A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco.

BACKGROUND: Early, precise and simultaneous identification of plant viruses is of great significance for preventing virus spread and reducing losses in agricultural yields. METHODS AND RESULTS: In this study, the identification of plant viruses from symptomatic samples collected from a cigar tobacco planting area in Deyang and a flue-cured tobacco planting area in Luzhou city, Sichuan Province, China, was conducted by deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform, and plant virus-specific contigs were generated based on virus-derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis were performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples, with a total of 105 contigs mapped to the closest plant viruses with lengths ranging from 34 ~ 1720 nt. The results indicated that the major viruses were potato virus Y, Chilli veinal mottle virus, tobacco vein banding mosaic virus, tobacco mosaic virus and cucumber mosaic virus. Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. CONCLUSIONS: These results provide a theoretical basis and convenient methods for the rapid detection and control of viruses in cigar- and flue-cured tobacco.

Cooperative roles of introns 1 and 2 of tobacco resistance gene N in enhanced N transcript expression and antiviral defense responses.

The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. The requirement of the native promoter, the elicitor, or the individual introns for enhanced expression of N has not been fully studied. Here, we determined that 35S promoter-driven N transcript expression could be enhanced in the presence of the four introns regardless of the co-expression of the virus elicitor in tobacco. Function analyses using a series of N transgenes with different combination of introns revealed that the presence of intron 1 more so than intron 2 allowed higher accumulation of premature and mature N transcripts; however, both introns were important for not only enhanced gene expression but also for induction of cell death in tobacco and induced local resistance to spread of virus in Nicotiana benthamiana. Our findings indicate that introns 1 and 2 cooperatively contribute to N expression and virus resistance.

Antiviral activity of glucosylceramides isolated from Fusarium oxysporum against Tobacco mosaic virus infection.

Natural elicitors derived from pathogenic microorganisms represent an ecologic strategy to achieve resistance in plants against diseases. Glucosylceramides (GlcCer) are classified as neutral glycosphingolipids. GlcCer were isolated and purified from Fusarium oxysporum mycelium. F. oxysporum is a plant pathogenic fungus, abundant in soil and causing severe losses in economically important crops such as corn, tobacco, banana, cotton and passion fruit. In this study we evaluate the capacity of GlcCer in inducing resistance in N. tabacum cv Xanthi plants against Tobacco mosaic virus (TMV). Spraying tobacco plants with GlcCer before virus infection reduced the incidence of necrotic lesions caused by TMV. In addition, plants already infected with the virus showed a reduction in hypersensitive response (HR) lesions after GlcCer treatment, suggesting an antiviral effect of GlcCer. Our investigations showed that GlcCer stimulates the early accumulation of H2O2 and superoxide radicals. In addition, the expression of PR-1 (pathogenesis-related 1, with suggested antifungal action), PR-2 (ß-1,3-glucanase), PR-3 (Chitinase), PR-5 (Osmotin), PAL (Phenylalanine ammonia-lyase), LOX (Lipoxygenase) and POX (Peroxidase) genes was highly induced after treatment of tobacco plants with GlcCer and induction levels remained high throughout a period of 6 to 120 hours. Our experiments demonstrate that GlcCer induces resistance in tobacco plants against infection by TMV.

Fructosan form Paenibacillus kribbensis PS04 enhance disease resistance against Rhizoctonia solani and tobacco mosaic virus

BACKGROUND: Rice sheath blight (caused by Rhizoctonia solani) and tobacco mosaic virus are very important plant diseases, causing a huge loss in global crop production. Paenibacillus kribbensis PS04 is a broad-spectrum biocontrol agent, used for controlling these diseases. Previously, extracellular polysaccharides (EPS) from P. kribbensis PS04 had been purified and their structure was inferred to be fructosan. This study aimed to evaluate the effects of exogenous EPS treatment on plant­pathogen interactions. RESULTS: Plant defense genes such as phenylalanine ammonia-lyase, catalase, chitinase, allene oxide synthase, and PR1a proteins were significantly induced by exogenous EPS treatment. Moreover, subsequent challenge of EPSpretreated plants with the pathogens (R. solani or tobacco mosaic virus) resulted in higher expression of defenseassociated genes. Increased activities of defense-associated enzymes, total phenols, and flavonoids were also observed in EPS pretreated plants. The contents of malondialdehyde in plants, which act as indicator of lipid peroxidation, were reduced by EPS treatment. CONCLUSIONS: This study comprehensively showed that EPS produced from P. kribbensis PS04 enhances disease resistance in plants by the activation of defense-associated genes as well as through the enhancement of activities of defense-related enzymes.

