Progress 09/01/23 to 08/31/24

Outputs
Target Audience: Nothing Reported Changes/Problems:The objective of this SBIR Phase II project is to expand on the successful outcomes of Phase I by advancing the development of a commercial process leveraging Silvec's innovative technology to effectively manage pathogenic fungi, with a particular focus on economically significant crops such as citrus and grapevines. Our research efforts have been primarily directed towards achieving the first two of four key objectives: 1) Design and optimize CYVaV-siRNA vectors incorporating multiple antifungal RNAi molecules to suppress B. cinerea and U. necator under controlled laboratory conditions and 2) Generate mother plants containing CYVaV-siRNA vectors that target B. cinerea and U. necator. As outlined in the preceding section, we have successfully designed and optimized CYVaV-siRNA vectors capable of inhibiting these fungal pathogens, including B. cinerea and the powdery mildew pathogen, in Nicotiana benthamiana. Moreover, we have successfully generated citrus plants infected with CYVaV-siRNA. However, subsequent investigations diverged from our preliminary observations--where CYVaV1 was detectable in systemic leaves six weeks post-infection in grapevines. We encountered difficulties in detecting CYVaV1 in systemic leaves after 24 weeks. Despite multiple independent attempts across various grape cultivars (Pinot Noir, Chardonnay, and Cabernet Franc), involving a total of 30 plants, we were unable to replicate the initial detection results. Comprehensive analyses, including RT-PCR and Northern blot assays, strongly indicate that CYVaV1 may not be suitable as a VIGS vector for grapevines. Consequently, we have initiated the investigation and development of alternative VIGS vectors tailored for grapevine applications. Actions taken to overcome this challenge: 1. Identification of Potential Grape VIGS Vectors: Through extensive literature review, four grape viruses causing asymptomatic infections have been identified as potential candidates for VIGS vector development: 1) Apple Latent Spherical Virus (ALSV), known as asymptomatic infections and effective RNAi activity in grapevines; 2) Grapevine Leaf Roll-associated Virus-2 SG isolate: known as asymptomatic infections and effective RNAi activity in grapevines; and 3) Umbra-like grape virus-4 (GULV-4): A newly identified virus from asymptomatic grapevines, related to Umbra viruses, showing promise as a VIGS vector. 2. Construction of Infectious Clones: We have successfully constructed full-length infectious clones for three viruses: ALSV (bipartite RNA genome ~4kb and ~7kb), GLRaV-2-SG (~16kb), and GULV-4 (~3.5kb). These constructs are currently being evaluated for infectivity in Nicotiana benthamiana and grapevines. 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?Key Upcoming Activities Objective 2: Generate Mother Plants Harboring Relevant VIGS Vectors Targeting Botrytis cinerea and Uncinula necator a) Grapevine Mother Plant Generation: Develop grapevine mother plants infected with alternative VIGS vectors, such as ALSV, GLRaV2, and GLRaV7, incorporating Silvec's proprietary RNAi fragments targeting host genes (DMR6, DND1) and pathogen-critical genes (SDHC, CYP51). b) Determine Gene Silencing Efficiency: Assess the efficiency of target gene silencing in these mother plants. Objective 3: Evaluate the Effectiveness of VIGS Vectors in Suppressing Economically Important Fungal Pathogens in Trees/Vines Under Greenhouse Conditions a) Assess Vector Efficacy: Evaluate the performance of alternative VIGS vectors in grapevines and/or sweet orange by measuring disease severity, yield, and quality in the greenhouse at the University of Maryland. b) Monitor Plant Health: Track the growth and overall health of grapevines carrying CYVaV-siRNA constructs. Objective 4: Assess Potential Off-Target Effects of RNAi and Environmental Impact of Alternative VIGS Platforms on Non-Target Organisms a) Bioinformatic and Environmental Analysis: Conduct bioinformatic analyses to identify potential off-target effects of the VIGS vectors (ALSV, GLRaV2/7-siRNA in grapevines, or CTV in sweet orange) on non-target organisms, including soil microorganisms and beneficial insects, using appropriate assays. b) Analyze and Report Results: Analyze the results of all experiments and compile comprehensive reports.

