Induced Gene Silencing Research Articles

The Exocyst Subunits EqSec5 and EqSec6 Promote Powdery Mildew Fungus Growth and Pathogenicity.

The exocyst complex in eukaryotic cells modulates secretory vesicle transportation to promote exocytosis. The exocyst is also required for the hyphal growth and pathogenic development of several filamentous phytopathogens. Obligate biotrophic powdery mildew fungi cause considerable damage to many cash crops; however, the exocyst's roles in this group of fungi is not well studied. To verify the functions of the exocyst in powdery mildew fungus, we identified two exocyst subunits, EqSec5 and EqSec6, from Erysiphe quercicola, a powdery mildew fungus that infects the rubber tree Hevea brasiliensis. When GFP-fused EqSec5 and EqSec6 were introduced into E. quercicola and another phytopathogenic fungus, Magnaporthe oryzae, they primarily localized to the hyphal tip region. Inducing gene silencing of EqSec5 or EqSec6 caused growth and infection defects, and those defects could not be fully restored under the NADPH oxidase inhibitor treatment to the plant. The silenced strains also induced the host defense response including reactive oxygen species accumulation and callose deposition. The silencing of EqSec5 or EqSec6 also inhibited the secretion of the effector protein EqIsc1, interrupting plant salicylic acid biosynthesis. Yeast two-hybrid and gene overexpression assays suggested that EqSec5 and EqSec6 interact with each other and can complement each other's function during host infection. Overall, our study provides evidence that the exocyst in this powdery mildew fungus facilitates effector secretion, hyphal growth, and infection.

Barley stripe mosaic virus-induced gene silencing for functional validation of abiotic stress in barley.

The barley stripe mosaic virus (BSMV) uses its genomic RNA components (alpha, beta, and gamma) as an efficient method for studying gene functions. It is a newly developed method that utilizes gene transcript suppression to determine the role of plant genes. BSMV derived from virus induced gene silencing (VIGS) is capable of infecting various key farming crops like barley, wheat, rice, corn, and oats. Nevertheless, the growing acceptance and enhancement of BSMV-VIGS will benefit all kinds of plants. Abiotic stresses such as drought and salt are highly affecting plant growth, development, and production. BSMV-induced temporal gene knockdown is performed during particular stressful situations to determine their specific function. The quick physiological and biochemical changes aid in confirming the role of the target genes. VIGS has a significant role to improve crop genetics and breeding, despite having certain restrictions. Thus, exploring the possible solution and addressing these difficulties will enhance the technology in the continuous advancement of plant manufacturing. BSMV-mediated VIGS has become popular in functional genomics; gene function can be determined without permanent transformation. In general, BSMV-mediated VIGS will be very helpful in the ongoing effort to develop resilient crops.

Cucurbit Crops Acquired Silencing: Virus-Induced Post-Transcriptional Silencing Is Transmitted Across the Graft Union

Plant grafting is one of the oldest agricultural technologies, capable of improving biotic and abiotic stress resistance, regulating plant growth, and increasing crop output and quality. Recent analytical technology has revealed that massive numbers of RNAs can move via the phloem to regulate the gene expression in the scion. However, there are currently no high-throughput methods for validating the role of these long-distance RNAs in grafting, which limits the development of rootstock resources. In this study, methodologies were developed for infection of TRSV vectors in germinating melon seeds. Efficient infection was achieved in Védrantais with completely white first-true leaves and above. Grafting was performed after the gene silencing phenotype appeared in the rootstock, resulting in gene silencing of cucurbit crops as the scion. Furthermore, different RNA viral vectors were utilized to demonstrate that the transfer of viral vectors across the graft union induced gene silencing of the scion. The approach established in this study can provide a valuable research tool for future transfer RNA function studies and rootstock breeding.

A Computational Approach for Designing and Validating Small Interfering RNA against SARS-CoV-2 Variants.

The aim of this study is to develop a novel antiviral strategy capable of efficiently targeting a broad set of SARS-CoV-2 variants. Since the first emergence of SARS-CoV-2, it has rapidly transformed into a global pandemic, posing an unprecedented threat to public health. SARS-CoV-2 is prone to mutation and continues to evolve, leading to the emergence of new variants capable of escaping immune protection achieved due to previous SARS-CoV-2 infections or by vaccination. RNA interference (RNAi) is a remarkable biological mechanism that can induce gene silencing by targeting complementary mRNA and inhibiting its translation. In this study, using the computational approach, we predicted the most efficient siRNA capable of inhibiting SARS-CoV-2 variants of concern (VoCs). The presented siRNA was characterized and evaluated for its thermodynamic properties, offsite-target hits, and in silico validation by molecular docking and molecular dynamics simulations (MD) with Human AGO2 protein. The study contributes to the possibility of designing and developing an effective response strategy against existing variants of concerns and preventing further.

