Cross-kingdom RNA interference has emerged as an important mechanism in plant-pathogen interactions, yet how fungal small RNAs coordinate host immune suppression remains unclear. Here, we demonstrate that Alternaria alternata f. sp. mali, the causal agent of apple leaf spot, suggests microRNA-like RNAs (milRNAs) into host tissues in association with extracellular vesicles (EVs). Small RNA sequencing of fungal EVs identified multiple milRNAs, among which AamilR251, AamilR292 and AamilR004 were the three most abundant species. These AamilRNAs target three apple (Malus domestica) immune-related kinases (MdMAPKKK1, MdRLK2 and MdMAPKK6). Molecular and genetic analyses revealed that these proteins form an interconnected signalling module and a linear phosphorylation cascade (MdRLK2-MdCRK10-MdMAPKKK1-MdMAPKK6) required for effective defence activation. Deletion of individual AamilRNAs significantly attenuated fungal virulence and restored expression of their respective host targets. Conversely, overexpression of MdMAPKKK1, MdRLK2 or MdMAPKK6 enhanced resistance to infection. Pull-down assays identified MdCRK10 as an interacting partner of MdRLK2, forming a complex with MAPKs that activates defence signalling. MdRLK2 phosphorylates MdCRK10 at S144 and S363, and mutation of these sites disrupts signal transduction and compromises disease resistance. These findings reveal a central immune signalling network in apple and identify key components for improving resistance against fungal pathogens.

Apple leaf spot severely threatens global apple production, and with chemical control posing environmental and health risks, resistance breeding necessitates exploration of a previously unknown regulatory mechanism that distinguishes ALT1-susceptible from -resistant apple cultivars. Small RNA sequencing of Alternaria alternata f. sp. mali (ALT1)-infected susceptible apple 'Golden Delicious' revealed a novel tRNA-Val-derived tsRNA (tsRVal) that negatively regulates MdTIR-1 expression to modulate ALT1 resistance. Our findings revealed that tsRVal biosynthesis required cleavage by the ribonuclease T2 family member MdRNS3a. Interestingly, we found that DNA methylation of the MdRNS3a promoter negatively regulates its expression in the resistant apple cultivar Han Fu, which in turn abrogates tsRVal biogenesis and maintains resistance to ALT1. Conversely, ALT1 inoculation induced promoter hypomethylation in GD, leading to robust MdRNS3a activation, significant tsRVal accumulation, and the subsequent development of pathogenic phenotypes. Our findings further support a regulatory model wherein MdbHLH3 functions as a methylation-sensitive transcriptional activator of MdRNS3a via promoter binding, thereby determining tsRVal biosynthesis and the resultant leaf spot sensitivity. Our study demonstrates that the methylation status of the MdRNS3a promoter can serve as a reliable epigenetic marker for distinguishing resistant and susceptible apple varieties, thus providing a valuable tool to facilitate disease-resistant apple breeding programs.

Alternaria leaf spot, a fungal disease affecting apple production globally, incurs significant economic losses annually. The pathogenic fungus, A. alternata f. sp. mali (ALT), produces the host-specific AM-toxin and secretory proteins, which disrupt plant metabolism, leading to increased defoliation, fruit drop, compromised fruit quality, and reduced production. In this study, we isolated eight novel ALT strains from Fuji apple leaves exhibiting leaf spot. The most aggressive strain, ALT7, was identified. To determine ALT7 secretory proteins, LC-MS analysis was performed, which showed that three secreted proteins were detected: AaAO (alcohol oxidase), AaPDE (alkaline phosphatase), and AaABC (ATP-binding cassette transporter). Agrobacterium-mediated transgenesis confirmed the extracellular localization of GFP-fused AaAO, AaABC, and AaPDE. To investigate their function, we used fungal transgenesis. Overexpression of AaABC, AaAO, and AaPDE in ALT7 increased its pathogenicity. Conversely, knocking them down decreased ALT7 pathogenicity. Additionally, AaABC was found to facilitate the secretion of AaAO and AaPDE. And AaAO and AaPDE facilitate plant susceptibility by degrading plant cell walls, while AaABC plays a crucial role in their transport, thereby participating in the plant pathogenic process. In conclusion, our findings suggest that AaABC in A. alternata f. sp. mali mediates the secretion of toxic proteins AaAO and AaPDE.

Glomerella leaf spot (GLS), incited by Colletotrichum gloeosporioides, poses a significant threat to apple production. While microRNAs (miRNAs) are known to be critical regulators of plant-pathogen interactions, their specific roles in the apple-C. gloeosporioides interaction are not fully understood. In this study, we identified two novel miR395 family members, designated miR395m and miR395n, which are significantly upregulated in susceptible apple varieties upon C. gloeosporioides infection. Both miRNAs were found to target and cleave MdTHESEUS1 (MdTHE1) and MdLaccase12 (MdLAC12). Overexpression of miR395m and miR395n in resistant variety 'Fuji' ('FJ') compromised resistance to C. gloeosporioides, while silencing these miRNAs in 'Royal Gala' ('GL-3') resulted in attenuated disease symptoms compared to control plants. Consistently, silencing of MdTHE1 and MdLAC12 in 'FJ' increased susceptibility to C. gloeosporioides, whereas their overexpression in 'GL-3' diminished disease severity. In addition, overexpression of miR395m/n suppressed the expression of pathogenesis-related (PR) proteins, the reactive oxygen species (ROS) scavenging system, and reduced lignin content. Taken together, miR395m/n modulate resistance to C. gloeosporioides by regulating MdLAC12 and MdTHE1. This study reveals a conserved miRNA-mediated regulatory module that fine-tunes defense responses in apple and provides new insights into GLS resistance breeding.

Apple early defoliation disease poses a serious threat to the sustainability of global apple production, causing substantial yield losses and reduced fruit quality. Chemical fungicides remain the primary means of disease control; however, their excessive use leads to environmental pollution and food safety concerns. Here, we identified a pathogen-induced tRNA-derived small RNA (tsRVal) that targets the disease-resistance gene Malus domestica TRANSPORT INHIBITOR RESPONSE 1 (MdTIR-1), thereby modulating apple susceptibility to early defoliation disease. To suppress tsRVal accumulation, we designed an artificial miRNA (si-tsRVal) and loaded it onto Fe3O4 magnetic nanoparticles (MNPs) to form stable spherical complexes (MNP-si-tsRVal). The MNP system efficiently delivered si-tsRVal plasmids into apple leaf cells via foliar spraying, as confirmed by transmission electron microscopy analyses and energy-dispersive spectroscopy. In both potted seedlings and field-grown trees, plants treated with MNP-EV exhibited severe leaf drop, whereas MNP-si-tsRVal-treated plants retained healthy foliage and vigor. Collectively, these results demonstrate that MNP-si-tsRVal enhances resistance to early defoliation disease, providing durable protection and a sustainable alternative to chemical fungicide application.

Apple (Malus domestica) is an economically important fruit crop, but its production is affected by Glomerella leaf spot, a devastating disease caused by the fungal pathogen Colletotrichum gloeosporioides. MicroRNA (miRNA) is a kind of non-coding RNA that plays an important role in the process of plant–pathogen interactions. However, little is known about the miRNAs that influence apple resistance against C. gloeosporioides. A novel miRNA, MIR396d-p3, was identified through small RNA sequencing (sRNA-seq). Functional analyses revealed that MIR396d-p3 negatively regulates apple resistance to C. gloeosporioides. In addition, MdUGT89A2 and MdRGA3 were confirmed as targets of MIR396d-p3 using 5′ RACE and heterologous expression assays. We further found that overexpressing MdUGT89A2 and MdRGA3 induce apple disease resistance to C. gloeosporioides, while silencing of MdUGT89A2 and MdRGA3 reduces resistance to C. gloeosporioides. These results indicate that MIR396d-p3 plays a role in the response to the infection of C. gloeosporioides through regulating the expressions of MdUGT89A2 and MdRGA3. This research provides a new perspective on the interaction between apples and C. gloeosporioides and offers possible targets for resistance breeding.

