Recent advancements in the field of plant–pathogen interactions have spotlighted the role of extracellular vesicles (EVs) as pivotal mediators of cross-kingdom communication, offering new vistas for enhancing crop protection strategies. EVs are instrumental in the transport of small regulatory RNAs (sRNAs) and other bioactive molecules across species boundaries, thus playing a critical role in the molecular warfare between plants and pathogens. This review elucidates the sophisticated mechanisms by which plants utilize EVs to dispatch sRNAs that silence pathogenic genes, fortifying defenses against microbial threats. Highlighting both eukaryotic and prokaryotic systems, this review delves into the biogenesis, isolation, and functional roles of EVs, illustrating their importance not only in fundamental biological processes but also in potential therapeutic applications. Recent studies have illuminated the significant role of EVs in facilitating communication between plants and pathogens, highlighting their potential in host-defense mechanisms. However, despite these advancements, challenges remain in the efficient isolation and characterization of plant-derived EVs. Overcoming these challenges is critical for fully harnessing their potential in developing next-generation crop protection strategies. This review proposes innovative strategies for utilizing RNA-based interventions delivered via EVs to bolster plant resilience against diseases. By integrating the latest scientific findings with practical applications in agriculture, this review aims to enhance the connection between fundamental plant biology and the development of innovative crop management technologies.

Background: Physcomitrium patens provides an evolutionary link between green algae and vascular plants. Although the genome of P. patens includes orthologs of all the core lignin biosynthetic enzymes, the occurrence of lignin in moss is very controversial. Besides, little information is available about the lignin enzymes in moss to date. For example, cinnamyl alcohol dehydrogenase (CAD) is a crucial enzyme that catalyzes the last step of the lignin biosynthetic pathway, suggesting an ideal way to study the evolutionary process. By investigating the functions of CAD in evolution, this study will elucidate the evolutionary roles of lignin-like in the early stage of land colonization. Results: CAD multigene family in P. patens is composed of four genes. The PpCADs contain a conserved glycine-rich domain to catalyze NADPH-dependent reduction to their corresponding alcohols, indicating that PpCADs have the potential to synthesize monolignols by bioinformatics analysis. Even though PpCAD1 could produce lignin in theory, no conventional monomer was detected in the cell wall or cytoplasm of PpCAD1_OE plants. However, the phenylpropanoids were promoted in PpCAD1_OE transformants to modify gametophore architecture and development, making the distribution of phyllids more scarcity and the moss colony more giant, possibly due to the enhanced expression of the AUX-IAA family. The transcripts of at least one gene encoding the enzyme in the lignin biosynthetic pathway were increased in PpCAD1_OE plants. In addition, the PpCAD1_OE gametophore inhibited the Botrytis cinerea assault mainly by enhanced phenylpropanoids in the cell wall instead of influencing transcripts of defense genes pathogenesis-related 10 (PR10) and nonexpresser of PR genes 1 (NPR1). Likewise, ectopic expression of PpCAD1 in Arabidopsis led to a significant increase in lignin content, exhibiting chunky roots, robust seedlings, advanced flowering, and efficient resistance against pathogens. Conclusion: PpCAD occurs in more than one copy, suggesting functional divergence in the ancestral plant. PpCAD1 catalyzes monolignol biosynthesis and has homologous functions with vascular plants. Despite no detected conventional monolignol, the increased phenylpropanoids in the PpCAD1_OE gametophore, possibly intermediate metabolites in the lignin pathway, had conserved functions during the evolution of terrestrial plants. The results inferred that the lignin enzyme of the early non-vascular plant played roles in stem elongation and resistance against pathogens of P. patens during the conquest of land.

Background: The diploid woodland strawberry (Fragaria vesca) is an excellent model plant for investigating economically significant traits and several genetic resources within the Rosaceae family. Agrobacterium rhizogenes-mediated hairy root transformation is an alternative for exploring gene functions, especially the genes specifically expressed in roots. However, the hairy root transformation has not been established in strawberry. Results: Here, we described an efficient and rapid hairy root transgenic system for strawberry using A. rhizogenes. Strain of A. rhizogenes MSU440 or C58C1 was the most suitable for hairy root transformation. The transformation efficiency was highest when tissues contained hypocotyls as explants. The optimal procedure involves A. rhizogenes at an optical density (OD₆₀₀) of 0.7 for 10 min and co-cultivation duration for four days, achieving a transgenic efficiency of up to 71.43%. An auxin responsive promoter DR5_ver2 carrying an enhanced green fluorescent protein (eGFP) marker was transformed by A. rhizogenes MSU440, thereby generating transgenic hairy roots capable of high eGFP expression in root tip and meristem of strawberry where auxin accumulated. Finally, this system was applied for functional analysis using jGCaMP7c, which could sense calcium signals. A significant upsurge in eGFP expression in the transgenic hairy roots was displayed after adding calcium chloride. The results suggested that this approach was feasible for studying specific promoters and could be a tool to analyze gene functions in the roots of strawberries. Conclusion: We established a rapid and efficient hairy root transformation in strawberry by optimizing parameters, which was adequate for promoter analysis and functional characterization of candidate genes in strawberry and other rosaceous plants.

