate submitting this paper to plant methods journal. The second paper, which we plan to submit to Plant Biotechnology, will describe the effects of the natural mutation upstream of the JMJ14 gene in 2032 plants on disease resistance and flowering time. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Aim1a. Recombinant expression and purification of antifungal peptides OeDef1, NCR044_v2, PDF2.2 and PDF4.3. We were successful in isolating and purifying some of these peptides. Aim 1b.Determinein vitroantifungal activity of OeDef1, NCR044_v2, PDF2.2 and PDF4.3 againstAlternaria japonicaandRhizoctonia solani. We found antifungal activity of PDF2.2 and NCR13 peptides. These peptides were of focus in the aim 2. Aim 2a. Constitutive gene expression in pennycress. A variety of transgenic pennycress lines were created in Phase I and in early Phase II, expressing PDF2.2, PDF4.3, NCR044_v2, and OeDef1 antifungal peptides under the control of the constitutive 35S, epidermis-specific A14, or pathogen-inducible PDF1.2 promoters. Unfortunately for all of these lines, expression of the transgene could never be reliably detected in Western blots. In October of 2023 we decided to design a new construct, this time with the expression and selection cassettes in opposite orientations with the expression cassette nearest to the left border. To demonstrate proof-of-concept for transgenic expression of antifungal peptides to control fungal diseases in pennycress, we decided to focus our efforts on constitutive overexpression of two antifungal peptides using the 35S promoter. Aim 2b. Pathogen-responsive gene expression in pennycress. NCR13 and PDF2.2 genes were cloned into the 35S promoter overexpression vector described above. Two NCR13 constructs were created, one with the genomic NCR13 (NCR13g) sequence, and one with the signal peptide of MtDef5.1 fused to the NCR13 peptide sequence (chimeric NCR13). These constructs were transformed into ARV1 plants (tt8 mutant that produces yellow, low fiber seeds) via Agrobacterium-mediated floral dip. T1 seeds were selected by DsRed fluorescence, and the presence of the transgene in T1 plants was confirmed by PCR. Seven 35S:chimeric NCR13 and two 35S:NCR13g T1 lines were isolated. All of these had detectable NCR13 transcript expression in leaves via RT-PCR, at varying levels of expression. Surprisingly, NCR13 protein could not be detected in any of these lines via Western blotting. This suggests that although RNA silencing of NCR13 is not occurring in these lines, the NCR13 protein is unstable in pennycress. Likely because of a lack of NCR13 expression, in detached leaf inoculations with S. sclerotiorum, the 35S:NCR13 T1 lines have equivalent susceptibility to ARV1 parental plants. Fifteen 35S:PDF2.2 T1 lines were isolated; 12 had PDF2.2 transcript expression higher than parental ARV1 plants. We do not yet have an antibody raised against PDF2.2 to check for protein expression. Leaves of T1 plants with PDF2.2 transcript overexpression are not more resistant to S. sclerotiorum infection than ARV1 plants, which suggests that overexpression of this antifungal peptide will not be a useful control strategy for engineering resistance to S. sclerotiorum in the vegetative stage. We tested the resistance of pods from the same 35S:PDF2.2 and 35S:NCR13 T1 plants to S. sclerotiorum. In the S. sclerotiorum spray assay, described above, not every pod develops necrotic symptoms. An average of 30.3 ± 13.8 % (SD) of parental ARV1 pods showed necrosis. One 35S:NCR13 T1 line (# 3), had only 11% of its pods develop symptoms. When 35S:PDF2.2 T1 pods were screened, 90.1 ± 8.4 % (SD) of parental ARV1 pods developed symptoms. 6 out of 13 35S:PDF2.2 T1 lines screened had fewer pods infected than the ARV1 mean - 1 SD suggesting that their pods are more resistant to S. sclerotiorum. However, these experiments were limited to testing individual T1 plants. Aim 2c. Epidermis-specific gene expression in pennycress. No work was performed as the results in the previous two sub-aims did not produce the level of resistance seen in in-vivo studies. Future work is required to address this topic. Aim 3. Identification of pathogen-responsive promoters of pennycress DNA-based real-time PCR analysis for the quantification of A. japonica and S. sclerotiorum growth after infection on pennycress lines and their resistance/susceptibility assessment have been established. ?Transcriptome sequencing of fungus-infected MN106 and 2032 leaves reveals genes that may contribute to the difference in susceptibility between varieties We infected leaves of MN106 and 2032 plants with agar disks containing S. sclerotiorum for 3 and 6 hr or A. japonica for 12 hr. These time points were chosen because they occur before the onset of necrotic symptoms, and therefore capture gene expression changes early in the infection process. Total RNA was isolated from these leaves and from control leaves (treated with