RNA Interference Transformation Research Articles

RNAi-mediated silencing of MAP kinase signalling genes (Fmk1, Hog1, and Pbs2) in Fusarium oxysporum reduces pathogenesis on tomato plants

Fusarium oxysporum is a soil-borne plant fungal pathogen, and causes colossal losses in several crop plants including tomato. Effective control measures include the use of harmful fungicides and resistant cultivars, but these methods have shown limited success. Conventional methods to validate fungal pathogenic genes are labour intensive. Therefore, an alternative strategy is required to efficiently characterize unknown pathogenic genes. RNA interference (RNAi) has emerged as a potential tool to functionally characterize novel fungal pathogenic genes and also to control fungal diseases. Here, we report an efficient method to produce stable RNAi transformants of F. oxysporum using Agrobacterium-mediated transformation (AMT). We have transformed F. oxysporum spores using RNAi constructs of Fmk1, Hog1, and Pbs2 MAP kinase signalling genes. Fmk1 RNAi fungal transformants showed loss of surface hydrophobicity, reduced invasive growth on tomato fruits and hypo-virulence on tomato seedlings. Hog1 and Pbs2 RNAi transformants showed altered conidial size, and reduced invasive growth and pathogenesis. These results showed that AMT using RNAi constructs is an effective approach for dissecting the role of genes involved in pathogenesis in F. oxysporum and this could be extended for other fungal systems. The obtained knowledge can be easily translated for developing fungal resistant crops by RNAi.

Inducible Repression of Nuclear-Encoded Subunits of the Cytochrome b6f Complex in Tobacco Reveals an Extraordinarily Long Lifetime of the Complex.

The biogenesis of the cytochrome b6f complex in tobacco (Nicotiana tabacum) seems to be restricted to young leaves, suggesting a high lifetime of the complex. To directly determine its lifetime, we employed an ethanol-inducible RNA interference (RNAi) approach targeted against the essential nuclear-encoded Rieske protein (PetC) and the small M subunit (PetM), whose function in higher plants is unknown. Young expanding leaves of both PetM and PetC RNAi transformants bleached rapidly and developed necroses, while mature leaves, whose photosynthetic apparatus was fully assembled before RNAi induction, stayed green. In line with these phenotypes, cytochrome b6f complex accumulation and linear electron transport capacity were strongly repressed in young leaves of both RNAi transformants, showing that the M subunit is as essential for cytochrome b6f complex accumulation as the Rieske protein. In mature leaves, all photosynthetic parameters were indistinguishable from the wild type even after 14 d of induction. As RNAi repression of PetM and PetC was highly efficient in both young and mature leaves, these data indicate a lifetime of the cytochrome b6f complex of at least 1 week. The switch-off of cytochrome b6f complex biogenesis in mature leaves may represent part of the first dedicated step of the leaf senescence program.

BEM46 shows eisosomal localization and association with tryptophan-derived auxin pathway in Neurospora crassa.

BEM46 proteins are evolutionarily conserved, but their functions remain elusive. We reported previously that the BEM46 protein in Neurospora crassa is targeted to the endoplasmic reticulum (ER) and is essential for ascospore germination. In the present study, we established a bem46 knockout strain of N. crassa. This Δbem46 mutant exhibited a level of ascospore germination lower than that of the wild type but much higher than those of the previously characterized bem46-overexpressing and RNA interference (RNAi) lines. Reinvestigation of the RNAi transformants revealed two types of alternatively spliced bem46 mRNA; expression of either type led to a loss of ascospore germination. Our results indicated that the phenotype was not due to bem46 mRNA downregulation or loss but was caused by the alternatively spliced mRNAs and the peptides they encoded. Using the N. crassa ortholog of the eisosomal protein PILA from Aspergillus nidulans, we further demonstrated the colocalization of BEM46 with eisosomes. Employing the yeast two-hybrid system, we identified a single interaction partner: anthranilate synthase component II (encoded by trp-1). This interaction was confirmed in vivo by a split-YFP (yellow fluorescent protein) approach. The Δtrp-1 mutant showed reduced ascospore germination and increased indole production, and we used bioinformatic tools to identify a putative auxin biosynthetic pathway. The genes involved exhibited various levels of transcriptional regulation in the different bem46 transformant and mutant strains. We also investigated the indole production of the strains in different developmental stages. Our findings suggested that the regulation of indole biosynthesis genes was influenced by bem46 overexpression. Furthermore, we uncovered evidence of colocalization of BEM46 with the neutral amino acid transporter MTR.

