Progress 09/01/24 to 08/31/25

Outputs
Target Audience:We had meetings with agricultural and scientific organizations interested in the RNAi technology for weed control in different cropping systems. For example, we met with the Agronomy Research Director of Cotton Inc., the coordinators ofRNAi technologies at Bayer CropScience and Pest Control solutions at BASF, and two university groups. In those meetings, we exchanged information about the challenges and possible solutions for the use of this technology. All the groups expressed their interested in receiving more information about the progress of the present project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral fellow assisted specialized in biotechnology assisted to the NortheasternWeed Science Society Conference to learn about weed control and use that knowledge for designing the RNAi technology. How have the results been disseminated to communities of interest?See comments in "Target audience" section. What do you plan to do during the next reporting period to accomplish the goals?We will be working on optimizing the sequence of the cell penetrating peptide in the buffer solution and the concentration of dsRNA, and this last piece of the RNAi technology will be our focus for the next few months.

Impacts
What was accomplished under these goals? In order to design dsRNA sequences for an RNAi technology, we began with a multiple sequence alignment of the amino acid sequences of the Blast hit genes (Table 1) using ClustalW and the Palmer genes clustered together with the Arabidopsis homologs were identified as candidate target genes. We identified 7 of the 8 proposed target genes, and cloned all these 7 identified target genes from Palmer amaranth via RT-PCR (Table 1). Two copies of FT, SnRK2, GA3ox2 and FRD3 genes were found in the Palmer amaranth genome, and only one copy was discovered for SOC1, GA3ox1, and FTSHI genes. The sequence identities between these Palmer genes and their Arabidopsis homologs were between 42% and 83% on amino acid sequence level. The DOG1 gene was not identified in any of the three available amaranth genomes studied, which was most likely due to sequencing or annotation flaws of these genomes. Besides these target genes, we have also identified and cloned the PDS gene from Palmer amaranth. Silencing the PDS gene will disrupt the biosynthesis of chlorophyll and the plants will exhibit a bleaching phenotype, thus the PDS gene could be used as a positive control which would make method development easier. In fact, due to this advantage, we have been mainly focusing on the PDS gene during the method development stage currently. Once a robust method is developed, we will shift our focus to silencing the 7 target genes we cloned from Palmer amaranth. Table 1. A summary of target genes cloned from Palmer amaranth. Target genes Pathways Arabidopsis homologs Length Expression patterns in Arabidopsis Candidate genes in Palmer FT Flowering AT1G65480.2 219aa siliques Ap4g185630 Ap4g185980 SOC1 Flowering AT2G45660.1 214aa N/A Ap12g062000* +Ap12g062010* DOG1 Seed dormancy AT5G45830.1 323aa seeds Not identified SnRK2 Seed dormancy AT3G50500.2 AT5G66880.1 369aa Ubiquitous, high in leaves Ap2g130650 Ap3g158460 GA3ox1 GA biosynthesis AT1G15550.1 358aa Ubiquitous, low Ap12g068910 GA2ox2 GA biosynthesis AT1G30040.1 341aa Flowers, siliques Ap11g051790* Ap2g117240* FTSHI1 Lethal AT4G23940.1 946aa Ubiquitous, high in young/immature leaves Ap12g055720* FRD3 Lethal AT3G08040.1 526aa roots Ap10g040060 Ap10g040230 PDS Chlorophyll biosynthesis AT4G14210.1 566aa Ubiquitous, high in leaves Ap15g099370* During literature review, we found that published protocols generally consisted of 4 parts, including pretreatment, which prepares leaves for treatment; application method, which is the way of applying dsRNA; buffer solution, which are constituents facilitating the application of dsRNAs; and the dsRNA itself, which induces RNA silencing. We have done 25 rounds of experiments (summarized in Table 2) on amaranth and tobacco plants, and reevaluated the most popular choices for each of the 4 parts. Tobacco was included in the experiment because of the robust genetic tools and abundant genetic information available to this model species, which made it much easier to test some of our ideas. Table 2. A summary of dsRNA application