Performing Department
(N/A)
Non Technical Summary
Palmer amaranth (Amaranthus palmeri) is the most serious weed in cotton mainly due to its rapid evolution of resistance to most mechanisms of action (MOA) and massive seed production that make control of its populations challenging. Therefore, there is an urgent need to develop innovative strategies that not only prevent interference with the crop but more importantly that reduce weed population growth. New molecular biology techniques can be incorporated into integrated programs to ensure the long-term viability of weed management strategies. RNAi technology has the potential to generate control solutions that complement existing weed management approaches. Although this technology has been widely investigated to reduce the economic impact of other pests, very limited research has been conducted for weed control which has mainly focused on substitution of herbicides. No work has been done to develop RNAi programs that target growth and fitness traits such as plant height and internode elongation, flowering, and seed dormancy. Genetic and physiological pathways controlling those traits have been widely characterized and are now well known. We also have identified new non-druggable targets that are critical for plant survival and can be used for weed control. Reducing the expression of genes related to those processes with RNAi technology will help reduce the survival, fitness, and seed production of Palmer amaranth escaping herbicide control in cotton fields. We propose: 1) to design RNAi systems to silence target genes in Palmer amaranth, 2) to characterize the effects of those RNAi systems on cotton, and 3) to determine dsRNA stability and persistence when exposed to different potential abiotic and biotic degradation agents. The proposed research will set the bases to regain control of herbicide resistant weeds in cotton while promoting numerous benefits to cotton growers by reducing reliance on herbicides.
Animal Health Component
0%
Research Effort Categories
Basic
40%
Applied
50%
Developmental
10%
Goals / Objectives
1) to design RNAi systems to silence target genes in Palmer amaranth2) to characterize the effect of those RNAi systems on cotton growth3) to determine dsRNA stability and persistence when exposed to different potential abiotic and biotic degradation agents.
Project Methods
Objective 1: To design RNAi systems to silence target genes in Palmer amaranthActivities. We selected genes from pathways related to 1) flowering time, 2) seed dormancy, 3) GA regulation, and 4) plant survival (Table 1). To gain confidence in understanding sequence similarities and genetic redundancy for genes of interest from Palmer amaranth, the genomic and cDNA corresponding to the selected genes will be sequenced from individuals from multiple populations. The Palmer amaranth reference sequence will be downloaded from CoGe (https://genomevolution.org/coge/). The sequence of major crop species will be retrieved from Ensembl Plants (https://plants.ensembl.org/index.html). Multiple sequence alignment and phylogenetic tree analysis will be performed by MEGA X software (Kumar et al. 2018). The sequences of the selected genes will be compared with those of major crop species and other plant species to identify conserved areas that can provide a multispecies effect or non-conserved areas that can provide selectivity.Table 1: List of selected genes for RNAi systems for Palmer amaranth management in cottonPathwayFirst preferenceSecond preferenceFloweringFT (Flowering Locus T)SOC1 (Suppressor of overexpression of Constans 1)Seed dormancyDOG1 (Delay of Germination 1)SnRK2 (SNF1-related protein kinase)GA regulationGA3ox (Gibberellin 3-oxidase)GA2ox (Gibberellin 2-oxidase)Lethal genesFtsHi1, Frd3n/aFoliar applications of dsRNA will be conducted on Palmer amaranth plants with a modified air brush gun testing different droplet sizes and leaf coverage. This will be done to deliver different amounts of the dsRNA to generate a dose-response curve.To evaluate the RNAi effect in Palmer amaranth, the experiment will be performed under greenhouse conditions.Greenhouse activity: Palmer amaranth will be grown in a greenhouse at standard ambient temperature favorable for its growth. The experiment will include dsRNA treated and non-treated controls, will have 6 replications with a single plant per replication, and will be conducted twice. The application of dsRNA for the first preference targets (FT, DOG1, GA3ox) will be performed separately and in combination using a full factorial design. Additionally, a dsRNA designed to reduce expression of PDS in Palmer amaranth will also be used as positive control to confirm dsRNA integrity and absorption during experiment establishment, and a negative control with the surfactant mix without dsRNA will be also included. A dose-response study will be performed with concentrations ranging from 0.1 to 100 µM for each target. The same approach will be used for the lethal genes FtsHi1 and Frd3. These two genes will be studied separately