Plant NLR immune receptor Tm-22 activation requires NB-ARC domain-mediated self-association of CC domain.

The nucleotide-binding, leucine-rich repeat-containing (NLR) class of immune receptors of plants and animals recognize pathogen-encoded proteins and trigger host defenses. Although animal NLRs form oligomers upon pathogen recognition to activate downstream signaling, the mechanisms of plant NLR activation remain largely elusive. Tm-22 is a plasma membrane (PM)-localized coiled coil (CC)-type NLR and confers resistance to Tobacco mosaic virus (TMV) by recognizing its viral movement protein (MP). In this study, we found that Tm-22 self-associates upon recognition of MP. The CC domain of Tm-22 is the signaling domain and its function requires PM localization and self-association. The nucleotide-binding (NB-ARC) domain is important for Tm-22 self-interaction and regulates activation of the CC domain through its nucleotide-binding and self-association. (d)ATP binding may alter the NB-ARC conformation to release its suppression of Tm-22 CC domain-mediated cell death. Our findings provide the first example of signaling domain for PM-localized NLR and insight into PM-localized NLR activation.

Identification of a Novel NtLRR-RLK and Biological Pathways That Contribute to Tolerance of TMV in Nicotiana tabacum.

Tobacco mosaic virus (TMV) infection can causes serious damage to tobacco crops. To explore the approach of preventing TMV infection of plants, two tobacco cultivars with different resistances to TMV were used to analyze transcription profiling before and after TMV infection. The involvement of biological pathways differed between the tolerant variety (Yuyan8) and the susceptible variety (NC89). In particular, the plant-virus interaction pathway was rapidly activated in Yuyan8, and specific resistance genes were enriched. Liquid chromatography tandem mass spectrometry analysis detected large quantities of antiviral substances in the tolerant Yuyan8. A novel Nicotiana tabacum leucine-rich repeat receptor kinase (NtLRR-RLK) gene was identified as being methylated and this was verified using bisulfite sequencing. Transient expression of TMV-green fluorescent protein in pRNAi-NtLRR-RLK transgenic plants confirmed that NtLRR-RLK was important for susceptibility to TMV. The specific protein interaction map generated from our study revealed that levels of BIP1, E3 ubiquitin ligase, and LRR-RLK were significantly elevated, and all were represented at node positions in the protein interaction map. The same expression tendency of these proteins was also found in pRNAi-NtLRR-RLK transgenic plants at 24 h after TMV inoculation. These data suggested that specific genes in the infection process can activate the immune signal cascade through different resistance genes, and the integration of signal pathways could produce resistance to the virus. These results contribute to the overall understanding of the molecular basis of plant resistance to TMV and in the long term could identify new strategies for prevention and control virus infection.

A Conserved Carboxylesterase Inhibits Tobacco mosaic virus (TMV) Accumulation in Nicotiana benthamiana Plants.

A carboxylesterase (CXE) or carboxylic-ester hydrolase is an enzyme that catalyzes carboxylic ester and water into alcohol and carboxylate. In plants, CXEs have been implicated in defense, development, and secondary metabolism. We discovered a new CXE gene in Nicotiana benthamiana that is related to virus resistance. The transcriptional level of NbCXE expression was significantly increased after Tobacco mosaic virus (TMV) infection. Transient over-expression of NbCXE inhibited TMV accumulation in N. benthamiana plants. Conversely, when the NbCXE gene was silenced with a Tobacco rattle virus (TRV)-based gene silencing system, TMV RNA accumulation was increased in NbCXE-silenced plants after infection. NbCXE protein was shown to interact with TMV coat protein (CP) in vitro. Additionally, the expressions of host defense-related genes were increased in transient NbCXE-overexpressed plants but decreased in NbCXE silenced N. benthamiana plants. In summary, our study showed that NbCXE is a novel resistance-related gene involved in host defense responses against TMV infection.

