Rvi6-mediated defense mechanisms against apple scab

Abstract

Background

Apple scab, caused by the hemibiotrophic fungus Venturia inaequalis (cooke) Wint., is a globally prevalent disease that severely threatens apple yield and fruit quality. Although the key resistance gene Rvi6 (resistance to Venturia inaequalis 6) has been widely deployed in apple scab-resistant breeding programs, the molecular mechanisms underlying its resistance phenotype remain poorly characterized.

Results

In this study, we generated transgenic apple calli overexpressing Rvi6 and systematically investigated both its resistance phenotype and underlying molecular mechanisms. The Rvi6 gene presented high expression levels in leaves and fruits throughout the growth cycle, and aligning with the infection window of V. inaequalis. Rvi6 overexpression significantly reduced the levels of IAA (indole-3-acetic acid), ABA (abscisic acid), and JA (jasmonic acid), and auxin signaling, as well as the callus growth, which directly evidencing the “growth-defense trade-off” hypothesis on Rvi6-mediated apple scab resistance. The marked inhibition of V. inaequalis infection in Rvi6-overexpressing calli was attributed to increased ROS (reactive oxygen species) scavenging capacity, increased osmolyte accumulation, and maintenance of plasma membrane integrity. Additionally, Rvi6 induction depressed apple growth by reducing auxin accumulation and attenuating auxin signaling. Transcriptome analysis revealed that multiple biological processes and signaling pathways are involved in Rvi6-mediated disease resistance. Pathways related to plant‒pathogen interactions, lipid and amino acid metabolism, and flavonoid biosynthesis were significantly enriched among the upregulated pathways. Conversely, plant hormone signal transduction, protein processing and modification, and carbohydrate metabolism were enriched predominantly in the downregulated pathways.

Conclusions

Rvi6 exhibits high expression in leaves and fruits across the growth cycle, aligning with the infection window of V. inaequalis. Rvi6 enhances ROS scavenging capacity, osmolyte accumulation, and plasma membrane integrity, as well as suppresses apple growth, thereby restricting V. inaequalis invasion. Plant immune responses mediated by Ca²⁺ or MAPK cascade reactions, plant hormone signaling and multiple secondary metabolic mechanisms, contribute to Rvi6-mediated resistance against apple scab. This study provides novel insights into the biological functions of Rvi6.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-07117-1.

Introduction

Apple scab is caused by the hemibiotrophic fungus Venturia inaequalis (cooke) wint. The pathogen primarily infects leaves and fruits, causing leaf and fruit abscission, cracked and deformed fruits, dark green mildew layers or black lignified lesions on fruit surfaces, and pulp lignification. These symptoms not only severely compromise fruit quality but also lead to weakened tree vigor and substantial yield losses. The natural resistance locus Vf was first identified in hybrid progeny using Malus floribunda 821 as a parent, from which the dominant single gene Rvi6 (formerly HcrVf2) was subsequently cloned [1]. Currently, MABs (molecular marker-assisted breeding) based on the Vf locus have been widely adopted in apple scab resistance breeding programs [2]. Notably, the genetic basis of most contemporary apple scab resistance breeding relies on the Vf resistance locus. Scab resistance traits derived from other wild apple species, such as Malus baccata and Malus prunifolia, are either directly attributed to the Vf locus or exhibit high homology to it [1].

The prolonged use of germplasms with a single resistance source often triggers resistance breakdown, primarily due to the emergence of pathogen variants capable of overcoming host resistance. For example, the Ahrensburg pathotype of V. inaequalis (subsequently designated race 6) induces disease symptoms in various resistant cultivars, such as ‘Prima’ (which relies on Vf-based resistance), whereas M. floribunda 821 and the ornamental crabapple ‘Evereste’ remain asymptomatic. Subsequent research revealed that M. floribunda 821 harbors an additional resistance locus, Vfh, which was unintentionally lost during breeding programs [1]. Later, Roberts and Crute identified race 7, which is virulent toward M. floribunda 821 and multiple Vf-resistant cultivars [3]. To date, the identified races of V. inaequalis are classified into approximately 7 categories, each displaying distinct virulence profiles against different resistance genes. In addition to Vf, other resistance loci have been identified, such as Vm (derived from Malus micromalus and Malus atrosanguinea), Vb, Vbj (both from M. baccata), and Vr (from Malus pumila) [1, 4]. These loci are increasingly being integrated into multiresistance gene breeding strategies. In recent years, the publication of complete genome sequences for plant-pathogenic fungi in the Venturia genus, including V. inaequalis, has significantly accelerated research into their pathogenic mechanisms [5].

The resistance derived from M. floribunda 821 is governed by two independent loci, Vf and Vfh. Vfh-mediated resistance is typified by the formation of “pinpoint pits”, primarily resulting from the hypersensitive response (HR) induced by pathogen infection. This defense mechanism triggers rapid necrosis in infected tissues and cells, restricting pathogen spread. In contrast, Vf-mediated resistance is characterized by the absence of visible chlorosis or necrosis symptoms, with only occasional formation of a minimal number of spores [1]. Current evidence indicates that Vf-mediated resistance does not act as the first line of defense against pathogen infection: V. inaequalis can penetrate the leaf epidermis of both Vf-resistant and Vf-susceptible cultivars to form primary stromata. The critical criterion for resistance assessment lies in the quantity and development of appressoria [6]. Valsangiacomo et al. further demonstrated no correlation between Vf-mediated resistance and cell wall degradation processes [7]. Koller et al. demonstrated that both Vf-mediated resistant and susceptible cultivars exhibited comparable inhibition of cellulase and polygalacturonase inhibitor synthesis during pathogen infection [8]. Perchepied et al. further revealed through transcriptome analysis that Vf-mediated resistance induces defense responses and systemic acquired resistance via the calcium signaling and auxin/brassinosteroid signaling pathways [2]. Additionally, while Vf-resistant cultivars present relatively high levels of phenolic compounds due to parental genetic effects, these compounds are not the primary determinants of their resistance phenotype [1].

