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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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., Competing Interests: Declaration of Competing Interest The Authors declare no conflict of interest in this manuscript. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The soluble solids content (SSC) is an important factor in the internal quality detection of apples. It is essential to have reliable and high-speed measurement of the SSC. However, almost all traditional equipment is inconvenient and expensive. We designed a handheld nondestructive SSC detector based on near-infrared (NIR) spectroscopy, which is composed of a portable NIR spectrometer, cloud server, smartphone app, and prediction model of SSC. We preprocessed the spectrum with multiplicative scatter correction (MSC), standard normal variable transformation (SNV), and Savitzky-Golay (S-G) smoothing algorithms. Besides, the linear weight reduction of the particle swarm optimization algorithm is carried out, and we establish the model of an extreme learning machine optimized with the improved particle swarm optimization (IPSO-ELM) algorithm. The R 2 , root mean square error of prediction (RMSEP), and residual prediction deviation (RPD) of the model are 0.993, 0.0155, and 11.6, respectively, which are better than the traditional model obviously. In addition, the number of wavelengths reduced from 228 to 70 as the model is simplified with the uninformative variable elimination (UVE) method. The time of training is reduced by 29.30% compared with the original spectrum. It can be verified that the IPSO-ELM model has good prediction performance, and the NIR diffuse reflectance spectroscopy is a reliable nondestructive measurement of SSC in apples.

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 CC R -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., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Apple (Malus domestica) exhibits classic S-RNase-mediated gametophytic self-incompatibility. Previous studies have shown that the S-RNase secreted from style cells could trigger signal transduction and defense responses mediated by Ca 2+ and reactive oxygen species (ROS) after entering into the pollen tube. In this study, we investigated the downstream genes activated by ROS during S-RNase-mediated gametophytic self-incompatibility in pollen tubes. A substantial increase in ROS, as well as up-regulated expression of a serine-threonine protein kinase gene, OXIDATIVE SIGNAL-INDUCIBLE1 (MdOXI1), was detected in apple pollen tubes treated with self-S-RNase. A kinase assay-linked phosphoproteomics (KALIP) analysis suggested that MdOXI1 could bind and phosphorylate the downstream protein kinase Pto-interacting protein 1-like (MdPTI1L). The phosphorylation level of MdPTI1L was significantly reduced after silencing MdOXI1 with antisense oligonucleotides in the pollen tube. Silencing of either MdOXI1 or MdPTI1L alleviated the inhibitory effect of self-S-RNase on pollen tube growth. Our results thus indicate that MdPTI1L is phosphorylated by MdOXI1 in the pollen tube and participates in the ROS signaling pathway triggered by S-RNase., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 'Golden Delicious' 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 'Hanfu' and at low levels in susceptible variety 'Golden Delicious' following ALT1 infection. MdhpRNA277 was strongly induced in 'Golden Delicious' but not 'Hanfu' following ALT1 inoculation. MdhpRNA277 promoter activity was much stronger in inoculated 'Golden Delicious' versus 'Hanfu'. We identified a single-nucleotide polymorphism (SNP) in the MdhpRNA277 promoter region between 'Golden Delicious' ( pMdhpRNA277-GD ) and 'Hanfu' ( pMdhpRNA277-HF ). The transcription factor MdWHy binds to pMdhpRNA277-GD , but not to pMdhpRNA277-HF Transgenic 'GL-3' 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 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., (© 2018 American Society of Plant Biologists. All rights reserved.)