Nutrient acquisition through symbiotic ectomycorrhizal fungi is carbon (C) costly but fundamental for plant growth, community, and ecosystem functioning. Here, we examined the functions of roots and mycorrhiza with respect to nutrient uptake after artificially inducing C limitation-seven months after girdling of an ectomycorrhizal tree, Pinus taeda. Root physiological activity (measured as root nitrogen content and root exudation) declined after girdling and was accompanied with 110% and 340% increases in mycorrhizal colonization and extramatrical hyphal length, respectively. Fungi colonizing roots switched to a community characterized by higher C efficiency (lower C cost) of nutrient acquisition (CENA, the amount of nutrient acquisition per unit C cost) and lower network complexity, indicating a tradeoff between CENA and stability of the fungal community. Root transcriptome analysis suggested a shift in metabolic pathways from a tricarboxylic acid cycle decomposition of carbohydrate to lipid biosynthesis to maintain closer associations with mycorrhiza for nutrient cycling after the girdling. By integrating multi-level evidence, including root transcriptome, fungal composition, and network complexity data, we demonstrate an increased dependence on mycorrhiza for nutrient acquisition under the C limitation condition, which is likely due to a shift to fungal community with higher CENA at the cost of lower stability.

Suppression of roots and/or their symbiotic microorganisms, such as mycorrhizal fungi and rhizobia, is an effective way for alien plants to outcompete native plants. However, little is known about how invasive and native plants interact with the quantity and activity of nutrient-acquisition agents. Here a pot experiment was conducted with monoculture and mixed plantings of an invasive plant, Xanthium strumarium, and a common native legume, Glycine max. We measured traits related to root and nodule quantity and activity and mycorrhizal colonization. Compared to the monoculture, fine root quantity (biomass, surface area) and activity (root nitrogen (N) concentration, acid phosphatase activity) of G. max decreased in mixed plantings; nodule quantity (biomass) decreased by 45%, while nodule activity in N-fixing via rhizobium increased by 106%; mycorrhizal colonization was unaffected. Contribution of N fixation to leaf N content in G. max increased in the mixed plantings, and this increase was attributed to a decrease in the rhizosphere soil N of G. max in the mixed plantings. Increased root quantity and activity, along with a higher mycorrhizal association was observed in X. strumarium in the mixed compared to monoculture. Together, the invasive plant did not directly scavenge N from nodule-fixed N, but rather depleted the rhizosphere soil N of the legume, thereby stimulating the activity of N-fixation and increasing the dependence of the native legume on this N source. The quantity-activity framework holds promise for future studies on how native legumes respond to alien plant invasions.

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.

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.

Understanding the relationships between ecosystem services (ES) and the factors driving their changes over long periods and multiple scales is key for landscape managers in decision-making. However, the widespread implementation of restoration programs has led to significant ES changes, with trade-offs across space and time that have been little explored empirically, making it challenging to provide effective experience for managers. We quantified changes and interactions among five ES across various stages of the Grain-to-Green Program in the eastern Loess Plateau, examining these dynamics at threefold spatial scales. We observed notable increases in soil retention and Net Ecosystem Production but declines in habitat quality and Landscape aesthetics under afforestation. Over time, and with more integrated restoration strategies, synergies between ES pairs weakened, and non-correlations (even trade-offs) increased. To avoid unnecessary trade-offs, we recommend incorporating socio-ecological factors driving ES changes and ES bundles, informed by empirical experience, into proactive spatial planning and environmental management strategies for multi-ES objectives. The temporal lags and spatial trade-offs highlighted by this study offer crucial insights for large-scale restoration programs worldwide.

