The dynamics of N6-methyladenine RNA modification in interactions between rice and plant viruses.

BACKGROUND: N6-methyladenosine (m6A) is the most common RNA modification in eukaryotes and has been implicated as a novel epigenetic marker that is involved in various biological processes. The pattern and functional dissection of m6A in the regulation of several major human viral diseases have already been reported. However, the patterns and functions of m6A distribution in plant disease bursting remain largely unknown. RESULTS: We analyse the high-quality m6A methylomes in rice plants infected with two devastating viruses. We find that the m6A methylation is mainly associated with genes that are not actively expressed in virus-infected rice plants. We also detect different m6A peak distributions on the same gene, which may contribute to different antiviral modes between rice stripe virus or rice black-stripe dwarf virus infection. Interestingly, we observe increased levels of m6A methylation in rice plant response to virus infection. Several antiviral pathway-related genes, such as RNA silencing-, resistance-, and fundamental antiviral phytohormone metabolic-related genes, are also m6A methylated. The level of m6A methylation is tightly associated with its relative expression levels. CONCLUSIONS: We revealed the dynamics of m6A modification during the interaction between rice and viruses, which may act as a main regulatory strategy in gene expression. Our investigations highlight the significance of m6A modifications in interactions between plant and viruses, especially in regulating the expression of genes involved in key pathways.

Rice yellow stunt virus activates polyamine biosynthesis to promote viral propagation in insect vectors by disrupting ornithine decarboxylase antienzyme function.

Intracellular polyamines (putrescine, spermidine, and spermine) have emerged as important molecules for viral infection; however, how viruses activate polyamines biosynthesis to promote viral infection remains unclear. Ornithine decarboxylase 1 (ODC1) and its antienzyme 1 (OAZ1) are major regulators of polyamine biosynthesis in animal cells. Here, we report that rice yellow stunt virus (RYSV), a plant rhabdovirus, could activate putrescine biosynthesis in leafhoppers to promote viral propagation by inhibiting OAZ1 expression. We observed that the reduction of putrescine biosynthesis by treatment with difluormethylornithine (DFMO), a specific nontoxic inhibitor of ODC1, or with in vitro synthesized dsRNAs targeting ODC1 mRNA could inhibit viral infection. In contrast, the supplement of putrescine or the increase of putrescine biosynthesis by treatment with dsRNAs targeting OAZ1 mRNA could facilitate viral infection. We further determined that both RYSV matrix protein M and ODC1 directly bind to the ODC-binding domain at the C-terminus of OAZ1. Thus, viral propagation in leafhoppers would decrease the ability of OAZ1 to target and mediate the degradation of ODC1, which finally activates putrescine production to benefit viral propagation. This work reveals that polyamine-metabolizing enzymes are directly exploited by a vector-borne virus to increase polyamine production, thereby facilitating viral infection in insect vectors.

Homo sapiens: The Superspreader of Plant Viral Diseases.

Plant viruses are commonly vectored by flying or crawling animals, such as aphids and beetles, and cause serious losses in major agricultural and horticultural crops. Controlling virus spread is often achieved by minimizing a crop's exposure to the vector, or by reducing vector numbers with compounds such as insecticides. A major, but less obvious, factor not controlled by these measures is Homo sapiens. Here, we discuss the inconvenient truth of how humans have become superspreaders of plant viruses on both a local and a global scale.

Synthesis of Four Optical Isomers of Antiviral Agent NK0209 and Determination of Their Configurations and Activities against a Plant Virus.

Previously, we reported for the first time that harmala alkaloids harmine and tetrahydroharmine exhibit activity against plant viruses, and we developed an analogue, designated NK0209, that efficiently prevents and controls plant virus diseases. Here, to investigate the influence of the spatial configuration of NK0209 on its antiviral activities, we synthesized its four optical isomers, determined their configurations, and evaluated their activities against tobacco mosaic virus. All four isomers were significantly more active than ningnanmycin, which is one of the most successful commercial antiviral agents, with in vivo inactivation, cure, and protection rates of 57.3 ± 1.9, 54.2 ± 3.3, and 55.0 ± 4.1% at 500 µg/mL. Furthermore, analysis of structure-activity relationships demonstrated for the first time that the spatial conformation of NK0209 is an important determinant of its antiviral activity, and our results provide information about the possible optimum configuration for interaction of this molecule with its target protein.

Seasonality of interactions between a plant virus and its host during persistent infection in a natural environment.

