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Arbuscular mycorrhizal fungi translocate polyphosphate through hyphae over a long distance to deliver to the host. More than three decades ago, suppression of host transpiration was found to decelerate phosphate delivery of the fungal symbiont, leading us to hypothesize that transpiration provides a primary driving force for polyphosphate translocation, probably via creating hyphal water flow in which fungal aquaporin(s) may be involved. The impact of transpiration suppression on polyphosphate translocation through hyphae of Rhizophagus clarus was evaluated. An aquaporin gene expressed in intraradical mycelia was characterized and knocked down by virus-induced gene silencing to investigate the involvement of the gene in polyphosphate translocation. Rhizophagus clarus aquaporin 3 (RcAQP3) that was most highly expressed in intraradical mycelia encodes an aquaglyceroporin responsible for water transport across the plasma membrane. Knockdown of RcAQP3 as well as the suppression of host transpiration decelerated polyphosphate translocation in proportion to the levels of knockdown and suppression, respectively. These results provide the first insight into the mechanism underlying long-distance polyphosphate translocation in mycorrhizal associations at the molecular level, in which host transpiration and the fungal aquaporin play key roles. A hypothetical model of the translocation is proposed for further elucidation of the mechanism.

Cape gooseberry (Physalis peruviana, L.) is a herbaceous plant belonging to the Solanaceae family that produces an edible berry appreciated for its nutraceutical and pharmaceutical properties. Its production is often limited by diseases and reproducible fruit quality. Recent studies have reported genes associated with fruit quality and resistance response to the root-infecting fungus Fusarium oxysporum f. sp. physali (Foph,) which causes vascular wilt. In order to standardize a method to validate the biological function of candidate genes in the non-model species P. peruviana, we tested the robust approach in reverse genetics, virus induced gene silencing (VIGS). In this study, we validated and optimized VIGS using an insert of the phytoenedesaturase (PDS) gene in a silencing viral vector generated from tobacco rattlevirus (TRV). Leaves infiltrated with Agrobacterium (GV3101 strain) showed photo-bleached segments, which were distinctive for PDS suppression at 7 days post-infection (dpi). More than half of the treated plants showed photo bleaching, indicating an efficiency rate of 50 % of the VIGS protocol. The results of this study showed that VIGS can be used for future functional gene characterization implicated in the immune response, disease resistance and fruit quality in capegooseberry.

Fusarium fungi are a pervasive threat to global agricultural productivity. They cause a spectrum of plant diseases that result in significant yield losses and threaten food safety by producing mycotoxins that are harmful to human and animal health. In recent years, the exploitation of the RNA interference (RNAi) mechanism has emerged as a promising avenue for the control of Fusarium‐induced diseases, providing both a mechanistic understanding of Fusarium gene function and a potential strategy for environmentally sustainable disease management. However, despite significant progress in elucidating the presence and function of the RNAi pathway in different Fusarium species, a comprehensive understanding of its individual protein components and underlying silencing mechanisms remains elusive. Accordingly, while a considerable number of RNAi‐based approaches to Fusarium control have been developed and many reports of RNAi applications in Fusarium control under laboratory conditions have been published, the applicability of this knowledge in agronomic settings remains an open question, and few convincing data on RNAi‐based disease control under field conditions have been published. This review aims to consolidate the current knowledge on the role of RNAi in Fusarium disease control by evaluating current research and highlighting important avenues for future investigation. We review biotechnology‐based crop protection against Fusarium diseases with novel RNA delivery technologies and RNA active ingredients.

• Virus-induced gene silencing (VIGS) can be harnessed to sequence-specifically degrade host transcripts and induce heritable epigenetic modifications referred to as virus-induced post-transcriptional gene silencing (ViPTGS) and virus-induced transcriptional gene silencing (ViTGS), respectively. Both ViPTGS and ViTGS enable manipulation of endogenous gene expression without the need for transgenesis. • Although VIGS has been widely used in many plant species, it is not always uniform or highly efficient. The efficiency of VIGS is affected by developmental, physiological and environmental factors. Here, we use recombinant Tobacco rattle viruses (TRV) to study the effect of temperature on ViPTGS and ViTGS using GFP as a reporter gene of silencing in N. benthamiana 16c plants. • We found that unlike ViPTGS, ViTGS was impaired at high temperature. Using a novel mismatch-small interfering RNA (siRNA) tool, which precisely distinguishes virus-derived (primary) from target-generated (secondary) siRNAs, we demonstrated that the lack of secondary siRNA production/amplification was responsible for inefficient ViTGS at 29°C. Moreover, inefficient ViTGS at 29°C inhibited the transmission of epigenetic gene silencing to the subsequent generations. • Our finding contributes to understanding the impact of environmental conditions on primary and secondary siRNA production and may pave the way to design/optimize ViTGS for transgene-free crop improvement.