The identification of the major resistance gene at the Vf locus represents a pivotal breakthrough in apple scab resistance research. Vinatzer et al. first cloned the primary Vf-mediated resistance gene HcrVf2 (representing Rvi6, abbreviated “resistance to V. inaequalis 6”), along with its homologous genes HcrVf1, HcrVf3, and HcrVf4 [9]. These genes exhibit high sequence homology to Cf-9 (Cladosporium fulvum resistance gene 9), which confers resistance to Cladosporium fulvum in tomato [10]. Among these genes, HcrVf1, HcrVf2, and HcrVf3 are expressed at higher levels in young leaves than in mature leaves, whereas HcrVf4 is active in young leaves and highly expressed in mature leaves. In addition to HcrVf3, which undergoes incomplete transcription due to sequence interruption, HcrVf1 and HcrVf4, which do not confer resistance, HcrVf2 mediates varying degrees of apple scab resistance [1]. Prediction of the HcrVf2 protein sequence revealed the presence of a signal peptide, an LRR (leucine-rich repeat) region, a domain of DUF (unknown function), an acidic domain, and a hydrophobic transmembrane domain with a basic C-terminus [9]. Although the protein structure of HcrVf2 is highly similar to that of Cf-9 [9], the infection strategies of the two pathogens differ fundamentally: V. inaequalis penetrates the cell membrane through specialized appressoria to acquire nutrients, whereas C. fulvum resides in the apoplast and relies on nutrients leaked from host cells [11]. The overexpression of HcrVf2 in the susceptible cultivar ‘Gala’ confers a resistance phenotype comparable to that of Vf-mediated resistant cultivars, effectively restricting V. inaequalis except races 6 and 7 by preventing the pathogen from forming extensive stromata and spores [1, 12]. The Rvi6 gene confers resistance to apple scab in ‘Gala’ apples. After inoculation with scab spores, the leaf symptoms of the resistant cultivar ‘Florina’[13], ‘Santana’ [14], and Rvi6-transgenic ‘Gala’ are significantly suppressed compared to the susceptible cultivar ‘Gala’. The leaves of wild-type ‘Gala’ inoculated with scab spores show typical symptoms and produce a large number of spores. Rvi6 significantly inhibits pathogen spore production, lesion expansion, and mycelial growth [13–15], however, symptoms such as pin-points, chlorotic and necrotic lesions, with or without leaf crinkling are also present [2].

The resistance mechanism mediated by Rvi6 in apple against the hemibiotrophic pathogen V. inaequalis aligns with the gene-for-gene hypothesis. Specifically, recognition between the plant R (resistance) gene and the pathogen Avr (avirulence) gene triggers ETI (effector-triggered immunity)-mediated resistance. This process likely involves the salicylic acid (SA) signaling pathway and is coordinated with multiple signaling networks, such as calcium signaling and brassinosteroid signaling, to orchestrate the resistance response [2]. As a result, the plant enhances pathogen resistance by upregulating PRs (pathogenesis-related proteins) and activating HR. Additionally, in line with typical fungal infection strategies, apple plants activate PTI (pattern-triggered immunity) by recognizing conserved elicitors from V. inaequalis through cell surface receptors.

Current research on apple scab resistance predominantly focuses on genetics-based MABs (molecular marker-assisted breeding), with resistant germplasms developed from the Vf locus already applied in commercial production. However, the molecular mechanisms underlying Rvi6-mediated apple scab resistance remain poorly characterized, with key metabolic and signaling pathways still undefined. By establishing a genetic transformation system for apple calli and integrating resistance phenotype characterization, this study revealed the physiological and biochemical mechanisms and elucidated the key metabolic/signaling pathways regulated by Rvi6. These findings not only enrich the understanding of plant disease resistance mechanisms, but also provide novel perspectives for genetic engineering-driven disease resistance breeding.

Materials and methods

Biological materials

Apple calli (Malus domestica ‘Orin’) were origin from Shandong Agricultural University, and maintained and subcultured at the College of Agriculture, Shihezi University [16]. Malus domestica ‘Xinping 1’ were cultured at the fruit tree germplasm nursery of Shihezi University. The V. inaequalis strain was collected and isolated from an apple orchard in Cocodala city, Xinjiang, and identified via DNA sequencing. The V. inaequalis strain was subcultured on potato dextrose agar (PDA) media at 20 °C in the dark.

Protein bioinformatics analysis

The physicochemical properties of the Rvi6 protein were analyzed via the ProtParam program (https://web.ExPASy.org/protparam/). Secondary structure prediction was performed via the SOPMA program (https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html), whereas transmembrane structure analysis was conducted via the TMHMM program (https://services.healthtech.dtu.dk/services/TMHMM-2.0/). Hydrophilicity profiling was carried out with the ProtScale program (https://web.ExPASy.org/protscale/), and protein motif analysis was executed via the MEME suite (https://meme-suite.org/meme/tools/meme).

Evolutionary relationship analysis

The Rvi6 coding sequence from M. floribunda was retrieved from the GenBank database (accession no. AJ297740). A total of 104 amino acid-coding sequences with high sequence identity to Rvi6 were extracted from the GenBank and GDR databases (https://www.rosaceae.org/) for evolutionary relationship analysis. Phylogenetic tree construction was performed via MEGA software (version 11).

Gene spatiotemporal expression pattern analysis

Seven-year-old M. domestica ‘Xinping 1’ (M. domestica ‘Rails’ × M. domestica ‘Line56193’) [17] served as the experimental material. During different growth stages in June, July, August, and September 2024, the relative expression levels of Rvi6 were detected via qRT‒PCR (quantitative real-time PCR) in absorptive roots, developing fruits, the 3rd to 4th leaves from the growth point, and new shoots. The 2^−ΔΔCT method was used to calculate relative expression levels.