A Novel Biological Agent Cytosinpeptidemycin Inhibited the Pathogenesis of Tobacco Mosaic Virus by Inducing Host Resistance and Stress Response.

Cytosinpeptidemycin (CytPM) is a microbial pesticide that displayed broad-spectrum antiviral activity against various plant viruses. However, the molecular mechanism underlying antiviral activity of CytPM is poorly understood. In this study, the results demonstrated that CytPM could effectively delay the systemic infection of tobacco mosaic virus (TMV) in Nicotiana benthamiana and significantly inhibit the viral accumulation in tobacco BY-2 protoplasts. Results of RNA-seq indicated that 210 and 120 differential expressed genes (DEGs) were significantly up- and down-regulated after CytPM treatment in BY-2 protoplasts, respectively. In addition, KEGG analysis indicated that various DEGs were involved in endoplasmic reticulum (ER) protein processing, suggesting a possible correlation between ER homeostasis and virus resistance. RT-qPCR was performed to validate the gene expression of crucial DEGs related with defense, stress responses, signaling transduction, and phytohormone, which were consistent with results of RNA-seq. Our works provided valuable insights into the antiviral mechanism of CytPM that induced host resistance to viral infection.

Purification and Structural Analysis of the Effective Anti-TMV Compound ε-Poly-l-lysine Produced by Streptomyces ahygroscopicus.

Microbial secondary metabolites produced by actinomycetes are important natural products widely applied to control plant diseases. A variety of actinomycetes were isolated from soil samples collected from Tianzhu Mountain in Shenyang, China. A Streptomyces strain Shenyang Tianzhu (STZ) exhibits effective antiviral activity against Tobacco mosaic virus (TMV). The isolate was identified as Streptomyces ahygroscopicus based on its cultural, morphological, physiological, biochemical characteristics as well as the phylogenetic analysis using 16S rRNA sequences. To obtain the pure anti-TMV compound from Streptomyces STZ, the culture broth was subjected to Amberlite IRC-50 ion-exchange resin, SX-8 macroporous adsorption resin and Sephadex G-25 gel column chromatography. The purified active compound was confirmed to be ε-poly-l-lysine (ε-PL), with molecular mass in the range of 3454⁻4352 Da by structural analysis with infrared (IR), matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF), thin-layer chromatography (TLC) and high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR). The protective and curative effects of the purified compound ε-PL were tested and the results showed that the compound exhibited significant protective and curative activity against TMV. The potential application of ε-PL as an efficient anti-plant virus agent was expected.

Naamines and Naamidines as Novel Agents against a Plant Virus and Phytopathogenic Fungi.

Naamines, naamidines and various derivatives of these marine natural products were synthesized and characterized by means of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The activities of these alkaloids against a plant virus and phytopathogenic fungi were evaluated for the first time. A benzyloxy naamine derivative 15d displayed excellent in vivo activity against tobacco mosaic virus at 500 µg/mL (inactivation activity, 46%; curative activity, 49%; and protective activity, 41%); its activities were higher than the corresponding activities of the commercial plant virucide ribavirin (32%, 35%, and 34%, respectively), making it a promising new lead compound for antiviral research. In vitro assays revealed that the test compounds exhibited very good antifungal activity against 14 kinds of phytopathogenic fungi. Again, the benzyloxy naamine derivative 15d exhibited broad-spectrum fungicidal activity, emerging as a new lead compound for fungicidal research. Additional in vivo assays indicated that many of the compounds displayed inhibitory effects >30%.

On the historical significance of Beijerinck and his contagium vivum fluidum for modern virology.

This paper considers the foundational role of the contagium vivum fluidum-first proposed by the Dutch microbiologist Martinus Beijerinck in 1898-in the history of virology, particularly in shaping the modern virus concept, defined in the 1950s. Investigating the cause of mosaic disease of tobacco, previously shown to be an invisible and filterable entity, Beijerinck concluded that it was neither particulate like the bacteria implicated in certain infectious diseases, nor soluble like the toxins and enzymes responsible for symptoms in others. He offered a completely new explanation, proposing that the agent was a "living infectious fluid" whose reproduction was intimately linked to that of its host cell. Difficult to test at the time, the contagium vivum fluidum languished in obscurity for more than three decades. Subsequent advances in technologies prompted virus researchers of the 1930s and 1940s-the first to separate themselves from bacteriologists-to revive the idea. They found in it both the seeds for their emerging virus concept and a way to bring hitherto opposing thought styles about the nature of viruses and life together in consensus. Thus, they resurrected Beijerinck as the founding father, and contagium vivum fluidum as the core concept of their discipline.