• Plants use their innate immune system to defend against phytopathogens. As a part of this, pattern triggered-immunity is activated via pattern recognition receptor (PRR) detection of pathogen-associated molecular patterns (PAMPs). Although an increasing number of PAMPs have been identified, the PRRs for their recognition remain largely unknown. • In the present study, we report a receptor-like protein RE02 (Response to VmE02) in Nicotiana benthamiana, which mediates the perception of VmE02, a PAMP previously identified from the phytopathogenic fungus Valsa mali, using virus-induced gene silencing (VIGS), co-immunoprecipitation, pull-down and microscale thermophoresis assays. • We show that silencing of RE02 markedly attenuated VmE02-triggred cell death and immune responses. RE02 specifically interacted with VmE02 in vivo and in vitro, and it displayed a high affinity for VmE02. Formation of a complex with the receptor-like kinases SOBIR1 and BAK1 was essential for RE02 to perceive VmE02. Moreover, RE02-silenced plants exhibited enhanced susceptibility to both the oomycete Phytophthora capsici and the fungus Sclerotinia sclerotiorum, while overexpression of RE02 increased plant resistance to these pathogens. • Together, our results indicate that the PAMP VmE02 and the receptor-like protein RE02 represent a new ligand–receptor pair in plant immunity, and that RE02 represents a promising target for engineering disease resistance.

Summary Sugarcane mosaic virus (SCMV) is a pathogen of worldwide importance that causes dwarf mosaic disease on maize (Zea mays). Until now, few maize genes/proteins have been shown to be involved in resistance to SCMV. In this study, we characterized the role of maize phenylalanine ammonia‐lyases (ZmPALs) in accumulation of the defence signal salicylic acid (SA) and in resistance to virus infection. SCMV infection significantly increased SA accumulation and expression of SA‐responsive pathogenesis‐related protein genes (PRs). Interestingly, exogenous SA treatment decreased SCMV accumulation and enhanced resistance. Both reverse transcription‐coupled quantitative PCR and RNA‐Seq data confirmed that expression levels of at least four ZmPAL genes were significantly up‐regulated upon SCMV infection. Knockdown of ZmPAL expression led to enhanced SCMV infection symptom severity and virus multiplication, and simultaneously resulted in decreased SA accumulation and PR gene expression. Intriguingly, application of exogenous SA to SCMV‐infected ZmPAL‐silenced maize plants decreased SCMV accumulation, showing that ZmPALs are required for SA‐mediated resistance to SCMV infection. In addition, lignin measurements and metabolomic analysis showed that ZmPALs are also involved in SCMV‐induced lignin accumulation and synthesis of other secondary metabolites via the phenylpropanoid pathway. In summary, our results indicate that ZmPALs are required for SA accumulation in maize and are involved in resistance to virus infection by limiting virus accumulation and moderating symptom severity.

Chrysanthemum is one of the most economically important flowers globally due to its high ornamental value. In recent years, a large percentage of the Chrysanthemum seticuspe genome has been determined, making this species useful as a model chrysanthemum plant. To fully utilize the genome’s information, efficient and rapid gene functional analysis methods are needed. In this study, we optimized the tomato aspermy virus (TAV) vector for virus-induced gene silencing (VIGS) in C. seticuspe. Conventional plant virus inoculation methods, such as the mechanical inoculation of viral RNA transcripts and agroinoculation into leaves, did not achieve successful TAV infections in C. seticuspe, but vacuum infiltration into sprouts was successful without symptoms. The TAV vector harboring 100 nucleotides of the phytoene desaturase (PDS) gene caused photobleaching phenotypes and a reduction in CsPDS expression in C. seticuspe. To our knowledge, this is the first report of VIGS in chrysanthemums.

Withania somnifera produces pharmacologically important triterpenoid withanolides that are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be elucidated. A jasmonate- and salicin-inducible WRKY transcription factor from W. somnifera (WsWRKY1) exhibiting correlation with withaferin A accumulation was functionally characterized employing virus-induced gene silencing and overexpression studies combined with transcript and metabolite analyses, and chromatin immunoprecipitation assay. WsWRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol pathway genes with corresponding reduction in phytosterols and withanolides in W. somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W. somnifera (phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover, WsWRKY1 binds to W-box sequences in promoters of W. somnifera genes encoding squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid pathway. Furthermore, while WsWRKY1 silencing in W. somnifera compromised the tolerance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco led to improved biotic stress tolerance. Together these findings demonstrate that WsWRKY1 has a positive regulatory role on phytosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its importance as a metabolic engineering tool for simultaneous improvement of triterpenoid biosynthesis and plant defense.

MicroRNAs (miRNAs) are a class of noncoding RNAs that play important roles in regulating gene expression. They are involved in various biological processes, including plant growth, development, hormone signalling pathways and defence responses. Numerous studies have demonstrated the crucial role of miRNA in modulating plant immunity against various pathogens, including fungi, bacteria, viruses, nematodes and oomycetes. In this review, we synthesise recent advances in defence‐related miRNAs in response to pathogens, highlighting their effects on plant–pathogen interactions and their functions in regulating hormone signalling pathways. Additionally, we explore the potential of small RNA‐based technology tools in protecting plants from pathogens, including artificial microRNA, synthetic trans‐acting small interfering RNA and RNA interference techniques, such as spray‐induced gene silencing, host‐induced gene silencing and virus‐induced gene silencing. miRNAs modulate plant immunity against various pathogens, including fungi, bacteria, viruses, nematodes and oomycetes, by targeting pathogen effectors and modulating hormonal signalling.