Engineering VIGS Vectors by Modifying Movement Proteins of the 30K Family.

Virus-induced gene silencing (VIGS) represents a particularly relevant tool in agricultural species for studying gene functionality. This study presents a novel approach for utilizing viruses belonging to the 30K family of movement proteins (MPs) as VIGS vectors. The method described here employs smaller inserts (54bp or less) than those commonly used (100-500bp). The developed strategy involves modifying 30K family MPs to introduce heterologous sequences of the gene of interest into their coding sequence. This approach enabled the successful induction of gene silencing in Nicotiana tabacum and Nicotiana benthamiana. Three representative viruses of the MP 30K family, alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV), and tobacco mosaic virus (TMV) were employed. The capacity to induce gene silencing of small inserts (18-54bp) was investigated, enabling to establish a correlation between insert size and silencing efficacy. This allowed the system to be calibrated to achieve partial silencing levels. The relationship between viral encapsidation and the level of gene silencing was also investigated, revealing that a high efficiency of viral encapsidation results in a reduction in the level of gene silencing achieved. Considering these findings, it can be concluded that the approach carried out with AMV, CMV, and TMV could be applied to other members of the MP 30K family. The MP 30K family comprises 20 viral genera and over 500 viral species, which can infect all agronomically significant plant species. Consequently, the strategy presented in this work could be applied to a wide range of relevant hosts.

Genome-wide identification of TaeGRASs responsive to biotic stresses and functional analysis of TaeSCL6 in wheat resistance to powdery mildew

BackgroundPowdery mildew is a devastating fungal disease that poses a significant threat to wheat yield and quality worldwide. Identifying resistance genes is highly advantageous for the molecular breeding of resistant cultivars. GRAS proteins are important transcription factors that regulate plant development and stress responses. Nonetheless, their roles in wheat-pathogen interactions remain poorly understood.ResultsIn this study, we used bioinformatics tools to identify and analyze wheat GRAS family genes responsive to biotic stresses and elucidated the function of TaeSCL6 within this family. A total of 179 GRAS genes in wheat were unevenly distributed on 7 chromosomes, and classified into 12 subfamilies based on phylogenetic relationship analysis. Gene duplication analysis revealed 13 pairs of tandem repeats and 142 pairs of segmental duplications, which may account for the rapid expansion of the wheat GRAS family. Expression pattern analysis revealed that 75% of the expressed TaeGRAS genes are responsive to biotic stresses. Few studies have focused on the roles of HAM subfamily genes. Consequently, we concentrated our analysis on the members of the HAM subfamily. Fourteen motifs were identified in the HAM family proteins from both Triticeae species and Arabidopsis, indicating that these motifs were highly conserved during evolution. Promoter analysis indicated that the promoters of HAM genes contain several cis-regulatory elements associated with hormone response, stress response, light response, and growth and development. Both qRT-PCR and RNA-seq data analyses demonstrated that TaeSCL6 responds to Blumeria graminis infection. Therefore, we investigated the role of TaeSCL6 in regulating wheat resistance via RNA interference and barley stripe mosaic virus induced gene silencing. Wheat plants with silenced TaeSCL6 exhibited increased susceptibility to powdery mildew.ConclusionsIn summary, this study not only validates the positive role of TaeSCL6 in wheat resistance to powdery mildew, but also provides candidate gene resources for future breeding of disease-resistance wheat cultivars.

SEC x IP two-dimensional LCMS for the analysis of non-denatured and denatured cyclic-peptide siRNAs in a single step

