The transcription factor PbeNAC83 positively regulates the Valsa canker resistance in apple and pear

【Objective】Valsa canker, caused by Valsa pyri (Vp), is a highly destructive trunk disease of pear trees, causing significant economic losses to the global pear industry. Characterized by a long incubation period, high pathogenicity, and rapid spread, this pathogen mainly invades the phloem of pear trees through wounds or stomata. It disrupts the transportation of water and nutrients, leading to the withering of trees, decline in yield, deterioration of fruit quality, the death of entire plants, and even the destruction of orchards. Existing prevention and control methods still have problems. Therefore, resistance breeding has become an effective solution to the problems. It is urgent to screen for major diseaseresistant genes and study their mechanisms. When the immune system of plants is activated upon pathogen infection during their growth and development, cellular transcriptional reprogramming occurs to regulate the expression of defense- related genes. As a vital plant- specific transcription factor family, NAC factors are significantly involved in this process. Previous studies have shown that NAC transcription factors are involved in a wide range of plant physiological processes and responses to various environmental stresses. They have achieved remarkable results in enhancing plant stress resistance. However, reports on the identification and analysis of NAC transcription factors in pears are scarce. This study aims to identify NAC transcription factors related to Valsa canker resistance, providing a theoretical basis for molecular breeding for disease resistance.【Methods】Based on transcriptome data, we screenedPbeNAC83 (Chr9.g47397) and performed bioinformatics analysis on it. The NAM-typical structural domains of PbeNAC83 were further identified using the online tools SMART. Predicting cis- acting elements using the online tool PlantCARE. Additionally, the phylogenetic relationship between PbeNAC83 and other plant homologous genes was analyzed by multiple sequence alignment and phylogenetic tree construction. To identify the potential roles of PbeNAC83 in resistance to Valsa canker, we treated the suspension cells of Duli-G03 with 20% VpM and analyzed them using qRT-PCR to detect the expression pattern of PbeNAC83 to signals from the Valsa canker pathogens. Subsequently, the CDS of PbeNAC83 was amplified and inserted into the pFGC5941 expression vector. The resulting recombinant plasmid was then introduced into Agrobacterium tumefaciens GV3101 using the freeze-thaw method. This approach was employed to elucidate the role of PbeNAC83 in conferring resistance to Valsa canker. To elucidate the role of PbeNAC83 in combating Valsa canker, we conducted transient expression experiments in the fruits of Huangguan pears (Pyrus bretschneideri) and Yanfu 3 apples (Malus×domestica), and stable expression in the suspension cells of“Duli-G03”. Moreover, we carried out infection tests on the fruits and suspension cells using Valsa pyri (Vp). The difference in PbeNAC83 overexpression in resistance to Valsa canker was studied. The Multifunctional Microplate Reader was used to assay its cellular activity after treatment with VpM. The expression levels of immune response- related genes, including those involved in pattern-triggered immunity (PTI), jasmonic acid (JA) signaling, phytoalexin production, and reactive oxygen species (ROS) pathways, were examined by qRT-PCR following the overexpression of this gene.【Results】The expression analysis results indicated that that the P. betulifolia transcription factor PbeNAC83 (Chr9.g47397) was significantly up-regulated in response to the induction by Valsa pyri Metabolites (VpM). Bioinformatics analysis indicated that PbeNAC83 belongs to the NAC family of transcription factors, and contains a typical NAM domain. In addition, the promoter region is rich in elements related to methyl jasmonate (MeJA), abscisic acid (ABA) signaling pathways, anaerobic induction, as well as other elements associated with plant disease resistance. Evolutionary analysis indicates that PbeNAC83 exhibits the highest homology with rna48275-v1.1-pbr of P. bretschneideri. Upon treatment with 20% VpM, the FPKM value of PbeNAC83 exhibited a rapid increase. As determined by qRT-PCR, the expression level of PbeNAC83 increased from 19.51 at 0 h to 211.2, 150.12, and 121.32 after 1, 3, and 6 h of treatment, respectively. Transient expression of PbeNAC83 in Huangguan pears and Yanfu3 apples significantly reduced the spreading rate of Valsa pyri (Vp). Further qRT-PCR assays confirmed that the target gene was overexpressed as we expected on the infiltrate site of fruits. PbeNAC83 was transfected into the DuliG03 suspension cells, and three wellgrown overexpression cell lines were obtained. Functional analysis demonstrated that PbeNAC83- OE cells were more resistant to Vp and VpM compared to the control. After inoculation with Vp, the spread rate of Valsa canker disease was significantly inhibited. Compared with the control, the PbeNAC83- OE1 cell line had at spread rate decreased by up to 57.9%, 67.6%, and 73.1% at 48, 60 and 72 h, respectively. Upon treatment with VpM, the activity levels in the PbeNAC83-overexpressing cell lines were elevated compared to those in the wild-type cells. Gene expression analysis revealed that key genes related to reactive oxygen species (ROS) and phytoalexin signaling pathways were significantly induced in the PbeNAC83 overexpression cell lines.【Conclusion】To summarize, PbeNAC83 is a typical NAC transcription factor associated with resistance to Valsa canker in pears, which is significantly upregulated under VpM induction, and its overexpression positively regulates the resistance of apples, pears, and DuliG03 suspension cells to Valsa canker. ROS and phytohormone signaling pathways mainly participate in the immune response regulated by PbeNAC83. These results are of great significance for a deeper un-derstanding of the mechanisms of Valsa canker disease resistance in pears. It will help to develop new disease resistance strategies, improve the disease resistance of pear trees, and promote the sustainable development of the pear industry.