Persistent infection, wherein a pathogen is continually present in a host individual, is widespread in virus-host systems. However, little is known regarding how seasonal environments alter virus-host interaction during such metastability. We observed a lineage-to-lineage infection of the host plant Arabidopsis halleri with Turnip mosaic virus for 3 years without severe damage. Virus dynamics and virus-host interactions within hosts were highly season dependent. Virus accumulation in the newly formed leaves was temperature dependent and was suppressed during winter. Transcriptome analyses suggested that distinct defence mechanisms, i.e. salicylic acid (SA)-dependent resistance and RNA silencing, were predominant during spring and autumn, respectively. Transcriptomic difference between infected and uninfected plants other than defence genes appeared transiently only during autumn in upper leaves. However, the virus preserved in the lower leaves is transferred to the clonal offspring of the host plants during spring. In the linage-to-linage infection of the A. halleri-TuMV system, both host clonal reproduction and virus transmission into new clonal rosettes are secured during the winter-spring transition. How virus and host overwinter turned out to be critical for understanding a long-term virus-host interaction within hosts under temperate climates, and more generally, understanding seasonality provides new insight into ecology of plant viruses.

Synthesis of Anthranilic Diamide Derivatives Containing Moieties of Trifluoromethylpyridine and Hydrazone as Potential Anti-Viral Agents for Plants.

A series of novel anthranilic diamide derivatives (5a-5ab) containing moieties of trifluoromethylpyridine and hydrazone was designed and synthesized. The synthesized compounds were evaluated in vivo for their activities against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). Most of the synthesized compounds displayed good to excellent antiviral activities. The compounds 5i, 5k, 5s, 5w, 5x, and 5z had the curative activity over 65% against TMV at the concentration of 500 µg/mL, which were significantly higher than those of ningnanmycin (55.0%) and ribavirin (37.9%). Notably, the curative activity of compound 5i was up to 79.5%, with the EC50 value of 75.9 µg/mL, whereas the EC50 value of ningnanmycin was 362.4 µg/mL. The pot experiments also further demonstrated the significantly curative effect of 5i. Meanwhile, compounds 5h, 5p and 5x displayed more protective activities on TMV than that of ningnanmycin. Moreover, compounds 5a, 5e, 5f, and 5i showed inactivation activity similar to ningnanmycin at 500 µg/mL, and the EC50 value of 5e (41.5 µg/mL) was lower than ningnanmycin (50.0 µg/mL). The findings of transmission electron microscopic (TEM) indicated that the synthesized compounds exhibited strong and significant binding affinity to TMV coat protein (CP) and could obstruct the self-assembly and increment of TMV particles. Microscale thermophoresis (MST) studies on TMV-CP and CMV CP revealed that some of the active compounds, particularly 5i, exhibited a strong binding capability to TMV-CP or CMV-CP. This study revealed that anthranilic diamide derivatives containing moieties of trifluoromethylpyridine and hydrazone could be used as novel antiviral agents for controlling the plant viruses.

Within-Host Multiplication and Speed of Colonization as Infection Traits Associated with Plant Virus Vertical Transmission.

Although vertical transmission from parents to offspring through seeds is an important fitness component of many plant viruses, very little is known about the factors affecting this process. Viruses reach the seed by direct invasion of the embryo and/or by infection of the ovules or the pollen. Thus, it can be expected that the efficiency of seed transmission would be determined by (i) virus within-host multiplication and movement, (ii) the ability of the virus to invade gametic tissues, (iii) plant seed production upon infection, and (iv) seed survival in the presence of the virus. However, these predictions have seldom been experimentally tested. To address this question, we challenged 18 Arabidopsis thaliana accessions with Turnip mosaic virus and Cucumber mosaic virus Using these plant-virus interactions, we analyzed the relationship between the effect of virus infection on rosette and inflorescence weights; short-, medium-, and long-term seed survival; virulence; the number of seeds produced per plant; virus within-host speed of movement; virus accumulation in the rosette and inflorescence; and efficiency of seed transmission measured as a percentage and as the total number of infected seeds. Our results indicate that the best estimators of percent seed transmission are the within-host speed of movement and multiplication in the inflorescence. Together with these two infection traits, virulence and the number of seeds produced per infected plant were also associated with the number of infected seeds. Our results provide support for theoretical predictions and contribute to an understanding of the determinants of a process central to plant-virus interactions.IMPORTANCE One of the major factors contributing to plant virus long-distance dispersal is the global trade of seeds. This is because more than 25% of plant viruses can infect seeds, which are the main mode of germplasm exchange/storage, and start new epidemics in areas where they were not previously present. Despite the relevance of this process for virus epidemiology and disease emergence, the infection traits associated with the efficiency of virus seed transmission are largely unknown. Using turnip mosaic and cucumber mosaic viruses and their natural host Arabidopsis thaliana as model systems, we have identified the within-host speed of virus colonization and multiplication in the reproductive structures as the main determinants of the efficiency of seed transmission. These results contribute to shedding light on the mechanisms by which plant viruses disperse and optimize their fitness and may help in the design of more-efficient strategies to prevent seed infection.