The increased effectiveness of small interfering RNAs (siRNAs) to induce gene silencing has brought a great therapeutic promise to many diseases. siRNAs are under highly active current research and development. Ligand conjugation and chemical modifications of the sense (SS) and antisense (AS) strands of the siRNA duplex improve stability and facilitate delivery, but significantly increase the complexity of the analytical requirements. Two chromatographic methods are needed to guide synthesis and formulation: (1) a non-denaturing method to analyze the duplex, residual sense and antisense strands, their impurities, and those of the duplex, and (2) a denaturing method for each strand and its impurities. In this work, ion-pair reversed phase (IP-RP) and strong anion exchange (SAX) methods were not successful in the analysis of a cyclic-peptide (CP) siRNA, in the non-denaturing mode. Selection of the most appropriate chromatographic method is greatly challenged by the chemical properties of the conjugated ligands. However, separation was possible by size exclusion chromatography (SEC). The non-denaturing SEC method was implemented, using a 2D-LC system, in the 1D dimension of the analysis, coupled with a denaturing IP-RP method in the 2D dimension. The 2D-LC system greatly simplified the siRNA analysis by combining, for the first time, the non-denaturing and denaturing methods into a single-instrument, one sample injection method. An additional benefit of the 2D-LC system is the interfacing of MS-incompatible methods (e.g., SAX, SEC) to a mass spectrometer, broadening thus the analytical options, by coupling with MS-compatible methods (IP-RP, HILIC) in the 2D dimension. Application of the approach was exemplified in a CP-siRNA duplex formulation study to determine the optimal mixing ratio of the individual strands. A duplex maximum was reached at a sample solution AS:SS ratio of 0.9. The method was found to be independent of the amount and concentration of sample injected. A duplex annealing study found no significant temperature or salt effects in the formulation of the CP-siRNA duplex.

Stimuli-Responsive Peptide/siRNA Nanoparticles as a Radiation Sensitizer for Glioblastoma Treatment by Co-Inhibiting RELA/P65 and EGFR.

To develop a novel approach for increasing radiosensitivity in glioblastoma (GBM) by using targeted nanoparticles to deliver siRNA aimed at silencing the EGFR and RELA/P65 genes, which are implicated in radioresistance. We engineered biodegradable, tumor-targeted, self-assembled, and stimuli-responsive peptide nanoparticles for efficient siRNA delivery. We evaluated the nanoparticles' ability to induce gene silencing and enhance DNA damage under radiation in vitro and in vivo. The nanoparticles were designed to exhibit pH-responsive endosomal escape and αvβ3 integrin targeting, allowing for preferential accumulation at tumor sites and traversal of the blood-brain tumor barrier. The application of these nanoparticles resulted in significant gene silencing, increased apoptosis, and decreased cell viability. The treatment impaired DNA repair mechanisms, thereby enhancing radiosensitivity in GBM cells. In a GBM mouse model, the combination of nanoparticle treatment with radiotherapy notably prolonged survival without apparent toxicity. Our findings suggest that nanoparticle-mediated dual gene silencing can effectively overcome GBM radioresistance. This strategy has the potential to improve clinical outcomes in GBM treatment, proposing a promising therapeutic avenue for this challenging malignancy.

Single-Stranded Hairpin Loop RNAs (loopmeRNAs) Potently Induce Gene Silencing through the RNA Interference Pathway.

Synthetic small interfering RNAs conjugated to trivalent N-acetylgalactosamine (GalNAc) are clinically validated drugs for treatment of liver diseases. Incorporation of phosphorothioate linkages and ribose modifications are necessary for stability, potency, and duration of pharmacology. Although multiple alternative siRNA designs such as Dicer-substrate RNA, shRNA, and circular RNA have been evaluated in vitro and in preclinical studies with some success, clinical applications of these designs are limited as it is difficult to incorporate chemical modifications in these designs. An alternative siRNA design that can incorporate chemical modifications through straightforward synthesis without compromising potency will significantly advance the field. Here, we report a facile synthesis of GalNAc ligand-containing single-stranded loop hairpin RNAs (loopmeRNAs) with clinically relevant chemical modifications. We evaluated the efficiency of novel loopmeRNA designs in vivo and correlated their structure-activity relationship with the support of in vitro metabolism data. Sequences and chemical modifications in the loop region of the loopmeRNA design were optimized for maximal potency. Our studies demonstrate that loopmeRNAs can efficiently silence expression of target genes with comparable efficacy to conventional double-stranded siRNAs but reduced environmental and regulatory burdens.

The MYB transcriptional factor BrMYB108 regulates Auxin-mediated delayed leaf senescence in postharvest Pak Choi

Auxin is the earliest discovered class of endogenous plant hormones, widely distributed in higher plants, and plays a key role in regulating plant growth and development. However, the molecular mechanism by which auxin affects postharvest leaf senescence in pak choi (Brassica rapa subsp. chinensis) remains unclear. In this study, we found that exogenous application of 2 mg L−1 of the auxin analog indoleacetic acid (IAA) could delay postharvest leaf senescence in pak choi. Importantly, we isolated an R2R3-MYB transcription factor (TF), BrMYB108, which was highly expressed in senescent leaves and whoes expression was suppressed by IAA treatment. Molecular evidence confirmed that BrMYB108 can bind to the promoter region of the chlorophyll metabolism gene BrSGR2 to activate its expression. Heterologous expression of BrMYB108 in Arabidopsis accelerated leaf senescence, while silencing BrMYB108 in pak choi using virus induced gene silencing (VIGS) delayed leaf senescence. In addition, we found that BrMYB108 is a direct target gene of the auxin response factor 17 (ARF17) homolog gene, BrARF17, in Arabidopsis. Our study reveals a model in which BrARF17 regulates postharvest leaf senescence in pak choi by targeting the expression of BrMYB108. These molecular insights into the auxin-mediated regulation of postharvest leaf senescence in pak choi have positive implications for manipulating BrMYB108 to improve the postharvest shelf life of vegetables.

Molecular approaches for the management of papaya ringspot virus infecting papaya: a comprehensive review.

Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.

Molecular characterization of cassava zinc finger-homeodomain (ZF-HD) transcription factors reveals their role in disease resistance

The production of cassava (Manihot esculenta Crantz) is constantly threatened by cassava bacterial blight (CBB), caused by Xanthomonas phaseoli pv. manihotis (Xpm). Zinc finger-homeodomain (ZF-HD) belongs to a family of homozygous heterotypic cassette genes widely implicated in various developmental and physiological processes in plants. Despite their importance, a comprehensive analysis of ZF-HD genes, particularly those involved in disease resistance, has not been performed for cassava. In the present study, we utilized bioinformatics methods to identify 21 ZF-HD genes distributed across 11 chromosomes of cassava genome, with the majority exhibiting gene structure without introns. Phylogenetic analysis categorized these genes into two major groups (MIF and ZHD) with five subgroups. We observed fourteen pairs of duplicated genes, suggesting that segmental duplication has likely facilitated the expansion of the cassava ZF-HD gene family. Comparative orthologous analyses between cassava and other plant species shed light on the evolutionary trajectory of this gene family. Promoter analyses revealed multiple hormone- and stress-related elements, indicative of a functional role in stress responses. Expression profiling through RNA-seq and quantitative real-time PCR (qRT-PCR) demonstrated that certain cassava ZF-HD genes are up-regulated in response to Xpm infection, suggesting their involvement in defense mechanisms. Notably, MeZHD7 gene was identified via virus induced gene silencing (VIGS) as potentially crucial in conferring resistance against CBB. Results from subcellular localization experiments indicated that MeZHD7 was localized in the nucleus. The Luciferase reporter assay demonstrated an interaction between MeZHD7 and MeMIF5. These findings may lay the foundation for further cloning and functional analyses of cassava ZF-HD genes, particularly those associated with pathogen resistance.

Enhancing photodynamic and radionuclide therapy by small interfering RNA (siRNA)-RAD51 transfection via self-emulsifying delivery systems (SNEDDS)

Background aimsGene-silencing by small interfering RNA (siRNA) is an attractive therapy to regulate cancer death, tumor recurrence or metastasis. Because siRNAs are easily degraded, it is necessary to develop transport and delivery systems to achieve efficient tumor targeting. Self-nanoemulsifying systems (SNEDDS) have been successfully used for pDNA transport and delivery, so they may be useful for siRNA. The aim of this work is to introduce siRNA-RAD51 into a SNEDDS prepared with Phospholipon-90G, Labrafil-M1944-CS and Cremophor-RH40 and evaluate its efficacy in preventing homologous recombination of DNA double-strand breaks caused by photodynamic therapy (PDT) and ionizing radiation (IR). MethodsThe siRNA-RAD51 was loaded into SNEDDS using chitosan. Transfection capacity was estimated by comparison with Lipofectamine-2000. ResultsSNEDDS(siRNA-RAD51) induced gene silencing effect on the therapies evaluated by cell viability and clonogenic assays using T47D breast cancer cells. ConclusionsSNEDDS(siRNA-RAD51) shown to be an effective siRNA-delivery system to decrease cellular resistance in PDT or IR.