Impacts
What was accomplished under these goals? The primary objective of the SBIR Phase II project is to advance and optimize a commercial process using Silvec's proprietary technology to effectively manage and control pathogenic fungi in economically significant trees and vines, particularly citrus and grapevines. This project specifically targets fungal pathogens like Botrytis cinerea (gray mold) and Erysiphe spp. (powdery mildew), which pose serious threats to these crops, causing substantial yield losses and diminishing produce quality.The two primary objectives for the first year are: (1) to design and optimize virus-induced gene silencing (VIGS) vectors, particularly CYVaV-based vectors, incorporating antifungal RNA interference (RNAi) sequences to suppress the genes responsible for susceptibility to gray mold and powdery mildew under controlled laboratory conditions; and (2) to generate mother plants that harbor these optimized vectors, targeting the pathogens in question. To achieve the first objective, extensive literature review identified 11 host susceptibility (S) genes as potential targets, including Downy Mildew Resistance 6 (DMR6), "Defense, No Death" (DND1 and DND2), Mildew Locus O 6 (MLO6), and others involved in fungal infection processes. Fungicide targets within the pathogens themselves, such as Succinate Dehydrogenase subunit C (SDHC) and Cytochrome P450 lanosterol 14α-demethylase (Cyp51/Erg11), were also considered. The project integrated 200 to 300 base pair (bp) regions of these target genes into tobacco rattle virus (TRV) VIGS vectors, which were then introduced into Nicotiana benthamiana plants to induce gene silencing. Gene silencing efficiency was quantified using quantitative reverse transcription PCR (qRT-PCR), revealing a 50-80% silencing efficiency for most target genes. Notably, silencing NbDMR6, NbMLO6, and NbDND1 significantly reduced powdery mildew severity by up to 95%. However, silencing NbDMR6 and NbMLO6 did not suppress Botrytis cinerea, whereas silencing NbEDR1 and NbDND1 effectively inhibited Botrytis growth, reducing disease severity by more than 80%. These findings highlight the complexity of plant-pathogen interactions and the need for targeting multiple genes to achieve broad-spectrum resistance. To optimize the silencing fragments for integration into Silvec's proprietary CYVaV1 vector, bioinformatics tools were used to predict optimal small interfering RNA (siRNA) regions within the NbDMR6, NbMLO6, and NbDND1 fragments. The 98-nucleotide (nt) regions with two potential siRNA sites were selected, optimized, and integrated into CYVaV1 VIGS vectors. These vectors were introduced into N. benthamiana plants, achieving up to 75% gene silencing. Disease evaluation indicated that the CYVaV1 VIGS vector containing the optimized NbDMR6 and NbMLO6 fragments could suppress powdery mildew by 40-50%. To achieve the second objective, efforts were made to generate mother plants for grapevine and citrus that harbor the optimized VIGS vectors targeting DMR6, MLO6, and DND1 genes. Initial attempts to introduce CYVaV1 vectors into grapevine were unsuccessful, likely due to the nonhost status of grapevines for the CYVaV virus or the plants' strong antiviral defenses. Despite multiple attempts, the novel vector could not be introduced into grapevines, underscoring the technical challenges of introducing foreign genetic material into nonhost species. In contrast, the project successfully obtained citrus mother trees infected with CYVaV1 carrying the DMR6 gene-silencing RNAi fragment. However, the gene silencing efficiency in citrus was lower than in N. benthamiana, suggesting the need for further optimization to enhance silencing efficiency in citrus. Factors such as differences in virus-host interactions or the stability of the VIGS vectors in citrus may have contributed to this lower efficiency. To overcome the challenges faced with grapevines and citrus, the project is adopting alternative VIGS vectors that have been successful in commercial plants. These vectors include citrus tristeza virus (CTV) for citrus and grapevine leafroll-associated viruses 2 and 7, as well as apple latent spherical virus (ALSV), which have been proven to infect grapevines. The team has successfully introduced CTV vectors targeting citrus DMR6 and DND1 genes into citrus plants. Additionally, efforts are underway to integrate optimized RNAi fragments into ALSV for gene silencing in grapevines. These alternative vectors offer a promising solution to the technical challenges encountered with CYVaV1 and may provide effective gene silencing in both citrus and grapevines. In summary, the SBIR Phase II project represents a significant advancement in developing biotechnological solutions for managing fungal diseases in high-value crops. The focus on designing and optimizing VIGS vectors, generating mother plants, and overcoming vector integration challenges is crucial for the commercial application of this technology. Silvec's approach has the potential to provide a sustainable and effective alternative to chemical fungicides, revolutionizing disease management practices in agriculture, enhancing crop productivity, reducing environmental impact, and improving economic outcomes for farmers and the agricultural industry.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Ying X, Bera S, Liu J, Toscano-Morales R, Jang C, Yang S, et al. (2024) Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins. PLoS Biol 22(4): e3002600. https://doi.org/10.1371/journal.pbio.3002600