experiments on Palmer amaranth and tobacco. Total number of experiments plants Target genes Pre-treatment application method Buffer solution dsRNA 17 Palmer amaranth PDS Silwet, silicon carbide treatment hand drop, infiltration Silwet L77, (NH4)2SO4, mannitol, MES Long dsRNA 8 tobacco PDS Silicon carbide treatment hand drop, infiltration Silwet L77, (NH4)2SO4, mannitol, MES, cell penetrating peptide Long dsRNA Diced dsRNA For the pre-treatment step, we found that the silicon carbide treatment was better than the Silwet L77 conditioning treatment for Palmer amaranth. Silicon carbide is a kind of abrasive, which could be used to make sand papers, so the silicon carbide treatment is essentially a gentle version of sanding. This suggested that the silicon carbide treatment probably damaged the cuticle (maybe even the cell wall) of amaranth leaves and allowed chemicals to penetrate. On the other hand, the Silwet L77 preconditioning didn'tto facilitate the penetration of ammonium sulfate into plant tissues efficiently, because only 33% (5/15 tested leaves, Figure 2) of leaves treated with Silwet L77 and ammonium sulfate exhibited some minor chemical burn damage. Since ammonium sulfate is a much smaller compound than the dsRNA molecules, it is extremely unlikely for the dsRNAs applied on leaf surface to get into plant cells without the cuticle being damaged. Other researchers also found that wounding of tissue was critical for RNA uptake through leaf surface application. Although the silicon carbide treatment is an efficient way to help topically applied chemicals penetrate into leaf tissues, when applied to tobacco, the treatment caused serious bruise (data not shown), which made it unsuitable for tobacco. Therefore, we used a different method to deliver dsRNA for tobacco. For application method, we found that infiltration is a reliable and easy method to inject the liquid solution into the apoplast space and cross the cuticle and cell wall, especially for tobacco. However, directly injecting the dsRNA solutions into tobacco leaves didn't induce the silencing of PDS gene (0/8 treated). This suggested that the even the dsRNA molecules successfully crossed the barriers of cuticle and cell wall, the last barrier, cell membrane, is able to stop the dsRNAs from further penetrating into the cytoplasm. Thus, we have uncovered this critical barrier which is largely neglected in previous researches. On the other hand, for Palmer amaranth, the cuticle is very thick and it is like a plastic layer wrapping around the leaves, which made it extremely difficult to do infiltration. Thus, a hand drop method was chosen for Palmer amaranth. For the buffer solution, we need to engineer an enabling mechanism for the dsRNA molecules to cross the cell membrane. Considering the biosafety, feasibility, price and our familiarity with the technology, we think cell penetrating peptides are our best choice. Cell penetrating peptides are small peptides that can carry cargos like protein, nucleotides and small molecules through the cell membrane. We have chosen the synthetic peptide KH9-Bp100 and Bp100-KH9 to test the efficiency of this method. These synthetic peptides contain a cell penetrating domain (Bp100, KKLFKKILKYL) and a RNA binding domain (KH9, KHKHKHKHKHKHKHKHKH), and they were shown to be able to carry siRNAs into plant cytoplasm across cell membrane previously. The last component of this technology are the dsRNA molecules which induce RNAi. We chose to synthesize the long dsRNAs first with the MEGAscript RNAi kit (Life Technologies), then these long dsRNAs were digested with a dicer-like enzyme from E.coli (ShortCut RNase III from NEB), generating small diced dsRNAs which were about 21 bp in size. Our approach combined the benefits of both long dsRNAs and siRNAs, which were lower costs, higher RNAi efficiencies and more permeable to plant cells. Taken together, our results suggest that for tobacco, the best combination of the 4 constituting parts of the RNAi technology is: no pretreatment, plus infiltration as the application method, plus cell penetrating peptides in the buffer solution, plus diced dsRNA molecules. For Palmer amaranth, the best combination is silicon carbide pretreatment, plus hand dropping as the application method, plus cell penetrating peptides in the buffer solution, plus diced dsRNA.

Publications