Apple leaf spot, caused by Alternaria alternata f. sp mali (ALT), poses a substantial threat to the global apple (Malus x 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.

OBJECTIVES:Circular RNAs (circRNAs) are involved in carcinogenesis, though their expression profile in renal cell carcinoma (RCC) is uncharacterized. The tumor suppressor gene miR-145-5p is expressed in RCC tissues, but its relationship with circRNAs is unknown. Thus, we aimed to identify differentially expressed circRNAs in RCC tissues and to explore the interaction between these circRNAs and miR-145 in the development of RCC.METHODS:We performed high-throughput sequencing and bioinformatics analyses to examine the expression pattern of circRNAs in RCC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to functionally annotate differentially expressed circRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for sequence verification. Small interfering RNAs were employed to investigate the function and mechanism of circRNAs in RCC. The relationship between miR-145-5p and circRNAs was confirmed using luciferase, RNA immunoprecipitation (RIP), and biotin-coupled probe RNA pull-down assays.RESULTS:Fifty-three circRNAs were significantly and differentially expressed in RCC compared to normal control tissue. Bioinformatic analyses indicated that two significantly upregulated circRNAs, circ-AFF2 and circ-ASAP1, had sequences corresponding to miR-145 response elements. Consistently, the luciferase reporter, RIP, and biotin-coupled probe RNA pull-down assays showed that circ-AFF2 and circ-ASAP1 may repress miR-145 by acting as sponges. circ-AFF2 and circ-ASAP1 were highly expressed in RCC patient-derived tumor samples; their overexpression correlated with poor prognosis and low miR-145 levels. Knockdown of circ-AFF2 or circ-ASAP1 in RCC cell lines inhibited proliferation, underscoring their oncogenic function. A circRNA-miRNA network was constructed for RCC using the differentially expressed circRNAs and projected miRNAs. Candidate genes were verified by RT-qPCR and western blot, indicating that circ-AFF2 and circ-ASAP1 may be connected to RCC proliferation and metastasis.CONCLUSION:circ-AFF2 and circ-ASAP1 were upregulated in RCC and likely promote tumor progression by sponging miR-145. Therefore, both circRNAs should be investigated further as potential diagnostic and therapeutic targets for RCC.

目的:总结妊娠期肾绞痛的诊治经验。方法:回顾性分析2016年11月至2020年1月南方医科大学深圳医院收治的38例妊娠期肾绞痛患者的临床资料。患者年龄(29.95±2.54)岁，妊娠各个时期均有发生肾绞痛，常见于中晚期(86.84%)。明确发现结石23例，结石合并尿脓毒血症1例。治疗上37例患者首选保守治疗，5例保守治疗失败及1例尿脓毒血症患者行局麻输尿管内支架管置入术。结果:首选保守治疗的患者中，32例症状缓解，6例置管患者术后症状均得到缓解。所有患者均未发生泌尿外科及产科相关并发症。结论:妊娠期肾绞痛的治疗方法以保守治疗为主，当需要外科干预时，首选局麻输尿管内支架管置入术，创伤小且安全有效。

目的 探讨非浸润性膀胱癌合并良性前列腺增生进行同期经尿道电切术的可行性.方法 选取2013年1月至2014年12月南方医科大学深圳医院诊治的92例非浸润性膀胱癌合并良性前列腺增生患者作为研究对象.根据手术方式不同,将92例患者分为研究组A(n =32)、研究组B(n=35)和对照组(n=25).研究组A进行传统的膀胱部分切除术、耻骨上前列腺摘除术.研究组B进行同期经尿道膀胱肿瘤电切术合并前列腺汽化电切术.对照组进行同期经尿道膀胱肿瘤电切术.比较各组临床疗效,术后前列腺症状改善情况及生活质量,术后性功能、并发症及复发情况.结果 研究组B与研究组A比较,具有术中出血量少,手术时间、膀胱冲洗时间、留置尿管时间、住院时间短的优势,差异具有统计学意义(P＜0.01);且术后前列腺症状改善情况、患者生活质量基本一致,IPSS评分、QOL评分、PVR值、MRF值差异均无统计学意义(均P＞0.05).各组术后性功能指标无差异无统计学意义(P＞0.05).研究组B的术后并发症发生率明显低于研究组A,差异具有统计学意义(P＜0.01),与对照组差异差异无统计学意义(P＞0.05).三组的复发时间差异无统计学意义(P＞0.05).结论 同期经尿道进行膀胱肿瘤与前列腺电切术能够明显改善患者的前列腺症状,提高患者生活质量,安全性较高.

