An Efficient Virus-Induced Gene Silencing System for Functional Genomics Research in Walnut ( L.) Fruits.

The Persian walnut ( L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV- recombinant clone. A co-culture inoculation method utilizing was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related polyphenol oxidase (PPO) genes and decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating fruit with efficient silence efficiency co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in fruits.