Molecular and biological characterization of a putative new sobemovirus infecting Physalis peruviana.

Physalis peruviana is a perennial solanaceous plant that has recently been established as a commercial crop in Brazil. This work reports the near-complete genome sequence, particle morphology, and plant host responses to a putative new sobemovirus, named "physalis rugose mosaic virus". The virus, characterized by isometric particles of ca. 30 nm in diameter, causes foliar symptoms of mosaic, malformation and blistering, accompanied by stunting. The near-complete genome sequence comprises 4175 nucleotides and contains five open reading frames that are similar to those of other sobemoviruses. In addition to P. peruviana, the new virus systemically infected Capsicum annuum, Nicotiana tabacum and Solanum lycopersicum by mechanical inoculation. Thus, this virus may cause disease in these crops in the field.

Evolution and ecology of plant viruses.

The discovery of the first non-cellular infectious agent, later determined to be tobacco mosaic virus, paved the way for the field of virology. In the ensuing decades, research focused on discovering and eliminating viral threats to plant and animal health. However, recent conceptual and methodological revolutions have made it clear that viruses are not merely agents of destruction but essential components of global ecosystems. As plants make up over 80% of the biomass on Earth, plant viruses likely have a larger impact on ecosystem stability and function than viruses of other kingdoms. Besides preventing overgrowth of genetically homogeneous plant populations such as crop plants, some plant viruses might also promote the adaptation of their hosts to changing environments. However, estimates of the extent and frequencies of such mutualistic interactions remain controversial. In this Review, we focus on the origins of plant viruses and the evolution of interactions between these viruses and both their hosts and transmission vectors. We also identify currently unknown aspects of plant virus ecology and evolution that are of practical importance and that should be resolvable in the near future through viral metagenomics.

Viruses in the phytobiome.

The phytobiome, defined as plants and all the entities that interact with them, is rich in viruses, but with the exception of plant viruses of crop plants, most of the phytobiome viruses remain very understudied. This review focuses on the neglected portions of the phytobiome, including viruses of other microbes interacting with plants, viruses in the soil, viruses of wild plants, and relationships between viruses and the vectors of plant viruses.

Virus-based pharmaceutical production in plants: an opportunity to reduce health problems in Africa.

BACKGROUND: Developing African countries face health problems that they struggle to solve. The major causes of this situation are high therapeutic and logistical costs. Plant-made therapeutics are easy to produce due to the lack of the safety considerations associated with traditional fermenter-based expression platforms, such as mammalian cells. Plant biosystems are easy to scale up and inexpensive, and they do not require refrigeration or a sophisticated medical infrastructure. These advantages provide an opportunity for plant-made pharmaceuticals to counteract diseases for which medicines were previously inaccessible to people in countries with few resources. MAIN BODY: The techniques needed for plant-based therapeutic production are currently available. Viral expression vectors based on plant viruses have greatly enhanced plant-made therapeutic production and have been exploited to produce a variety of proteins of industrial, pharmaceutical and agribusiness interest. Some neglected tropical diseases occurring exclusively in the developing world have found solutions through plant bioreactor technology. Plant viral expression vectors have been reported in the production of therapeutics against these diseases occurring exclusively in the third world, and some virus-derived antigens produced in plants exhibit appropriate antigenicity and immunogenicity. However, all advances in the use of plants as bioreactors have been made by companies in Europe and America. The developing world is still far from acquiring this technology, although plant viral expression vectors may provide crucial help to overcome neglected diseases. CONCLUSION: Today, interest in these tools is rising, and viral amplicons made in and for Africa are in progress. This review describes the biotechnological advances in the field of plant bioreactors, highlights factors restricting access to this technology by those who need it most and proposes a solution to overcome these limitations.

Direct and indirect influences of virus-insect vector-plant interactions on non-circulative, semi-persistent virus transmission.