Construction of Rvi6 transgenic apple callus lines

The open reading frame (ORF) of Rvi6 was amplified and inserted into the pBI121 vector to generate the binary recombinant vector pBI121-CaMV 35S::Rvi6-HA, which was subsequently transformed into ‘Orin’ apple calli via Agrobacterium [16]. Calli transfected with the empty vector (pBI121-CaMV 35S:: HA) served as negative controls. Positive transgenic calli were selected on MS media supplemented with agar (15 g·L⁻¹), 2,4-D (1.5 mg·L⁻¹), 6-BA (0.5 mg·L⁻¹), and kanamycin A (50 mg·L⁻¹) and confirmed via qRT‒PCR. Transgenic calli were maintained in the dark at 24 °C and subcultured every 15 days [16]. The primers used are listed in Supplementary Table S1.

Subcellular localization analysis of Rvi6

The ORF of Rvi6 was amplified to generate the recombinant vector pBI121-CaMV 35S::Rvi6-GFP for transient genetic transformation. The recombinant vector-transformed Agrobacterium was infiltrated into the leaves of 30-day-old tobacco seedlings [18]. The subcellular localization of the Rvi6 protein was visualized via a Nikon® AXR NSPARC laser confocal microscope two days postinfiltration.

Determination of morphological and physicochemical indicators

Transgenic calli (0.2 g fresh weight) were inoculated on MS subculture media. The growth phenotypes were recorded, and the fresh weight was measured every 2 days, with 18 biological replicates per line. Endogenous levels of IAA (indole-3-acetic acid), ABA (abscisic acid), JA (jasmonic acid), SA (salicylic acid), and GA₃ (gibberellic acid 3) were quantified via the liquid chromatography‒mass spectrometry (LC‒MS) method [16, 19] in 8-day-old subcultured calli, with three biological replicates. For pathogen inoculation assays, equal amounts of V. inaequalis hyphae were placed at the center of 4-day-old calli, and infection phenotypes and lesion areas (calculated from diameters, three independent biological replicates) were recorded daily. At 12 days postinoculation, callus tissues 5–10 mm from the lesion peripheries were collected for physicochemical analyses, with 6 independent biological replicates. SOD (superoxide dismutase), POD (peroxidase), and CAT (catalase) activities were determined via the nitrogen blue tetrazole photochemical reduction, guaiacol oxidation, and ammonium molybdate methods, respectively. Total soluble protein was measured via a Coomassie brilliant blue protein assay kit (Jining, China). The malondialdehyde (MDA), total soluble sugar, and proline contents were determined following Chang et al. [20]. The relative electrical conductivity was measured according to Li et al. [21]. The glycine betaine content was assayed as described by Gupta et al. [22]. The total flavonoid content was quantified via the method of Zhang et al. [23].

Transcriptome analysis of transgenic apple calli

Rvi6-overexpressing (OE) and control (CT) transgenic calli were selected for RNA-Seq analysis, with three independent biological replicates. Total mRNA extraction, cDNA library construction, next-generation sequencing, and transcript annotation were performed by Novogene Co., Ltd. (China). The apple reference genome and annotation data were derived from the GDR database and published by Daccord et al. [23]. Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and gene set enrichment analysis (GSEA) of the differentially expressed genes (DEGs) were conducted via NovoMagic online tools (https://magic-plus.novogene.com/).

Statistical analysis

One-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test was performed via IBM SPSS Statistics (version 23) software for multiple comparisons. Pairwise comparisons were conducted via Student’s t test (paired, two-tailed) via Microsoft Excel (version 2019) software. Homoscedasticity and normality were assessed via Levene’s test and the Shapiro‒Wilk test, respectively. Column charts were generated via GraphPad Prism (version 9.5.0) and Microsoft Excel (version 2019) software. PCA (principal component analysis), correlation, and cluster analysis were conducted via OriginPro (version 2021) software. All the data are presented as the means ± SEs (standard errors) from biological replicates.

Results

Phylogenetic analysis of the Rvi6 protein

Protein structure plays a decisive role in determining biological function. Using the amino acid sequence of Rvi6 as a reference, 103 highly conserved orthologous proteins were identified and subjected to phylogenetic analysis (Fig. 1).

Fig. 1.

Phylogenetic relationships of Rvi6 orthologous proteins. A total of 103 Rvi6 orthologous proteins were selected for phylogenetic analysis to determine their evolutionary relationships. Highlighted colors denote distinct clades. The species of origin for each ortholog are labeled at the branch terminals, with symbols indicating genera containing two or more orthologous proteins. Rvi6 is indicated by a blue arrow. Phylogenetic trees were inferred via the neighbor‒joining method, and evolutionary distances were calculated via the Poisson correction model

On the basis of genetic distance, these orthologs were classified into four major clades, with the majority derived from perennial plants. Notably, 20 orthologous proteins were clustered into clade III, including 10 Rosaceae species (containing Rvi6), 4 Malvaceae species, 2 Salicaceae species, and others. The presence of both pear/apple species among the 10 Rosaceae members suggests a potentially conserved resistance mechanism against scab in some apple and pear germplasms. Notably, 15 additional orthologs from Rosaceae species formed a distinct clade with distant genetic relatedness, possibly resulting from genetic drift or natural selection. This divergence may be correlated with different resistance loci and specialized resistance mechanisms across germplasms. Protein structure prediction further revealed that Rvi6 is a hydrophobic transmembrane protein containing multiple α-helices, β-sheets, and coils (Fig. S1A-D), strongly implying its role as a transmembrane receptor. Conserved domains of Rvi6 were identified in closely related species, which may be related to its functional relevance to disease resistance (Fig. S1E).