Cloning of Rhamnosyl Transferase Gene and Construction of Its RNAi Vector in Potato

Steroidal glycoalkaloids (SGAs) are potentially harmful metabolites found in potatoes and other Solanaceous plants. SGAs accumulation affects food quality and safety, we hope to use molecular biology methods to reduce SGAs content and assist breeding efforts to ensure food safety, develop new and improved varieties of potatoes. To investigate the relationship between the gene expression of the rhamnosyltransferase (sgt3) and the biosynthesis of steroidal glycoalkaloid (SGAs), and reveal the role of sgt3 gene in biosynthetsis of potato steroidal glycoalkaloid, similar gene cDNA sequence of the potato sgt3 was obtained from the total RNA of potato tubers by reverse transcription polymerase chain reaction (RT-PCR) using specific primers synthesized according to the conserved domain of GenBank No: DQ266437 sequence. The protein function and structure of the similar sgt3 gene cDNA sequence were predicted and analyzed by related software of bioinformatics technology. Interference expression vector based on sgt3 gene was constructed. The Blast result showed that the gene sequence shared a high level of similarity with the sgt3 gene in GenBank (accession No: DQ266437) and its homology was 99.54%, and similarity of the amino acid sequence was 99%. The full-length of cDNA was 1 500 bp, which contained 505 amino acids, UDPG glycosyltransferase conserved domain and many important functional sites. The 3D structure of protein was predicted by homology comparative modeling in Swiss-Model, the results showed that the 3D structure of SGT3 was highly similar to that of the glycosyltransferase, so it was inferred that SGT3 should be a member of glycosyltransferase superfamily that has function of steroidal glycoalkaloid. Sgt3 similar gene obtained here was rhamnosyl transferase gene, and its sequence was submitted with GenBank No: HM188447. The RNA interference transformation expression vector in which sgt3 gene was regulated by Actin and CIPP promoters was constructed, which will lay a solid foundation for the synthesis of alkaloids glycosides, further research of metabolism and cultivation of transgenic potato varieties with low indican alkaloids character.

Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

BackgroundSporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii.ResultsThe presence of RNA interference (RNAi) mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G) plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1) gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90) as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM), an inhibitor of HSP90.ConclusionsUsing the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These results confirmed SSCMK1 as an important enzyme involved in the dimorphism of S. schenckii, necessary for the development of the yeast phase of this fungus. Also this study constitutes the first report of the transformation of S. schenckii and the use of RNAi to study gene function in this fungus.

Suppression of polygalacturonase gene expression in the phytopathogenic fungus Ophiostoma novo-ulmi by RNA interference

The fungal pathogen, Ophiostomo novo-ulmi, has been responsible for the rapid decline of American elm (Ulmus americana) across North America and remains a serious threat to surviving elm populations. The production of pectinolytic polygalacturonase enzymes has been implicated as a virulence factor for many fungal pathogens, including O. novo-ulmi. Previous work has shown that the targeted disruption of the endopolygalacturonase gene locus epg1 of O. novo-ulmi reduced, but did not eliminate pectinase activity. In the present study, we evaluated the use of RNA interference (RNAi) as a method of suppressing expression of the epg1 locus in O. novo-ulmi and compared its efficiency to the gene disruption method. While there was a reduction in epg1-specific mRNA transcripts and in the amount of polygalacturonase enzyme secreted for both methods of gene regulation, neither method completely suppressed the expression of pectinase activity. There was, however, a significantly greater reduction in both transcript levels and secreted enzyme observed for some of the RNAi transformants. As the first demonstration of RNAi in O. novo-ulmi, this method of gene regulation shows promise in future studies of gene expression and pathogenicity.

Gene silencing by RNA Interference in the white rot fungus Phanerochaete chrysosporium.

The effectiveness of RNA interference (RNAi) is demonstrated in the lignin-degrading fungus Phanerochaete chrysosporium. The manganese-containing superoxide dismutase gene (MnSOD1) was used as the target for RNAi. The plasmid constructed for gene silencing contained a transcriptional unit for hairpin RNA expression. Significantly lower MnSOD expression at both the mRNA and protein activity levels was detected in RNAi transformants. Furthermore, even though P. chrysosporium possesses three copies of the MnSOD gene, this RNAi construct was sufficient to decrease the enzymatic activity by as much as 70% relative to control levels. Implementation of the RNAi technique in P. chrysosporium provides an alternative genetic tool for studies of gene function, particularly of essential genes or gene families.

Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity

Peanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct. A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant (P < 0.05) reduction in Ara h 2 content in several transgenic seeds. Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T(0) plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut. The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology.