Apple leaf spot caused by the Alternaria alternata f. sp. mali (ALT1) fungus is one of the most devastating diseases of apple (Malus × domestica). We identified a hairpin RNA (hpRNA) named MdhpRNA277 that produces small RNAs and is induced by ALT1 infection in 9Golden Delicious9 apple. MdhpRNA277 produces mdm-siR277-1 and mdm-siR277-2, which target five resistance (R) genes that are expressed at high levels in resistant apple variety 9Hanfu9 and at low levels in susceptible variety 9Golden Delicious9 following ALT1 infection. MdhpRNA277 was strongly induced in 9Golden Delicious9 but not 9Hanfu9 following ALT1 inoculation. MdhpRNA277 promoter activity was much stronger in inoculated 9Golden Delicious9 vs. 9Hanfu9. We identified a single nucleotide polymorphism (SNP) in the MdhpRNA277 promoter region between 9Golden Delicious9 (pMdhpRNA277-GD) and 9Hanfu9 (pMdhpRNA277-HF). The transcription factor MdWHy binds to pMdhpRNA277-GD, but not to pMdhpRNA277-HF. Transgenic 9GL-39 apple expressing pMdhpRNA277-GD:MdhpRNA277 was more susceptible to ALT1 infection than plants expressing pMdhpRNA277-HF:MdhpRNA277 due to induced mdm-siR277 accumulation and reduced expression of the five target R genes. We confirmed that the SNP in pMdhpRNA277 is associated with Alternaria leaf spot resistance by crossing. This SNP could be used as a marker to distinguish between apple varieties that are resistant or susceptible to Alternaria leaf spot.

Alternaria leaf spot seriously threatens the sustainable development of the global apple industry, causing significant losses and reducing fruit quality and yield. The causal agent Alternaria alternata f. sp. mali (Alternaria mali, ALT) produces various molecules to modulate infection, such as cell wall-degrading enzymes, toxins, and elicitor-like molecules. ALT produces the host-specific AM-toxin, an important pathogenicity factor. ALT also releases effectors into apple cells that modify host defense, but these proteins have not yet been described. Here, we identified the pathogenic fungal types responsible for early defoliation from diseased leaves of Fuji (Malus domestica cv. ‘Fuji’) apple collected from five districts in Shandong Province, China. The ALT isolates ALT2 to ALT7 were pathogenic to four apple cultivars, with ALT7 being the most aggressive. We extracted mycotoxins (AM-toxin-2 to AM-toxin-7) from each isolate and used them to treat different apple varieties, which led to leaf-spot symptoms and damaged chloroplasts and nuclear membranes, followed by cell death. AM-toxin-7 produced the most severe symptoms, but chloroplasts remained intact when the mycotoxin was inactivated. Mass spectrometry identified 134 secretory proteins in ALT7 exosomes, and three secreted proteins (AltABC, AltAO, and AltPDE) were confirmed to be involved in apple pathogenesis. Therefore, ALT secretes AM-toxin and secretory proteins as an infection strategy to promote fungal invasion and overcome the host defense system.

Apple leaf spot, caused by Alternaria alternata f. sp mali (ALT), poses a substantial threat to the global apple (Malus × domestica Borkh.) industry. Fungal effectors promote pathogen infestation and survival by interfering with plant immune responses. In our study, we investigated the secretion of effector proteins by the virulent ALT7 strain. Using mass spectrometry, we identified the effector AaAlta1, which belongs to the Alt a 1 protein family (AA1s). Further analysis confirmed that ALT7 secretes AaAlta1. AaAlta1 knockdown mutants displayed reduced pathogenicity in apple tissue culture seedlings, while overexpression strains exhibited enhanced pathogenicity compared to the wild-type ALT7 strain. Using immunoprecipitation followed by mass spectrometry, we isolated pathogenesis-related protein 10-2 (PR10-2) as an interaction partner of AaAlta1 in apple. Knockdown mutants of AaAlta1 showed increased PR10-2-mediated callose deposition in apple, a critical plant defense response. The enhanced defense responses in apple substantially reduced their susceptibility to infection by these ALT7 mutants. Our findings delineate an infection strategy whereby ALT7 secretes AaAlta1 to suppress PR10-2, thereby circumventing the apple defense system.

Distant hybridization, such as between apple (Malus domestica Borkh.) and pear (Pyrus communis L.), can introduce unique genetic traits that aren’t possible through traditional hybridization methods. However, these hybrids often display maternal traits, making it difficult to visually identify hybrid offspring in the early stages. Thus, identifying these hybrids has been a longstanding challenge. To address this challenge, this study performed whole-genome resequencing of apple ‘Golden Delicious’ and pear ‘Yan Zhuang’, along with 62 of their hybrid offspring, to develop effective molecular markers. Genomic variation analysis revealed significant genetic diversity between apple and pear. By selecting markers based on notable parental differences and high polymorphism among offspring, 51 effective markers were identified from 340 apple-specific InDel markers. These markers could reliably identify the 62 offspring as distant hybrids with genetic material from both apple and pear. Among them, InDel1-7, InDel2-3, InDel3-1, and InDel4-3 proved to be the most efficient, achieving a 100% identification rate for hybrid offspring when used in combination in the ‘Golden Delicious’ × ‘Yan Zhuang’ population. Furthermore, marker universality tests showed that 88.2%, 76.5%, and 70.6% of the 51 markers were transferable to the ‘Golden Delicious’ × ‘Jin Zhui’, ‘Fuji’ × ‘Yan Zhuang’, and ‘Fuji’ × ‘Jin Zhui’ hybrid populations, respectively. The identification efficiencies in these three validation populations were 82.6%, 80.3%, and 85.0%, with the four highly efficient markers exceeding a 70% identification rate. This study developed an efficient molecular marker system for identifying apple and pear distant hybrid offspring based on InDel variation, providing a valuable tool for breeding new varieties of Rosaceae fruit trees.