Spatiotemporal expression specificity of the Rvi6 gene

To investigate the spatiotemporal expression pattern of Rvi6, its expression levels across four consecutive developmental stages were detected in the cultivated apple ‘Xinping 1’ (Fig. 2). In June, Rvi6 presented the highest expression in leaves. At this stage, the functional leaves of new shoots are essentially mature, and young fruits are in the expansion phase. In July, Rvi6 presented high expression levels in roots, leaves, and fruits. At this stage, the fruits were in the full expansion phase, and the leaves had entered a stable functional state. Leaf diseases frequently occur from June–July, whereas fruits remain largely disease free. In August, Rvi6 presented the highest expression level in fruits, followed by leaves, new shoots, and roots. At this stage, fruits are in the late expansion phase and gradually undergo color changes. In September, Rvi6 expression was highest in leaves, followed by fruits. At this stage, fruits had entered the mature phase, whereas new shoot leaves maintained relatively stable functions. Both fruits and leaves were susceptible to infection from August–September. Overall, Rvi6 exhibited consistently high expression in leaves and fruits throughout the developmental stages.

Fig. 2.

Spatiotemporal expression analysis of Rvi6. The relative expression levels of Rvi6 in the cultivated apple ‘Xinping 1’ were determined via qRT‒PCR. Images above the bars depict fruits and leaves at four developmental stages. One-way ANOVA followed by Fisher’s LSD test was conducted for statistical analysis, with different letters above the bars indicating significant differences (p < 0.05)

Specifically, Rvi6 was continuously highly expressed in leaves, with low expression in young fruits but increased expression during late fruit expansion and color transformation, which is largely consistent with the infection window of V. inaequalis. Notably, Rvi6 expression decreases during fruit maturity, potentially through regulatory mechanisms governing fruit ripening and senescence, which are often associated with reduced disease resistance in mature fruits.

Resistant phenotype and physicochemical mechanism of Rvi6

To investigate the effects of Rvi6 on the phenotypic and physicochemical traits of apple fruits, we generated stable transgenic apple calli overexpressing Rvi6 via Agrobacterium-mediated transformation (Fig. 3A). Hormone profiling revealed that Rvi6 overexpression significantly reduced the levels of IAA, ABA, and JA while markedly increasing the level of GA₃, whereas SA levels remained unaffected (Fig. 3B-F). Consistent with the IAA level, Rvi6 overexpression also significantly inhibited apple callus growth, and differences were observed after culture for 2 days (Fig. 3G), which indicates that Rvi6 may inhibit apple growth via the suppression of aux signaling. In addition, subcellular localization assays revealed that Rvi6 localizes to the plasma membrane, where it performs its biological functions (Fig. 3H), which is also consistent with the results of bioinformatics analysis (Fig. S1C).

Fig. 3.

Functional analysis of the effects of Rvi6 overexpression on the phenotypic and physicochemical traits of apple. Three independent transgenic calli lines were validated for Rvi6 overexpression via qRT‒PCR (A). OE represents Rvi6-overexpressing calli lines transfected with the recombinant pBI121-CaMV 35S::Rvi6-HA vector, and CT represents control calli lines transfected with the empty pBI121-CaMV 35S::HAvector. The levels of plant hormones, including IAA (B), JA (C), ABA (D), GA₃ (E), and SA (F), in the CT and OE callus lines were determined. The biomass of the CT and OE transgenic callus lines subcultured for 0, 2, 4, 6, 8, 10, 12, and 14 days was measured, and representative phenotypes are shown below (G). Subcellular localization analysis was performed using tobacco mesophyll cells transfected with pBI121-CaMV 35S::Rvi6-GFP or pBI121-CaMV 35S::GFP (H). One-way ANOVA with Fisher’s LSD test was used for multiple comparisons, and different letters above columns indicate significant differences (p < 0.05). Student’s t test (paired, two-tailed) was used for pairwise comparisons, and asterisks denote significant differences (*p < 0.05; **< 0.01)

To explore Rvi6-mediated apple scab resistance, multiple key phenotypic and physicochemical indicators were determined. V. inaequalis was inoculated onto transgenic apple calli, and lesion areas were significantly reduced in Rvi6-overexpressing calli starting from 4 days of inoculation (Fig. 4A-B). Furthermore, the natural expression level of Rvi6 was markedly induced by V. inaequalis infection, and a clear dose‒effect relationship was detected between Rvi6 expression and V. inaequalis infection intensity (Fig. 4C). In terms of ROS (reactive oxygen species) scavenging capacity, V. inaequalis infection significantly increased SOD activity in the Rvi6-overexpressing lines, whereas no significant change was observed under noninoculated conditions (Fig. 4D). POD activity did not significantly change upon infection, and Rvi6 overexpression significantly increased POD activity under noninoculated conditions (Fig. 4E). CAT activity was significantly increased by Rvi6 regardless of infection status (Fig. 4F). In terms of the accumulation of osmotic adjustment substances, while V. inaequalis infection significantly reduced the contents of total soluble proteins, flavonoids, and proline, these compounds remained at relatively high levels in the Rvi6-overexpressing lines (Fig. 4G, I, J). Additionally, Rvi6 overexpression significantly reduced the content of total soluble sugars (Fig. 4H). Although Rvi6 did not affect the oxidative damage (Fig. 4 K) or integrity (Fig. 4L) of the plasma membrane, the induction of Rvi6 significantly enhanced plasma membrane integrity under normal conditions.

Fig. 4.

