MicroRNAs (miRNAs), a class of small non-coding regulatory RNAs, are key molecules in many biological and metabolic processes of plant growth, development and stress response via targeting mRNAs. The phloem-feeding insect whitefly Bemisia tabaci (Hemiptera, Aleyrodidae) is a serious pest that causes devastating harm to agricultural production worldwide. However, the function of host miRNAs in the response to whitefly infestation remains unclear. Here, we sequenced the small RNA and degradome of tobacco (Nicotiana tabacum L.), after and before infestation by B. tabaci. We identified 1291 miRNAs belonging to 138 miRNA families including 706 known miRNAs and 585 novel miRNAs. A total of 47 miRNAs were differentially expressed, of which 30 were upregulated and 17 were downregulated by whitefly exposure. Then, computational analysis showed that the target genes of differential miRNAs were involved in R gene regulation, plant innate immunity, plant pathogen defense, the plant hormone signal pathway and abiotic stress tolerance. Furthermore, degradome analysis demonstrated that 253 mRNAs were cleaved by 66 miRNAs. Among them, the targets cleaved by upregulated miR6025, miR160, miR171, miR166 and miR168 are consistent with our prediction, suggesting that pathogen-related miRNAs may function in plant defense against whitefly. Moreover, our results show that plant miRNA response and miRNA-mediated post-transcriptional regulation for phloem-feeding insect infestation are similar to pathogen invasion. Our study provides additional data to further elucidate how host plants respond and defend the phloem-feeding insects.

The molecular mechanisms of plant responses to phytophagous insect eggs are poorly understood, despite their importance in insect–plant interactions. This study investigates the plant defense mechanisms triggered by the eggs of whitefly Bemisia tabaci, a globally significant agricultural pest. A transcriptome comparison of tobacco plants with and without eggs revealed that whitefly eggs may activate the response of defense-related genes, including those involved in the salicylic acid (SA) signaling pathway. SA levels are induced by eggs, resulting in a reduction in egg hatching, which suggests that SA plays a key role in plant resistance to whitefly eggs. Employing Agrobacterium-mediated transient expression, virus-induced gene silencing assays, DNA–protein interaction studies, and bioassays, we elucidate the regulatory mechanisms involved. Pathogenesis-related proteins NtPR1-L1 and NtPR5-L2, downstream of the SA pathway, also affect whitefly egg hatching. The SA-regulated transcription factor NtWRKY70a directly binds to the NtPR1-L1 promoter, enhancing its expression. Moreover, NtPR1-L1 promotes callose deposition, which may impede the eggs’ access to water and nutrients. This study establishes the SA-WRKY70-PR-callose axis as a key mechanism linking plant responses and defenses against whitefly eggs, providing new insights into the molecular interactions between plants and insect eggs.

Herbivore insects deploy salivary effectors to manipulate the defense of their host plants, however, whether insect small RNAs (sRNAs) act as effectors to regulate plant-insect interaction is currently unclear. Here, we report that a microRNA (miR29-b) from the saliva of phloem-feeding insects can transfer into the host plant phloem and fine-tune the host defense. The salivary gland’s abundant miR29-b was produced by insect Dicer 1 and insect salivary exosome is involved in its transferring and releasing into the host plant. Insect miR29-b effector hijacks plant Argonaute 1 to silence host defense gene Bcl-2-associated athanogene 4 ( BAG4 ). Silencing of BAG4 suppressed the expression of phenylalanine ammonia-lyase and the accumulation of salicylic acid (SA), therefore negatively regulating host defense against herbivore insects. miR29-b is highly conserved in Hemiptera, Coleoptera, Hymenoptera, Orthoptera, and Blattaria insects and also targets the BAG4 gene. Notably, BAG4 orthologs exist in a wide range of plant species and may as the target of insect miR29-b. Our work provides new insight into the intriguing defense and counter-defense between herbivores and plants. Teaser: Phloem-feeding insects produce and transfer small RNA into the host plants to fine-tune plant basal defense by cross-kingdom gene silencing.