Assessing transgenerational gene editing capacity for enhancing aquaculture productivity in decapod crustaceans

AbstractIn the wake of global population increases and stagnating capture fishery production, there exists a requirement for novel biotechnologies to increase aquaculture productivity to offset demand. Through recognition of limitations imposed by aquaculture—increased incidence of disease, stress, cannibalism, and mortality in captivity—researchers continue to investigate RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR) technologies for their potential to alleviate these at a genetic level. In decapods, with the exception of generating desirable all‐male monosex populations in Macrobrachium rosenbergii, RNAi and CRISPR technologies have yet to provide a significant commercial use case. This literature review provides a comprehensive examination of RNAi and CRISPR in decapods, along with popular delivery methods (injection/microinjection; electroporation; receptor‐mediated ovary transduction of cargo [ReMOT]; and nanoparticles). It identifies a number of limitations including scalability bottlenecks imposed by RNAi delivery methods, and deferred gene editing progress as a result of CRISPR's infancy. Finally, it recognises gene editing induced gene silencing (GEiGS®) as a novel method for silencing gene expression, and suggests an interim examination of transgenerational gene silencing (TGS) (‘parental RNAi’) in decapods to enhance aquaculture productivity.

The β-galactosidase gene AtrBGAL2 regulates Akebia trifoliata fruit cracking

Cracking of Akebia trifoliata fruit at maturity is problematic for the cultivation of the horticultural crop, shortening shelf-life quality and compromising commercial value. However, the molecular mechanisms underlying this feature of A. trifoliata are not known. Genes involved in cell wall metabolism were identified by genome and transcriptome sequencing, which may play important roles in fruit cracking. One of the galactose metabolism related genes, β-galactosidase (AtrBGAL2), was identified in A. trifoliata, and overexpression (OE) of AtrBGAL2 resulted in early fruit cracking, higher water-soluble pectin contents, and lower acid-soluble pectin, cellulose, and hemicellulose content compared to the wild type. Whereas silencing of AtrBGAL2 in trifoliata by virus induced gene silencing showed opposite trends. The levels of AtrBGAL2 transcripts were 24.6 and 66.0-fold higher in OE A. trifoliata and tomato fruits, respectively, and the cell wall-related genes were also gradually greater than in control plants during fruit ripening. Whereas the expression levels of AtrBGAL2 was significantly down-regulated by 54.1 % and 73.7 % in gene silenced A. trifoliata and CRISPR/Cas9 tomato mutant plants, respectively, and cell wall-related genes were also significantly reduced. These results demonstrate that AtrBGAL2 plays important roles in regulating fruit cracking during fruit ripening.

RhMED15a-like, a subunit of the Mediator complex, is involved in the drought stress response in Rosa hybrida

BackgroundRose (Rosa hybrida) is a globally recognized ornamental plant whose growth and distribution are strongly limited by drought stress. The role of Mediator, a multiprotein complex crucial for RNA polymerase II-driven transcription, has been elucidated in drought stress responses in plants. However, its physiological function and regulatory mechanism in horticultural crop species remain elusive.ResultsIn this study, we identified a Tail module subunit of Mediator, RhMED15a-like, in rose. Drought stress, as well as treatment with methyl jasmonate (MeJA) and abscisic acid (ABA), significantly suppressed the transcript level of RhMED15a-like. Overexpressing RhMED15a-like markedly bolstered the osmotic stress tolerance of Arabidopsis, as evidenced by increased germination rate, root length, and fresh weight. In contrast, the silencing of RhMED15a-like through virus induced gene silencing in rose resulted in elevated malondialdehyde accumulation, exacerbated leaf wilting, reduced survival rate, and downregulated expression of drought-responsive genes during drought stress. Additionally, using RNA-seq, we identified 972 differentially expressed genes (DEGs) between tobacco rattle virus (TRV)-RhMED15a-like plants and TRV controls. Gene Ontology (GO) analysis revealed that some DEGs were predominantly associated with terms related to the oxidative stress response, such as ‘response to reactive oxygen species’ and ‘peroxisome’. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment highlighted pathways related to ‘plant hormone signal transduction’, in which the majority of DEGs in the jasmonate (JA) and ABA signalling pathways were induced in TRV-RhMED15a-like plants.ConclusionOur findings underscore the pivotal role of the Mediator subunit RhMED15a-like in the ability of rose to withstand drought stress, probably by controlling the transcript levels of drought-responsive genes and signalling pathway elements of stress-related hormones, providing a solid foundation for future research into the molecular mechanisms underlying drought tolerance in rose.