sterile agar disks for the same time points) and sent for Illumina mRNA sequencing at Novogene. Differential gene expression analysis was performed in R using the DESeq2 package, using cutoffs of log2(fold change) >1 and p-adjusted values <0.05. Fungal infection led to more differential gene expression in pennycress the longer the infections proceeded. At 3 hr of S. sclerotiorum infection, there were no differentially expressed genes (DEGs) in infected versus control MN106 plants. However, 104 genes were differentially expressed by 3 hr infection in 2032 plants. More robust transcriptional responses were apparent at later time points: in both genotypes approximately 2800 genes were differentially expressed by 6 hr S. sclerotiorum infection. By 12 hr of A. japonica infection, 3232 and 6276 genes are differentially expressed in MN106 and 2032 leaves, respectively. For all time points and for both pathogens, fewer genes are downregulated by infection than upregulated. Most of the genes induced by infection are common to both pathogens and both genotypes, whereas the downregulated genes are more unique to each pathogen and genotype than shared. More work is required to identify thepathogen-responsive promoters of pennycress and we have the data that is needed to help us in that direction. Aim4.1. Identification of pennycress Pep gene family. Elicitor peptides (PEPs) are endogenous peptides in plants that can activate immunity against fungi, bacteria, and insects. When cells are damaged, PEPs are cleaved from precursor proteins called PROPEPs, allowing the PEPs to bind PEP receptors (PEPRs) on neighboring cells and activate immune signaling (Bartels and Boller 2015). Since the submission of this grant, we have found 7 putative PROPEP genes in the pennycress genome. We have numbered the pennycress PROPEPs according to their closest homolog in Arabidopsis, and have not found a homolog of Arabidopsis PROPEP8 in pennycress. We had the pennycress PEPs chemically synthesized, and first examined their ability to elicit an oxidative burst, a sign of pattern-triggered immunity (PTI) activation. PEP recognition by a PEPR triggers a phosphorylation cascade that phosphorylates respiratory burst oxidase protein D (RBOHD), activating ROS production (Lee et al. 2018). Treatment of 1 µM PEP3 elicited an oxidative burst, although it is less than half the magnitude of that of flg22, a bacterial elicitor included as a positive control. Other PEPs have a small oxidative burst not much above background levels. 4.2. Expression of TaPeps in transgenic pennycress. Results observed in aim 4.2 did not show any evidence of the pennycress PEPs providing the resistance to these fungal pathogens in the in-vivo experiments and therefore we did not generate the transgenic lines for the PEP peptides.
Publications
- Type:
Other
Status:
Other
Year Published:
2024
Citation:
1. Development of visual and molecular assays to screen for S. sclerotiorum and A. japonica resistance in pennycress.
2. Identification of a key disease susceptibility allele in the breeding germplasm.
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Progress 09/30/21 to 09/29/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We are most excited by the fact that using the assay for Sclerotinia sclerotiorum leaf infection developed with the funds from this award, we were able to consistently demonstrate difference in susceptibility of MN106 and 2032 genotypes. We initiated the effort to map the susceptibility locus (or loci) in 2032, an early maturing line that is highly prevalent in CoverCress' existing product candidates and its breeding pipeline. Preliminary results suggest that the trait is recessive (or semi-dominant), and we should be able to identify the molecular nature of genetic difference(s) between the germplasms in 2023. Achieving this goal will lead to the development of a molecular marker that can be used to counter select against susceptibility loci in all future CoverCress products, a milestone which will have immediate positive commercial impact and broad implications. ?We are testing different pennycress accessions for resistance to Sclerotinia sclerotiorum and Alternaria japonica. Identification of resistant germplasm will facilitate identification of underlying QTLs through GWAS studies. We have established pathogen infection assays for pennycress to allow us to begin screening pennycress germplasm for resistance to these pathogens. We are also conducting RNA-seq studies to identify pennycress S genes that respond to fungal infection early. These genes will be potential targets for editing to develop disease resistant pennycress. In addition, the promoters of these genes will be useful for expression of antifungal genes in transgenic pennycress. Preliminary studies indicate that pennycress elicitor peptides have the potential to provide resistance to white mold.
Impacts
What was accomplished under these goals?
