Feng Li

RNA silencing plays a central role in plant antiviral defence, in which viral RNA is processed into small RNAs (sRNAs) that guide Argonaute-mediated cleavage of viral transcripts. Among these, 22-nucleotide (nt) sRNAs are unique in their ability to trigger secondary sRNA production, thereby amplifying transitive silencing. In tomato, this 22-nt antiviral pathway is strongly activated upon virus infection through the upregulation of DCL2b, yet the underlying mechanism has remained unclear. miR6026, generated by DCL2b and targeting DCL2b itself, forms a feedback loop that fine-tunes this response. Here we show that tobacco mosaic virus (TMV) and tomato bushy stunt virus (TBSV) activate DCL2b transcription via the Dof16 transcription factor. Transcription of MIR6026 is also activated by both TMV and TBSV, while viral infection promotes DCL2b relocalization towards the cytoplasm, preventing pri-miR6026 processing into mature miR6026. This redistribution is associated with reduced phosphorylation of DCL2b at Tyr69, which weakens its interaction with importin-α and limits its nuclear localization. Notably, Arabidopsis DCL2 exhibits a similar virus-induced relocalization pattern, highlighting a conserved mechanism that enhances antiviral silencing during infection while minimizing off-target effects on host genes under normal conditions.

SUMMARY: Tomato is an important vegetable crop that is rich in genetic variation, but published high‐quality tomato reference genomes are limited. Small RNAs play ubiquitous roles in plants, but their biogenesis in tomatoes is not well studied. We assembled a high‐quality chromosome‐level reference genome for the tomato cultivar Ailsa Craig 57 (A57) and generated CRISPR mutants deficient in double‐stranded RNA‐binding protein 1 ( SlDRB1 ) in this cultivar. Mutations in SlDRB1 led to severely abnormal development (e.g., loss of leaf polarity, pollen abortion, and infertility). High‐throughput sRNA sequencing analysis revealed that SlDRB1, similar to its Arabidopsis ortholog Hyponastic Leaves 1 ( HYL1 ), is required for the biogenesis of most miRNAs. Interestingly, SlDRB1 is also required for the accumulation of a large population of small RNAs derived from rRNAs and negatively regulates miR6026, which is dependent on DCL2 instead of DCL1 . Comprehensive protein–protein interaction studies between SlDRB1 and SlDCL1 uncovered detailed interaction mechanisms involving subdomains of these two proteins. Their potential role in miRNA biogenesis is discussed.

Pepper is an economically important crop. Owing to its recalcitrance to genetic transformation, virus-induced gene silencing (VIGS) is currently the major technique available for validating gene function in pepper. However, the low efficiency and difficulty of silencing genes in reproductive organs remain major challenges in pepper VIGS studies. To address these limitations, we developed an optimized VIGS system by structure-guided truncation of the Cucumber mosaic virus 2b (C2b) silencing suppressor. A silencing suppression assay revealed that the C2bN43 mutant retained systemic silencing suppression while abrogated local silencing suppression activity in systemic leaves. The engineered TRV-C2bN43 system significantly enhanced VIGS efficacy in pepper, providing a powerful tool for functional genomics studies in pepper. By leveraging transcriptomic profiles, we identified CaAN2, an anther-specific MYB transcription factor, whose suppression via TRV-C2bN43 perturbation resulted in coordinated downregulation of structural genes in anthocyanin biosynthesis pathway and abolished anthocyanin accumulation in anthers establishing its essential regulatory role in pigmentation. This study validated and provided mechanistic insight for a further optimized VIGS system in pepper.

Plant viruses impose serious threats to agriculture in China and worldwide

SUMMARY: The accessory proteins Hyponastic‐like 1 (HYL1) and Serrated (SE) enhance the precise and efficient processing of miRNAs by Dicer‐like 1 (DCL1), which is important for proper miRNA function. However, other factors determining the precision and efficiency of miRNA biogenesis are not well‐known. Here, we found that an asymmetric bulge (AB) at the 3′ end of miR‐5p (produced from the 5′ arm of the pre‐miRNA) reduced the precision of the second cleavage, whereas an AB at other sites of miR‐5p mainly affected the accumulation level of miR‐5p in transient expression in Nicotiana benthamiana. In contrast, many ABs in miR‐3p (produced from the 3′ arm of the pre‐miRNA) impose strong negative impact on the processing precision and the accumulation level of miR‐5p in N. benthamiana. Arabidopsis DCL1/SE/HYL1 complex‐mediated miRNA processing was reconstituted in Saccharomyces cerevisiae to further investigate AB‐mediated interference with DCL1 processing. With this system, the positional effect of AB on miRNA processing was tested. The results showed that ABs on the middle of miR‐5p have less of an impact on DCL1 cleavage efficiency and precision, whereas those on miR‐3p or near the ends of miR‐5p strongly reduce DCL1 cleavage activity, precision or both. Studies using the yeast miRNA processing system and transgenic Arabidopsis also revealed the importance of the interaction between the 2‐nt 3′ overhang of pre‐miRNA and the 3′ overhang binding pocket (3′BP) on the precision of the second cleavage reaction for many endogenous miRNAs. These findings provide new insights into the mechanism of miRNA biogenesis.

Key message CaPCR1 (Capana12g002165) was a candidate gene regulating fruit concave/pointed tip shape in pepper. Abstract The concave shape of the fruit tip in pepper plants is highly susceptible to drought and low temperature stresses, resulting in the appearance of a pointed tip fruit, which affects its commercial value. However, few studies on the process of fruit tip development and regulatory genes in pepper have been reported. Herein, the developmental process of the ovary before anthesis, especially changes in the shape of the ovary tip, was studied in detail. The results showed that the final fruit tip shape was consistent with the ovary tip shape before anthesis, and a concave tip shape gradually developed. F₄ recombinant inbred lines (RILs) were constructed to map the genes regulating fruit tip shape through hybridization of the LRS and SBS pepper inbred lines. CaPCR1 (Capana12g002165), an OFP (OVATE Family Protein) family gene, was located in the candidate region on chr12. Three SNPs were found in the protein coding sequence of CaPCR1 between SBS and LRS, but only one SNP led to amino acid variation. Sequence variations, including base replacements, deletions and insertions, were also detected in the gene promoter region. The relative expression level of the CaPCR1 gene was significantly greater in the concave tip ovary than in the pointed tip ovary. qRT‒PCR analysis revealed that the CaPCR1 gene was expressed mainly in the gynoecium, placenta and green fruit pericarp, which was consistent with its function in ovary and fruit development. Taken together, these results suggested that CaPCR1 is a candidate gene involved in fruit tip shape determination in pepper.

Coverage quantification is required in many sequencing datasets within the field of genomics research. However, most existing tools fail to provide comprehensive statistical results and exhibit limited performance gains from multithreading. Here, we present PanDepth, an ultra-fast and efficient tool for calculating coverage and depth from sequencing alignments. PanDepth outperforms other tools in computation time and memory efficiency for both BAM and CRAM-format alignment files from sequencing data, regardless of read length. It employs chromosome parallel computation and optimized data structures, resulting in ultrafast computation speeds and memory efficiency. It accepts sorted or unsorted BAM and CRAM-format alignment files as well as GTF, GFF and BED-formatted interval files or a specific window size. When provided with a reference genome sequence and the option to enable GC content calculation, PanDepth includes GC content statistics, enhancing the accuracy and reliability of copy number variation analysis. Overall, PanDepth is a powerful tool that accelerates scientific discovery in genomics research.