Cucumber mosaic virus-induced gene and microRNA silencing in water dropwort (Oenanthe javanica (Blume) DC)

Abstract

Water dropwort (Oenanthe javanica (Blume) DC), an aquatic perennial plant from the Apiaceae family, rich in dietary fibert, vitamins, and minerals. It usually grows in wet soils and water. Despite accumulating the transcriptomic data, gene function research on water dropwort is still far behind than that of the other crops. The cucumber mosaic virus (CMV) induced gene silencing was established to study the functions of gene and microRNA (miRNA) in the water dropwort. CMV Fast New York strain (CMV-Fny) genomic RNAs 1, 2, and 3 were individually cloned into pCB301 vectors. We deleted part of the ORF 2b region and introduced recognition sites. A CMV-induced gene silencing vector was employed to suppress the expression of endogenous genes, including phytoene desaturase (PDS). In order to assess the efficacy of gene silencing, we also cloned conserved sequence of gibberellin insensitive dwarf (GID1) cDNA sequences into the vector and inoculated the water dropwort. The height of CMV-GID1-infected plants was marginally reduced as a result of GID1 gene silencing, and their leaves were noticeably longer and thinner. Additionally, we also used a CMV-induced silencing vector to analyze the roles of endogenous miRNAs. We used a short tandem target mimic approach to clone miR319 and miR396 from water dropwort into the CMV vector. Plants with CMV-miRNA infection were driven to exhibit the distinctive phenotypes. We anticipate that functional genomic research on water dropwort will be facilitated by the CMV-induced gene silencing technique.

Keywords: Cucumber mosaic virus (CMV); Gibberellin-insensitive dwarf 1; Oenanthe javanica; Virus-induced gene silencing (VIGS); miRNA.

Fig. 1

Construction of the CMV VIGS vector. A, Construct map of CMV-Fny. B, Symptoms of CMV-Fny in O. javanica (4 wpi). Scale bars are 2 cm. C, Detection of CMV-Fny-infected O. javanica by agro-infection (4 wpi). The vector plasmid with the CMV RNA3 insert was amplified as the positive (lane P) control. D, Construct map of the CMV-Fny 2b deletion mutant. E, Symptoms of the CMV-Fny 2b deletion mutant in O. javanica. Scale bars are 2 cm. F, Detection of CMV-Fny 2b deletion mutant-infected O. javanica by agro-infection (4 wpi). The vector plasmid with the CMV RNA3 insert was amplified as the positive (lane P) control

Fig. 2

Silencing of the OjPDS gene in O. javanica using the CMV VIGS vector. A, Construct diagram of infectious clones of pCB301CMV-OjPDS. B, RT‒PCR detection of the PDS^N and PDS^C of O. javanica. C, Phenotypes of pCB301CMV-OjPDS in O. javanica (4 wpi). Scale bars are 2 cm. D, Detection of pCB301CMV-OjPDS-infected ‘Fq1’ by agro-infection (4 wpi). The vector plasmid with the CMV RNA3 insert was amplified as the positive (lane P) control. E, Detection of pCB301CMV-OjPDS-infected ‘Yzcbq’ by agro-infection (4 wpi). The vector plasmid with the CMV RNA3 insert was amplified as the positive (lane P) control

Fig. 3

Silencing of the OjGID1 gene in O. javanica using the CMV VIGS vector. A, Construct diagram of infectious clones of pCB301CMV-OjGID1; B, RT‒PCR detection of GID1 of ‘Yzcbq’. C, Phenotypes of pCB301CMV-OjGID1 in ‘Yzcbq’ (30 dpi). Scale bars are 2 cm. D, Systematic leaf morphology analysis of pCB301CMV-OjGID1 in ‘Yzcbq’ (30 dpi). E Detection of pCB301CMV-OjGID1-infected O. javanica by agro-infection (30 dpi). F RT-qPCR analysis of OjGID1 expression levels in CMV△2b- or CMV-OjGID1-infected plants. G RT-qPCR analysis of GA-related genes expression levels in CMV△2b- or CMV-OjGID1-infected plants

Fig. 4

Silencing of miR319 in O. javanica using the CMV-based VbMS vector. A, Schematic representation of VbMS of miR319. B, Phenotypes of plants inoculated with CMV-STTM319 at 40 dpi. C, Systematic leaf morphology analysis of pCB301CMV-STTM319 in ‘Fq1’ (40 dpi). Scale bars are 2 cm. D, RT-PCR detection confirmed the expression of STTM319 in CMV-STTM319-infected plants. E, Stem-loop RT-qPCR analysis of miR319 expression levels in CMV△2b- or CMV-STTM319-infected plants. F, Detection of the relative expression levels of the miR319 target genes TCP2 and TCP4

Fig. 5

Silencing of miR396 in O. javanica using the CMV-based VbMS vector. A, Schematic representation of VbMS of miR396. B, Phenotypes of plants inoculated with CMV-STTM396 at 40 dpi. C, Systematic leaf morphology analysis of pCB301CMV-STTM396 in ‘Fq1’ (40 dpi). Scale bars are 2 cm. D, RT‒PCR detection confirmed the expression of STTM396 in CMV-STTM396-infected plants. E, Stem‒loop RT‒qPCR analysis of miR396 expression levels in CMV△2b- or CMV-STTM396-infected plants. F, Detection of the relative expression level of the miR396 target gene Growth-Regulating Factor 9-like