Summary MicroRNAs play important roles in the regulation of gene expression in plants and animals. However, little information is known about the action mechanism and function of fungal microRNA‐like RNAs (milRNAs). In this study, combining deep sequencing, molecular and histological assays, milRNAs and their targets in the phytopathogenic fungus Valsa mali were isolated and identified. A critical milRNA, Vm‐milR16, was identified to adaptively regulate the expression of virulence genes. Fourteen isolated milRNAs showed high expression abundance. Based on the assessment of a pathogenicity function of these milRNAs, Vm‐milR16 was found to be a critical milRNA in V. mali by regulating sucrose non‐fermenting 1 (VmSNF1), 4,5‐DOPA dioxygenase extradiol (VmDODA), and a hypothetical protein (VmHy1). During V. mali infection, Vm‐milR16 is downregulated, while its targets are upregulated. Overexpression of Vm‐milR16, but not mutated Vm‐milR16, significantly reduces the expression of targets and virulence of V. mali. Furthermore, deletion of VmSNF1, VmDODA and VmHy1 significantly reduce virulence of V. mali. All three targets seem to be essential for oxidative stress response and VmSNF1 is required for expression of pectinase genes during V. mali–host interaction. Our results demonstrate Vm‐milRNAs contributing to the infection of V. mali on apple trees by adaptively regulating virulence genes.

Lateral flow immunoassay (LFIA) has emerged as an effective technique in the field of food safety and environmental monitoring. However, sensitive and quantitative detection is still challenging for LFIAs in complex environments. In this work, a dual-model colorimetric/SERS lateral flow immunoassay for ultrasensitive determination of clenbuterol was constructed based on a metallic core–shell Au/Au nanostar acting as a multifunction tag. Raman reporter molecules are located between the core (AuNP) and shell (Au nanostar) to form a sandwich structure, which contributes to eliminate the environmental interference and improve the detection stability. In addition, the Au/Au nanostar provides a much higher Raman enhancement due to the presence of sharp tips and larger surface roughness in comparison with gold nanoparticles (AuNPs). Thus, on the basis of the antibody–antigen interaction, the dual-model immunoassay can produce strong colorimetric and surface-enhanced Raman spectroscopy (SERS) signals for highly sensitive detection of the target analyte, clenbuterol. Under optimal conditions, clenbuterol could be detected by the colorimetric model with a visual detection limit of 5 ng/mL. Meanwhile, the SERS signal of the Au/Au nanostar was accumulated on the test line for the SERS model detection with a quantitative detection limit as low as 0.05 ng/mL, which is at least 200-fold lower than that of the traditional AuNPs-based immunoassay. Furthermore, recovery rates of the proposed method in food samples were 86–110%. This dual-model immunoassay provides an effective tool for antibiotic residues analysis and demonstrates a broad potential for future applications in food safety monitoring.

Summary Small RNAs (sRNAs) play important roles in various biological processes by silencing their corresponding target genes in most eukaryotes. However, cross‐kingdom regulation mediated by fungal microRNA‐like RNAs (milRNAs) in plant–pathogen interactions is still largely unknown. Using molecular, genetic, histological, and biochemical approaches, we found that the apple tree Valsa canker pathogen Valsa mali milRNA Vm‐milR1 could suppress the host immunity by silencing two host receptor‐like kinase genes, MdRLKT1 and MdRLKT2. Vm‐milR1 was highly induced during V. mali infection. Deletion of Vm‐milR1 precursor abolished the generation of Vm‐milR1 and reduced the virulence of V. mali. Inoculation of Vm‐milR1 deletion mutants induced the host defence responses, including reactive oxygen species (ROS) accumulation, callose deposition, and high expression of defence‐related genes. Furthermore, Vm‐milR1 was confirmed to be able to suppress the expression of MdRLKT1 and MdRLKT2 in a sequence‐specific manner. Moreover, overexpression of either MdRLKT1 or MdRLKT2 enhanced apple resistance to V. mali by activating the host defence responses. Furthermore, knockdown of MdRLKT1 or MdRLKT2 compromised the host resistance to V. mali. Our study revealed that V. mali was equipped with Vm‐milR1 as an sRNA effector to silence host receptor‐like kinase genes, suppress the host defence responses, and facilitate pathogen infection.

Expanding sensing modes and improving catalytic performance of nanozyme-based analytical chemistry are beneficial to realizing the desired biosensing of analytes. Herein, Schiff-base chemistry coupled with a novel catechol oxidase-like nanozyme (CHzyme) is designed and constructed, exhibiting two main advantages, including (1) improving catalytic performance by nearly 2-fold compared with only the oxidase-like role of CHzyme; (2) increasing the designability of the output signal by signal transduction of cascade reaction. Thereafter, the substrate sensing modes based on a cascade reaction between the CHzyme-catalyzed reaction and Schiff-base chemistry are proposed and comprehensively studied, containing catalytic substrate sensing mode, competitive substrate sensing mode, and generated substrate sensing mode, expecting to be employed in environmental monitoring, food analyses, and clinical diagnoses, respectively. More meaningfully, the generated substrate sensing mode is successfully applied to construct a cascade reaction coupling ratiometric fluorescent immunoassay for the detection of clenbuterol, increasing 15-fold in detection sensitivity compared with the traditional enzyme-linked immunosorbent assay. It is expected that the expanded universal substrate sensing modes and the Schiff-base chemistry-enhanced nanozyme can enlighten the exploration of innovative biosensors.

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