RNAi System Research Articles

Establishment of RNAi-Mediated Pest Control Method for Red Imported Fire Ant, Solenopsis invicta.

RNAi plays a crucial role in insect gene function research and pest control field. Nonetheless, the variable efficiency of RNAi across diverse insects and off-target effects also limited its further application. In this study, we cloned six essential housekeeping genes from Solenopsis invicta and conducted RNAi experiments by orally administering dsRNA. Then, we found that mixing with liposomes significantly enhanced the RNAi efficiency by targeting for SiV-ATPaseE. Additionally, we observed a certain lethal effect of this dsRNA on queens by our established RNAi system. Furthermore, no strict sequence-related off-target effects were detected. Finally, the RNAi effect of large-scale bacteria expressing dsRNA was successfully confirmed for controlling S. invicta. In summary, this study established an RNAi system for S. invicta and provided a research template for the future development of nucleic acid drugs based on RNAi.

Kaolinite nanoclay-shielded dsRNA drenching for management of Globodera pallida: An environmentally friendly pest management approach.

Globodera pallida, an obligate sedentary endoparasite, is a major economic pest that causes substantial potato yield losses. This research aimed to study the effects of gene silencing of three FMRFamide-like peptides (FLPs) genes to reduce G. pallida infestation on potato plants by using kaolinite nanoclay as a carrier to deliver dsRNAs via drenching. A dsRNA dosage of 2.0mg/ml silenced flp-32c by 89.5%, flp-32p by 94.6%, and flp-2 by 94.3%. J2s incubated for 5 and 10h showed no phenotypic changes. However, J2s of G. pallida efficiently uptake dsRNA of all targeted genes after 15h of incubation. On the other hand, J2s that had been kept for 24h had a rigid and straight appearance. Under fluorescence microscopy, all dsRNA-treated nematodes showed fluorescein isothiocyanate (FITC) signals in the mouth, nervous system, and digestive system. The untreated population of J2s did not show any FITC signals and was mobile as usual. The drenching of potato cultivar Kufri Jyoti with the dsRNA-kaolinite formulations induced deformation and premature death of J2s, compared with untreated J2s that entered J3 or J4 stages. This study validates that the nanocarrier-delivered RNAi system could be employed effectively to manage G. pallida infestations.

Naturally segregating genetic variants contribute to thermal tolerance in a Drosophila melanogaster model system.

Thermal tolerance is a fundamental physiological complex trait for survival in many species. For example, everyday tasks such as foraging, finding a mate, and avoiding predation are highly dependent on how well an organism can tolerate extreme temperatures. Understanding the general architecture of the natural variants within the genes that control this trait is of high importance if we want to better comprehend thermal physiology. Here, we take a multipronged approach to further dissect the genetic architecture that controls thermal tolerance in natural populations using the Drosophila Synthetic Population Resource as a model system. First, we used quantitative genetics and Quantitative Trait Loci mapping to identify major effect regions within the genome that influences thermal tolerance, then integrated RNA-sequencing to identify differences in gene expression, and lastly, we used the RNAi system to (1) alter tissue-specific gene expression and (2) functionally validate our findings. This powerful integration of approaches not only allows for the identification of the genetic basis of thermal tolerance but also the physiology of thermal tolerance in a natural population, which ultimately elucidates thermal tolerance through a fitness-associated lens.

Inducible Expression of dsRNA in Escherichia coli.

Double-stranded RNA (dsRNA) is a valuable tool for reverse genetics research and gene silencing applications. It is also an important management method for pests and diseases in agriculture. It can be synthesized both in vivo and in vitro. The latter presents the drawback of high production cost, the former is less expensive and suitable for scalable production. In general, dsRNAs are obtained in vivo from Escherichia coli heterologous systems that require the IPTG-inducible T7 RNA polymerase. In this report, we describe the construction of an RNAi system for the expression of dsRNA using the HT115 bacterial strain and the L4440 plasmid, and the extraction and identification of dsRNA.

