Although a large number of genomic data and transcriptome data of rubber tree can be obtained [9–11], but few functional genes have been proved in rubber tree[12–15]. Functional gene analysis is still a major challenge lacking efficient and reliable genetic transformation system[15, 16]. In this study, we examined Agrobacterium-mediated TRV-based VIGS in rubber tree. VIGS is an effective tool to study gene function in vivo and more than 40 viruses have been applied as VIGS vectors [17, 28–30]. TRV has been used in VIGS to silence genes in many plants [19–25]. Rubber tree is the most important rubber-producing crops, it is no reported that VIGS was applied in rubber tree. An efficient and reliable TRV-VIGS system for gene function analysis in rubber would be extremely valuable.
Phytoene desaturase (PDS) gene, encoding the firs enzyme catalyzing in the carotenoid biosynthetic pathway [31], has been applied as a reporter gene in VIGS systems. The silencing of PDS transcript will result in easily-detected photo-bleaching symptoms [19–25]. Thus, we used pTRV-HbPDS to silence HbPDS in rubber tree. Inoculation of Agrobacterium strain carrying pTRV was applied to silence HbPDS. Compare to some plants, it is difficult that Agrobacterium inoculated into leaves by a syringe. We employed rubbed leaves for agroinfiltration. Agroinfiltration of rubbed leaves resulted in higher VIGS efficiency and obvious albino symptoms in rubber tree. Agroinfiltration of rubbed leaves might increase the percentage of infected rubber tree cells, resulted higher silencing efficiencies.
In pTRV2-HbPDS infiltrated rubber tree plant developed leaves showed partial bleaching phenotypes at 60 days post infiltration. The all photo-bleaching could be observed on the upper newly developed at leaves at 80 days post infiltration. In contrast, pTRV2-MePDS-infiltrated cassava showed a photo-bleaching in the distal leaves at 20 days post infiltration [24], suggesting that different plants have different sensitivity to TRV. The typical photo-bleaching was observed in the tobacco top young leaves [32]. But in rubber tree, the photo-bleaching appeared in fully expanded leaves, and then were observed in the upper newly leaves. This is consistent with in poplar plants infiltrated with TRV2-PePDS [20], suggesting that the virus transfers from the inoculation site to the upper newly developing leaves.
In Addition, the transcript level of HbPDS in the upper leaves of HbPDS-VIGS plants was 5.1% -15.3% of that in the pTRV-GFP plants (Fig. 7), and 1.5–16.5% of chlorophyll and carotenoids content in the upper plant leaves (Fig. 8). In TRV-VIGS system, the silencing of reporter gene was 30–70% in rose [23], 28–38% in tomato [22], 37.9–53.1% in cassava [24], and 55–64% of in poplar [20]. Although reporter genes could not silenced completely, the photo-bleaching was obvious. Consistently, the transcript level of HbPDS in HbPDS-VIGS rubber trees was 5.1% -15.3% of that in the control, with photo-bleaching phenotype and a 1.5–16.5% level of chlorophyll and carotenoids content.