Progress 05/15/22 to 01/14/23

Outputs
Target Audience:Silvec Biologics achieved both milestones of the phase I proposal and successfully demonstrated the efficacy of their CYVaV platform in suppressing B. cinerea. They identified nine siRNA candidate genes and generated CYVaV vectors compatible with siRNA. In vitro testing showed that dsRNA and siRNA targeting Sdhc and Cyp51 were effective in inhibiting botrytis growth. Milestone 2 involved delivering CYVaV vectors with siRNA targeting essential Sdhc genes of B. cinerea into model plants challenged with botrytis fungi, leading to an approximately 80% reduction in disease. The study highlights the potential of using the novel ula-like CYVaV vector in controlling fungal pathogens. Milestone 1: Identify siRNAs compatible with Silvec's proprietary CYVaV vector and are able to silence the expression of critical genes encoding the important targets by commercial synthetic fungicides in B. cinerea in vitro Six isolates of B. cinerea were evaluated for fungicide resistance against four active ingredients: azoxystrobin, pyraclostrobin, boscalid, and fluxapyroxad. The isolates, obtained from Texas A&M University (Bc05.10 and Bc.T4), the University of Maryland (Gp18-165 and Gp18-205), and Clemson University (7CCR11B and 7CCR27), were subjected to an in vitro mycelial growth assay on Potato Dextrose Agar (PDA) plates. The plates were supplemented with 25 μg/mL of each fungicide and 0.25% DMSO as the mock control. Bc05.10 and Bc.T4 showed significant inhibition in the presence of all four fungicides, while the other four isolates remained unaffected (Fig 1). The six isolates were further evaluated for resistance by determining their Minimal Inhibition Concentration (MIC) in Potato Dextrose Broth (PDB) medium using a liquid growth assay. Fungicide concentrations ranging from 0 to 128 μg/mL were used, and the final inoculum concentration was 1x104 CFU/mL of fungal spores. The optical density at 600 nm of the fungal growth was measured using a plate reader, and the data were analyzed using Microsoft Excel. Bc05.10 and Bc.T4 were found to be sensitive to all four fungicides, with their 90% inhibition concentrations (MIC90) less than 8 μg/mL. In contrast, the other four isolates were resistant to the tested fungicides, with none of their MIC90 values less than 128 μg/mL (Table 1). The resistance of B. cinerea was also evaluated on plant tissues. Azoxystrobin and Boscalid were sprayed onto detached leaves from 3-week-old N. benthamiana plants at a final concentration of 200 μg/mL, followed by deposition of 10 μL of a 1x106 CFU/mL spore suspension of the 7CCR27 isolate onto the leaves. Four days after inoculation, data showed that both Azoxystrobin and Boscalid were ineffective in controlling the disease caused by the resistant isolate. These findings suggest that fungicide resistance in Botrytis is a severe issue in the field. Based on recent reports, nine genes, enlisted in table 2, have been identified from the Botrytis genome, including β-Tubulin targeted by MBC fungicides; BOS1 targeted by DC fungicides; Sdha, Sdhb, and Sdhc targeted by SDHI fungicides; Cyp51 targeted by DMI fungicides; Cytb targeted by QoI fungicides; and Erg27 involved in cell wall synthesis and MRR1 involved in multidrug resistance. To investigate the inhibition of B. cinerea growth through RNA-induced silencing, a set of primers with T7 promoters were designed for amplification of fragments from various Botrytis genomic genes using high-fidelity DNA polymerase. The primers were listed in Table 2. The potential siRNA target mRNAs of the candidate genes were predicted using the sidirect2.rnai.jp website. The amplicon was designed to contain siRNA motifs predicted as functional and off-target reduced, with a seed duplex Tm below 15°C. In vitro transcription of dsRNAs was performed using T7 DNA-dependent RNA polymerase, and the siRNAs were generated using ShortCut RNase III (NEB). The resulting siRNAs, dsRNAs, and DNAs were used to assess the efficacy of RNA-induced inhibition of B. cinerea growth in a 96-well plate format, using concentrations of DNAs (0.5 to 50 ng/μL), dsRNAs (0.5 to 50 ng/μL), and siRNAs (0.1 to 10 ng/μL) with a final concentration of 1x104 CFU/mL of freshly prepared spore resuspension of the multidrug-resistant 7CCRisolates. The results, as presented in Fig. 2a, indicated that dsRNAs (~560 nt for Sdhc and ~300 nt