Modeling Retinoblastoma in adult Drosophila by reducing Rbf gene expression

Retinoblastoma is an eye cancer that begins in the retina due to genetic mutations in the retinal cells, typically of the Rb1 tumor suppressor gene, which encodes for the Rb protein. Rb inhibits the E2F transcription factor, which drives the expression of genes needed for entry into S-phase of the cell cycle. Loss of function of Rb dysregulates E2F, causing uninhibited cell-cycle proliferation and tumor formation. Current treatments for retinoblastoma are highly invasive such as surgery to remove the eye or chemotherapy impacting patients' lives. If an animal model of retinoblastoma is constructed, less invasive therapies can be tested. Drosophila melanogaster have a short life cycle, are cost-effective, and have an Rb1 homologue called Rbf1, making them an efficient model. An RNAi system was used to knock down the expression of Rbf1 protein via the gsGAL4-UAS gene switch system of gene regulation. A fly line in which the gsGAL4 transcription factor is expressed under the eye-specific elav promoter was crossed with a line in which an siRNA targeting Rbf1 mRNA is expressed under the control of an upstream activating sequence (UAS). When the offspring of this cross were fed RU486, the normally cytoplasmic gsGAL4 entered the nucleus of the retinal cells in which it was expressed and bound to the UAS, driving the expression of the siRNA, which was expected to knock down levels of Rbf1 protein in these flies. RU486-treated flies exhibited a change in eye morphology, suggestive of tumor formation, as well as differential Kaplan-Meier survival curves.

Physical exercise ameliorates age-related deterioration of skeletal muscle and mortality by activating Pten-related pathways in Drosophila on a high-salt diet.

The phosphatase and tensin congeners (Pten) gene affects cell growth, cell proliferation, and rearrangement of connections, and it is closely related to cellular senescence, but it remains unclear the role of muscle-Pten gene in exercise against age-related deterioration in skeletal muscle and mortality induced by a high-salt diet (HSD). In here, overexpression and knockdown of muscle Pten gene were constructed by building MhcGAL4 /PtenUAS-overexpression and MhcGAL4 /PtenUAS-RNAi system in flies, and flies were given exercise training and a HSD for 2 weeks. The results showed that muscle Pten knockdown significantly reduced the climbing speed, climbing endurance, GPX activity, and the expression of Pten, Sirt1, PGC-1α genes, and it significantly increased the expression of Akt and ROS level, and impaired myofibril and mitochondria of aged skeletal muscle. Pten knockdown prevented exercise from countering the HSD-induced age-related deterioration of skeletal muscle. Pten overexpression has the opposite effect on skeletal muscle aging when compared to it knockdown, and it promoted exercise against HSD-induced age-related deterioration of skeletal muscle. Pten overexpression significantly increased lifespan, but its knockdown significantly decreased lifespan of flies. Thus, current results confirmed that differential expression of muscle Pten gene played an important role in regulating skeletal muscle aging and lifespan, and it also affected the adaptability of aging skeletal muscle to physical exercise since it determined the activity of muscle Pten/Akt pathway and Pten/Sirt1/PGC-1α pathway.

Antiviral mechanism of Interferon-like and RNAi system in oyster

Antiviral mechanism of Interferon-like and RNAi system in oyster

Engineered symbiotic bacteria interfering Nosema redox system inhibit microsporidia parasitism in honeybees

Nosema ceranae is an intracellular parasite invading the midgut of honeybees, which causes serious nosemosis implicated in honeybee colony losses worldwide. The core gut microbiota is involved in protecting against parasitism, and the genetically engineering of the native gut symbionts provides a novel and efficient way to fight pathogens. Here, using laboratory-generated bees mono-associated with gut members, we find that Snodgrassella alvi inhibit microsporidia proliferation, potentially via the stimulation of host oxidant-mediated immune response. Accordingly, N. ceranae employs the thioredoxin and glutathione systems to defend against oxidative stress and maintain a balanced redox equilibrium, which is essential for the infection process. We knock down the gene expression using nanoparticle-mediated RNA interference, which targets the γ-glutamyl-cysteine synthetase and thioredoxin reductase genes of microsporidia. It significantly reduces the spore load, confirming the importance of the antioxidant mechanism for the intracellular invasion of the N. ceranae parasite. Finally, we genetically modify the symbiotic S. alvi to deliver dsRNA corresponding to the genes involved in the redox system of the microsporidia. The engineered S. alvi induces RNA interference and represses parasite gene expression, thereby inhibits the parasitism significantly. Specifically, N. ceranae is most suppressed by the recombinant strain corresponding to the glutathione synthetase or by a mixture of bacteria expressing variable dsRNA. Our findings extend our previous understanding of the protection of gut symbionts against N. ceranae and provide a symbiont-mediated RNAi system for inhibiting microsporidia infection in honeybees.

