Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco

Abstract

Background: The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results: Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion: We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

Keywords: Apoplast; Plants; RNA viruses; RNases; Resistance.

Fig. 1

Virus resistance in transgenic (ESR) and control plants. Two typical plants are shown for each ESR line and the control. Disease symptoms were observed in control plants, whereas ESR plants had normal phenotypes

Fig. 2

Western blot analysis of the accumulation of CMV after inoculation in ESR and control plants. Protein samples from uninoculated upper leaves were subjected to electrophoresis and blotting. Immunological detection using an anti-CMV coat protein antibody was examined using protein samples from 6 plants in each line

Fig. 3

RNase activity in the crude extract and apoplast fractions from ESR and control plants. Relative activity is shown with standard errors (n = 8 for the crude extract and n = 3 for apoplasts). Significant differences at P < 0.01 and no significant differences between ESR and control plants, calculated using the t-test, are indicated by ** and ns, respectively