Genome-Wide Analysis of DCL, AGO, and RDR Gene Families in Pepper (Capsicum Annuum L.).

RNA silencing is an evolutionarily conserved mechanism that regulates variety of cellular processes in plants. Argonaute protein (AGO), Dicer-like protein (DCL) and RNA-dependent RNA polymerase (RDR) are critical components of RNA silencing. These efficient and indispensable components of the RNAi pathway have not been identified and characterized in pepper. In this study, we identified 12 CaAGO, 4 CaDCL and 6 CaRDR genes in pepper and compared them with those of Arabidopsis, tobacco, potato and tomato. Detailed phylogenetic analyses revealed that each CaAGO, CaDCL and CaRDR protein family were classified into four clades. The tissue specific expression and respond to abiotic or biotic stress were studied. The real-time quantitative polymerase chain reaction (PCR) results demonstrated that CaAGO2, CaAGO10b, CaDCL2 and CaDCL4 were upregulated with cucumber mosaic virus (CMV), potato virus Y (PVY) and tobacco mosaic virus (TMV) infections, whereas they showed difference expression patterns in response to abiotic stress. In addition, we found that many of the candidate genes were induced by phytohormones and H2O2 treatment. Our results provide useful information for further elucidation of gene silencing pathways and RNAi-mediated host immunity in pepper.

Does adaptation to vertebrate codon usage relate to flavivirus emergence potential?

Codon adaptation index (CAI) is a measure of synonymous codon usage biases given a usage reference. Through mutation, selection, and drift, viruses can optimize their replication efficiency and produce more offspring, which could increase the chance of secondary transmission. To evaluate how higher CAI towards the host has been associated with higher viral titers, we explored temporal trends of several historic and extensively sequenced zoonotic flaviviruses and relationships within the genus itself. To showcase evolutionary and epidemiological relationships associated with silent, adaptive synonymous changes of viruses, we used codon usage tables from human housekeeping and antiviral immune genes, as well as tables from arthropod vectors and vertebrate species involved in the flavivirus maintenance cycle. We argue that temporal trends of CAI changes could lead to a better understanding of zoonotic emergences, evolutionary dynamics, and host adaptation. CAI appears to help illustrate historically relevant trends of well-characterized viruses, in different viral species and genetic diversity within a single species. CAI can be a useful tool together with in vivo and in vitro kinetics, phylodynamics, and additional functional genomics studies to better understand species trafficking and viral emergence in a new host.

Lethal mutagenesis of an RNA plant virus via lethal defection.

Lethal mutagenesis is an antiviral therapy that relies on increasing the viral mutation rate with mutagenic nucleoside or base analogues. Currently, the molecular mechanisms that lead to virus extinction through enhanced mutagenesis are not fully understood. Increasing experimental evidence supports the lethal defection model of lethal mutagenesis of RNA viruses, where replication-competent-defectors drive infective virus towards extinction. Here, we address lethal mutagenesis in vivo using 5-fluorouracil (5-FU) during the establishment of tobacco mosaic virus (TMV) systemic infections in N. tabacum. The results show that 5-FU decreased the infectivity of TMV without affecting its viral load. Analysis of molecular clones spanning two genomic regions showed an increase of the FU-related base transitions A → G and U → C. Although the mutation frequency or the number of mutations per molecule did not increase, the complexity of the mutant spectra and the distribution of the mutations were altered. Overall, our results suggest that 5-FU antiviral effect on TMV is associated with the perturbation of the mutation-selection balance in the genomic region of the RNA-dependent RNA polymerase (RdRp). Our work supports the lethal defection model for lethal mutagenesis in vivo in a plant RNA virus and opens the way to study lethal mutagens in plant-virus systems.

Over-expression of Oryza sativa Xrn4 confers plant resistance to virus infection.

