Head blight, which is caused by mycotoxin-producing fungi of the genus Fusarium, is an economically important crop disease. We assessed the potential of host-induced gene silencing targeting the fungal cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes, which are essential for ergosterol biosynthesis, to restrict fungal infection. In axenic cultures of Fusarium graminearum, in vitro feeding of CYP3RNA, a 791-nt double-stranded (ds)RNA complementary to CYP51A, CYP51B, and CYP51C, resulted in growth inhibition [half-maximum growth inhibition (IC50) = 1.2 nM] as well as altered fungal morphology, similar to that observed after treatment with the azole fungicide tebuconazole, for which the CYP51 enzyme is a target. Expression of the same dsRNA in Arabidopsis and barley rendered susceptible plants highly resistant to fungal infection. Microscopic analysis revealed that mycelium formation on CYP3RNA-expressing leaves was restricted to the inoculation sites, and that inoculated barley caryopses were virtually free of fungal hyphae. This inhibition of fungal growth correlated with in planta production of siRNAs corresponding to the targeted CYP51 sequences, as well as highly efficient silencing of the fungal CYP51 genes. The high efficiency of fungal inhibition suggests that host-induced gene-silencing targeting of the CYP51 genes is an alternative to chemical treatments for the control of devastating fungal diseases.

https://www.pnas.org/content/pnas/110/48/19324.full.pdf

Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species

Plant pathogenic fungi represent the largest group of disease-causing agents on crop plants, and are a constant and major threat to agriculture worldwide. Recent studies have shown that engineered production of RNA interference (RNAi)-inducing dsRNA in host plants can trigger specific fungal gene silencing and confer resistance to fungal pathogens1-7. Although these findings illustrate efficient uptake of host RNAi triggers by pathogenic fungi, it is unknown whether or not such an uptake mechanism has been evolved for a natural biological function in fungus-host interactions. Here, we show that in response to infection with Verticillium dahliae (a vascular fungal pathogen responsible for devastating wilt diseases in many crops) cotton plants increase production of microRNA 166 (miR166) and miR159 and export both to the fungal hyphae for specific silencing. We found that two V. dahliae genes encoding a Ca2+-dependent cysteine protease (Clp-1) and an isotrichodermin C-15 hydroxylase (HiC-15), and targeted by miR166 and miR159, respectively, are both essential for fungal virulence. Notably, V. dahliae strains expressing either Clp-1 or HiC-15 rendered resistant to the respective miRNA exhibited drastically enhanced virulence in cotton plants. Together, our findings identify a novel defence strategy of host plants by exporting specific miRNAs to induce cross-kingdom gene silencing in pathogenic fungi and confer disease resistance.

/pdf/cotton-plants-export-micrornas-to-inhibit-virulence-gene-1c7kv0q19v.pdf

Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen

In most eukaryotes, double-stranded RNA is processed into small RNAs that are potent regulators of gene expression. This gene silencing process is known as RNA silencing or RNA interference (RNAi) and, in plants and nematodes, it is associated with the production of a mobile signal that can travel from cell-to-cell and over long distances. The sequence-specific nature of systemic RNA silencing indicates that a nucleic acid is a component of the signalling complex. Recent work has shed light on the mobile RNA species, the genes involved in the production and transport of the signal. This review discusses the advances in systemic RNAi and presents the current challenges and questions in this rapidly evolving field.

/pdf/intercellular-and-systemic-movement-of-rna-silencing-signals-4aayd7lc3v.pdf

Intercellular and systemic movement of RNA silencing signals

Summary RNA interference (RNAi) has emerged as a powerful genetic tool for scientific research over the past several years. It has been utilized not only in fundamental research for the assessment of gene function, but also in various fields of applied research, such as human and veterinary medicine and agriculture. In plants, RNAi strategies have the potential to allow manipulation of various aspects of food quality and nutritional content. In addition, the demonstration that agricultural pests, such as insects and nematodes, can be killed by exogenously supplied RNAi targeting their essential genes has raised the possibility that plant predation can be controlled by lethal RNAi signals generated in planta. Indeed, recent evidence argues that this strategy, called host-induced gene silencing (HIGS), is effective against sucking insects and nematodes; it also has been shown to compromise the growth and development of pathogenic fungi, as well as bacteria and viruses, on their plant hosts. Here, we review recent studies that reveal the enormous potential RNAi strategies hold not only for improving the nutritive value and safety of the food supply, but also for providing an environmentally friendly mechanism for plant protection.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12226