because mortality is the target phenotype. However, they will be tested alone and in combination to see if their combined action favors a synergistic response. Further, to identify the timing of application, we will perform dsRNA applications at different growth stages, namely, cotyledon to 2-leaf stage, 4- to 6-leaf stage, 8- to 10-leaf stage, flowering, and seed production. After application, plants will be monitored weekly to determine plant height and width, internode length, number of days to flowering, seed number, and seed weight. Seed germinability will be determined before and after stratification to quantify dormancy, and non-germinated seeds will be analyzed to determine viability. The results will be further validated by checking the expression of genes by qRT-PCR in different parts of the plant.Objective 2: To characterize the effect of RNAi systems on cotton growth. ? Glufosinate resistant cotton plants will be treated with the optimum doses of the different dsRNA constructs and at the respective stages to reduce the fitness of Palmer amaranth based on the previous objective. This study will be conducted under greenhouse conditions. Cotton will be treated with single dsRNA targets including the lethal genes and with the integrated sequential program (GA, followed by flowering+seed dormancy inhibitors). Height, biomass, leaf number and area, branching architecture, flowering time, flower number, square number, boll number, open boll number, and fiber and cottonseed yield and germinability will be evaluated. The experiment will be a completely randomized design with 6 replications of a single plant, and it will be conducted twice. A dsRNA will also be designed to target the bar gene (causal gene for glufosinate resistance); this will serve as positive control for this experiment. In case of negative results, plants will be treated with glufosinate to determine whether the lack of response was due to the construct used or limited uptake of the construct. The survival and injury after glufosinate application of nontreated (negative control) plants will be compared with bar dsRNA treated plants. Additionally, qRT-PCR experiments will be performed from tissues from root, shoot, internode and stem to check whether there is an impact on cotton or not for each construct.Objective 3: Determine dsRNA stability and persistence when exposed to different potential abiotic and biotic degradation agents Off-target negative effects of molecules used for pest control is a major concern (Dubelman et al. 2014; Albright et al. 2017). Despite the existence of multiple forms of RNA in all organisms and in the environment, understanding the persistence of dsRNA can help anticipate potential off-target effects. Stable molecules that are highly persistence in the environment are more likely to affect other organisms than those that are quickly degraded. The main mechanisms for degradation of pesticides and organic molecules are UV light and microbial activity (via enzymes) (Albright et al. 2017). Researchers reported that 90% dsRNA dissipated in 35 to 96 hours in microcosms with sterile water and water from ponds, and dissipation was considerably faster in sediments (Fischer et al. 2017).Activities: We will conduct a series of laboratory experiments to evaluate the stability of the dsRNA constructs used in the previous experiments. Aliquots of dsRNA solutions with biologically active concentrations based on the dose-response studies, will be added to sterile aqueous solutions with pH from 4 to 9. Also, dsRNA will be maintained under white light and UV light at ambient intensities. To evaluate not only chemical but also biological factors, dsRNA will be added to aqueous extracts of from soil types of representative agricultural areas, crop systems, and non-agricultural land from North Carolina based on the methodology used by Fischer et al. (2017). We will sample a larger number of conditions compared with published research including 1) soil types: mock, sandy, loam, and clay soils, 2) crop systems: row crop rotations including cotton, sweet potato, wheat, and pastures systems, 3) aquatic systems: natural lakes, large artificial reservoirs, urban ponds, rivers, and marsh systems. Samples will be split, and one half will be autoclaved to quantify the role of living microbial communities on dsRNA dissipation. For soil samples, extracts will be obtained by adding soil samples to water in beakers and maintaining them in a shaker for 48 hours. An aliquot of the dsRNA constructs will be added to each beaker. After that, samples will be allowed to settle and decanted. Subsamples will be collected periodically 6 hour-intervals from each original condition and sediment and liquid phases for analyzes using specific primers and qtPCR. If needed, PCR will be conducted, and samples run on an agarose gel to visualize dsRNA degradation and identify potential cross amplification with microbial RNA. This approach will allow creating time dependent dissipation curves (Fischer et al. 2017; Albright et al. 2017).?