Functional characterization of Rvi6-mediated apple scab resistance. The CT and OE callus lines were inoculated with V. inaequalis, phenotye (B) and lesion area (A) measurements were recorded daily, and multiple key physicochemical indicators were measured (D-L). After V. inaequalis was inoculated for 10 days, the relative expression level of Rvi6 in wild-type calli was determined (C). NT represents untreated wild-type cllus, and IS, MS, and OS represent the callus tissues 0–5 mm, 5–10 mm, and 10–15 mm from the hyphal margin, respectively. One-way ANOVA with Fisher’s LSD test was used for multiple comparisons, and different letters indicate significant differences (p < 0.05)

Transcriptional regulation modulated by Rvi6 in apple call

To investigate the effect of Rvi6 on the transcriptional regulatory mechanism, RNA-Seq (RNA sequencing) analysis was performed using the CT and OE callus lines. The gene expression distributions of the two groups were similar overall, but there were certain differences in distribution (Fig. S2A-B). Data quality assessment revealed that the average proportion of clean reads was 96.6%, with average Q20 and Q30 values of 98.66% and 95.91%, respectively (Fig. S2C-D). The high-quality data constituted a substantial proportion and demonstrated excellent quality, accurately reflecting gene expression patterns and being suitable for subsequent data analysis.

In terms of gene expression levels, significant intergroup differences were observed between the CT and OE groups (Fig. 5A), with intragroup correlations exceeding intergroup correlations (Fig. 5B), demonstrating that Rvi6 overexpression markedly influenced apple transcriptional regulation. Retrieved within log₂ (fold change) ≥ 1 or ≤ −1 and p < 0.05, a total of 4492 DEGs were classified into two distinct clusters. Cluster 1 included 2,139 upregulated DEGs (Fig. 5C-D), which were enriched primarily in plant‒pathogen interactions, the MAPK (mitogen‒activated protein kinase) signaling pathway, multiple amino acid metabolic processes, and the biosynthesis of secondary metabolites such as phenylpropanoids, flavonoids, cutin, suberin, and wax (Fig. S4A, C). Cluster 2 included 2353 downregulated DEGs (Fig. 5C-D), which were predominantly enriched in plant hormone signal transduction, sugar metabolism, photosynthesis, and biosynthesis of secondary metabolites such as terpenoids (Fig. S4B, D). Additionally, 344 differentially expressed TFs (transcription factors) spanning 18 families were screened using log₂ (fold change) ≥ 3 or ≤ −3 and p < 0.05 (Fig. 5E). TFs, including many genes related to MYB, NAC, bHLH, ERF, C2H2, MADS, WRKY, and bZIP, are directly or indirectly associated with plant stress resistance (particularly immune responses). In addition, the expression patterns of several representative TFs were experimentally validated via qRT‒PCR (Fig. S3).

Fig. 5.

RNA-Seq analysis of Rvi6-overexpressing apple calli. Total mRNA from Rvi6-overexpressing (OE) and control (CT) apple calli was subjected to RNA-Seq analysis, with three independent biological replicates. A PCA was performed on the basis of gene expression levels. B Correlation analysis was conducted via Spearman correlation coefficients to evaluate pairwise sample relationships on the basis of expression profiles. C Volcano plots of up-(red) and downregulated (green) DEGs with log₂ (fold change) ≥ 1 or ≤ -1 and p < 0.05 are shown. D Cluster analysis was conducted on DEGs whose log₂ (fold change) ≥ 1 or ≤ -1 and p < 0.05. E Analysis of annotated differentially expressed TFs with log₂ (fold change) ≥ 3 or ≤ -3 and p < 0.05. Red and green denote up-and downregulated TFs, respectively, upon Rvi6 overexpression. The color intensity corresponds to the magnitude of the fold change, and the circle size reflects the negative logarithm of the p value. The fold change is defined as the ratio of fragments per kilobase of transcript per million mapped reads (FPKM) values of the OE group to those of the CT group. The FPKM values were standardized via z score normalization for cluster analysis

To investigate the key transcriptional regulatory mechanisms, KEGG and GO analyses were performed. Rvi6 overexpression significantly enriched pathways related to the biosynthesis of phenylpropanoids, diterpenoids, and other plant secondary metabolites; plant hormone signal transduction; plant‒pathogen interactions; and pentose and glucuronate interconversions (Fig. 6A). Concurrently, it markedly impacted biological processes such as ROS scavenging mechanisms, protein hydrolysis, ion channel activity, and transcriptional regulatory activity (Fig. 6B). To further explore the key pathways and biological functions, GSEA was performed on the basis of KEGG and GO annotations. The major upregulated pathways were significantly enriched in plant‒pathogen interactions, lipid and amino acid metabolism, and flavonoid biosynthesis, which involve biological processes such as protein and enzyme activity regulation, protein synthesis, and calcium ion signaling responses (Fig. 6C‒D). Conversely, major downregulated pathways were significantly enriched in plant hormone signal transduction, protein processing and modification, and carbohydrate metabolism, which are associated with biological processes, including chromatin/nucleosome assembly and cellular signal transduction (Fig. 6C-D).

Fig. 6.

KEGG and GO enrichment analyses of DEGs. DEGs with log₂ (fold change) ≥1 or ≤-1 and p <0.05 were subjected to KEGG (A) and GO (B) enrichment analyses. The top 20 KEGG terms and the top 40 GO terms with the lowest p values were selected. The color intensity reflects the p value, and the circle size represents the number of genes enriched in each term. GSEA was performed on the basis of KEGG (C) and GO (D) annotations, with the top 10 significantly enriched pathways and functional categories shown. Red and green bars indicate the number of DEGs enriched in up-and downregulated terms, respectively, whereas numerical labels denote the number of core enriched DEGs. The dots at the end of each bar represent the FDR (false discovery rate) q value for the corresponding pathway or function

Metabolisms and signaling pathways responsive to Rvi6 overexpression

On the basis of the GSEA results, three key metabolic and signaling pathways with statistically significant enrichment (characterized by low FDR q values), namely, the plant‒pathogen interaction, plant hormone signal transduction, and MAPK signaling pathways, were markedly induced by Rvi6 overexpression. These pathways are critically associated with the molecular mechanisms underlying pathogen infection responses, highlighting their central role in mediating host defense pathways triggered by Rvi6 (Fig. 7).