Plant viruses that are transmitted in a non-circulative, semi-persistent (NCSP) manner have determinants on, and/or accessories to, their capsids that facilitate virion binding to specific retention sites in their insect vectors. Bilateral interactions and interactions occurring at the nexus of all three partners (virus, vector and plant) also contribute to transmission by influencing virus acquisition and inoculation. Vector feeding behavior lies at the core of this trio of virus transmission processes (retention-acquisition-inoculation), but transmission may also be mediated by virus infection-triggered and/or vector feeding-triggered plant cues that influence behavioral responses such as vector attraction, deterrence and dispersal. Insights into the multiphasic interactions and coordinated processes will lead to a better understanding of the mechanisms of NCSP transmission.

Long-distance movement of helical multipartite phytoviruses: keep connected or die?

All living organisms have to preserve genome integrity to ensure the survival of progeny generations. Viruses, though often regarded as 'non living', protect their nucleic acids from biotic and abiotic stresses, ranging from nuclease action to radiation-induced adducts. When the viral genome is split into multiple segments, preservation of at least one copy of each segment is required. While segmented and monopartite viruses use an all-in-one strategy, multipartite viruses have to address in the cell at least one of each viral particle in which the split positive stranded RNA genome is individually packaged. Here, we review and discuss the biology of multipartite helical RNA phytoviruses to outline our current hypothesis on a coordinated genomic RNA network RNP complex that preserves an all-in-one strategy and genome integrity.

Susceptibility Genes to Plant Viruses.

Plant viruses use cellular factors and resources to replicate and move. Plants respond to viral infection by several mechanisms, including innate immunity, autophagy, and gene silencing, that viruses must evade or suppress. Thus, the establishment of infection is genetically determined by the availability of host factors necessary for virus replication and movement and by the balance between plant defense and viral suppression of defense responses. Host factors may have antiviral or proviral activities. Proviral factors condition susceptibility to viruses by participating in processes essential to the virus. Here, we review current advances in the identification and characterization of host factors that condition susceptibility to plant viruses. Host factors with proviral activity have been identified for all parts of the virus infection cycle: viral RNA translation, viral replication complex formation, accumulation or activity of virus replication proteins, virus movement, and virion assembly. These factors could be targets of gene editing to engineer resistance to plant viruses.

Citrus Psorosis Virus Movement Protein Contains an Aspartic Protease Required for Autocleavage and the Formation of Tubule-Like Structures at Plasmodesmata.

Plant virus cell-to-cell movement is an essential step in viral infections. This process is facilitated by specific virus-encoded movement proteins (MPs), which manipulate the cell wall channels between neighboring cells known as plasmodesmata (PD). Citrus psorosis virus (CPsV) infection in sweet orange involves the formation of tubule-like structures within PD, suggesting that CPsV belongs to "tubule-forming" viruses that encode MPs able to assemble a hollow tubule extending between cells to allow virus movement. Consistent with this hypothesis, we show that the MP of CPsV (MPCPsV) indeed forms tubule-like structures at PD upon transient expression in Nicotiana benthamiana leaves. Tubule formation by MPCPsV depends on its cleavage capacity, mediated by a specific aspartic protease motif present in its primary sequence. A single amino acid mutation in this motif abolishes MPCPsV cleavage, alters the subcellular localization of the protein, and negatively affects its activity in facilitating virus movement. The amino-terminal 34-kDa cleavage product (34KCPsV), but not the 20-kDa fragment (20KCPsV), supports virus movement. Moreover, similar to tubule-forming MPs of other viruses, MPCPsV (and also the 34KCPsV cleavage product) can homooligomerize, interact with PD-located protein 1 (PDLP1), and assemble tubule-like structures at PD by a mechanism dependent on the secretory pathway. 20KCPsV retains the protease activity and is able to cleave a cleavage-deficient MPCPsV in trans Altogether, these results demonstrate that CPsV movement depends on the autolytic cleavage of MPCPsV by an aspartic protease activity, which removes the 20KCPsV protease and thereby releases the 34KCPsV protein for PDLP1-dependent tubule formation at PD.IMPORTANCE Infection by citrus psorosis virus (CPsV) involves a self-cleaving aspartic protease activity within the viral movement protein (MP), which results in the production of two peptides, termed 34KCPsV and 20KCPsV, that carry the MP and viral protease activities, respectively. The underlying protease motif within the MP is also found in the MPs of other members of the Aspiviridae family, suggesting that protease-mediated protein processing represents a conserved mechanism of protein expression in this virus family. The results also demonstrate that CPsV and potentially other ophioviruses move by a tubule-guided mechanism. Although several viruses from different genera were shown to use this mechanism for cell-to-cell movement, our results also demonstrate that this mechanism is controlled by posttranslational protein cleavage. Moreover, given that tubule formation and virus movement could be inhibited by a mutation in the protease motif, targeting the protease activity for inactivation could represent an important approach for ophiovirus control.