for Cyp51) and siRNAs (~21 nt) targeting the fungal Sdhc and Cyp51 were capable of inhibiting the growth of 7CCR in PDB medium, while the fungal growth in wells containing DNAs and mock solution was not significantly suppressed. Fig. 2b further demonstrated the minimum inhibitory concentration (MIC) at 90% inhibition level 3 days post inoculation, revealing that the longer dsRNA for Sdhc (~560 nt, MIC90 at approximately 50 ng/μL) had reduced inhibition capability compared to the shorter dsRNA for Cyp51 (~300 nt, MIC90 at ~ 5 ng/μL); the MIC90 of both siRNAs for Sdhc and Cyp51 was less than 0.5 ng/μL. The efficacy of siRNA targeting the Sdhc gene in reducing fungal disease caused by Botrytis was also evaluated using a detached leaf assay. The experiment utilized fresh 7CCR isolate spores, with a final concentration of 1x104 CFU/mL, that were incubated in various concentrations of Sdhc-targeting siRNA (0 ~ 10 ng/uL) for 12 hours at 4°C. The treated spores were then deposited onto 3-week-old detached N. benthamiana leaves, and data was collected three days after inoculation. The results indicated that a concentration of 5-10 ng/uL of siRNA was able to reduce fungal damage by more than 80% (Fig 2). However, the inhibition rate induced by the siRNA was significantly reduced in a spray application assay. The assay involved spraying siRNA targeting the Botrytis Sdhc gene at concentrations of 0 ~ 80 ng/uL onto detached leaves from 3-week-old N. benthamiana plants and then depositing a 10 μL spore suspension, with a concentration of 1x106 CFU/mL. Data was collected three days after inoculation and the results, presented in Fig2, showed that even at the highest test concentration of 80 ng/uL, spraying of siRNA was unable to efficiently control the disease caused by Botrytis (less than 50% control). Milestone 2: Demonstrate that CYVaV-derived siRNAs are able to silence targeted B. cinerea critical genes and suppress pathogenicity of B. cinerea when infecting N. benthamiana The delivery of siRNAs into plants was accomplished using the CYVaV vector. Three RNA sequences (Fig 3) targeting Botrytis Sdhc gene were modified according to Silvec's proprietary structures and integrated into the CYVaV genome by replace the region from 2220-2280 nt and confirmed through Sanger sequencing. The resultant vectors were delivered into 3-week-old N. benthamiana plants via agrobacterium infiltration and the presence of the siRNA inserts was verified through sequencing 4 weeks post-infiltration. The CYVaV-infected plants were then challenged with a suspension of Botrytis spores (1x106CFU/mL) and lesions caused by the fungal pathogen were monitored over time. Data analysis revealed that the siRNA targeting the essential Sdhc gene resulted in a significant reduction by ~ 80% in disease severity, while no reduction was observed in the two other mimics targeting different regions of the Sdhc gene, indicating that the growth inhibition of the fungal pathogen is dependent on the targeted region. 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? Nothing Reported

Impacts
What was accomplished under these goals? Silvec Biologics has successfully achieved the proposed milestones in the phase I proposal and demonstratedthe concept of utilizing the proprietary CYVaV platform to suppress the destructive disease caused by B. cinerea. For Milestone 1, we conducted an investigation into the resistance of B. cinerea to commonly used fungicides and identified nine siRNA candidate genes that encode important targets of commercial synthetic fungicides in B. cinerea. We demonstrated the efficacy of both dsRNA and siRNA in targeting Botrytis Sdhc (SDHI fungicide targets) and Cyp51 (DMI fungicide targets), resulting in efficient inhibition of botrytis growth in vitro. Additionally, we successfully generated CYVaV vectors that are compatible with siRNA using proprietary structures. For Milestone 2, we delivered CYVaV vectors containing siRNA targeting B. cinerea essential Sdhc genes into model plants and subsequently challenged them with botrytis fungi. The results of this study showed that the disease caused by B. cinerea in plants infected with CYVaV siRNA vectors targeting botrytis genes was significantly reduced by approximately 80%. This work has paved the way for further utilization of the novel ula-like CYVaV vector in defeating economically importantfungal pathogens.

Publications