Development of a robust and efficient virus-induced gene silencing system for reverse genetics in recalcitrant Camellia drupifera capsules

Abstract

Background: Virus-induced gene silencing (VIGS) is a rapid and powerful method for gene functional analysis in plants that pose challenges in stable transformation. Numerous VIGS systems based on Agrobacterium infiltration has been widely developed for tender tissues of various plant species, yet none is available for recalcitrant perennial woody plants with firmly lignified capsules, such as tea oil camellia. Therefore, there is an urgent need for an efficient, robust, and cost-effective VIGS system for recalcitrant tissues.

Results: Herein, we initiated the Tobacco rattle virus (TRV)-elicited VIGS in Camellia drupifera capsules with an orthogonal analysis including three factors: silencing target, virus inoculation approach, and capsule developmental stage. To facilitate observation and statistical analysis, two genes predominantly involved in pericarp pigmentation were selected for silencing efficiency: CdCRY1 (coding for a key photoreceptor affecting light-responsive perceivable anthocyanin accumulation in exocarps) and CdLAC15 (coding for an oxidase catalyzing the oxidative polymerization of proanthocyanidins in mesocarps, resulting in unperceivable red-hued mesocarps). The infiltration efficiency achieved for each gene was ~ 93.94% by pericarp cutting immersion. The optimal VIGS effect for each gene was observed at early (~ 69.80% for CdCRY1) and mid stages (~ 90.91% for CdLAC15) of capsule development.

Conclusions: Using our optimized VIGS system, CdCRY1 and CdLAC15 expression was successfully knocked down in Camellia drupifera pericarps, leading to fading phenotypes in exocarps and mesocarps, respectively. The established VIGS system may facilitate functional genomic studies in tea oil camellia and other recalcitrant fruits of woody plants.

Keywords: Camellia drupifera; CdCRY1; CdLAC15; Infiltration; Pericarp pigmentation; Virus-induced gene silencing.

Fig. 1

Simplified flow chart of agrobacteria preparation and infiltration into C. drupifera capsules by four approaches. TRV2-w/o vector was used as control

Fig. 2

Different infiltrating approaches to the capsules of C. drupifera at five different developmental stages. (I) Peduncle injection. (II) Direct pericarp injection. (III) Pericarp cutting immersion. (IV) Fruit-bearing shoot infusion. Five developmental stages indicated by numbers in parentheses: 1. 64 DAP, 2. 95 DAP, 3. 126 DAP, 4. 156 DAP, 5. 187 DAP

Fig. 3

Flavonoid-based pigmentation in C. drupifera ‘Hongpi’ exocarps and ‘Hongrou’ mesocarps throughout capsule development. A Capsule phenotype from the day after pollination to maturation (322 days) Scale bar (64 DAP) = 300 µm; Scale bar (other developmental stages) = 1 cm; The development of C. drupifera capsules occurs in four stages: Fruitlet, Enlargement, Maturation, and Ripening. The Fruitlet stage spans from 64 to 126 days after pollination (DAP), followed by the Enlargement stage from 156 to 217 DAP. The Maturation stage spans from 248 to 279 DAP, and finally, the Ripening stage occurs between 301 and 332 DAP. B Variations of ACN content at 10 different developmental stages of ‘Hongpi’ exocarps. C Variations of insoluble PA content at 10 different developmental stages of ‘Hongrou’ mesocarps

Fig. 4

Phylogenetic analyses of two target genes (CdCRY1 and CdLAC15) involved in percarp pigmentation and their expression profiles at different capsule developmental stages. A Maximum likelihood trees of 6 CdCRY homologs (upper) and 25 CdLAC homologs (lower) bootstrap values > 50% are shown on each clade. B Conserved domains shared by CdCRYs (upper) and CdLACs (lower) generated by PfamScan; a specific fragment of each gene (indicated by red dashed rectangles) was selected to construct recombinant TRV2. C Expression profiles of CdCRYs (upper) and CdLACs (lower) showing the relative expression level of each gene at 10 different developmental stages of capsules

Fig. 5

Variable silencing efficiency of CdCRY1 and CdLAC15. A Three main groups of pericarp morphology for TRV2-CdCRY1 (upper) and TRV2-CdLAC15 (lower) at 279 DAP. B Boxplot diagram of CdCRY1 (left) and CdLAC15 (right) showing the relative expression level in three groups. Histogram analysis of ACNs (left) and insoluble PAs (right) in three groups. C Orthogonal analyses with three factors illustrating the effects of TRV2-CdCRY1 (left) and TRV2-CdLAC15 (right) across five different developmental stages through four different infiltrating approaches to capsules