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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.