Delivery of progeny virus from the infectious clone of cucumber green mottle mosaic virus and quantification of the viral load in different host plants

Abstract

Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is a widely occurring tobamovirus in cucurbits. The genome of CGMMV has been used previously for the expression of foreign genes in the plant. High throughput delivery and high viral titer are important requirements of foreign protein expression in plant through virus genome-based vector, in this study, Agrobacterium containing infectious construct of CGMMV was infiltrated through syringe, vacuum and high-speed spray to N. benthamiana, cucumber and bottle gourd leaves. The success rate of systemic infection of CGMMV agro-construct through all three methods was higher (80-100%) in N. benthamiana compared to the cucurbits (40-73.3%). To determine the high-throughput delivery of CGMMV in the plant system, four delivery methods viz. rubbing, syringe infiltration, vacuum infiltration and high-speed spray using the progeny virus derived through CGMMV agro-construct were compared in the three different plant species. Based on the rate of systemic infection and time required to perform delivery by different methods, vacuum infiltration was found most efficient for the high-throughput delivery of CGMMV. The quantification of CGMMV through qPCR revealed that CGMMV load varied considerably in leaf and fruit tissues depending with the time of infection. Immediately after expression of symptoms, a high load of CGMMV (~ 1 µg/100 mg of tissues) was noticed in young leaves of N. benthamiana and cucumber. In bottle gourd leaves, the CGMMV load was far low compared to N. benthamiana and cucumber plants. In the fruit tissues of cucumber and bottle gourd higher virus load was observed in mature fruit but not in immature fruit. The findings of the present study will serve as an important base line information to produce foreign protein through CGMMV genome-vector.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-023-03630-y.

Keywords: Bottle gourd; Cucumber; Cucumber green mottle mosaic virus; High-speed spray; Nicotiana benthamiana; Progeny virus; Syringe infiltration; Vacuum infiltration; Viral titer.

Fig. 1

Delivery of CGMMV by various methods to three different plant species i.e., Nicotiana benthamiana, Cucumber cv. Hilton and Bottle gourd cv. Pusa Santhusht. A Proportion of plants expressed symptoms following delivery of CGMMV BP4 agro-infectious construct through various delivery methods i.e., syringe infiltration, high-speed spray and vacuum infiltration at 12–15 days post inoculation. Different letters i.e., a, b, c, d and e indicate significant difference between their mean value at, p < 0.05. B Average time taken for the delivery of CGGMV to three different plant species by various methods. Different letters i.e., a, b, c, d and e, indicate significant difference between their mean value at, p < 0.01. *Vacuum infiltration pressure = 8.07psi, *High-speed spray pressure = 4.1psi, *Sec = seconds

Fig. 2

Agro-infiltration of N. benthamiana with agrobacterium culture, EHA105 containing BP4 in pCambia2300 to generate progeny virus. a N. benthamiana leaf showing a symptom of CGMMV following inoculation of infectious agro-construct, b Electron microscopy showing the presence of progeny virion of agro-construct BP4 and c RT-PCR amplified product of CP gene of CGMMV (lane 1–2) from infected N. benthamiana plant; H: healthy; + ve: positive control; –ve: negative control;1 kb ladder

Fig. 3

A Syringe infiltration of progeny virus from the infectious construct of CGMMV, a: the inoculum of progeny virus maintained on ice, b, c, and d: is infiltration using 2 ml syringe to cucumber, bottle gourd and N. benthamiana respectively, e, f and g: is CGMMV infected cucumber, bottle gourd and N. benthamiana respectively, showing systemic mottling symptoms at 15 dpi. B and C RT-PCR using CP gene primers of syringe infiltrated cucumber and bottle gourd plants respectively with progeny virus sap at 15 dpi: lane 1–13 and lane 1–10 PCR amplified plant samples (amplicon size of 500 bp). + ve: positive control; −ve: negative control; H: healthy; 1 kb ladder

Fig. 4

A Vacuum infiltration of progeny virus from the infectious construct of CGMMV, a: the inoculum of progeny virus in 500 ml beaker; b: inoculum containing beaker placed inside vacuum infiltration instrument; c: plants uprooted for infiltration; d: uprooted plants were placed inside inoculum containing beaker; e: vacuum infiltrating plants in vacuum infiltration instrument by generating the pressure of 8.07psi; f, g and h: infiltrated cucumber, bottle gourd and N. benthamiana respectively; i, j and k: infected cucumber, bottle gourd and N. benthamiana respectively, showing systemic mottling symptoms at 15 dpi. B and C RT-PCR with CGMMV CP gene primers of vacuum infiltrated cucumber and bottle gourd plants respectively with progeny virus sap at 15 dpi: lane 1–11 and lane1-7 PCR amplified plant samples (amplicon size of 500 bp); + ve: positive control; –ve: negative control; H: healthy; 1 kb ladder

Fig. 5

A High-speed spray of progeny virus from the infectious construct of CGMMV, a: spraying inoculum of progeny virus, b, c and d: spraying inoculum with the pressure of 4.1psi to cucumber, bottle gourd and N. benthamiana respectively; e, f and g: infected cucumber, bottle gourd and N. benthamiana respectively, showing systemic mottling symptoms at 15 dpi. B and C RT-PCR amplification with CP gene of high-speed sprayed cucumber and bottle gourd plants respectively with progeny virus sap at 15 dpi: lane 1–12 and lane 1–7 PCR amplified plant samples (amplicon size of 500 bp); + ve: positive control; –ve: negative control; H: healthy; 1 kb ladder