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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. [ABSTRACT FROM AUTHOR]

SUMMARY: Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide‐binding, and leucine‐rich repeat domain CCR‐NB‐LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB‐LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1‐inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB‐ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad‐spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple. Significance Statement: Apple leaf spot includes three major fungal diseases affecting the apple industry: Alternaria leaf spot, caused by Alternaria alternata f. sp. mali, Glomerella leaf spot, caused by Glomerella cingulata, and Marssonina leaf spot, caused by Marssonina coronaria. These three devastating diseases of apple (Malus × domestica) cause early severe defoliation, weaken tree vigor, and reduce apple production. In the present study we identified the mode of action of two CCR‐NB‐LRR R proteins, MdRNL2 and MdRNL6, to enhance resistance to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot infection in the resistant apple cultivar HF. The identification and cloning of these novel broad‐spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple. Also, we generated genetically transformed HF plantlets to prove that suppression of MdRNL2 expression results in susceptibility to three apple leaf spot pathogens, similar to the effect of suppressing MdRNL6 in transgenic apple plantlets. Taken together, our data provide detailed evidence for a previously unknown regulatory mechanism in which three apple leaf spot pathogens induce the production of MdRNL2 and MdRNL6, which form a heterocomplex at the NB‐ARC domain and contribute to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot resistance. Our results provide a basis for deciphering the R‐protein‐based defense mechanism against three apple leaf spot pathogens. [ABSTRACT FROM AUTHOR]

Alternaria leaf spot in apple (Malus x domestica), caused by the fungal pathogen Alternaria alternata f. sp. mali (also called A. mali), is a devastating disease resulting in substantial economic losses. We previously established that the resistance (R) protein MdRNL2, containing a coiled-coil, nucleotide-binding, and leucine-rich repeat (CCR-NB-LRR) domain, interacts with another CCR-NB-LRR protein, MdRNL6, to form a MdRNL2–MdRNL6 complex that confers resistance to A. mali. Here, to investigate the function of the MdRNL2–MdRNL6 complex, we identified two novel pathogenesis-related (PR) proteins, MdPR10-1 and MdPR10-2, that interact with MdRNL2. Yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays confirmed that MdPR10-1 and MdPR10-2 interact with MdRNL2 and MdRNL6 at the leucine-rich repeat domain. Transient expression assays demonstrated that accumulation of MdPR10-1 and MdPR10-2 enhanced the resistance of apple to four strains of A. mali that we tested: ALT1, GBYB2, BXSB5, and BXSB7. In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternaria leaf spot resistance by inhibiting fungal growth. Our data provide evidence for a novel regulatory mechanism in which MdRNL2 and MdRNL6 interact with MdPR10-1 and MdPR10-2 to inhibit fungal growth, thereby contributing to Alternaria leaf spot resistance in apple. The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple. [ABSTRACT FROM AUTHOR]

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 'NGR196' apple. MdhpRNA277 produces mdm-siR277-1 and mdm-siR277-2, which target five resistance (R) genes that are expressed at high levels in the resistant apple variety 'Hanfu' and at low levels in the susceptible variety 'NGR196' following ALT1 infection. MdhpRNA277 was strongly induced in 'NGR196' but not 'Hanfu' following ALT1 inoculation. MdhpRNA277 promoter activity was much stronger in inoculated 'NGR196' versus 'Hanfu'. We identified a single-nucleotide polymorphism (SNP) in the MdhpRNA277 promoter region between 'NGR196' (pMdhpRNA277-NGR196) and 'Hanfu' (pMdhpRNA277-HF). The transcription factor MdWHy binds to pMdhpRNA277-NGR196 , but not to pMdhpRNA277-HF. Transgenic 'GL-3' apple expressing pMdhpRNA277-NGR196: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 A. alternata leaf spot resistance by crossing. This SNP could be used as a marker to distinguish between apple varieties that are resistant or susceptible to A. alternata leaf spot. [ABSTRACT FROM AUTHOR]

Plants are frequently attacked by a variety of pathogens and thus have evolved a series of defense mechanisms, one important mechanism is resistance gene (R gene)-mediated disease resistance, but its expression is tightly regulated. NBS-LRR genes are the largest gene family of R genes. microRNAs (miRNAs) target to a number of NBS-LRR genes and trigger the production of phased small interfering RNAs (phasiRNAs) from these transcripts. phasiRNAs cis or trans regulate NBS-LRR genes, which can result in the repression of R gene expression. In this study, we screened for upregulated miR482 in the susceptible apple cultivar 'Golden Delicious' (GD) after inoculation with the fungal pathogen Alternaria alternata f. sp. mali (ALT1). Additionally, through combined degradome sequencing, we identified a gene targeted by miR482, named MdTNL1 , a gene encoding a TIR-NBS-LRR (Toll/interleukin1 receptor-nucleotide binding site-leucine-rich repeat) protein. This gene exhibited a significant down-regulation post ALT1 inoculation, suggesting an impact on gene expression mediated by miRNA regulation. miR482 could cleave MdTNL1 and generate phasiRNAs at the cleavage site. We found that overexpression of miR482 inhibited the expression of MdTNL1 and thus reduced the disease resistance of GD, while silencing of miR482 increased the expression of MdTNL1 and thus improved the disease resistance of GD. This work elucidates key mechanisms underlying the immune response to Alternaria infection in apple. Identification of the resistance genes involved will enable molecular breeding for prevention and control of Alternaria leaf spot disease in this important fruit crop. • MdTNL1 belonging to the TIR type significantly down-regulated after infection with ALT1. • miR482 generates phasi RNAs after cleavage of MdTNL1. • miR482- MdTNL1 pathway involved in apple resistance to apple Alternaria leaf spot disease. • Overexpression of MdTNL1 or silencing of miR482 improves apple resistance to Alternaria leaf spot disease. [ABSTRACT FROM AUTHOR]