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The Persian walnut ( Juglans regia  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- JrPDS  recombinant clone. A co-culture inoculation method utilizing  Agrobacterium  was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for  J. regia  fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the  JrPDS  transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related  J. regia  polyphenol oxidase (PPO) genes  JrPPO1  and  JrPPO2  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  J. regia  fruit with efficient silence efficiency  via  co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in  J. regia  fruits.

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