Establishment of a VIGS system for the CpPDS gene in Manaohong cherry seedlings

【Objective】Cherry trees are woody perennials characterized by high genetic heterogeneity and a extended juvenile phase, which typically lasts 5-7 years. These attributes considerably constrain the efficiency of conventional hybrid breeding for trait improvement. Furthermore, the establishment of homologous genetic transformation systems in cherries remains technically challenging due to difficulties in plant regeneration and protracted transformation cycles, which significantly impede in-depth functional genomic research. Cerasus pseudocerasus Lindl. (Manaohong cherry), a distinctive fruit tree resource native to Guizhou Province, has been suggested by phylogenetic studies to potentially represent a hybrid progeny derived from crosses between European sweet cherry and Chinese sour cherry. The high genetic diversity of Manaohong cherry renders it a highly valuable system for a wide range of research applications, including cultivar improvement, stress resistance studies, and comparative functional genomics between Chinese and European cherry varieties. Virus-induced gene silencing (VGS) is a posttranscriptional gene regulatory technique that utilizes modified viral vectors to introduce host- derived gene sequences into plant tissues, leading to sequence-specific degradation of target mRNAs. This method facilitates rapid and transient suppression of endogenous gene expression without introducing heritable genetic changes. As a potent reverse genetics tool, VIGS allows high-throughput functional analysis of candidate genes across diverse species, and is particularly suited for studying gene function in recalcitrant perennial plants that are difficult to transform stably. Compared to stable transgenic approaches or CRISPR-based gene editing, VIGS provides distinct advantages such as shorter experimental timelines and broader host compatibility. While VIGS has been widely implemented in herbaceous model plants such as tomato and tobacco, its application in woody Rosaceae species, especially Manaohong cherry, has remained unexplored, primarily due to challenges such as viral vector–host incompatibility and low infection efficiency. In this study, we established for the first time an efficient TRV-mediated VIGS system in Manaohong seedlings. This breakthrough demonstrates the feasibility of using VIGS for functional gene studies in this woody fruit crop and provides a robust new tool for investigating molecular mechanisms in woody plants.【Methods】The TRV-mediated VIGS vector pTRV2-CpPDS was constructed using seamless cloning technology. A 300 bp fragment of the CpPDS gene was amplified by PCR with specific primers CpPDS-F and CpPDS-R, designed to include 21 bp homologous arms matching the termini of the pTRV2 vector linearized by Xba Ⅰ and Kpn Ⅰ. The PCR-amplified fragment was subsequently inserted into the linearized pTRV2 vector via seamless assembly, yielding the recombinant plasmid pTRV2-CpPDS. Next, the recombinant pTRV2-CpPDS vector harboring the Phytoene desaturase (PDS) gene was successfully introduced into competent cells of Agrobacterium tumefaciens strain GV3101 using a freeze-thaw transformation method. This genetic transformation procedure employed cyclic temperature shocks between liquid nitrogen/-80 ℃ and 37 ℃ to facilitate plasmid-membrane fusion, thereby achieving highly efficient transfer of the exogenous genetic material. Subsequently, Agrobacterium cultures carrying pTRV2-CpPDS were adjusted to an OD600 of approximately 1.0 and mixed at a 1∶1 volume ratio with cultures containing the helper plasmid pTRV1. After static incubation at 28 ℃, the Agrobacterium culture was used to infect Manaohong cherry seedlings. During the inoculation procedure, a sterile needleless syringe was used to aspirate the bacterial suspension, which was then slowly injected into the abaxial surface of all true leaves of the Manaohong cherry until the entire leaf surface was fully infiltrated. After inoculation, the plants were cultured in darkness at 24 ℃, followed by normal culture with 16h of light and 8h of darkness. The efficiency of TRV-mediated VIGS was assessed through phenotypic analysis, semi- quantitative RT-PCR, and quantitative real-time PCR (qRT-PCR).【Results】At 7 days post- infection (dpi), newly developed leaves of pTRV2- CpPDS- infiltrated Manaohong cherry plants began to exhibit chlorosis, whereas leaves of wild-type (WT) and empty vector (EV)-transformed plants showed no phenotypic alterations. The chlorotic symptoms progressively intensified and expanded over the subsequent three weeks, indicating effective induction of gene silencing. By 45 dpi, WT and EV control plants maintained normal green pigmentation, whereas the newly emerged leaves of pTRV2- CpPDS-silenced plants had displayed severe photobleaching. To quantitatively evaluate the efficiency of TRV-mediated VIGS, semi-quantitative RT-PCR was performed to analyze the transcript levels of endogenous CpPDS in leaves collected at 45 dpi from photobleached regions of the silenced plants. Compared with wild-type (WT) and empty vector (EV) controls, the expression of CpPDS was significantly downregulated in pTRV2-CpPDS-infiltrated plants.【Conclusion】The CpPDS gene was effectively silenced in Manaohong cherry seedlings infected with pTRV2-CpPDS. Compared with the WT and EV controls, the newly developed leaves of the pTRV2-CpPDS-infiltrated plants exhibited pronounced chlorotic phenotypes, confirming the successful implementation of a TRV-mediated VIGS system in this woody species. These results demonstrate the considerable potential of VIGS as a powerful tool for functional gene studies in fruit trees and offer a viable alternative to stable genetic transformation. In the future, this system may become an important tool for studying molecular regulatory mechanisms and functional genes of developmental metabolic networks in Manaohong cherries.