OsMADS1 Regulates Grain Quality, Gene Expressions, and Regulatory Networks of Starch and Storage Protein Metabolisms in Rice.

OsMADS1 plays a vital role in regulating floret development and grain shape, but whether it regulates rice grain quality still remains largely unknown. Therefore, we used comprehensive molecular genetics, plant biotechnology, and functional omics approaches, including phenotyping, mapping-by-sequencing, target gene seed-specific RNAi, transgenic experiments, and transcriptomic profiling to answer this biological and molecular question. Here, we report the characterization of the 'Oat-like rice' mutant, with poor grain quality, including chalky endosperms, abnormal morphology and loose arrangement of starch granules, and lower starch content but higher protein content in grains. The poor grain quality of Oat-like rice was found to be caused by the mutated OsMADS1Olr allele through mapping-by-sequencing analysis and transgenic experiments. OsMADS1 protein is highly expressed in florets and developing seeds. Both OsMADS1-eGFP and OsMADS1Olr-eGFP fusion proteins are localized in the nucleus. Moreover, seed-specific RNAi of OsMADS1 also caused decreased grain quality in transgenic lines, such as the Oat-like rice. Further transcriptomic profiling between Oat-like rice and Nipponbare grains revealed that OsMADS1 regulates gene expressions and regulatory networks of starch and storage protein metabolisms in rice grains, hereafter regulating rice quality. In conclusion, our results not only reveal the crucial role and preliminary mechanism of OsMADS1 in regulating rice grain quality but also highlight the application potentials of OsMADS1 and the target gene seed-specific RNAi system in improving rice grain quality by molecular breeding.

Function of Tryptophan 2,3-Dioxygenase in Monochamus alternatus Hope Revealed by RNA Interference

Monochamus alternatus Hope (Coleoptera: Cerambycidae), an invasive beetle that has caused billions of dollars of economic losses, is a serious pest of Pinus massoniana in many Asian countries. An efficient RNAi system is helpful for functional genomics research on M. alternatus. In this study, a tryptophan 2,3-dioxygenase (TDO) related to the ommochrome synthesis in insects was identified. Using RNAi technology, the M. alternatus TDO gene was silenced by injecting dsRNA into pupae, and individuals were analyzed by phenotype and expression of the TDO gene by RT-qPCR. The results show that TDO is expressed in different developmental stages of M. alternatus, having its peak expression during the prepupal stage. White-eye phenotypes were observed in the pupal and adult stages after dsRNA injection, and a significant 81% decrease in TDO mRNA levels 48 h after injection was determined by RT-qPCR. This gene can be used as a genetic marker and is an important discovery for future genetic engineering tools to control M. alternatus populations.

Application of Novel Molecular Biology in Cancer Therapy

Cancer is a genetic illness that develops for various reasons, including the activation of oncogenes, the failure of tumor suppressor genes, or mutagenesis induced by environmental stimuli. This article was produced using PubMed, Nature, Science Direct, Springer, and Elsevier data. Oncogenes are altered forms of normal proto-oncogenic genes that are important for cell proliferation, development, and regulation. The transformation of a gene to an oncogene is caused by chromosomal translocation or gene mutation due to addition, deletion, duplication, or viral infection. These oncogenes are targeted by medications or the RNAi system to limit malignant cell development. Various molecular biology methods for cancer detection and treatment have been developed, including targeting cancer stem cell pathways for cancer therapy, retroviral therapy, oncogene silencing, and alterations in tumor suppressor genes. Among all the techniques used, RNAi, zinc finger nucleases, and CRISPR have a greater chance of reaching a cancer-free planet.

Molecular Strategies to Target Protein Aggregation in Huntington's Disease.

Huntington’s disease (HD) is a neurodegenerative disorder caused by the aggregation of the mutant huntingtin (mHTT) protein in nerve cells. mHTT self-aggregates to form soluble oligomers and insoluble fibrils, which interfere in a number of key cellular functions. This leads to cell quiescence and ultimately cell death. There are currently still no treatments available for HD, but approaches targeting the HTT levels offer systematic, mechanism-driven routes towards curing HD and other neurodegenerative diseases. This review summarizes the current state of knowledge of the mRNA targeting approaches such as antisense oligonucleotides and RNAi system; and the novel methods targeting mHTT and aggregates for degradation via the ubiquitin proteasome or the autophagy-lysosomal systems. These methods include the proteolysis-targeting chimera, Trim-Away, autophagosome-tethering compound, autophagy-targeting chimera, lysosome-targeting chimera and approach targeting mHTT for chaperone-mediated autophagy. These molecular strategies provide a knowledge-based approach to target HD and other neurodegenerative diseases at the origin.

