Progress 05/01/24 to 04/30/25
Outputs
Target Audience:The intended audience for this research is comprised of professionals in the field of agricultural science. We have primarily disseminated our findings to this audience through presentations at WSU and at annual scientific meetings. Additionally, undergraduate students, graduate students, and postdoctoral researchers have been exposed to our research through formal classroom instruction, lab rotation experiences, and lectures within the Plant Pathology department and School of Molecular Biosciences at WSU. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training to a total of three personnel in the Gleason lab: one Master's student. student Kamryn Clements. The training included practicing and improving different scientific techniques and presenting results and data to the department. Kamryn is expected to graduate in May 2025. A School of Molecular Biosciences PhD student, Brook Baker, did a rotation in the Gleason for 6 weeks to learn plant and root-knot nematode techniques. She presented her result in a presentation to the lab. 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 will continue with our efforts to silence the fat-5/fat-6/fat-7 genes in M. javanica both with host-induced gene silencing and with silencing by soaking. For the plant sterol work, we will obtain and analyze the data for the root sterol analysis that is being performed at the WSU Metabolomics facility. This data will guide us in selecting additional sterol mutants to test in nematode infection assays. We will also further investigate the molting behaviors we've observed in two of the Arabidopsis mutant lines. Finally, we are expanding our sterol research to tomatoes by generating sterol mutants in tomato roots, which will be tested with nematodes.
Impacts
What was accomplished under these goals?
Root-knot nematodes are significant agricultural pests. The goal of this project is to pioneer innovative controls for root-knot nematodes by focusing on two specific aspects of their biology: their diet and the processing of nutrients, particularly sterols. This endeavor aims to benefit the agricultural community, especially growers nationwide grappling with root-knot nematode infestations. Given the broad host range of these nematodes and their ability to infect various crops, this research has the potential to help diverse agricultural production systems. The first goal is to alter plants so they naturally interfere with nematode nutrition, crops can become more resistant to these pests. In the previous report, the Meloidogyne javanica gene encoding a delta 9 desaturase (homologous to C. elegans fat-5/fat-6/fat-7 genes) was cloned into two vectors for gene silencing: one for dsRNA generation and the second is for the generating transgenic plants that silence the nematode gene. The major accomplishment in this reporting period is that the host-induced gene silencing construct was introduced into the model Arabidopsis thaliana by floral dip. The transgenic plants were confirmed by PCR and are currently being grown for seed. In addition, a pilot experiment was performed in which M. javanica juveniles were soaked in small dsRNA to silence expression of the fat-5/fat-6/fat-7 gene. Gene expression analysis confirmed that soaking nematodes in a special solution for the essential fat-producing genes affected its expression. These experiments pave the way for generating plants that interfere with the nematodes' ability to synthesize the nutrients they need to survive. The second objective is to study how nematodes rely on certain plant compounds, called sterols, to develop. By modifying these compounds in plants, we aim to prevent the nematodes from maturing and reproducing. To accomplish this, we obtained 19 Arabidopsis plant lines with genetic changes that affect sterol production So far, 13 of these plant lines have been confirmed to have the desired genetic modifications. These 13 lines were subjected to gene expression analysis to confirm that they were knocked down in expression. Of these lines, 7 were sent for mass spectrometry at the MJMurdockMetabolomics Laboratory to determine their root sterol profiles. Results from the mass spec analysis are expected in March 2025. To better understand how a specific gene affects plant growth, we introduced a part of a gene from Arabidopsis, known as the catalytic domain of HMGR (CD-HMGR), into the plant. We created four separate lines of transgenic Arabidopsis plants with this gene and confirmed that the gene was actively being expressed in these plants using a technique called qRT-PCR. The two lines with the highest levels of gene expression were selected for further study. These plants will be analyzed for their root sterol and lipid content using mass spectrometry. These experiments help ensure that the plants with altered gene expression are accurate and ready for further research on how their root sterols are affected. One of the significant milestones in this study involved examining how certain sterol mutations affect plant response to nematode infections. We have tested two types of sterol mutant lines, one with a mutation in sterol C24 methyltransferase and the other in C14 reductase, along with two lines where CD-HMGR was overexpressed. We were particularly interested in how these