Abstract
Tomato curly stunt virus (ToCSV) is a geminivirus from the begomovirus genus. This genus comprises tomato leaf curl virus, which have been reported to be one of the most devasting viral plant diseases worldwide. The ToCSV is a small, circular, single-stranded DNA virus. It has six open reading frames that encode proteins that are essential for its replication, encapsidation, and movement within its host plants. It does not encode for any accessory replication proteins such as DNA polymerase. As a result, it manipulates the plant’s natural physiology to create an environment that is suitable for its replication and movement. The ToCSV was initially reported in South Africa in 1997 at the Onderberg, Mpumalanga. Over 20 years it had spread to other provinces, namely, Limpopo, North West, Gauteng, KwaZulu-Natal, North Cape, and Eastern Cape. This virus has been detected in Mozambique, South Africa’s neighbouring country, and the South African strain shares more than 90% nucleotide identity with the Mozambican isolates. During viral infections, the plants will exhibit physical symptoms such as foliar chlorosis, leaf curling, plant stunting, and decreased fruit set. The ToCSV host range is not limited to tomato plants, but it can infect other plants such as Nicotiana benthamiana.
During geminivirus infections, plants will activate numerous defence systems in an attempt to stop infection. One such defence strategy is innate immunity. Innate immunity is a multitiered plant defence strategy that includes pathogen-associated molecular patterns (PAMPs)-triggered immunity and effector-triggered immunity eventually resulting in a hypersensitive response or programmed cell death. This type of immunity is characterized by an accumulation of reactive oxygen species and plant hormones, a calcium influx, and the phosphorylation and dephosphorylation of proteins. Pathways activated during innate immunity can be triggered by the use of priming agents. This type of resistance is called induced resistance (IR). IR can be achieved by priming plants with agents such as volatile organic compounds, beneficial microorganisms and phytohormones. Plant priming boosts the plant's resistance by keeping the plant in a state of alertness before infection and it reduces the lag time required for the plant to mount defence. In this study, the exogenous effects of exogenous salicylic acid and methyl-jasmonate were investigated on plant hormone-responsive pathogenesis-related genes in response to ToCSV infections. The objectives of this study were to activate IR by exogenous application with naturally occurring compounds in susceptible and
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tolerant tomato plants. The molecular of IR was determined by quantifying gene expression of a total of eight phytohormone-responsive genes.
Firstly, an infectivity assay was conducted to assess the phenotypic effects of phytohormone treatment followed by ToCSV infection in susceptible (cv. Moneymaker) and tolerant (cv. Rodade) tomato cultivars. Tomato seedlings were treated with deionised water (ddH2O), 0.05mM methyl-jasmonate (MeJA), 0.1mM MeJA, 1mM salicylic acid (SA), and 2mM SA as well as 1mM SA combined with 0.1mM MeJA. An untreated ToCSV inoculated group was set up in parallel to the treatment groups. The seedlings were treated for 3 consecutive days and infected with ToCSV infectious clones via Agrobacterium-based inoculation. The symptom severity and presence were determined at 8 days post-inoculation (dpi) (early infection), 15 dpi (onset of symptoms), and 35 dpi (fully symptomatic) using a disease severity index (DSI) and using conventional polymerase chain reaction (PCR). For the susceptible cultivar, none of the pretreated plants had a DSI higher than the untreated control group. In the tolerant plants, the MeJA plants had DSI higher than the untreated group at 15 dpi. Screening for the presence of the viral ToCSV DNA revealed that only the untreated susceptible plants inoculated with ToCSV were positive for infection. Those were the plants that were subjected to qPCR for relative quantification against 18S (internal control). The viral ToCSV titre continued to grow with the progression of the study.
One of the markers of plant defence is the increased accumulation of reactive oxygen species. In this study, hydrogen peroxide (H2O2) was measured using luminol chemiluminescence. In both cultivars, the treated and untreated ToCSV infected plants had a higher concentration of H2O2 than the mock inoculated. In the tolerant cultivar, an increase in H2O2 correlated with an increase in plant height. The susceptible plants had an H2O2 concentration peak. Additionally, superoxide dismutase (SOD) gene expression was quantified against Actin 7 (ACT 7). The expression of SOD was upregulated by MeJA treatment at all time points in the tolerant cultivar. However, the gene was differentially regulated, across timepoints, in the ToCSV-infected susceptible plants that were pretreated, as well as the SA- treated tolerant plants.
Eight candidate genes, known to be involved in the JA pathway (LoxD, MYC2, PR3, and PR4) and SA pathway (NPR1, WRKY40, PR1, and GRX) were investigated. To achieve this objective, total RNA was isolated from 4 samples per group. The RNA was reverse-transcribed to complementary DNA and subjected to qPCR for gene expression quantification. ACT 7 served as the endogenous control. qPCR results were analyzed using the 2^-ΔΔCt Gene
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expression was quantified for each time point. The genes were differentially expressed. However, treatment with 0.1mM MeJA induced all genes studied and at all time points.
Hexanoic acid (HA) is a fatty acid that has been used as a priming agent against bacterial, fungal, and viral infections. A preliminary study was conducted to investigate the effects of HA on susceptible and resistant tomatoes infected with ToCSV. The plants were treated with ddH2O, 0.6mM, and 1.2mM HA and thereafter agro-inoculated with ToCSV 24 hours after the regime. Phenotypical assessment and leaf harvesting were conducted at 15 and 30 dpi. Both tomato cultivars showed milder symptom scores. The susceptible plants had the ToCSV viral DNA detected by conventional PCR at 30 dpi. The resistant cultivar has one positive plant per treatment and at 15 dpi only. Viral load was quantified for the susceptible plant at 35 dpi. The ddH2O treated plants had the highest viral load compared to the HA-treated plants. Three candidate genes (LoxD, PR1, and GRX) were investigated. GRX and LoxD were upregulated in both cultivars and time points. However, PR1 expression was differential.
In conclusion, IR was mounted in the tomato seedlings by treatment with phytohormones and HA. Exogenous pretreatment resulted in milder symptoms and delayed detection of ToCSV. IR was achieved by the differential regulation of the genes studied. Both SA and JA pathways play significant roles in the defence against the ToCSV.