Fig. 7.

Metabolic and signaling pathways responsive to Rvi6overexpression. According to GSEA, three pathways with significant enrichment within low FDR (false discovery rate) q values were retrieved, closely associated with plant disease immune resistance, including plant‒pathogen interaction (upregulated, A), MAPK signaling pathway-plant (upregulated, C), and plant hormone signal transduction (downregulated, B). Numerical values and filled colors within boxes denote gene expression levels as log₂(fold change), whereas border colors represent GSEA-derived rank metric scores. The core enriched DEGs are labeled with their gene IDs beside the corresponding nodes. The enrichment plots for each pathway are displayed on the right side of the diagrams

The plant‒pathogen interaction pathway was identified as an upregulated type according to GSEA, with multiple core enriched genes also showing upregulation (Fig. 7A). Cf-9 encodes a plant resistance protein that specifically recognizes the C. fulvum effector avr9 (avirulence protein 9). Notably, the secondary and tertiary protein structures of Cf-9 are similar to those of Rvi6 [9]. Rvi6 overexpression also activated the Cf-9-mediated CDPK (calcium-dependent protein kinase)-Rboh (respiratory burst oxidase homolog)-ROS pathway, which induced HR and cell wall reinforcement. Rvi6 additionally activated Ca²⁺-mediated plant‒pathogen interaction pathways, including CNGCs (cyclic nucleotide-gated channels)-CaM/CML (calmodulin/calmodulin-like protein)-NO (nitric oxide) and CNGCs-MPK4 (mitogen-activated protein kinase 4)-WRKY33 (WRKY DNA-binding protein 33)-PR1 (pathogenesis-related protein 1). Furthermore, AvrRpm1 (avirulence protein Rpm1) and AvrB (avirulence protein B)-mediated posttranslational modifications of RIN4 were proposed to activate the RPM1 (resistance to Pseudomonas syringae 1)-HSP90 (heat shock protein 90) resistance mechanism [24], and Rvi6-mediated disease resistance may involve this pathway.

Plant hormones play crucial roles in regulating plant disease resistance. GSEA revealed downregulation of plant hormone signal transduction, with multiple core enriched genes involved in the auxin, cytokinin, and jasmonic acid signaling pathways (Fig. 7B). Rvi6 inhibited cell enlargement, plant growth, cell division, and shoot initiation by downregulating the auxin and cytokinin signaling pathways. Additionally, MYC2 (myelocytomatosis 2) mediates senescence and stress responses by inducing dissociation of the JAZ-MYC2 (jasmonate ZIM-domain-myelocytomatosis 2) complex and releasing resistance-related transcription factors [25], a process in which Rvi6 may be involved.

The MAPK signaling pathway is involved in multiple plant stress responses, including recognition and defense against bacterial and fungal invasions. GSEA revealed upregulation of the MAPK signaling pathway. BAK1 (brassinosteroid-insensitive 1-associated receptor kinase 1), FLS2 (flagellin-sensing 2), and their interaction induced by flg22 (flagellin 22) were also upregulated (Fig. 7C). In the MPK4-related phosphate signaling cascade, MKS1 was significantly upregulated, inducing WRKY33 (WRKY DNA-binding protein 33)-PAD3 (phytoalexin deficient 3)-mediated camalexin synthesis. In the MPK3/6-related phosphate signaling cascade, ACS6 (1-aminocyclopropane-1-carboxylic acid synthase 6) and PR1 are markedly upregulated, contributing to ethylene biosynthesis and late-stage pathogen defense responses, respectively.

Discussions

Phylogeny and resistance application of Rvi6 gene

Four receptor-like sequences have been identified at the apple Vf locus, designated HcrVf1-HcrVf4 [26]. Among these, HcrVf3 is a truncated pseudogene. HcrVf1 and HcrVf2 confer partial resistance to V. inaequalis in cultivated varieties, whereas HcrVf4 is not involved in resistance [10, 26, 27]. A new international naming system was subsequently proposed, stipulating that resistance (R) genes are prefixed with “R” followed by the pathogen abbreviation. Subsequent studies reclassified HcrVf2 as Rvi6 [26]. The Vf-mediated resistance locus has been utilized in apple breeding for decades. For example, 22 Rvi6 derivatives were identified in M. domestica ‘Florina’ [14], and orthologous sequences were detected in M. micromalus and M. baccata [28]. Approximately 90% of cultivars, including ‘Honeycrisp’, ‘Antonovka’, ‘Braeburn’, ‘Florina’, ‘Jonagold’, and ‘Topaz’, harbor Rvi6 homologs [29, 30]. Phylogenetic analysis of Rvi6 amino acid sequences revealed that M. floribunda, M. sylvestris, M. sieversii, M. micromalus, M. baccata, and several domestic cultivars (‘Antonovka’ and ‘Golden Delicious’) clustered into group III, exhibiting close genetic relationships (Fig. 1), which is consistent with prior studies [29, 30]. In contrast, varieties such as ‘MM106’ and ‘Fuji’ (group IV) and ‘Hanfu’ (group I) presented distant relationships (Fig. 1), suggesting that Rvi6 may have undergone polyphyletic differentiation and evolution.