mutations might influence nematode molting--a critical part of their life cycle. They infected the roots of the plants with nematodes and measured how many of the parasitic juvenile nematodes (called J2) were able to complete their molting process. The results from the CD-HMGR overexpression lines were not consistent, but they did show a general trend toward reduced molting, suggesting that this gene might play a role in regulating nematode development. Interestingly, the mutant with the altered C24 methyltransferase gene was more susceptible to the nematodes and showed higher molting rates. This finding makes sense when considering that the plant's altered auxin responses and increased levels of a specific sterol, 24-methylenecholesterol, might help the nematodes thrive. However, the mutant in C14 reductase showed a clear impact on molting, but not on the nematodes' ability to penetrate the roots, which points to the idea that certain sterols, like sitosterol and campesterol glucosides, may be critical for nematodes to molt and progress in their life cycle. In addition to genetic manipulation, we also used a chemical approach to alter the sterol composition of Arabidopsis plants. The plants were treated with three different chemicals--lovastatin, Hyemeglusin, and phosphomevalonate--each known to influence sterol biosynthesis in various ways. To ensure that these chemicals would not cause any major side effects, such as stunted growth or other unwanted effects on the plants, we determined concentrations that didn't harm the plants. We also tested the chemicals for any negative effects on the viability of juvenile nematodes to ensure the treatments wouldn't skew their results. By manipulating the plant's sterol composition through these chemicals, we can further explore how specific sterols influence plant defense mechanisms, particularly their ability to resist nematode infection and molting. This combined genetic and chemical approach allows for a more nuanced understanding of plant-nematode interactions and could open new pathways for improving crop resistance to these harmful pests.
Publications
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:The intended audience for this research is comprised of professionals in the field of agricultural science. We have primarily disseminated our findings to this audience through presentations at WSU. Additionally, undergraduate students, graduate students, and postdoctoral researchers have been exposed to our research through formal classroom instruction, lab rotation experiences, and lectures within the Plant Pathology department and School of Molecular Biosciences at WSU. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training to a total of three personnel in the Gleason lab: one Postdoc (Scott Anderson, formerly a PhD student in the Gleason lab), one Master's student (Leah Morrison), and one Ph.D. student (Kamryn Clements). The training included practicing and improving different scientific techniques and presenting results and data to the department. In the Watts lab, a PhD rotation student Tanner Badigian participated in the project and received training in Gateway cloning and plant tissue culture. Scott Anderson, who initially joined as a Ph.D. student, successfully defended his thesis titled "NOVEL MOLECULAR APPROACHES TO IDENTIFY AND CONTROL PLANT PARASITIC NEMATODES" before transitioning to a post-doc position within the Gleason lab. He also delivered a seminar to the WSU Plant Pathology department during his doctoral studies. Leah Morrison is set to graduate in May 2024 with her thesis titled "ROOT KNOT NEMATODES AND INSIGHTS INTO THEIR PARASITISM AND FATTY ACID DIET." She has presented a seminar on her work concerning the HMGR mutants to the WSU Plant Pathology department. All personnel paid from this project have completed CITI training in Responsible Conduct of Research (RCR). How have the results been disseminated to communities of interest?The main avenue for dissemination of the products has been through publications of academic theses (Scott Anderson and Leah Morrison) and presentations to the Plant Pathology Department at WSU. What do you plan to do during the next reporting period to accomplish the goals?We will proceed with our efforts to silence the fat-5/fat-6/fat-7 genes in M. javanica. Initially, we will synthesize dsRNA through in vitro transcription, allowing us to conduct "silencing by soaking," where J2 worms are exposed to dsRNA for gene suppression. Additionally, we will transform wildtype Arabidopsis plants with the delta9 desaturase gene inserted into the Gateway pB7GWIWG2-7F2,1 vector. Transformed plants will be challenged with nematodes to see if host silencing of the nematode delta9 desaturase gene will result in the inhibition of growth or reproduction of the parasitic nematodes.For the plant sterol work, we will finish characterizing the available mutant lines. Our previous experience with one Salk line of HMGR1 revealed that a T-DNA insertion does not consistently result in a knockout phenotype. Therefore, it is imperative to assess gene expression across all knockout and overexpression lines to verify the anticipated changes. Once confirmed through qRT-PCR analysis, we will proceed with characterizing the root sterol content, a task to be undertaken by the WSU Proteomics lab.