Interference Efficiency and Effects of Bacterium-mediated RNAi in the Fall Armyworm (Lepidoptera: Noctuidae).

RNAi is an effective tool for gene function analysis and a promising strategy to provide environmentally friendly control approaches for pathogens and pests. Recent studies support the utility of bacterium-mediated RNAi as a cost-effective method for gene function study and a suitable externally applied delivery mechanism for pest control. Here, we developed a bacterium-mediated RNAi system in Spodoptera frugiperda based on four target genes, specifically, Chitinase (Sf-CHI), Chitin synthase B (Sf-CHSB), Sugar transporter SWEET1 (Sf-ST), and Hemolin (Sf-HEM). RNAi conducted by feeding larvae with bacteria expressing dsRNAs of target genes or injecting pupae and adults with bacterially synthesized dsRNA induced silencing of target genes and resulted in significant negative effects on growth and survival of S. frugiperda. However, RNAi efficiency and effects were variable among different target genes and dsRNA delivery methods. Injection of pupae with dsCHI and dsCHSB induced a significant increase in wing malformation in adults, suggesting that precise regulation of chitin digestion and synthesis is crucial during wing formation. Injection of female moths with dsHEM resulted in lower mating, fecundity, and egg hatching, signifying a critical role of Sf-HEM in the process of egg production and/or embryo development. Our collective results demonstrate that bacterium-mediated RNAi presents an alternative technique for gene function study in S. frugiperda and a potentially effective strategy for control of this pest, and that Sf-CHI, Sf-CHSB, Sf-ST, and Sf-HEM encoding genes can be potent targets.

Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans.

Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that function in the nucleus. We previously found that erroneous rRNAs induce the generation of antisense ribosomal siRNAs (risiRNAs) which silence the expression of rRNAs via the nuclear RNAi defective (Nrde) pathway. To further understand the biological roles and mechanisms of this class of small regulatory RNAs, we conducted forward genetic screening to identify factors involved in risiRNA generation in Caenorhabditis elegans. We found that risiRNAs accumulated in the RNA exosome mutants. risiRNAs directed the association of NRDE proteins with pre-rRNAs and the silencing of pre-rRNAs. In the presence of risiRNAs, NRDE-2 accumulated in the nucleolus and colocalized with RNA polymerase I. risiRNAs inhibited the transcription elongation of RNA polymerase I by decreasing RNAP I occupancy downstream of the RNAi-targeted site. Meanwhile, exosomes mislocalized from the nucleolus to nucleoplasm in suppressor of siRNA (susi) mutants, in which erroneous rRNAs accumulated. These results established a novel model of rRNA surveillance by combining ribonuclease-mediated RNA degradation with small RNA-directed nucleolar RNAi system.

Down-regulation of TNF-α via macrophage-targeted RNAi system for the treatment of acute inflammatory sepsis

Sepsis is a systemic inflammatory response syndrome caused by bacterial infection. The sepsis therapy has involved prescription of adequate antibiotics, requiring several days to determine the proper type without reducing the inflammatory response. Thus, it is necessary to rapidly decrease fundamental inflammation, which can induce serious organ damage. In the inflammatory mechanism, tumor necrosis factor-alpha (TNF-α) produced by macrophages has an important role in infiltration of macrophages into infected sites and as a trigger for secretion of pro-inflammatory cytokines. However, commercialized TNF-α antibody medicines have limits such as fibrosis, cytokine storms, and high production costs. There is a growing need for anti-inflammatory sepsis treatment free from side effects. For this reason, TNF-α converting enzyme (TACE) could be an innovative target to break the positive feedback loop of inflammatory mediators (TNF-α) since it converts the inactive TNF-α membrane bound form to the activated soluble form in macrophages. A non-viral gene delivery system was developed in this study to deliver siRNA into inflammation-mediated macrophages without toxicity. The peptide-based gene carrier created by conjugating positively-charged nine arginine (9R) and the TKPR (Thr-Lys-Pro-Arg) sequence from the Fc region of Immunoglobulin G (IgG) specifically binds to the neuropilin-1 (NRP-1) receptor on the macrophage surface. Our results demonstrated that siTACE/TKPR-9R complexes were internalized in macrophages and successfully down-regulated TACE mRNA level. Finally, RNA interference with cell-targeted peptide carriers indicates a fundamental therapy for acute inflammatory sepsis free of off-target effects.

