An efficient virus-induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl virus (CaLCuV)-based vector

Abstract

Background: Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, represents an effective technology for the analysis gene functions in plants. However, no VIGS vectors are available for Brassica oleracea till now. The gene silencing vectors TRV, pTYs and CaLCuV were chose to improve the VIGS system in cabbage, using phytoene desaturase (PDS) gene as an efficient visual indicator for VIGS. Results: We successfully silenced the expression of PDS and observed the photobleaching phenomenon in cabbage by pTYs and CaLCuV, with the latter displaying higher efficiency and lower cost. Then, the factors potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including targeting fragment strategy, inoculation method and incubation temperature, were compared. The optimized CaLCuV-based VIGS system is as follows: approximately 500 bp insert sequence, Agrobacterium OD 600 =1.0, vacuum osmosis method at the bud stage, and the incubation temperature of 22 °C, through which we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected two other genes in cabbage including Mg-chelataseH subunit (ChlH) and lobed-leaf 1 (LL1) to knock down their expression, and we observed the expected yellow or lobed leaves. In addition, we successfully applied the CaLCuV-based VIGS system in two other representative Brassica crops including B. rapa and B. nigra and thus expanded its application scope. Conclusion: These results demonstrate that CaLCuV-based VIGS effectively work in cabbage and can be used for analyzing gene function. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crops. B. oleracea accession '87-534', B. rapa cultivar 'Zhongbai 76', B. nigra accession 'Juntus' and B. campestris cultivar '49 Caixin' were provided by Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (IVF-CAAS). Plants were incubated in a mixture of compost: vermiculite (1:1) in a growth chamber, with a 16 h photoperiod and 50% relative humidity. Roots, stems and leaves with a photobleaching phenotype were harvested and stored at − 80

Advancing Virus-Induced Gene Silencing in Sunflower: key factors of VIGS spreading and a novel simple protocol

“…Notably, the most commonly used agroinfiltration method for VIGS is the syringe infiltration of leaves. This methods has been applied in VIGS studies on a broad range of plant species: Brassica oleracea [ 3 ], Gerbera hybrid [ 45 ], Solanum lycopersicum [ 46 ], lettuce [ 47 ], Oryza sativa [ 48 ], Triticum aestivum [ 31 ], Eschscholzia californica [ 49 ], Capsicum annuum [ 50 ], Gossypium hirsutum [ 51 ]. In sunflower [ 38 ], using syringe infiltration, the authors observed the photo-bleaching phenotype in expanded leaves and leaflets near the inflorescence.…”

“…Virus-Induced Gene Silencing (VIGS) has emerged as a powerful tool in many areas of crop research, including functional gene analysis [ 1,2,3 ], pathogen resistance studies [4, 5, 6 ], abiotic stress tolerance [ 7, 8, 9 ], and general crop improvement [ 10 ]. The number of search projects dedicated to the VIGS technology is booming, highlighting its widespread adoption and recognition as a versatile tool for advancing crop science and biotechnology.…”

Geminivirus-Derived Vectors as Tools for Functional Genomics

“…CaLCuV-based GIGS involved the use of Agrobacterium and was relatively inexpensive and had a higher batch efficiency target. This geminiviral vector system has also been said to be equally efficient in other Brassica species, B. rapa, and B. nigra, and therefore a potent functional genomic tool for crop plants (Xiao et al, 2020).…”

Genome-Wide Identification of the U-Box E3 Ubiquitin Ligase Gene Family in Cabbage (Brassica oleracea var. capitata) and Its Expression Analysis in Response to Cold Stress and Pathogen Infection

“…Moreover, Brassica oleracea is also considered to be a model for the study of the genomic evolution of Brassica species due to its typical diploid genome structure [ 26 ]. The molecular mechanisms underlying many significant biological processes, including pigment accumulation [ 27 ], self-incompatibility [ 28 ], and flowering [ 29 ], have been dissected in Brassica oleracea , while a lot of classic biotechnologies, including virus-induced gene silencing (VIGS) [ 30 ] and CRISPR/Cas9-based gene editing [ 31 ], have also been applied in Brassica oleracea . Thus, the discovery of functional genes in Brassica oleracea is required for the genetic improvement of desired traits by the most advanced molecular breeding strategies.…”