Biology of viral satellites and their role in pathogenesis.

Extraviral components that can influence the accumulation and pathogenesis of their associated helper viruses are known as 'satellites'. The maintenance of satellites requires their ability to associate with their helper viruses. Satellites can be categorized as either satellite viruses or satellite nucleic acids based on their ability to encode capsid proteins. Understanding the biology of satellites is important since they are pathogenic to a wide range of plant, animal, and yeast organisms. Most satellites influence the pathogenesis of their helper viruses by altering the interaction between the host and helper virus. However, the molecular mechanism that governs the trilateral interaction between host, satellites, and helper virus remains largely unexplored. This review comprehensively describes details of the association and interaction of helper viruses with satellite viruses, satellite RNAs, and satellite DNAs, and their implications for pathogenesis.

The Role of Viruses in the Phytobiome.

Viruses are an important but sequence-diverse and often understudied component of the phytobiome. We succinctly review current information on how plant viruses directly affect plant health and physiology and consequently have the capacity to modulate plant interactions with their biotic and abiotic environments. Virus interactions with other biota in the phytobiome, including arthropods, fungi, and nematodes, may also impact plant health. For example, viruses interact with and modulate the interface between plants and insects. This has been extensively studied for insect-vectored plant viruses, some of which also infect their vectors. Other viruses have been shown to alter the impacts of plant-interacting phytopathogenic and nonpathogenic fungi and bacteria. Viruses that infect nematodes have also recently been discovered, but the impact of these and phage infecting soil bacteria on plant health remain largely unexplored.

CRISPR-Cas13a: Prospects for Plant Virus Resistance.

CRISPR-Cas13a is an efficient RNA targeting and editing tool characterized recently in prokaryotes. This system can be recruited to engineer resistance against plant RNA viruses and regulate gene expression. We discuss some important achievements in using the CRISPR-Cas13a system to confer resistance against plant RNA viruses.

Glycolate oxidase gene family in Nicotiana benthamiana: genome-wide identification and functional analyses in disease resistance.

Glycolate oxidase (GOX)-dependent production of H2O2 in response to pathogens and its function in disease resistance are still poorly understood. In this study, we performed genome-wide identification of GOX gene family in Nicotiana benthamiana and analyzed their function in various types of disease resistance. Sixteen GOX genes were identified in N. benthamiana genome. They consisted of GOX and HAOX groups. All but two NbGOX proteins contained an alpha_hydroxyacid_oxid_FMN domain with extra 43-52 amino acids compared to that of FMN-dependent alpha-hydroxyacid oxidizing enzymes (NCBI-CDD cd02809). Silencing of three NbGOX family genes NbHAOX8, NbGOX1 and NbGOX4 differently affected resistance to various pathogens including Tobacco rattle virus, Xanthomonas oryzae pv. oryzae (Xoo) and Sclerotinia sclerotiorum. Effect of these genes on resistance to Xoo is well correlated with that on Xoo-responsive H2O2 accumulation. Additionally, silencing of these genes enhanced PAMP-triggered immunity as shown by increased flg22-elicited H2O2 accumulation in NbGOX-silenced plants. These NbGOX family genes were distinguishable in altering expression of defense genes. Analysis of mutual effect on gene expression indicated that NbGOX4 might function through repressing NbHAOX8 and NbGOX1. Collectively, our results reveal the important roles and functional complexity of GOX genes in disease resistance in N. benthamiana.

A simple method for obtaining rice black-streaked dwarf virus-infected small brown planthopper nymphs.

Rice black-streaked dwarf virus (RBSDV), an important rice virus, is transmitted by vector small brown planthopper (SBPH) in a persistent manner, but not transovarial transmission. In order to obtain viruliferous SBPH nymphs for relevant research, a simple and reliable method was developed, through allowing SBPH adults laying eggs on RBSDV-infected rice plants. The results showed the hatching nymphs on diseased plants could early acquire virus, and the virus was detected in 2nd-instar nymphs from the spawning method, which was earlier than insect feed on diseased plant. The average viruliferous rate of SBPH from the spawning method was 32.9%, which was not lower than the feeding diseased plant method. The novel method was very easy to operate and time-saving, facilitating the study on the interaction between RBSDV and SBPH nymphs (especially young 2nd-4th instar nymphs), such as, the effect of RBSDV on nymph development, host plant orientation preference of viruliferous nymph, identification of viral interacting protein in nymph, etc.