Plant Xrn4 is a cytoplasmic 5' to 3' exoribonuclease that is reported to play an antiviral role during viral infection as demonstrated by experiments using the Xrn4s of Nicotiana benthamiana and Arabidopsis thaliana. Meanwhile, little is known about the anti-viral activity of Xrn4 from other plants. Here, we cloned the cytoplasmic Xrn4 gene of Oryza sativa (OsXrn4), and demonstrated that its over-expression elevated the 5'-3' exoribonuclease activity in rice plants and conferred resistance to rice stripe virus, a negative-sense RNA virus causing serious losses in East Asia. The accumulation of viral RNAs was also decreased. Moreover, the ectopic expression of OsXrn4 in N. benthamiana also conferred plant resistance to tobacco mosaic virus infection. These results show that the monocotyledonous plant cytoplasmic Xrn4 also has an antiviral role and thus provides a strategy for producing transgenic plants resistant to viral infection.

Delivery of Pesticides to Plant Parasitic Nematodes Using Tobacco Mild Green Mosaic Virus as a Nanocarrier.

Plant parasitic nematodes are a major burden to the global agricultural industry, causing a $157 billion loss each year in crop production worldwide. Effective treatment requires large doses of nematicides to be applied, putting the environment and human health at risk. Challenges are to treat nematodes that are located deep within the soil, feeding on the roots of plants. To attack the problem at its roots, we propose the use of tobacco mild green mosaic virus (TMGMV), an EPA-approved herbicide as a carrier to deliver nematicides. TMGMV self-assembles into a 300 × 18 nm soft matter nanorod with a 4 nm-wide hollow channel. This plant virus is comprised of 2130 identical coat protein subunits, each of which displays solvent-exposed carboxylate groups from Glu/Asp as well as Tyr side chains, enabling the functionalization of the carrier with cargo. We report (1) the successful formulation and characterization of TMGMV loaded with ∼1500 copies of the anthelmintic drug crystal violet (CV), (2) the bioavailability and treatment efficacy of CVTMGMV vs CV to nematodes in liquid cultures, and (3) the superior soil mobility of CVTMGMV compared to free CV.

A Novel Protein Elicitor (PeBA1) from Bacillus amyloliquefaciens NC6 Induces Systemic Resistance in Tobacco.

Here we reported a novel protein elicitor from Bacillus amyloliquefaciens NC6 induced systemic resistance (ISR) in tobacco. The purification was executed by ion-exchange chromatography, native-page extraction and HPLC, and the amino acid sequence was identified by mass spectrometry. This recombinant elicitor protein, expressed in Escherichia coli by an E1 expression vector, had good thermal stability, and the elicitor caused a clearly defined hypersensitive response (HR) necrosis in tobacco leaves. It could also trigger early defence events, including generation of reactive oxygen species (H2O2 and O2 (-)) and phenolic-compound accumulation. Quantitative real-time PCR (Q-RT-PCR) results indicated that several plant defence genes, including the salicylic acid (SA)-responsive PR1a, PR1b, PR5, and phenylalanine ammonia lyase (PAL), as well as the jasmonic acid (JA)-responsive PDF1.2 and CORONATINE INSENSITIVE 1 (COI1), were all up-regulated. Moreover, infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus (TMV) and the fungal pathogen Botrytis cinerea.

Antiviral activity of glycoprotein GP-1 isolated from Streptomyces kanasensis ZX01.

Plant virus diseases have seriously damaged global food security. However, current antiviral agents are not efficient enough for the requirement of agriculture production. So, developing new efficient and nontoxic antiviral agents is imperative. GP-1, from Streptomyces kanasensis ZX01, is a new antiviral glycoprotein, of which the antiviral activity and the mode of action against Tobacco mosaic virus (TMV) were investigated in this study. The results showed that GP-1 could fracture TMV particles, and the infection and accumulation of TMV in host plants were inhibited. Moreover, GP-1 could induce systematic resistance against TMV in the host, according to the results of activities of defensive enzymes increasing, MDA decreasing and overexpression of pathogenesis-related proteins. Furthermore, GP-1 could promote growth of the host plant. In conclusion, GP-1 showed the ability to be developed as an efficient antiviral agent and a fertilizer for agriculture.