New wind in the sails: improving the agronomic value of crop plants through RNAi-mediated gene silencing

Rust fungi cause devastating diseases of wheat and other cereal species globally. Genetic resistance is the preferred method to control rusts but the effectiveness of race-specific resistance is typically transient due to the genetic plasticity of rust populations. The advent of RNA interference (RNAi) technology has shown promise for the engineering of resistance to some biotrophic pathogens in plants by altering the expression of essential pathogens' genes. Gene fragments from the rust fungi Puccinia striiformis f. sp. tritici or P. graminis f. sp. tritici were delivered to plant cells through the Barley stripe mosaic virus system, and some reduced the expression of the corresponding genes in the rust fungus. The ability to detect suppression was associated with the expression patterns of the fungal genes because reduction was only detected in transcripts with relatively high levels of expression in fungal haustoria. The results indicate that an in planta RNAi approach can be used in functional genomics research for rust fungi and that it could potentially be used to engineer durable resistance.

https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-10-10-0229

Development of a host-induced RNAi system in the wheat stripe rust fungus Puccinia striiformis f. sp. tritici.

Self-complementary RNA transcripts form a double-stranded RNA (dsRNA) that triggers a sequence-specific mRNA degradation, in a process known as RNA interference (RNAi), leading to gene silencing. In vascular plants, RNAi molecules trafficking occur between cells and systemically throughout the plant. RNAi signals can spread systemically throughout a plant, even across graft junctions from transgenic to non-transgenic stocks. There is also a great interest in applying RNAi to pathogenic fungi. Specific inhibition of gene expression by RNAi has been shown to be suitable for a multitude of phytopathogenic filamentous fungi. However, double-stranded (ds)RNA/small interfering (si)RNA silencing effect has not been observed in vivo. This study demonstrates for the first time the in vivo interference phenomenon in the pathogenic fungus Fusarium verticillioides, in which expression of an individual fungal transgene was specifically abolished by inoculating mycelial cells in transgenic tobacco plants engineered to express siRNAs from a dsRNA corresponding to the particular transgene. The results provide a powerful tool for further studies on molecular plant-microbe and symbiotic interactions. From a biotechnological perspective, silencing of fungal genes by generating siRNAs in the host provides a novel strategy for the development of broad fungi-resistance strategies in plants and other organisms.

/pdf/in-vivo-trans-specific-gene-silencing-in-fungal-cells-by-in-5029ulm499.pdf

In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA

The bone morphogenetic protein BMP2 plays a crucial role in the formation and regeneration of bone and cartilage, which is critical for maintaining skeletal integrity and bone fracture repair. Because of its important role in osteogenic properties it has been commercially produced for clinical use. Here we report attempts to express human BMP2 using two plant systems (lettuce chloroplast and soybean seeds). The rhBMP2 gene (coding for the 13 kDa active polypeptide) was introduced in two regions of the lettuce chloroplast genome. Two homoplasmic events were achieved and RT-PCR demonstrated that the BMP2 gene was transcribed. However, it was not possible to detect accumulation of rhBMP2 in leaves. Two soybean events were achieved to express a full-length hBMP2 gene (coding for the 45 kDa pro-BMP2) fused with the α-coixin signal peptide, under control of the β-conglycinin promoter. Pro-BMP2 was expressed in the transgenic seeds at levels of up to 9.28% of the total soluble seed protein as determined by ELISA. It was demonstrated that this recombinant form was biologically active upon administration to C2C12 cell cultures, because it was able to induce an osteogenic cascade, as observed by the enhanced expression of SP7 (osterix) and ALPI (alkaline phosphatase) genes. Collectively, these results corroborated our previous observation that soybean seeds provide an effective strategy for achieving stable accumulation of functional therapeutic proteins, such as BMP2.

Rebeca C. D’Allastta

Papers

In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA

Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2