Location: Vegetable Crops Research
Project Number: 5090-21220-006-037-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2022
End Date: Jul 31, 2025
Objective:
Early blight (EB) caused by the fungus Alternaria solani, is one of the most significant and common diseases of potatoes in most potato growing regions worldwide. EB incidence throughout the world and here in the US has increased, possibly due to changes in climate that allow the pathogen to persist in areas that were previously free of the disease. In an effort to mitigate fungicide resistance build-up and effective control of EB, this proposed study focuses on the development of bio-control alternatives including small RNAs and virus-derived peptides for EB disease management.
Recent studies have shown that peptides and small RNAs can be trafficked across kingdoms, likely through extracellular vesicles that are small cellular membranes that bud inwardly or outwardly, often carrying cargo from one cell to another. Using such a system, plants can naturally transfer small RNAs to fungal pathogens to inhibit their virulence by turning off, or silencing, genes essential for pathogenicity and growth. Taking advantage of such a delivery system, the application of small RNAs is being explored to control some fungal diseases. Similarly, small peptides including plant and viral-derived proteins have shown to exhibit activity against plant fungal pathogens. In this study, we propose to characterize special small RNAs and viral-derived proteins for anti-fungal properties against EB.
Our specific objectives are to: 1)Identify RNA interference targets in A. solani, test their ability to silence target genes, and assay disease phenotypes following small RNA treatment; 2) Identify virus proteins that can affect A. solani virulence.
Approach:
Objective 1: To test whether small RNA treatment can affect the expression of genes in A. solani, we will first treat fungal spores with double stranded and small RNAs corresponding to A. solani genes. We have already determined that A. solani can take up small RNAs from the environment, so we will focus on testing whether the translocation of these molecules leads to the silencing of target genes. We propose to test multiple genes involved in ergosterol, chitin, and programmed cell death pathways. Targeting these pathways in other fungal plant pathogens has resulted in effective reduction in pathogen virulence. The RNA synthesis process will follow an established procedure in the cooperator lab that was previously performed with Sclerotinia sclerotiorum. We will test if the external application of RNAs leads to silencing of the gene target(s) in A. solani using quantitative reverse transcription PCR (qRT-PCR). Spores of A. solani will be incubated with dsRNA or a water control and allowed to germinate. Germinated spores will be collected 24 hours later and used for RNA extraction. This RNA will be used as a template in qRT-PCR to determine if transcription levels differ between dsRNA treated samples and the water or unrelated dsRNA control. Following confirmation of the reduction in gene expression, we will use larger scale dsRNA applications to monitor disease progression following A. solani inoculation. Detached leaves of susceptible potato will be treated (sprayed) with dsRNA and then inoculated with A. solani spores. Leaves will be photographed daily up to 7 days after inoculation and lesion areas will be calculated to determine if dsRNA applications influence A. solani growth.
Objective 2: We hypothesize that one of the 10 PVY viral-encoded proteins is a determinant of reduced virulence against A. solani. Each of the PVY encoded proteins will be cloned into our established B. subtilis secretory protein expression system for recombinant protein expression and its direct secretion into culture media. We will rapidly screen the library of B. subtilis clones encoding each PVY encoded protein cloned in the pBE-S construct, fused to a HIS-tag and the signal peptide. The level of expression and secretion of the virus-derived protein in the culture media will be tested by ELISA assay on the culture media using HIS-tag specific antibodies. We will assess the suppressive effects of cell-free filtrates containing PVY-encoded proteins on A. solani. We will compare the mycelial growth daily on PDA plates following exposure to the cell-free filtrates, and next the virulence of the fungus on potato detached leaves. To complement this experiment, we will test the anti-fungal property of the REP viral protein on EB. We hypothesize that the REP could have a broad-spectrum effect on necrotrophic pathogens like white mold and EB.
