In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention as new and crucial players in the regulation of numerous cellular processes and disease responses. In this review, we describe how diverse ncRNAs, including both small RNAs and long ncRNAs, may be used to engineer resistance against plant viruses. We discuss how double-stranded RNAs and small RNAs, such as artificial microRNAs and trans-acting small interfering RNAs, either produced in transgenic plants or delivered exogenously to non-transgenic plants, may constitute powerful RNA interference (RNAi)-based technology that can be exploited to control plant viruses. Additionally, we describe how RNA guided CRISPR-CAS gene-editing systems have been deployed to inhibit plant virus infections, and we provide a comparative analysis of RNAi approaches and CRISPR-Cas technology. The two main strategies for engineering virus resistance are also discussed, including direct targeting of viral DNA or RNA, or inactivation of plant host susceptibility genes. We also elaborate on the challenges that need to be overcome before such technologies can be broadly exploited for crop protection against viruses.

Plant-virus interactions are greatly influenced by environmental factors such as temperatures. In virus-infected plants, enhanced temperature is frequently associated with more severe symptoms and higher virus content. However, the mechanisms involved in controlling the temperature regulation of plant-virus interactions are poorly characterised. To elucidate these further, we analysed the responses of potato plants cv Chicago to infection by potato virus Y (PVY) at normal (22 °C) and elevated temperature (28 °C), the latter of which is known to significantly increase plant susceptibility to PVY. Using RNAseq analysis, we showed that single and combined PVY and heat-stress treatments caused dramatic changes in gene expression, affecting the transcription of both protein-coding and non-coding RNAs. Among the newly identified genes responsive to PVY infection, we found genes encoding enzymes involved in the catalysis of polyamine formation and poly ADP-ribosylation. We also identified a range of novel non-coding RNAs which were differentially produced in response to single or combined PVY and heat stress, that consisted of antisense RNAs and RNAs with miRNA binding sites. Finally, to gain more insights into the potential role of alternative splicing and epitranscriptomic RNA methylation during combined stress conditions, direct RNA nanopore sequencing was performed. Our findings offer insights for future studies of functional links between virus infections and transcriptome reprogramming, RNA methylation and alternative splicing.

In this work we developed and exploited a spray-induced gene silencing (SIGS)-based approach to deliver double-stranded RNA (dsRNA), which was found to protect potato against potato virus Y (PVY) infection. Given that dsRNA can act as a defence-inducing signal that can trigger sequence-specific RNA interference (RNAi) and non-specific pattern-triggered immunity (PTI), we suspected that these two pathways may be invoked via exogeneous application of dsRNA, which may account for the alterations in PVY susceptibility in dsRNA-treated potato plants. Therefore, we tested the impact of exogenously applied PVY-derived dsRNA on both these layers of defence (RNAi and PTI) and explored its effect on accumulation of a homologous virus (PVY) and an unrelated virus (potato virus X, PVX). Here, we show that application of PVY dsRNA in potato plants induced accumulation of both small interfering RNAs (siRNAs), a hallmark of RNAi, and some PTI-related gene transcripts such as WRKY29 (WRKY transcription factor 29; molecular marker of PTI), RbohD (respiratory burst oxidase homolog D), EDS5 (enhanced disease susceptibility 5), SERK3 (somatic embryogenesis receptor kinase 3) encoding brassinosteroid-insensitive 1-associated receptor kinase 1 (BAK1), and PR-1b (pathogenesis-related gene 1b). With respect to virus infections, PVY dsRNA suppressed only PVY replication but did not exhibit any effect on PVX infection in spite of the induction of PTI-like effects in the presence of PVX. Given that RNAi-mediated antiviral immunity acts as the major virus resistance mechanism in plants, it can be suggested that dsRNA-based PTI alone may not be strong enough to suppress virus infection. In addition to RNAi- and PTI-inducing activities, we also showed that PVY-specific dsRNA is able to upregulate production of a key enzyme involved in poly(ADP-ribose) metabolism, namely poly(ADP-ribose) glycohydrolase (PARG), which is regarded as a positive regulator of biotic stress responses. These findings offer insights for future development of innovative approaches which could integrate dsRNA-induced RNAi, PTI and modulation of poly(ADP-ribose) metabolism in a co-ordinated manner, to ensure a high level of crop protection.

Potato virus Y, an important viral pathogen of potato, has several genetic variants and geographic distributions which could be affected by environmental factors, aphid vectors, and reservoir plants. PVY is transmitted to virus-free potato plants by aphids and passed on to the next vegetative generations through tubers, but the effects of tuber transmission in PVY is largely unknown. By using high-throughput sequencing, we investigated PVY populations transmitted to potato plants by aphids in different climate zones of Russia, namely the Moscow and Astrakhan regions. We analyzed sprouts from the tubers produced by field-infected plants to investigate the impact of tuber transmission on PVY genetics. We found a significantly higher diversity of PVY isolates in the Astrakhan region, where winters are shorter and milder and summers are warmer compared to the Moscow region. While five PVY types, NTNa, NTNb, N:O, N-Wi, and SYR-I, were present in both regions, SYRI-II, SYRI-III, and 261-4 were only found in the Astrakhan region. All these recombinants were composed of the genome sections derived from PVY types O and N, but no full-length sequences of such types were present. The composition of the PVY variants in the tuber sprouts was not always the same as in their parental plants, suggesting that tuber transmission impacts PVY genetics.

Virus infections in plants are a major threat to crop production and sustainable agriculture, which results in significant yield losses globally. The past decade has seen the development and deployment of sophisticated high-throughput omics technologies including genomics, transcriptomics, proteomics, and metabolomics, in order to try to understand the mechanisms underlying plant–virus interactions and implement strategies to ameliorate crop losses. In this review, we discuss the current state-of-the-art applications of such key omics techniques, their challenges, future, and combinatorial use (e.g., single cell and spatial omics coupled with super-resolution high-throughput imaging methods and artificial intelligence-based predictive models) to obtain new mechanistic insights into plant–virus interactions, which could be exploited for more effective plant disease management and monitoring.

The external application of double-stranded RNA (dsRNA) has recently been developed as a non-transgenic approach for crop protection against pests and pathogens. This novel and emerging approach has come to prominence due to its safety and environmental benefits. It is generally assumed that the mechanism of dsRNA-mediated antivirus RNA silencing is similar to that of natural RNA interference (RNAi)-based defence against RNA-containing viruses. There is, however, no direct evidence to support this idea. Here, we provide data on the high-throughput sequencing (HTS) analysis of small non-coding RNAs (sRNA) as hallmarks of RNAi induced by infection with the RNA-containing potato virus Y (PVY) and also by exogenous application of dsRNA which corresponds to a fragment of the PVY genome. Intriguingly, in contrast to PVY-induced production of discrete 21 and 22 nt sRNA species, the externally administered PVY dsRNA fragment led to generation of a non-canonical pool of sRNAs, which were present as ladders of ~18–30 nt in length; suggestive of an unexpected sRNA biogenesis pathway. Interestingly, these non-canonical sRNAs are unable to move systemically and also do not induce transitive amplification. These findings may have significant implications for further developments in dsRNA-mediated crop protection.