Prosystemin overexpression induces transcriptional modifications of defense-related and receptor-like kinase genes and reduces the susceptibility to Cucumber mosaic virus and its satellite RNAs in transgenic tomato plants

Abstract

Systemin is a plant signal peptide hormone involved in the responses to wounding and insect damage in the Solanaceae family. It works in the same signaling pathway of jasmonic acid (JA) and enhances the expression of proteinase inhibitors. With the aim of studying a role for systemin in plant antiviral responses, a tomato (Solanum lycopersicum) transgenic line overexpressing the prosystemin cDNA, i.e. the systemin precursor, was inoculated with Cucumber mosaic virus (CMV) strain Fny supporting either a necrogenic or a non-necrogenic satellite RNA (satRNA) variant. Transgenic plants showed reduced susceptibility to both CMV/satRNA combinations. While symptoms of the non-necrogenic inoculum were completely suppressed, a delayed onset of lethal disease occurred in about half of plants challenged with the necrogenic inoculum. RT-qPCR analysis showed a correlation between the systemin-mediated reduced susceptibility and the JA biosynthetic and signaling pathways (e.g. transcriptional alteration of lipoxygenase D and proteinase inhibitor II). Moreover, transgenically overexpressed systemin modulated the expression of a selected set of receptor-like protein kinase (RLK) genes, including some playing a known role in plant innate immunity. A significant correlation was found between the expression profiles of some RLKs and the systemin-mediated reduced susceptibility to CMV/satRNA. These results show that systemin can increase plant defenses against CMV/satRNA through transcriptional reprogramming of diverse signaling pathways.

Fig 1. Cucumber mosaic virus/satellite RNA infections alter prosystemin expression in tomato plants.

Expression of prosystemin transcript measured with RT-qPCR in tomato plants cv. Moneymaker at 9 days post-inoculation with Cucumber mosaic virus (CMV-Fny) in combination with its satellite RNAs, either necrogenic (FN) or non-necrogenic mutant (FNNmut). Gene expression is relative to mock and normalized by ubiquitin gene (ubi3). Bars on the columns represent the standard deviation (n = 6). Columns with different letters are significantly different according to LSD test (P<0.05).

Fig 2. Severity of disease symptoms induced by Cucumber mosaic virus/satellite RNA infections is reduced in transgenic tomato plants overexpressing prosystemin.

Severity of symptoms caused by Cucumber mosaic virus (CMV) combined with two satellite RNAs, either necrogenic (77-satRNA) or non-necrogenic (the mutant variant NNmut-satRNA), on tomato plants cv. Better Boy, wild type (BB) or overexpressing the prosystemin transgene (BBP+). In each graph, the disease severity scores observed on 14 plants is shown. In the BBP+/FN graph (bottom right), two groups of plants that displayed significantly different disease development are indicated with a red (8 plants showing rapid necrosis or RN) and a blue (6 plants showing delayed necrosis or DN) line.

Fig 3. Disease symptoms induced by Cucumber mosaic virus/satellite RNA infections in transgenic tomato plants overexpressing prosystemin.

Disease phenotypes in transgenic plants cv. Better Boy overexpressing prosystemin (BBP+) 21 days post-inoculation with CMV/satRNA combinations. Top Left panel, mock-inoculated healthy control (Mock); Top Right, plants inoculated with CMV/NNmut-satRNA showing suppression of symptoms (FNNmut); Bottom Left, plants inoculated with CMV/77-satRNA showing rapid and lethal necrosis [FN (RN)]; Bottom Right, plants inoculated with CMV/77-satRNA showing partial resistance and delayed necrosis [FN (DN)];

Fig 4. Both ectopic overexpression of prosystemin and Cucumber mosaic virus/satellite RNA infections trigger extensive transcriptional reprogramming.

Gene expression measured with RT-qPCR in tomato plants cv. Better Boy, wild type (BB) or overexpressing the prosystemin transgene (BBP+), at 9 days post-inoculation with Cucumber mosaic virus (CMV) in combination with the necrogenic 77-saRNA (FN) or the non-necrogenic mutant NNmut-satRNA (FNNmut). A: prosystemin; B: CMV titer, expressed as RNA2 accumulation in leaf tissues, and relative to BB/FN plants; C: marker-genes of salicylate and jasmonate signaling pathways; D: jasmonate-biosynthesis genes; E: receptor-like protein kinases. Gene expression is relative to mock-inoculated BB plants, and normalized by ubiquitin gene (ubi3). Bars on the columns represent the standard deviation (n = 6). Per each individual gene analyzed separately, columns with different letters are significantly different according to LSD test (P<0.05). Disease symptoms: ‘-‘ = no symptoms; LD = leaf distortion; RN = rapid necrosis; DN = delayed necrosis.