Virus-induced gene silencing for in planta validation of gene function in cucurbits

Abstract

Virus-induced gene silencing (VIGS) is a powerful tool for high-throughput analysis of gene function. Here, we developed the VIGS vector pCF93, from which expression of the cucumber fruit mottle mosaic virus genome is driven by the cauliflower mosaic virus 35S promoter to produce viral transcripts in inoculated plants. To test the utility of the pCF93 vector, we identified candidate genes related to male sterility (MS) in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. Specifically, we exploited previously reported reference-based and de novo transcriptome data to define 38 differentially expressed genes between a male-sterile line and its fertile near-isogenic line in the watermelon cultivar DAH. We amplified 200- to 300-bp fragments of these genes, cloned them into pCF93, and inoculated DAH with the resulting VIGS clones. The small watermelon cultivar DAH enabled high-throughput screening using a small cultivation area. We simultaneously characterized the phenotypes associated with each of the 38 candidate genes in plants grown in a greenhouse. Silencing of 8 of the 38 candidate genes produced male-sterile flowers with abnormal stamens and no pollen. We confirmed the extent of gene silencing in inoculated flowers using reverse transcription-qPCR. Histological analysis of stamens from male-fertile and male-sterile floral buds and mature flowers revealed developmental defects and shrunken pollen sacs. Based on these findings, we propose that the pCF93 vector and our VIGS system will facilitate high-throughput analysis for the study of gene function in watermelons.

Figure 1

VIGS of the PDS gene results in a photobleaching phenotype in N. benthamiana. A, Schematic diagrams of the vectors pCF93-NbPDS and pCF157-NbPDS and the nucleotide sequence around the MCS. XhoI and PmeI are the restriction sites to clone the insert of interest. 35S, cauliflower mosaic virus 35S promoter; MP, triple gene block. B and C, comparison of VIGS phenotypes in N. benthamiana plants infected by pCF93-NbPDS and pCF93K-NbPDS (B) or pCF157-NbPDS and pCF157K-NbPDS (C) at 12 dpi. Of the four constructs tested, pCF93 exhibited the strongest silencing. Scale bars, 2 cm.

Figure 2

Photobleaching was observed not only in leaves but also in stems and flowers in cucurbit plants. A, Photobleaching phenotypes and relative PDS expression levels in N. benthamiana leaves infected with pCF93-NbPDS or wild-type CFMMV. Scale bars, 2 cm. B, Comparison of gene silencing efficiency in melon leaves infected with p35SCF-Cm_flc (top, left) or pCF93-CmPDS (top, right). pCF93-CmPDS-induced VIGS phenotypes in melon grown in the greenhouse (bottom). Relative PDS expression levels are shown on the right. Scale bars, 3 cm. C, Photobleaching phenotypes on watermelon leaves and stems infected with pCF93-CcPDS (left) and relative PDS expression levels in leaves inoculated with pCF93-CcPDS or wild-type CFMMV. Scale bars, 3 cm. D, Photobleaching of leaves and flowers from cucumber plants inoculated with pCF93-CsPDS (left). Scale bars, 3 cm (leaf) and 1 cm (flower). Right: relative PDS expression levels in leaves and flowers from cucumber plants inoculated with p35SCF-Cm_flc (CF-leaf and CF-flower) and pCF93-CsPDS (PDS-leaf and PDS-flower). Relative PDS expression levels were determined using PDS-specific primers and normalized to GAPDH (A) or 18S rRNA expression levels by RT–qPCR (B, C, and D). The relative expression levels in CFMMV-Cm samples were set to 1. Significant differences were determined using a Student’s t test (^****P < 0.0001 and 0.001 <^**P <  0.01).

Figure 3

Estimation of lycopene and β-carotene contents in watermelon fruits silenced for PDS. A, VIGS phenotypes in the fruits of three watermelon cultivars. Control fruits are on the left, and VIGS fruits are on the right. Scale bars, 2 cm. B and C, Lycopene (B) and β-carotene (C) contents in the flesh of watermelon fruits inoculated with p35SCF-Cm_flc (CF) or pCF93-CcPDS (PDS). D, Lycopene and β-carotene contents, as determined by HPLC. E, Semi-quantitative RT–PCR analysis of PDS expression levels using a CcPDS-specific primer set. F, Contents of chlorophyll a and b in the peels of watermelon fruits inoculated with p35SCF-Cm_flc (CF) or pCF93-PDS vectors. G, Total chlorophyll contents. Watermelon cultivars “Chris cross,” “DAH,” and “2401” infected by p35SCF-Cm_flc are denoted as “CC-CF,” “DAH-CF,” and “2401-CF,” respectively. “Chris cross,” “DAH,” and “2401” infected by pCF93-ccpds are denoted as “CC-pds,” “DAH-pds,” and “2401-pds,” respectively.

Figure 4

Male-sterile flowers develop on watermelon plants silenced for candidate genes via VIGS. Complete male sterility was observed in some silenced plants, while other plants exhibited partial male sterility, with flowers having various degrees of male sterility. Mock, representative flowers from watermelon plants inoculated with pCF93-PDS-int; MS phenotype, watermelon plants silenced for EXPA9. Partially abnormal male-sterile flowers developed on LIM-, MAD2-, FAP5-, and PG-silenced plants (from left to right). Arrows indicate aborted stamen. Scale bars, 0.5 cm.

Figure 5

Histological analysis of stamens from floral buds and mature flowers of mock-inoculated and silenced plants. The floral buds of plants inoculated with pCF93-PDS-int (mock) formed normal pollen sacs containing pollen grains. In contrast, floral buds of plants silenced for a candidate gene (EXPA9 here) had no pollen. Pollen sacs of mature mock flowers displayed a normal morphology, but those of silenced plants were atrophied (arrow). Scale bars, 200 and 500 µm.

Figure 6

Relative expression levels of male sterility-related candidate genes in VIGS plants. Relative transcript levels were determined by RT–qPCR and normalized to 18S rRNA levels. The relative transcript levels of the eight candidate genes were measured in inoculated plants and mock controls. Relative expression levels in the mock control inoculated with pCF93-PDS-int were set to 1. Significant differences were determined using one-way ANOVA, followed by Tukey’s post-hoc test for multiple comparisons.