Impacts
What was accomplished under these goals?
Root-knot nematodes are significant agricultural pests. The goal of this project is to pioneer innovative controls for root-knot nematodes by focusing on two specific aspects of their biology: their diet and the processing of nutrients, particularly sterols. This endeavor aims to benefit the agricultural community, especially growers nationwide grappling with root-knot nematode infestations. Given the broad host range of these nematodes and their ability to infect various crops, this research has the potential to help diverse agricultural production systems. The first objective is to interfere with nematode fatty acid synthesis using RNA interference (RNAi) of parasitic nematode lipid biosynthetic genes. The major accomplishment under this objective has been the cloning of three Meloidogyne javanica genes encoding putative delta 9 desaturases (fat-5/fat-6/fat-7). These genes were cloned into a yeast expression vector and tested for rescue of the yeast delta 9 desaturase mutant ole1. GC/MS analysis of the transgenic yeast strains showed that all of the delta 9 desaturase genes converted 16:0 to 16:1, demonstrating that their activity is most similar to the C. elegans FAT-5 desaturase. The M. javanica genes did not show activity in converting 18:0 to 18:1. We then cloned a region of DNA that is identical in all three genes and inserted this sequence into two vectors. The first vector is for creating dsRNA for nematode soaking and the second vector is for generating transgenic plants that silence the nematode gene. In this experiment, all three of the putative delta 9 desaturase genes are predicted to be silenced. So far we demonstrated successful cloning of a region corresponding to the delta 9 desaturase and have generated sequencing data to confirming the correct sequence in the two silencing clones that were generated. The second objective is to manipulate the sterol diet of the nematodes. To accomplish this, we initially focused on three Arabidopsis mutant lines targeting 3-hydroxy-3-methylglutaryl Coenzyme A reductase (HMGR), which governs the conversion of HMG-CoA to mevalonate, a critical step in the mevalonic acid pathway. We obtained one mutant line for HMGR1 and two mutant lines for HMGR2 from the Arabidopsis Biological Resource Center (ABRC). PCR analysis confirmed homozygosity in the hmgr1 line. However, quantitative gene expression analysis revealed no significant knockdown of HMGR1 expression in the roots, contrary to expectations. In addition, plants displayed no discernible growth phenotype as anticipated for this mutant. Moreover, nematode infection assays demonstrated no alterations in galling compared to the control. This led us to conclude that the mutant line we received was not adequately silenced, perhaps due to the position of the T-DNA insertion, and as a result, showed a wildtype phenotype in our assays. In the two hmgr2 lines, only one line showed a significant decrease in HMGR2 expression in the roots. Interestingly, this line also had more galls after M. javanica inoculation, compared to the control. All statistics (Welchs's t tests) were performed using GraphPad software. Overall, the data indicated that HMGR2 may be influencing plant defenses and that mevalonic acid may have a role in plant immune responses. Furthermore, we obtained 17 additional sterol mutant lines from the ABRC, anticipated to exhibit modified sterol content. These encompass mutations in HMGR1 and sterol methyltransferase (SMT) 1, 2, and 3, along with mutants in sterol glucosyltransferase and sterol C-14 reductase. Each line underwent PCR analysis to confirm the presence of the mutation and was cultivated under selective conditions to attain homozygosity. Additionally, we requested two overexpressing lines, SMT2OE and CD-HMGROE, from Dr. Herbert Schaller. However, due to unavailability, we cloned SMT2 and catalytic domain of HMGR (CD-HMGR) independently. The wildtype Arabidopsis (Col-0) was transformed by floral dip transformation with either SMT2OE or CD-HMGROE. At the time of writing, there were 12 independent CD-HMGROE lines. These lines were in the process of being tested for gene expression by qRT-PCR. There was only 1 line of SMT2OE and an additional screen of T1 seeds is underway to identify additional transgenic lines. These experiments serve as crucial validation steps, ensuring the accuracy of plant materials (i.e., mutants with altered gene expression) for subsequent root sterol analysis.
Publications
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