Small auxin-up-regulated RNAs (SAURs) are genes rapidly activated in response to auxin hormones, significantly affecting plant growth and development. However, there is limited information available about the specific functions of SAURs in rice due to the presence of extensive redundant genes. In this study, we found that OsSAUR10 contains a conserved downstream element in its 3′ untranslated region that causes its transcripts to be unstable, ultimately leading to the immediate degradation of the mRNA in rice. In our investigation, we discovered that OsSAUR10 is located in the plasma membrane, and its expression is regulated in a tissue-specific, developmental, and hormone-dependent manner. Additionally, we created ossaur10 mutants using the CRISPR/Cas9 method, which resulted in various developmental defects such as dwarfism, narrow internodes, reduced tillers, and lower yield. Moreover, histological observation comparing wild-type and two ossaur10 mutants revealed that OsSAUR10 was responsible for cell elongation. However, overexpression of OsSAUR10 resulted in similar phenotypes to the wild-type. Our research also indicated that OsSAUR10 plays a role in regulating the expression of two groups of genes involved in auxin biosynthesis (OsYUCCAs) and auxin polar transport (OsPINs) in rice. Thus, our findings suggest that OsSAUR10 acts as a positive plant growth regulator by contributing to auxin biosynthesis and polar transport.

Gibberellins (GAs) play indispensable roles in the fruit development of horticultural plants. Unfortunately, the molecular basis behind GAs regulating fruit development in R. roxburghii remains obscure. Here, GA₃ spraying to R. roxburghii ‘Guinong 5’ at full-bloom promoted fruit size and weight, prickle development, seed abortion, ascorbic acid accumulation, and reduction in total soluble sugar. RNA-Seq analysis was conducted to generate 45.75 Gb clean reads from GA₃- and non-treated fruits at 120 days after pollination. We obtained 4275 unigenes belonging to differently expressed genes (DEGs). Gene ontology and the Kyoto Encyclopedia of Genes and Genomes displayed that carbon metabolism and oxidative phosphorylation were highly enriched. The increased critical genes of DEGs related to pentose phosphate, glycolysis/gluconeogenesis, and citrate cycle pathways might be essential for soluble sugar degradation. Analysis of DEGs implicated in ascorbate revealed the myoinositol pathway required to accumulate ascorbic acid. Finally, DEGs involved in endogenous phytohormones and transcription factors, including R2R3 MYB, bHLH, and WRKY, were determined. These findings indicated that GA₃-trigged morphological alterations might be related to the primary metabolites, hormone signaling, and transcription factors, providing potential candidate genes that could be guided to enhance the fruit development of R. roxburghii in practical approaches.

Background: Pectate lyase (PL, EC 4.2.2.2), as an endo-acting depolymerizing enzyme, cleaves α-1,4-glycosidic linkages in esterified pectin and involves a broad range of cell wall modifications. However, the knowledge concerning the genome-wide analysis of the PL gene family in Fragaria vesca has not been thoroughly elucidated. Results: In this study, sixteen PLs members in F. vesca were identified based on a genome-wide investigation. Substantial divergences existed among FvePLs in gene duplication, cis-acting elements, and tissue expression patterns. Four clusters were classified according to phylogenetic analysis. FvePL6, 8 and 13 in cluster II significantly contributed to the significant expansions during evolution by comparing orthologous PL genes from Malus domestica, Solanum lycopersicum, Arabidopsis thaliana, and Fragaria×ananassa. The cis-acting elements implicated in the abscisic acid signaling pathway were abundant in the regions of FvePLs promoters. The RNA-seq data and in situ hybridization revealed that FvePL1, 4, and 7 exhibited maximum expression in fruits at twenty days after pollination, whereas FvePL8 and FvePL13 were preferentially and prominently expressed in mature anthers and pollens. Additionally, the co-expression networks displayed that FvePLs had tight correlations with transcription factors and genes implicated in plant development, abiotic/biotic stresses, ions/Ca²⁺, and hormones, suggesting the potential roles of FvePLs during strawberry development. Besides, histological observations suggested that FvePL1, 4 and 7 enhanced cell division and expansion of the cortex, thus negatively influencing fruit firmness. Finally, FvePL1-RNAi reduced leaf size, altered petal architectures, disrupted normal pollen development, and rendered partial male sterility. Conclusion: These results provide valuable information for characterizing the evolution, expansion, expression patterns and functional analysis, which help to understand the molecular mechanisms of the FvePLs in the development of strawberries.