目的 探究酪蛋白激酶2相互作用蛋白1(CKIP-1)表达在前列腺部尿道少瘢痕愈合中的作用机制.方法 收集小儿尿道瘢痕组织和正常尿道组织.通过酶消化法联合组织块法建立原代的人尿道瘢痕成纤维细胞.通过质粒转染沉默或过表达CK-IP-1.通过qPCR和Western blot检测mRNA和蛋白的表达水平.CCK-8法检测细胞活力.免疫荧光染色检测α-SMA的表达.结果 与正常尿道组织相比,瘢痕组织中CKIP-1水平降低而ROCK升高(P<0.05).OE-CKIP-1组的CKIP-1 mRNA和蛋白水平升高,细胞活力、α-SMA、COLⅠ、COLⅢ、TGF-β1和ROCK蛋白水平均显著低于对照组(P<0.05).siCKIP-1组的CKIP-1 mRNA和蛋白水平降低,其他上述指标均显著高于对照组(P<0.05).结论 CKIP-1蛋白可能通过下调TGF-β1抑制ROCK2相关通路,并抑制COLⅠ、COLⅢ和α-SMA的表达,从而抑制人尿道瘢痕成纤维细胞纤维化.

Abstract Purpose: The aim of the present study was to assess whether SARS-CoV-2 can be detected in the expressed prostatic secretion (EPS) of patients with corona virus disease 2019 (COVID-19). Methods: 18 cases of COVID-19, and 5 suspected cases, were selected from three medical centers to detect the RNA expression of SARS-CoV-2 in their EPS with RT-PCR. Results: Results were negative in all EPS samples for SARS-CoV-2 of suspected and confirmed patients. Conclusions: No SARS-CoV-2 was expressed in EPS of patients with COVID-19.

Mycorrhizal fungi symbiosis is an important strategy for plant to uptake soil nutrients. Alien plants could thwart the symbiotic relationship between native plants and mycorrhizal fungus, and thus suppress the growth of native plants, which is an important mechanism for alien plant invasion, and has been increasingly emphasized in recent studies. In the present review, we summarized several key aspects of such mycorrhizal-related mechanism of plant invasion: 1) the impacts of alien plants on mycorrhizal fungi of native plants (i.e. mycorrhizal colonization rate, internal structures of mycorrhizal hyphae, amount of external hyphae, mycorrhizal and non-mycorrhizal composition, and mycorrhizal network); 2) mechanisms of alien-plant impacts on mycorrhizal fungi of native plants, including ecological mechanisms such as resource competition, allelopathy and edaphic fertility, as well as their molecular mechanisms; 3) variations of the above mentioned alien-plant impacts and related mechanisms at different durations of plant invasion. Despite thwarting mycorrhizal fungi of native plants is an important mechanism for alien plant invasion, researches on such mechanism are still scarce comparing to other mechanisms such as natural enemy release and new weapon hypothesis. Therefore, we proposal several research areas that need to be focused on in future studies: 1) how do global changes affect the alien-plant-invasion impacts on mycorrhizal fungi of native plants; 2) what are the relationships among different mechanisms including the mycorrhizal-related mechanism; 3) how does the mycorrhizae-related mechanism change at large spatio-temporal scales.