Melatonin is responsible for rice resistance to rice stripe virus infection through a nitric oxide-dependent pathway

Abstract

Rice stripe virus (RSV) causes one of the most important rice virus diseases of plants in East Asia. However, the molecular mechanisms controlling rice resistance to RSV infection are largely unknown. Recently, several studies presented a novel model that melatonin (MT) and nitric oxide (NO) participate in the plant-pathogen interaction in a synergetic manner. In this study, there was a difference in MT content between two rice varieties that correlated with one being susceptible and one being resistant to RSV, which suggested that MT is related to RSV resistance. In addition, a test with two NO biosynthesis inhibitors revealed that NO inhibitor were able to increase the disease incidence of RSV. A pharmacological experiment with exogenous MT and NO showed that increased MT and NO in the MT-pretreated plants led to lower disease incidences; however, only NO increased in a NO-releasing reagent [sodium nitroprusside (SNP)] pretreated plants. The expressions level of OsPR1b and OsWRKY 45 were significantly induced by MT and NO. These results suggest that rice resistance to RSV can be improved by increased MT through a NO-dependent pathway.

Keywords: Melatonin; Nitric oxide; Rice; Rice stripe virus.

Fig. 1

Different phenotypes of Nipponbare (susceptible) and Zhendao 88 (resistant) inoculated with RSV. a Symptoms of the two varieties at 28 dpi. The lower panel of (a) shows the symptomatic leaves of the representative plants. Bars = 5 cm. b Relative expression of RSV CP in the two varieties at 7, 14, 21, 28 and 35 dpi. c Disease incidences of the two varieties at 30 dpi. Thirty plants were used for each treatment in the experiment of disease incidence. All the experiments were repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. Different letters represent significantly difference at P < 0.05 according to Duncan’s multiple tests

Fig. 2

The endogenous MT content in rice plants after RSV infection. a Relative endogenous MT level of the two varieties in response to RSV pathogen infection within 24 h. The value of the control group was set at 1.0. b Disease incidence of the two varieties pretreated with 200 μM L-NAME (NOS inhibitor), or the combination of treatments with 200 μM L-NAME and 200 μM Tungstate (NR inhibitor). c Effect of inhibitors on the expressions of OsNOA1 in Nipponbare plants under normal conditions. Mock: water treated. L-NAME: 200 μM L-NAME treated. L + T: 200 μM L-NAME and 200 μM Tungstate together. All the experiments were: repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. Different letters or asterisks represent significantly difference at P < 0.05 according to Duncan’s multiple tests

Fig. 3

The resistance to RSV can be improved by the increased production of MT and NO. The high disease incidence of Nipponbare inoculated with RSV can be reduced by exogenous MT and NO. The disease incidence of Nipponbare was recorded at 30 dpi pretreated with: (a) The disease incidence of Nipponbare inoculated with RSV pretreated with four different concentrations of MT (0.1, 1, 10 and 100 μM) or (b) with five different concentrations of SNP (10, 50, 100, 500 and 1000 μM) for 12 h and inoculated with viruliferous for 3 d. c The disease incidence of Nipponbare pretreated with deionized water, 10 μM MT, 100 μM SNP, 100 μM Old-SNP, 100 μM cPTIO and 10 μM MT + 100 μM cPTIO for 12 h and then inoculation with RSV for 3 d. All data were recorded at 30 dpi. Thirty plants were used for each treatment in the experiment of disease incidence. d and (e) Nipponbare plant treated with virus-free SBPH, RSV (viruliferous SBPH), 10 μM MT or 50 μM SNP. The rice plants were inoculated by virus-free SBPH or viruliferous SBPH for 3 days, then all SBPHs were removed from the plants, the time point when SBPHs were removed was set as 0 hpi. After 0, 6, 12, 18 and 24 h, plants were taken immediately for the melatonin or NO assays. All the experiments were repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. Different letters or asterisks represent significantly difference at P < 0.05 according to Duncan’s multiple tests

Fig. 4

Transcriptional profiles of OsPR1b and OsWRKY 45. Nipponbare plants were pretreated with different treatments and then were inoculated with virus-free SBPH or RSV (viruliferous SBPH). Total RNA was extracted from rice leaves at 18 hpi. The assay was performed by qPCR and normalized against the combination of UBQ 10 and GAPDH. The values of the water-treated group were set at 1.0. All the experiments were repeated three times, and similar results were obtained. The data represent the means ± SD of triplicate measurements. Different letters or asterisks represent significantly difference at P < 0.05 according to Duncan’s multiple tests