Molecular Dissection of Cucumis metuliferus Resistance against Papaya Ringspot Virus by Grafting

Abstract

Vegetable crops of the genus Cucumis are very popular worldwide and have great market value. However, their fruit quality and yield are hindered by viral diseases. C. metuliferus is considered a wild species with resistance to viral diseases that is lacking in cultivated crops of the Cucumis genus, such as melon. The C. metuliferus line L37 shows extreme resistance against Papaya ringspot virus (PRSV-HA), whereas line L35 is a susceptible line. In this study, reciprocal grafting experiments between L35 and L37 were performed, and the PRSV-HA strain was pre-inoculated in the rootstock leaves. The results revealed that the resistance signal in the L37 rootstock could transmit and provide resistance to the L35 scion. Subsequently, double sandwich grafting was performed using the pre-inoculated L35 as the rootstock, which was then grafted onto the L37 intermediate and the L35 scion. The results showed that PRSV-HA RNA accumulated in the L35 rootstock leaf, petiole, and stem tissues, whereas PRSV-HA RNA accumulated in some intermediate and scion petiole and stem tissues. No HCPro RNA was detected in the L35 scion leaves. The results showed that the suppression of the virus occurred in the leaves, and the resistance effect spread from the rootstock in the scion direction. Hence, this study has demonstrated that RNA silencing of systemic signals is responsible for L37 resistance against PRSV. C. metuliferus L37 could provide a valuable resistance source for crops of the Cucumis species against viral diseases through grafting.

Keywords: Cucumis metuliferus; RNA silencing; grafting; immune; virus resistance.

Figure 1

Nongrafting and reciprocal grafting of C. metuliferus against PRSV inoculation. (A) Schematic representation of reciprocal grafting between resistant C. metuliferus line PI292190 (L37) and susceptible line Acc.2459 (L35). 1–4 are nongrafted controls. 5–6 and 7–8 were self-grafting and reciprocal grafting, respectively. The PRSV-HA strain was inoculated at the rootstock, and the scion was grafted onto the rootstock. As a control, 1–2 were nongrafted, and no PRSV inoculation was performed. White plants indicate L35, and black plants indicate L37. The syringe indicates the PRSV-HA inoculation of the rootstock and the slash plug represents the grafted site. The white bar indicates 30 cm. (B) Analysis of PRSV HCPro RNA accumulation in the systemic leaves of the rootstock (Rts) and scion (Sci) grafting plants. Actin indicates the internal control; M indicates the molecular marker. n = 3.

Figure 2

PRSV induced systemic resistance in L37 rootstock. (A) Schematic and grafted plants after PRSV-HA inoculation. Nos. 1 and 2 are L35 and L37 nongrafted plants inoculated with the mock control. Nos. 3 and 4 are L35 and L37 nongrafted plants inoculated with PRSV-HA. Nos. 5–9 are L35 scions grafted onto L37 rootstocks. No. 5 is a grafted plant with a noninoculated control, and No. 6 is a grafted plant inoculated with a mock control. No. 7–9 are L35 scions grafted onto L37 rootstock and then inoculated into L37 rootstock one to three times by PRSV, and the L35 scions were inoculated the same time as the last L37 rootstock inoculation. Schematic drawing and symbols are as in Figure 1. ×2 and ×3 indicate PRSV-HA inoculated in the L37 rootstock twice and three times at two-day intervals, respectively. The white bar indicates 30 cm. (B) qRT-PCR analysis of PRSV-HA HCPro RNA accumulation in the scion L35 systemic leaves. Total RNA was extracted from the systemic leaves of four-week-old nongrafted or grafted C. metuliferus plants 7 dpi. Relative amount of HCPro RNA was normalized to Actin mRNA. Values represent the fold change, relative to the negative control, as means ± standard deviation from three biologically independent experiments. Asterisks indicate statistically significant difference (** p < 0.01 by Student’s t-test).

Figure 3

Graft transmission of virus suppression in the scions. (A) Schematic and double sandwich grafted on PRSV-HA pre-inoculated L35 rootstock. 1 is the nongrafted L35 inoculated with PRSV-HA. 2 is the L37 scion grafted onto the pre-inoculated L35 rootstock. 3 is a double sandwich grafting plant. The intermediate L37 and scion L35 were grafted onto the pre-inoculated L35 rootstock in turn. The schematic drawing and symbols are as represented in Figure 1. Rts, Int, and Sci indicate the rootstock, intermediate, and scion, respectively. The white bar indicates 10 cm. (B) Analysis of PRSV HCPro RNA accumulation in the double sandwich grafting plants. Actin indicates the internal control; M indicates the molecular marker.

Figure 4

Dissection of systemic PRSV suppression transmitted in the vascular system of double sandwich grafting plants. (A) Schematic of double sandwich grafting. The intermediate L37 and scion L35 were grafted onto the pre-inoculated L35 rootstock in turn. Each plant part was subdivided into leaf (Lf), petiole (Pe), and stem (St) tissues. The schematic drawing and symbols are represented as in Figure 1. (B) Analysis of PRSV HCPro RNA accumulation in double sandwich grafting plants. Actin indicates the internal control; M indicates the molecular marker.

Figure 5

Three PRSV RNA accumulation patterns were revealed in the double sandwich grafting plants. The intermediate (Int) L37 and scion (Sci) L35 were grafted onto pre-inoculated L35 rootstock (Rts) in turn. Each plant part was subdivided into leaf (Lf), petiole (Pe), and stem (St) tissues. Analysis of PRSV HCPro RNA accumulation in double sandwich grafting plants revealed three patterns in which three plants are shown (-1, -2, -3): (i) PRSV accumulated in the L35 rootstock and in petiole and stem tissues of intermediate L37 and scion L35. (ii) PRSV accumulated in the pre-inoculated L35 rootstock and in the petiole and stem tissues of the intermediate L37. In the ii-3 grafting plants, trace amounts of the virus were detected in the leaves of the L37 intermediate. The gel image was strengthened by inverting it. (iii) PRSV accumulated only in the pre-inoculated L35 rootstocks, and no virus was detected in the intermediate L37 and scion L35. Each gel photo represents a different biological replicate. Lf* indicates severe malformation of the leaf and petiole were combined into one sample. Actin indicates the internal control; M indicates the molecular marker.