Establishment of virus-induced gene silencing (VIGS) system in Luffa acutangula using Phytoene desaturase (PDS) and tendril synthesis related gene (TEN)

Abstract

Background: Virus-induced gene silencing (VIGS) is a reverse genetics technology that can efficiently and rapidly identify plant gene functions. Although a variety of VIGS vectors have been successfully used in plants, only a few reports on VIGS technology in Luffa exist.

Results: In the present study, a new cucumber green mottle mosaic virus (CGMMV)-based VIGS vector, pV190, was applied to establish the CGMMV-VIGS to investigate the feasibility of the silencing system for Luffa. Phytoene desaturase (PDS) gene was initially selected as a VIGS marker gene to construct a recombinant vector. Plants infected with Agrobacterium harboring pV190-PDS successfully induced effective silencing in Luffa, and an effective gene silencing phenotype with obvious photobleaching was observed. To further validate the efficiency, we selected TEN for gene-silencing, which encodes a CYC/TB1-like transcription factor and is involved in tendril development. Luffa plants inoculated with the pV190-TEN exhibited shorter tendril length and nodal positions where tendrils appear are higher compared to those of non-inoculated plants. RT-qPCR showed that the expression levels of PDS and TEN were significantly reduced in the CGMMV-VIGS plants. Moreover, we evaluated the CGMMV-VIGS efficiency in three cucurbits, including cucumber, ridge gourd, and bottle gourd.

Conclusion: We successfully established a CGMMV-based VIGS system on ridge gourd and used marker genes to identify the feasibility of the silencing system in Luffa leaves and stems.

Keywords: Cucumber green mottle mosaic virus (CGMMV); Luffa; PDS; TEN; VIGS.

Fig. 1

Evaluation of TRV-VIGS in ridge gourd using PDS gene. A PDS gene structure in ridge gourds and pTRV2-PDS vector map used in this study. White boxes represent 5′- and 3′-UTRs, black boxes represent exons, and the black line represents introns, red boxes represent target silent fragment. B Agroinfiltration of ridge gourd leaves. C Detection of pTRV2-PDS recombinant vector fragments after VIGS treatment (Lane 1 is Marker; Lane 2 is empty pTRV2 negative control; TRV RNA2 was detected in the upper newly emerged leaves tissue of ridge gourds infiltrated with pTRV2-PDS Agrobacterium suspension (Lane 3–7). D RT-qPCR analysis of PDS in ridge gourd leaves of inoculated with pTRV1 and pTRV2 or inoculated with pTRV1 and pTRV2-PDS. Error bar indicates SE. * Stands for significant difference (P < 0.05). n = 3 biological replicates

Fig. 2

Silencing effect induced by CGMMV-VIGS vector in ridge gourd leaves. A The pV190-PDS vector map used in this study. B Photobleaching phenotype of ridge gourd leaves after inoculation with pV190-PDS for 22 d and 34 d. C Photobleaching phenotypes in different leaves of ridge gourd at 34 d. No. 2-1, 2-2, 2-3; 5-1, 5-2; 6-1, 6-2 represent different leaf positions on a single plant, whereas No. 1, 3 and 4 represent leaves from different plants. D Detection of pV190-PDS recombinant vector fragments after VIGS treatment [M: marker; Lane 1 is empty pV190 negative control; CGMMV RNA was detected in leaf tissues of ridge gourd that showed PDS silencing symptoms (lanes 2–12)]. E The relative expression of PDS after pV190-PDS recombinant vector was agroinfiltrated into ridge gourd leaves. Error bar indicates SE. * Stands for significant difference (P < 0.05). n = 3 biological replicates

Fig. 3

The utility of CGMMV-VIGS in ridge gourd using TEN gene. A The pV190-TEN vector map used in this study. B Structure diagram of cucumber and Luffa TEN gene; amino acid alignment of the TEN partial sequence of ridge gourd cloned for silencing; and the sequence of cucumber TEN gene. C Phenotype of plants with Wild type plants (WT) and infected pV190-TEN recombinant vectors at 10 and 20 dpi. D Length of tendrils at the fourth node of WT; length of tendrils at the third node of pV190-TEN recombinant vector-infected plants. E, F Length of tendrils and number of nodes of tendrils in plants with WT and infected pV190-TEN recombinant vectors at 20 dpi (due to the different nodes of each plant, the length of tendrils = the sum of the lengths of tendrils at each node/the number of nodes in the plant). G Relative expression levels of TEN genes after the pV190-TEN recombinant vector was agroinfiltrated into ridge gourd leaves. *** Stands for significant difference (P ＜0.001)

Fig. 4

Evaluation of CGMMV-VIGS efficiency in three cucurbits. A Photobleaching phenotype of ridge gourd, cucumber, and bottle gourd after silencing the PDS genes with the CGMMV-VIGS vector. B The earliest time at which photobleaching occurred in three cucurbits. (C) Number of photobleached plants and treated plants in the three cucurbits. D and E The relative expression of the PDS gene after CGMMV-VIGS recombinant vector was agroinfiltrated into cucumber and bottle gourd leaves. Error bar indicates SE. ** indicates significant difference (P < 0.01)