?Progress toward major goals of the project Aim1. Characterize T1 transgenic pennycress lines constitutively expressing OeDef1, NCR044_v2, PDF2.2 and PDF4.3 to identify homozygous lines and determine expression of each peptide. Expression and gene copy analyses of OeDef1, NCR044_v2_PDF2.2 and PDF4.3 in transgenic pennycress lines. Disease resistance evaluation of transgenic lines. We generated multiple constructs designed to achieve overexpression of OeDef1, NCR044_v2, PDF2.2 and PDF4.3 by placing them under the control of the constitutive CaMV35S promoter. These chimeric genes were introduced into pennycress using the floral dip transformation method. Each chimeric construct also contains a cassette containing DsRed fluorescent protein ORF under control of Cassava Vein Mosaic Virus (CVMV) promoter as a reporter gene to enable easy visualization and sorting of transgenic seed from null segregant seed. Using DsRed-positive lines containing chimeric OeDef1 and NCR044_v2 genes, we conducted Western blot analysis to determine if these lines are expressing OeDef1 and NCR044_v2 proteins. A strong expression of OeDef1 was achieved in one of the T1 lines and a weak expression of NCR044 was detected in a few lines. However, in T2 generation, no expression of this protein was detected. Following these findings, we have redesigned our expression vectors to increase/optimize the expression of each of the four antifungal peptides. The strategy that we are undertaking to overcome this issue is to use different configuration of the expression cassettes (in case the original configuration is prone to gene silencing) and to mitigating potential risk of cell toxicity resulting from high constitutive Def protein expression by switching to tissue-specific or pathogen-inducible promoters that will deliver protein expression only at the right time and tissues affected by the pathogen. The optimized expression vectors were designed, produced and transformed into plants. Aim 2. Characterize T1 transgenic pennycress lines expressing OeDef1, NCR044_v2, PDF2.2 and PDF4.3 from the epidermis-specific promoter A14 or pathogen-responsive PDF1.2 promoter. Expression and gene copy analyses of OeDef1, NCR044_v2_PDF2.2 and PDF4.3 in transgenic pennycress lines. Disease resistance evaluation of transgenic lines. The T1 transgenic lines we have generated with the pathogen-responsive PDF1.2 promoter/AFP gene constructs and epidermis-specific A14/AFP gene constructs. Further characterization of these lines will not be pursued until we have enhanced the expression of transgenes using the optimized expression vector. Aim3. Generate transgenic pennycress lines expressing TaPep1, TaPep2 and TaPep3 constitutively and test the lines for resistance to fungal pathogens. Determine expression of TaPep1, TaPep2 and TaPep3 in transgenic pennycress Determine JA-responsive defense gene expression in transgenic Pep lines. Determine resistance of homozygous Pep lines to fungal pathogens. Plant elicitor peptides (Peps) are endogenous regulators of pathogen defense responses. These peptides of 23-27 amino acids are hypothesized to bind to PEPR receptors and activate expression of a diverse suite of protective defense responses. TaPeps thus have the potential to be used as a mechanism for manipulating resistance to fungal pathogens in crops. We have tested TaPep1, TaPep2 and TaPep3 for their ability to confer resistance to the field isolate of the fungal pathogen Sclerotinia sclerotiorum. The seed of 2032 WG genotype were treated with each peptide at a concentration of 5 µM for 15 hr. The control seed were treated with equal volume of water. The control and peptide-treated seed were sown in soil and plants were allowed to grow for 38 days. The detached leaves of these plants were inoculated with 2 mm plug of S. sclerotiorum isolate CA1 and the infection was allowed to proceed for 72 hr in high humidity Ziploc boxes. In leaves of plants generated from seed treated with TaPep1 and TaPep2 50-70%reduction in lesions caused by white mold was observed compared with plants generated from the control and TaPep3-treated seed . These results indicate that TaPep1 and TaPep2 have the potential as host defense activating peptides capable of conferring resistance to white mold. TaPeps are cationic peptides carrying a net charge of +5 to +7. In addition, TaPep1 and TaPep2 also contain 17.4 and 22% hydrophobic amino acids. The presence of cationic and hydrophobic amino acid residues are hallmarks of antimicrobial peptides. We tested the antifungal activity of TaPep2 and TaPep3 against a fungal pathogen Botrytis cinerea in vitro. TaPep1 could not be purified to homogeneity on reverse phase HPLC and thus could not be tested in this assay. We found that TaPep2 and TaPep3 inhibited the growth of this pathogen with the MIC values of 12 and 6 µM, respectively. Thus, TaPeps are the first plant Peps demonstrated to have direct antifungal activity.During the USDA_SBIR phase II funding, we will express all three TaPeps in transgenic pennycress using the optimized expression vector. Aim 4. Identify pathogen-responsive genes in pennycress and characterize their promoters. We have developed a reproducible stem infection assay for S. sclerotiorum isolate CA1using the pennycress genotype 2032 WG and 2 mm plug of S. sclerotiorum mycelium. We have also developed A. japonica leaf infection assay. Using S. sclerotiorum leaf infection assay, we performed RNA-seq analysis on pathogen infected tissue and identified genes that are activated early (at 6hrs) after challenge with S. sclerotiorum. Aim 5. Screen pennycress germplasm for resistance to white mold and Alternaria leaf spot. Using the validated white mold leaf spot infection assays developed above, we initiated mapping of S. sclerotiorum relative resistance/susceptibility genes in MN106 and 2032 germplasms. The results suggest that susceptibility trait in commercially important 2032 germplasm obeys simple Mendelian rules of inheritance, giving us hope that the underlying locus/mutation can be identified relatively quickly using standard bulk segregant (BSA) analysis. We have successfully identified several important traits in pennycress using BSA in the past and have in our possession multiple populations from MN106/2032 crosses that can be used to clone/characterize and validate the underlying mutation(s) resulting in this difference in phenotypes. Alternaria pod infection assays also demonstrate that 2032 appears to be more susceptible to this pathogen than MN106 (Figure 8). Once we optimize this assay, we will use it to pursue Alternaria susceptibility genes using the same BSA approach that is being used for Sclerotinia. We also plan to screen several accessions of pennycress germplasm for resistance to each disease. Identification of resistant germplasm will allow us to identify the QTL(s) underlying resistance to each pathogen and perform genome wide association studies to identify the disease resistance genes.
Publications
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