Engineering Virus Resistance In Plants Research Articles

Engineering plant virus resistance: from RNA silencing to genome editing strategies.

SummaryViral diseases severely affect crop yield and quality, thereby threatening global food security. Genetic improvement of plant virus resistance is essential for sustainable agriculture. In the last decades, several modern technologies were applied in plant antiviral engineering. Here we summarized breakthroughs of the two major antiviral strategies, RNA silencing and genome editing. RNA silencing strategy has been used in antiviral breeding for more than thirty years, and many crops engineered to stably express small RNAs targeting various viruses have been approved for commercial release. Genome editing technology has emerged in the past decade, especially CRISPR/Cas, which provides new methods for genetic improvement of plant virus resistance and accelerates resistance breeding. Finally, we discuss the potential of these technologies for breeding crops, and the challenges and solutions they may face in the future.

Editing plants for virus resistance using CRISPR-Cas.

This minireview summarizes recent advancements using the clustered regularly interspaced palindromic repeats-associated nuclease systems (CRISPR-Cas) derived from prokaryotes to breed plants resistant to DNA and RNA viruses. The CRISPR-Cas system represents a powerful tool able to edit and insert novel traits into plants precisely at chosen loci offering enormous advantages to classical breeding. Approaches to engineering plant virus resistance in both transgenic and non-transgenic plants are discussed. Iterations of the CRISPR-Cas system, FnCas9 and C2c2 capable of editing RNA in eukaryotic cells offer a particular advantage for providing resistance to RNA viruses which represent the great majority of known plant viruses. Scientists have obtained conflicting results using gene silencing technology to produce transgenic plants resistant to geminiviruses. CRISPR-Cas systems engineered in plants to target geminiviruses have consistently reduced virus accumulation providing increased resistance to virus infection. CRISPR-Cas may provide novel and reliable approaches to control geminiviruses and other ssDNA viruses such as Banana bunchy top virus (BBTV).

Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

Artificial miRNAs for Specific Gene Silencing and Engineering Virus Resistance in Plants

Artificial miRNAs for Specific Gene Silencing and Engineering Virus Resistance in Plants

Resistance of non-transgenic papaya plants to papaya ringspot virus (PRSV) mediated by intron-containing hairpin dsRNAs expressed in bacteria.

RNA-mediated virus resistance based on natural antiviral RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants. In this study, a conserved 3'-region (positions 9839-10117, 279 nt) of the capsid protein (CP) gene of papaya ringspot virus (PRSV), designated CP279, was used to generate an intron-containing hairpin RNA (ihpRNA) construct by one-step, zero-background ligation-independent cloning (OZ-LIC). The RNaseIII-deficient Escherichia coli strain M-JM109lacY was identified as the best choice for producing large quantities of specific ihpRNA-CP279. Resistance analyses and ELISA data verified that most papaya plants mechanically co-inoculated with TRIzol-extracted ihpRNA-CP279 and PRSV were resistant to PRSV, and resistance was maintained throughout the test period (>2 months post-inoculation). In contrast, a 1-2 day interval between sequential inoculation of PRSV and ihpRNA-CP279 did not result in complete protection against PRSV infection, but delayed the appearance of viral symptoms by 3 to 4 days. These findings indicate that direct mechanical inoculation of papaya plants with bacterially-expressed ihpRNA-CP279 targeting the PRSV CP gene can interfere with virus infection. This work lays a foundation for developing a non-transgenic approach to control PRSV by directly spraying plants with ihpRNA or crude bacterial extract preparations.

RNA Silencing and Plant Viral Diseases

RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.

植物のRNAサイレンシングとウイルスの病徴誘導

RNA silencing plays an important role in plant resistance against viruses. As a counter-defense against RNA silencing, plant viruses have evolved RNA silencing suppressors (RSSs). RNA silencing is likely to play a major role in disease development. For example, RSSs have been found to disturb the gene expression controlled by miRNAs in plant tissue and organ development, resulting in plant malformation. Mosaic symptoms, which are typical in virus-infected plants, are actually a consequence of local arms race between host RNA silencing and viral RSSs. In addition, recent studies revealed that viral siRNAs could induce RNA silencing even against a certain host gene and thus a disease symptom through a complementary (homologous) sequence coincidentally found between virus and host gene. RNA silencing is the principal mediator of viral pathogenicity and disease induction and therefore should be exploited as a powerful tool for engineering virus resistance in plants as well as in animals.

Artificial MicroRNA-Mediated Virus Resistance in Plants

RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.

Novel strategies for engineering virus resistance in plants

Virus resistance in plants has been engineered by transgenic expression of viral and non-viral factors. Recent reports suggest that these strategies will lead to the development of highly effective and broad-spectrum resistance to virus disease.

Prospects for engineering virus resistance in plants with antisense RNA

The use of antisense RNA to manipulate plant metabolism has already been shown to be successful in altering pigment production in flowers and manipulating the ripening and softening processes in fruit. The major losses in crop yields due to viral pathogens have led to investigations of many strategies for developing virus-resistant plants. By inhibiting viral replication, the use of antisense may permit the engineering of such resistant plants.