Spatiotemporal expression of Rvi6 is consistent with the pathogenesis regularity

As leaf age increases, the incidence and lesion area of apple scab decrease, indicating that older leaves develop increased resistance to V. inaequalis [30]. MacHardy hypothesized that this age-related resistance primarily stems from reduced cellular tissue pH, inactivation of cell wall-degrading enzymes, and production of antimicrobial metabolites [31, 32]. Additionally, increasing leaf age is correlated with upregulated Rvi6 expression [33] and increased transcription of resistance-related genes involved in redox homeostasis, defense metabolite biosynthesis, and cell wall component synthesis (e.g., callose, wax, and lignin precursors) [30, 32]. Our field observations confirmed significantly lower scab severity in older leaves than in young leaves (data not shown). Although we did not directly measure Rvi6 expression between leaf age groups, Rvi6 transcripts were highly abundant in both leaves and fruits, remaining consistently elevated throughout leaf development and peaking at fruit maturity, and the patterns largely coincided with the infection timeline of susceptible apple varieties (Fig. 2). Furthermore, given that the parentage of ‘Xinping 1’ apple is of natural hybrid origin, the genetic origin of Rvi6 remains elusive, thus necessitating phylogenetic analysis in conjunction with sequence polymorphism assessment. Collectively, these results reinforce the roles of Rvi6 in conferring age dependent, autonomous resistance against V. inaequalis infection.

Rvi6 significantly enhances apple scab resistance

Most apple scab-resistant cultivars are resistant primarily to the Vf locus [1]. Genetic transformation studies have demonstrated that Rvi6 confers high resistance to V. inaequalis in the susceptible cultivar ‘Gala’ [14, 15]. When heterologously expressed in pear trees or homologously expressed in tetraploid apple lines with elevated Rvi-related gene expression, robust defense responses and severely restricted pathogen sporulation are observed [2, 15, 34]. While ‘Gala’ plantlets have been used to validate Rvi6-mediated resistance, direct phenotypic observation of whole plants is challenging. Instead, apple callus transgenic technology has emerged as an efficient and convenient system for gene function analysis [35]. In this study, three transgenic callus lines overexpressing Rvi6 presented 870–1697-fold higher relative expression levels than did the empty vector transgenic controls (Fig. 3A). Following inoculation with V. inaequalis, the callus phenotypes were monitored for one month until signs of natural senescence became apparent. Notably, the natural expression level of Rvi6 was markedly induced by V. inaequalis infection, and Rvi6 significantly inhibited scab lesion expansion (Fig. 4A-B), which demonstrated that Rvi6 mediates active immune resistance responses and recapitulates the resistance phenotype previously observed in transgenic ‘Gala’ plants, reinforcing the conserved functional role of Rvi6 across different experimental germplasms. These results not only validate the utility of callus-based assays for rapid resistance screening but also provide molecular evidence that Rvi6 is a key determinant of broad-spectrum scab resistance in rosaceous crops.

Multiple hormones and TFs are involved in Rvi6-mediated apple resistance

Rvi6 shares amino acid sequence homology with the S. lycopersicum resistance protein Cf-9 [26]. Cf-9 is a transmembrane receptor protein containing leucine-rich repeats (LRRs) that mediates resistance responses to C. fulvum. Subcellular localization assays confirmed that Rvi6 localizes to the plasma membrane (Fig. 3H), which is consistent with its classification as a transmembrane hydrophobic protein. Structural analyses revealed that Rvi6 harbors diverse secondary structures (Fig. S1), which aligns with the findings of Vinatzer et al. [9]. Rvi6 overexpression led to significant inhibition of cell growth (Fig. 3G), which was accompanied by reduced cellular IAA levels (Fig. 3B) and downregulated auxin signaling (Fig. 7B). These observations align with the “growth-defense trade-off” hypothesis [36]. Pathogens often employ the strategy of activating host auxin signaling pathways via effectors to suppress plant resistance [18], whereas plant resistance proteins such as Rvi6 may counteract this by inhibiting auxin signaling to increase defensive capacity. The SA and JA/ET (jasmonic acid/ethylene) pathways are known to be mutually inhibitory, with SA signaling primarily enhancing resistance against biotrophic pathogens and JA/ET signaling conferring protection against necrotrophic pathogens [2, 37]. For the hemibiotrophic fungus V. inaequalis, Rvi6 overexpression did not significantly affect SA levels (Fig. 3F), whereas JA hormone levels (Fig. 3C) and JA signaling pathways (Fig. 7B) were notably downregulated. These observations are inconsistent with the classical model of SA/JA-ET antagonism in plant defense. However, these findings align with previous findings suggesting that apple scab resistance mechanisms may operate independently of SA signaling and are dissociated from the canonical JA/SA defense hierarchy [2, 38]. Recent studies have demonstrated that GA₃ upregulates pathogen-related genes, enhances antioxidant enzyme activity, reduces leaf ROS levels, and positively regulates tomato resistance to tomato yellow leaf curl virus [39]. While most plant hormones tested were downregulated by Rvi6, GA₃ levels were significantly upregulated (Fig. 3E). Concurrently, pathogen-related genes such as WRKYs, NACs, MYBs, and AP2-EREBPs presented marked transcriptional changes (Fig. 5E, S3), accompanied by increased accumulation of osmotic adjustment substances (Fig. 4 K, M), maintenance of ROS homeostasis (Fig. 4D-F), and preservation of plasma membrane integrity (Fig. 4L). These findings suggest that GA₃ may directly or indirectly modulate Rvi6-mediated resistance mechanisms against apple scab. Furthermore, Mansoor et al. reported that the activities of apple ROS-scavenging enzymes, including SOD, POD, and CAT, were significantly increased by inoculation with V. inaequalis spores [40]. However, these findings differed from our findings (Fig. 4D–F), which might be attributed to variations in apple tissue types and germplasms, as well as differences in V. inaequalis inocula.

The potential signaling mechanism of Rvi6-mediated apple scab resistance

Rvi6 overexpression profoundly affected global transcriptional regulation, activating numerous disease resistance-related TFs and MAPKs (Fig. 5E, S3). These components likely play pivotal roles in orchestrating disease resistance signaling networks, which aligns with findings in transgenic ‘Gala’ plantlets [34, 41]. The pathways upregulated under Rvi6 induction included plant‒pathogen interactions, flavonoid biosynthesis, tropane/piperidine/pyridine alkaloid metabolism, and lipid/amino acid/biotin metabolism (Fig. 6C). Concurrently, Rvi6 significantly increased flavonoid accumulation (Fig. 4 J), which is consistent with previous observations in ‘Gala’ plantlets [2]. These changes underscore the roles of Rvi6 in coordinating multilayered defense responses, from pathogen perception to secondary metabolite-mediated resistance. With respect to plant‒pathogen interactions, the majority of functional genes involved in Avr9-Cf-9-mediated CDPK-Rboh-ROS tomato leaf mold resistance mechanisms, as well as several relative expression levels of CDPKs (Fig. S3), were markedly upregulated (Fig. 7A), which suggested that not only do Cf-9 and Rvi6 share structural similarities but also that they may utilize similar signal transduction pathways and that they may have evolved from a common ancestor. In addition, in response to Rvi6 overexpression, Ca²⁺-CNGC-mediated MPK4-WRKY33-PR1 and CaM/CML-NO signal transduction mechanisms were also upregulated, which enhanced HR, cell wall reinforcement, and stomatal closure (Fig. 7A). In the context of plant‒pathogen interactions, most functional genes involved in the Avr9‒Cf-9-mediated CDPK‒Rboh‒ROS resistance pathway against tomato leaf mold, along with several CDPKs with increased relative expression (Fig. S3), were significantly upregulated by Rvi6 overexpression (Fig. 7A). These findings suggest that in addition to structural similarities, Cf-9 and Rvi6 may share conserved signal transduction mechanisms, potentially evolving from a common ancestral resistance gene. Additionally, Rvi6 overexpression activated Ca²⁺-CNGC-mediated MPK4-WRKY33-PR1 and CaM/CML-NO signaling cascades, which synergistically increase HR activation, cell wall fortification, and stomatal closure, which are key components of the early defense response (Fig. 7A). Furthermore, our results are also essentially consistent with those of Perchepied et al. [2], indicating that the Ca²⁺ and auxin signaling pathways mediate Rvi6-driven resistance (Fig. 7). These findings highlight the evolutionary conservation of core resistance modules across solanaceous and rosaceous species. Plant MAPK signaling pathways exert broad functions in immune responses through typical MAPK cascade reactions, where activated MAPKs phosphorylate diverse substrates, including TFs, kinases, and enzymes, to induce immune resistance [42]. Rvi6 upregulated flg22-induced FLS2-BAK1 interaction and downstream signaling, a canonical immune mechanism in plants. This activation led to marked upregulation of three key functional proteins, which drive camalexin and ethylene biosynthesis and orchestrate late-stage pathogen defense responses (Fig. 7). The observed upregulation of both early (receptor complex) and late (secondary metabolite) components highlights the roles of Rvi6 in synchronizing temporal immune phases to increase disease resistance.

Furthermore, based on transcriptomic studies, this research has initially identified that Rvi6 may be involved in the Ca²⁺-CDPK-MAPK cascade, a defense mechanism shared with tomato Cf-9. However, the key downstream signaling mechanisms remain unclear. Subsequent studies will investigate the mechanism at the level of AvrRvi6-Rvi6 protein interaction, explore the Ca²⁺-CDPK-mediated signal transduction mechanism by which Rvi6 regulates apple scab resistance from the perspectives of protein phosphorylation modification and protein–protein interaction, and simultaneously identify key transcription factors that regulate Rvi6 expression. This will facilitate an in-depth understanding of the key molecular mechanisms by which Rvi6 responds to the effector AvrRvi6 of V. inaequalis to activate the ETI immune resistance pathway in apples.

Conclusion

Rvi6 enhances resistance to V. inaequalis in apple by improving ROS scavenging capacity, promoting the accumulation of osmolytes, maintaining plasma membrane integrity, as well as inhibiting apple growth. Furthermore, Rvi6 induces the upregulation of pathways related to plant-pathogen interactions, lipid/amino acid metabolism, and flavonoid biosynthesis, while downregulating those involved in plant hormone signal transduction, protein processing/modification, and carbohydrate metabolism. The roles of the Ca²⁺-CDPK and hormone signaling pathways in this resistance mechanism will be the focus of future research.

Authors’ contributions

AC: Writing-original draft, Investigation, Formal analysis, Methodology, Data curation. HG: Investigation, Formal analysis. HJ: Methodology, Investigation. WY: Methodology, Investigation. XW: Conceptualization, Writing-review & editing, Project administration, Supervision, Resources, Funding acquisition. LX: Project administration, Supervision, Resources, Funding acquisition. All authors read and approved the manuscript.

Funding

This work was supported by grants from the Xinjiang Tianchi Doctoral Project (CZ006012), the Funding Project of the Postdoctoral Mobile Station of Crop Science of Shihezi University (351762), the Science and Technology Project of Xinjiang Production and Construction Corps (2024DA053), and the Natural Science Foundation of China (32260722).

Data availability

Supplementary material associated with this article can be found in supplementary information files. The transcriptome data have been deposited in the Sequence Read Archive (SRA) database of NCBI within BioProject PRJNA1273063. All materials are available through corresponding authors upon reasonable request.

Declarations

In accordance with the task requirements and permissions of the Xinjiang Tianchi Doctoral Project (CZ006012), all plant materials and V. inaequalis used in this study were preserved and propagated at the College of Agriculture, Shihezi University. The V. inaequalis strain was collected, and its DNA sequence identification was performed by Aoxing Chen. The acquisition and preservation of all materials used in this study comply with institutional, national, and international guidelines.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.