Development of novel management tool to mitigate spotted wilt disease.

In our 2021 study, we applied dsRNA against Tomato Spotted Wilt Virus (TSWV) on tobacco plants under controlled conditions. We induced resistance in tobacco plants by targeting the N and NSs genes. We observed the movement of dsRNA from inoculated leaves to systemic leaves using semi-quantitative RT-PCR. Both dsRNAs reduced viral titer and symptom severity. Notably, dsRNA against the N gene resulted in only 16%, and dsRNA against NSs led to 54% of disease incidence. Overall, both dsRNAs outperformed no treatment controls, with dsRNA against N showing superior results (fig 4) (Konakalla et al, 2021).

We explored two approaches on tobacco plants: curative (TSWV inoculation followed by dsRNA application) and preventive (dsRNA followed by TSWV inoculation), targeting the N and NSs genes of TSWV. Statistical analysis reveals a substantial reduction in virus titer after treating inoculated plants with dsRNA. In both approaches, dsRNA against the N gene outperformed the NSs gene. Notably, preventive dsRNA application against the N gene significantly decreased TSWV titer in both inoculated and systemic leaves (figs 5 and 6) (Unpublished).

To further expand this research, we are aiming to target the NSm and Gn/Gc of TSWV. This will help to better understand the essentiality of these genes in the pathogenicity of TSWV. All the experiments will be conducted in the Crop Virology laboratory on the Tifton campus of the University of Georgia

The method for this project would be the following:

Objective 1:

1. To find the conserved regions to construct dsRNA against NSm and Gn/Gc genes of TSWV.

Sequences of NSm and Gn/Gc will be retrieved from NCBI GenBank as well as from different isolates collected by our lab members from various counties of Georgia, US. MEGA-X software will be used to align multiple sequences. Conserved regions of each gene will be used to design primers (Konakalla et al, 2021).

2. To produce dsRNA molecules targeting the NSm and Gn/Gc genes of TSWV.

dsRNA molecules will be produced through a two-step PCR approach followed by in vitro transcription. To begin with, total RNA from TSWV-infected N. tabaccum plants will be extracted using the Quick RNA miniprep kit (ZYMO Research, US) according to the manufacturer’s instruction. 2 μg of total RNA will be used as a template for cDNA synthesis using SuperScript™ IV Reverse Transcriptase (Thermo Scientific, US). Viral genes will be amplified with specifically designed primers. The T7 RNA polymerase promoter sequence will be added to the 5’ end of both forward and reverse primers. PCR will be performed using about 100 ng of cDNA as a template. To synthesized sense and antisense viral derived ssRNA, in vitro transcription system named T7 Ribomax^TM Express large-scale RNA production kit (Promega, Madison, US) will be used with 1 microgram of purified DNA template. After removing all the DNA from the reaction mix, dsRNA will be obtained by mixing the specific sense and antisense ssRNA. The formation of dsRNA will be confirmed by Mung Bean Nuclease treatment (NEB, US). As a negative control, dsRNA against the coat protein of cucurbit chlorotic yellows virus (CCYV) will be used.

3. To apply dsRNA molecules along with TSWV onto the tabacum.

TSWV inoculum stock will be prepared from the N. tabacum plants inoculated with isolates obtained from the field samples. Each inoculation will be prepared by homogenizing 1 g of infected plant tissue in 25 ml of phosphate inoculation buffer (pH 7.0). The plants will be inoculated at the four-leaf stage. Each leaf will receive 50 μl of inoculation buffer with TSWV on the adaxial side. 10 μg of dsRNA will be added during inoculation in treatment. There will be a total of three replicates with 10 plants for treatment (TSWV with dsRNA), 5 plants as untreated control (only TSWV), 5 as negative control (TSWV with dsRNA against coat protein of CCYV), 5 as a mock plant (only inoculation buffer with dsRNA) and 5 plants as negative control (only with inoculation buffer). This experiment will be repeated three times using both dsRNA against NSm and Gn/Gc genes of TSWV.

4. To measure the symptom severity and viral titer in both treated and untreated plants.

Three samples from each replicate will be collected at 6, 9, and 12 DPI. TSWV titer will be measured using qPCR using primers for the N gene of TSWV. The plants will be observed till 30 DPI. A severity scale of 0 to 5 will be used where, 0= no symptom, 1= chlorotic and necrotic spots in inoculated leaves, 2= initiation of necrotic spots in systemic leaves, 3= spread of necrotic spots in systemic leaves, 4= necrotic spots and vein necrosis in systemic leaves, and 5= stunted growth with severe necrosis in systemic leaves.

Objective 2:

1. To evaluate dsRNA movement inside tabacum plants.

Two leaf disks from each inoculation and the systemic leaf from the mock plants will be taken at 1-, 3-, 6-, and 9 days post-inoculation (DPI). Three plants will be used from each time point. The leaves will be washed with Triton X-100 (0.05%) and water to remove residual dsRNA from the leaf surface. cDNA will be performed using random primers and dsRNA will be quantified using q-PCR with the help of primers specific for dsRNA against NSm and Gn/Gc genes (Tabein et al, 2020).

2. To evaluate small RNA (sRNA) populations originating from dsRNA against NSm and Gn/Gc genes.

The samples used for dsRNA movement at 1 DPI will be subjected to high throughput sequencing for sRNA analysis. Small RNA libraries preparation and sequencing will be performed by BGI genomics (China). The Qiagen CLC genomics workbench will be used to process the raw data.

Objective 3:

We’ll disseminate the results to stakeholders through diverse platforms like UGA extension networks, county agents, growers’ meetings, and field tours, along with prioritizing inclusion, and equity.