Construction of a recombinant vector containing SAI gene using Gateway-based RNAi system: a preliminary study in the development of high sucrose sugarcane

Recombinant plasmid construction that produces hairpin molecules for gene knockdown is not an easy method. Gateway cloning system is a technology to transfer DNA fragments utilizing clonase and specific sites. This study aimed to construct RNAi expression vector containing soluble acid invertase (SAI) gene using Gateway system to produce high-sugar content sugarcane. PCR was conducted to generate specific SAI fragment using cDNA template from two sugarcane varieties: Bululawang and PSJK922. Of the two specific primer pairs designed, only one pair amplified the SAI 161 bp gene fragment. Amplification occurred in both cDNA samples. TA (Thymine-Adenine pair) cloning was performed with pCR8/GW/TOPO plasmid and a clonase reaction was carried out to insert the specific fragments into the pHELLSGATE 8 hairpin plasmid. The results showed that two of the four colonies analyzed by associated restriction enzymes had plasmid pHELLSGATE8::hpSAI that contained a hairpin component of the SAI fragment. One attempt to construct the hairpin RNA using Gateway system in this study indicates the easiness of the system when compared to traditional cloning. The DNA sequence in the hairpin portion of this plasmid will be analysed to confirm SAI gene fragment, and it will be followed by transformation of sugarcane and SAI characterization.

Alteration in the Culex pipiens transcriptome reveals diverse mechanisms of the mosquito immune system implicated upon Rift Valley fever phlebovirus exposure.

Rift Valley fever phlebovirus (RVFV) causes an emerging zoonotic disease and is mainly transmitted by Culex and Aedes mosquitoes. While Aedes aegypti-dengue virus (DENV) is the most studied model, less is known about the genes involved in infection-responses in other mosquito-arboviruses pairing. The main objective was to investigate the molecular responses of Cx. pipiens to RVFV exposure focusing mainly on genes implicated in innate immune responses. Mosquitoes were fed with blood spiked with RVFV. The fully-engorged females were pooled at 3 different time points: 2 hours post-exposure (hpe), 3- and 14-days post-exposure (dpe). Pools of mosquitoes fed with non-infected blood were also collected for comparisons. Total RNA from each mosquito pool was subjected to RNA-seq analysis and a de novo transcriptome was constructed. A total of 451 differentially expressed genes (DEG) were identified. Most of the transcriptomic alterations were found at an early infection stage after RVFV exposure. Forty-eight DEG related to immune infection-response were characterized. Most of them were related with the RNAi system, Toll and IMD pathways, ubiquitination pathway and apoptosis. Our findings provide for the first time a comprehensive view on Cx. pipiens-RVFV interactions at the molecular level. The early depletion of RNAi pathway genes at the onset of the RVFV infection would allow viral replication in mosquitoes. While genes from the Toll and IMD immune pathways were altered in response to RVFV none of the DEG were related to the JAK/STAT pathway. The fact that most of the DEG involved in the Ubiquitin-proteasome pathway (UPP) or apoptosis were found at an early stage of infection would suggest that apoptosis plays a regulatory role in infected Cx. pipiens midguts. This study provides a number of target genes that could be used to identify new molecular targets for vector control.

A truncated intracellular Dicer-like molecule involves in antiviral immune recognition of oyster Crassostrea gigas

The enzyme Dicer is best known for its role as an endoribonuclease in the small RNA pathway, playing a crucial role in recognizing viral double-stranded RNA (dsRNA) and inducing down-stream cascades to mediate anti-virus immunity. In the present study, a truncated Dicer-like gene was identified from oyster Crassostrea gigas, and its open reading frame (ORF) encoded a polypeptide (designed as CgDCL) of 530 amino acids. The CgDCL contained one N-terminal DEAD domain and a C-terminal helicase domain, but lack the conserved PAZ domain, ribonuclease domain (RIBOc) and dsRNA binding domain. The mRNA transcripts of CgDCL were detected in all the examined tissues with high expression levels in lip, gills and haemocytes, which were 62.06-fold, 48.91-fold and 47.13-fold (p < 0.05) of that in mantle, respectively. In the primarily cultured oyster haemocytes, the mRNA transcripts of CgDCL were significantly induced at 12 h after poly(I:C) stimulation, which were 4.04-fold (p < 0.05) of that in control group. The expression level of CgDCL mRNA in haemocytes was up-regulated significantly after dsRNA and recombinant interferon-like protein (rCgIFNLP) injection, which was 12.87-fold (p < 0.01) and 3.22-fold (p < 0.05) of that in control group, respectively. CgDCL proteins were mainly distributed in the cytoplasm of haemocytes. The recombinant CgDCL protein displayed binding activity to dsRNA and poly(I:C), but no obvious dsRNA cleavage activity. These results collectively suggest that truncated CgDCL from C. gigas was able to be activated by poly(I:C), dsRNA and CgIFNLP, and functioned as an intracellular recognition molecule to bind nucleic acid of virus, indicating a potential mutual cooperation between RNAi and IFN-like system in anti-virus immunity of oysters.

