Performing Department
(N/A)
Non Technical Summary
Successful outcomes from this research will open the door to applying this innovative methodology to reverse herbicides resistance in agronomically relevant and problematic weeds known to evolve NSTR to herbicides (e.g., Palmer amaranth and Lolium spp.). The ability to address herbicide resistance will benefit farmers across the United States as well as around the world.
Animal Health Component
0%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
0%
Goals / Objectives
Most cases of resistance can be grouped into two broad categories, namely target site resistance (TRS) and non-target site resistance (NSTR) mechanisms. Non-target-site herbicide resistance (NTSR) is a problem that has plagued crop production for many years and trends suggest that this will become the most pressing issue in the coming years. NTSR is particularly problematic because these mechanisms of resistance can extend to diverse chemical groups with affecting target sites and even impart resistance to herbicides that have never been used on these weeds.Cytochrome P450 monooxygenases (P450s) are often implicated in NTSR because they can catalyze a myriad of reactions using herbicides as substrates.Unfortunately, P450s belong to one of the largest enzyme families for all organisms, with high diversity in plants ranging from 200 to more than 900 genes in some weeds. Consequently, it is difficult to identify which P450 has changed over time to recognize a particular herbicide as a substrate, making it impossible to target this enzyme to reverse herbicide resistance. A common method to overcome NTSR has been the use of P450 monooxygenase inhibitors. While these inhibitors restore the efficacy of certain herbicides by preventing their metabolism by the weed, this often leads to crop injury by reducing the natural ability of the crop to metabolize the herbicide as well.While there are hundreds of P450s in plants, most P450s must work in concert with cytochrome P450 reductases (CPRs) to function properly. Indeed, while the P450s catalyze oxidative reactions, CPRs are necessary partners to complete the redox cycle for the P450s to continue their function in subsequent reactions. While P450s form a very large enzyme family, most plants only have a few (one to three) CPR genes, which makes manipulation of the activity of all P450 genes feasible via modulating the activities of a few CPR genes.Therefore, we propose that CPRs may be a good target to develop innovative, synergistic, and selective technologies that would prolong the activity of herbicides that are susceptible to metabolic degradation.
Project Methods
Experiment 1: Designing CRISPR/Cas9 systemSince the two ATR genes are linked, we have devised a new set of experiments to generate plants with both ATR genes knocked out. To do this, we will use CRISPR/Cas9 technologies available in our laboratory. The CRISPR associated protein 9 (Cas9) is directed to its DNA target by base pairing between the guide RNA (gRNA) and DNA. A protospacer-adjacent motif (PAM) sequence 5'-NGG-3' (PAM motif) downstream of the gRNA-binding region is required for Cas9 recognition and cleavage. Cas9/gRNA cuts both strands of the target DNA, triggering endogenous DNA double-strand breaks (DSB) repair. For a knockout experiment, the DSB is repaired via the error-prone non-homologous end joining (NHEJ) pathway, which introduces an inDel, insertion or any SNPat the DSB site that knocks out gene function (desirable). In a knock-in experiment, the DSB is repaired by homologous recombinant repair (HDR) using the donor template present, resulting in the donor DNA sequence integrating into the DSB site (non-desirable for the proposal).Objective 2. Gene and metabolomic networks associated with CPRs in plantsExperiment 1: RNA-seq analysisSeeds of Arabidopsis thaliana Col0, ATR1, ATR2 and ATR1?ATR2 knockout lines will be grown in growth chambers. The experimental design includes four biological replications. Samples will be collected at the rosette stage and flash frozen in liquid nitrogen for RNAseq analysis. Total RNA will be extracted from leaf tissues (40 mg). Gene expression differences between log2 and early stationary phase will be obtained from a package from DESeq2 package (26) using the adjusted p-values from the Benjamini-Hochberg Procedure (27). This method evaluates the weight of each factor considered in the analysis and its impact on DEGs. We simply defined genes with at least 2-fold change between two samples and FDR (false discovery rate) less than 0.001 as differential expressed genes. Transcripts with log2FC >0 and statistical significance at P ≤ 0.05 will be assumed to have change in their expression levels (28).Experiment 2: Metabolomic analysisNon-targeted metabolomics analysis of the samples will be performed by the CSU ARC Bio analytical resources core.Objective 3. Gene-silencing of the CPRs as an innovative approach to reverse metabolism-based NTSR to herbicidesExperiment 1: Reversing metabolism-based resistance to herbicidesArabidopsis lines with atr1atr1 x atr2atr2 homozygous double knockouts generated with CRISPR/Cas9 will be crossed with a transgenic line of Arabidopsis expressing with a P450 gene known to metabolize several herbicides including bensulfuron-methyl as done before for the atr1atr1 and atr2atr2 T-DNA knockouts described in the preliminary data.Mesotrione and its hydroxy metabolite will be detected using a LC-MS/MS system consisting of a Nexera X2 UPLC with 2 LC-30AD pumps, a SIL-30AC MP autosampler, a DGU-20A5 Prominence degasser, a CTO-30A column oven, and SPD-M30A diode array detector coupled to an 8040 quadrupole mass-spectrometer with ESI. For mesotrione, the MS will be in positive mode [M+H]+ with a MRM optimized for: 340.00>227.95 set for 100 ms dwell time with a Q1 pre-bias of -16V, a collision energy of -18V and a Q3 pre-bias of -16V.For the hydroxy-metabolite, the parameters will be in positive mode [M+H]+ with a MRM optimized for: 356.00>55.00 set for 100 ms dwell time with a Q1 pre-bias of -16V, a collision energy of -18V and a Q3 pre-bias of -16V; 356.00>228.05 set for 100 ms dwell time with a Q1 pre-bias of -16V, a collision energy of -18V and a Q3 pre-bias of -16V.Experiment 2: Innovative method to silence expression of ATRsWhile we will develop atr1?atr2 double knockout in experiment 1, our preliminary data demonstrates that knocking out ATR1 is sufficient to dramatically reduce herbicide metabolism. Therefore, we will focus on repressing ATR1 gene expression in a number of weeds in Experiment 2. While the availability of weed genomes has been lacking in the past, we have access to the genomes of many weed species through the International Weed Genomic Consortium that is hosted by our laboratory.We have previously shown that FANA ASOs have biological activity in plants as measured by gene expression knockdown and growth inhibition (Gaines lab, unpublished). In a current grant from DARPA that ends in early 2024, we are improving the delivery of FANA ASOs into plants by complexing them with nanoparticles. While the DARPA grant focuses on improving foliar delivery for spray-on gene silencing applications and a recently awarded USDA ARA NACA focuses on reversing glyphosate resistance in Palmer amaranth through EPSPS over-expression, we are proposing to use the results of this delivery research to support an innovative approach to reverse all P450-dependent herbicide resistance by targeting plant CPRs for gene silencing. We will design 21 nucleotide FANA ASOs to target the mRNA for ATR1 (most important target based on our preliminary data) or the combination of ATR1 and ATR2.