Mutations alter pll gene promoter from constitutive to inducible, coffering palmately lobed leaf trait of melon

The leaves of the common melon are approximately round and uncut. A natural mutant of melon named bm7, which has palmately-lobed leaves. We found that bm7 plants were more conducive to increasing planting density and achieving higher yields, and showed better disease resistance. Therefore, the germplasm resources of muskmelon split leaves have the potential of application. Previous study showed that the palmately-lobed leaf trait was controlled by a single recessive gene, pll (numbered MELO3C010784). By cloning and sequencing the pll genes of Jiashi (wide-type) and bm7 (mutant) plants, we noticed that there was no mutation in this gene, but its expression level in bm7 was far below than that in Jiashi. The silencing of pll gene with virus induced gene silencing (VIGS) confers palmately-lobed leaf trait of Jiashi plants. We found three mutations in the pll gene promoter of bm7 plants, located at 373, 493, 506 bp upstream the translation initiation codon. Then, we had found the pll gene promoter activity of bm7 was significantly lower than that of Jiashi. In addition, the expression level and promoter activity of pll gene in Jiashi plants were not affected by environmental factors, but in bm7 plants, the expression level and promoter activity of pll gene, all decreased with the increase in light intensity and/or temperature. All the results indicated that the mutations convert the pll gene promoter from constitutive to inducible, and results in significantly reduced expression of pll gene, conferring the palmately-lobed leaf trait of melon.

Unveiling novel anti-viral mechanisms of ε-poly-l-lysine on tobacco mosaic virus-infected Nicotiana tabacum through microRNA and transcriptome sequencing

MicroRNAs (miRNAs) play important roles in plant defense against various pathogens. ε-poly-l-lysine (ε-PL), a natural anti-microbial peptide produced by microorganisms, effectively suppresses tobacco mosaic virus (TMV) infection. To investigate the anti-viral mechanism of ε-PL, the expression profiles of miRNAs in TMV-infected Nicotiana tabacum after ε-PL treatment were analyzed. The results showed that the expression levels of 328 miRNAs were significantly altered by ε-PL. Degradome sequencing was used to identify their target genes. Integrative analysis of miRNAs target genes and gene-enriched GO/KEGG pathways indicated that ε-PL regulates the expression of miRNAs involved in critical pathways of plant hormone signal transduction, host defense response, and plant pathogen interaction. Subsequently, virus induced gene silencing combined with the short tandem targets mimic technology was used to analyze the function of these miRNAs and their target genes. The results indicated that silencing miR319 and miR164 reduced TMV accumulation in N. benthamiana, indicating the essential roles of these miRNAs and their target genes during ε-PL-mediated anti-viral responses. Collectively, this study reveals that microbial source metabolites can inhibit plant viruses by regulating crucial host miRNAs and further elucidate anti-viral mechanisms of ε-PL.

Host-Induced Gene Silencing of Effector AGLIP1 Enhanced Resistance of Rice to Rhizoctonia solani AG1-IA

Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing (TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into siRNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.

The development of an egg-soaking method for delivering dsRNAs into spider mites

Recently, the first sprayable RNAi biopesticide, Ledprona, against the Colorado potato beetle, Leptinotarsa decemlineata, has been registered at the United States Environmental Protection Agency. Spider mites (Acari: Tetranychidae), a group of destructive agricultural and horticultural pests, are notorious for rapid development of insecticide/acaricide resistance. The management options, on the other hand, are extremely limited. RNAi-based biopesticides offer a promising control alternative to address this emerging issue. In this study, we i) developed an egg-soaking dsRNA delivery method; ii) evaluated the factors influencing RNAi efficiency, and finally iii) investigated the potential mode of entry of this newly developed egg-soaking RNAi method. In comparison to other dsRNA delivery methods, egg-soaking method was the most efficient, convenient/practical, and cost-effective method for delivering dsRNAs into spider mites. RNAi efficiency of this RNAi method was affected by target genes, dsRNA concentration, developmental stages, and mite species. In general, the hawthorn spider mite, Amphitetranychus viennensis, is more sensitive to RNAi than the two-spotted spider mite, Tetranychus urticae, and both of them have dose-dependent RNAi effect. For different life stages, egg and larvae are the most sensitive life stages to dsRNAs. For different target genes, there is no apparent association between the suppression level and the resultant phenotype. Finally, we demonstrated that this egg-soaking RNAi method acts as both stomach and contact toxicity. Our combined results demonstrate the effectiveness of a topically applied dsRNA delivery method, and the potential of a spray induced gene silencing (SIGS) method as a control alternative for spider mites.