Functional analysis of mosquito and Drosophila Na+‐dependent cation‐chloride cotransporters

Dipteran Na+‐dependent cation‐chloride cotransporters (CCCs) group into two clades. Drosophila melanogaster Ncc69 is a bona fide Na+‐K+‐Cl− cotransporter and has a single ortholog in Aedes aegypti (aeNKCC1). Drosophila Ncc83 has not been physiologically characterized and has two orthologs in Ae. aegypti (aeCCC2 and aeCCC3). Previous work showed that oocytes expressing aeCCC2 have higher Na+ conductances than controls. Moreover, in flux assays using Li+ as a Na+ tracer, Li+ transport by aeCCC2 was not dependent upon extracellular Cl− and not inhibited by the loop diuretics furosemide or bumetanide. These are surprising results, since previously characterized CCCs are electroneutral, Cl−‐dependent, and sensitive to the loop diuretics. We have further characterized aeCCC2 and performed an initial analysis of Ncc83, using two‐electrode voltage clamp. Following a hypotonic pre‐incubation, changes in membrane potential upon Na+ replacement were 20 ± 3 mV in Ncc83 and 26 ± 4 mV in aeCCC2, approximately two‐fold greater than before swelling. Na+‐dependent currents in oocytes expressing aeCCC2 depended on extracellular Na+ with saturable Michaelis‐Menton kinetics. The apparent affinity for Na+ was 4.6 ± 0.5 mM (mean, SEM, n=5). To characterize the function of Ncc83 in Drosophila Malpighian tubules (MT), we developed procedures to assess the cation concentrations of secreted fluid in Ramsay secretion assays using cation‐exchange chromatography. The physiological cations were readily detectable in 10,000–50,000 fold dilutions of secreted fluid, and Li+ and Rb+ were also detectable when added to the bathing saline as tracers for Na+ and K+. In w118‐control flies, fluid secreted by MT measured using these procedures had a [K+] of 104.1 ± 8.8 mM (mean, SEM, n=7) and [Na+] of 61.2 ± 7.8 mM. In preliminary experiments, [K+] was 112.7 ± 1.3 mM (n=4) and [Na+] was 48.2 ± 4.1 mM in fluid secreted by MTs of flies with MT‐specific knockdown of Ncc83 using the GAL4‐UAS RNAi system. Further work is needed to determine the physiological roles of Ncc83 and aeCCC2. Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.Support or Funding InformationFunding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.

A novel transcriptional regulator RXE1 modulates the essential transactivator XYR1 and cellulase gene expression in Trichoderma reesei.

XYR1 is the key transcription activator for cellulase gene expression in the model filamentous fungus Trichoderma reesei, which is widely applied in the industry due to its excellent capability of secreting a large quantity of cellulases. Despite the essential role of XYR1, the regulation of its expression in T. reesei cellulolytic response is poorly understood. In this study, we identified a transcription factor RXE1 exhibiting strong binding activity to the xyr1 promoter using yeast one-hybrid screen. RXE1 homologs exist in quite a few filamentous fungi but none of them have been assessed regarding their functional involvement in plant cell wall degradation. Knockdown of rxe1 in T. reesei using a copper-mediated RNAi system not only abrogated conidiation, but also remarkably compromised xyr1 and cellulase gene expression. The defective cellulase but not conidia production in the rxe1-knockdown strain was fully rescued by the constitutive expression of XYR1. Our study thus identified a novel transcriptional regulator controlling xyr1 and cellulase gene expression, which will contribute to elaborating the intricate network of cellulase gene regulation in T. reesei.