syn-tasiRnas targeting the coat protein of potato virus Y confer antiviral resistance in Nicotiana benthamiana.

Trans-acting small interfering RNAs (tasiRNAs) are 21-nt phased (phased siRNAs) resulting from successive DCL-catalyzed processing from the end of a double-stranded RNA substrate originating from the RDR of an AGO-catalyzed cleaved RNA at a micro RNA target site. Plant tasiRNAs have been synthesized to produce synthetic tasiRNAs (syn-tasiRNAs) targeting viral RNAs that confer viral resistance. In this study, we engineered syn-tasiRNAs to target potato virus Y (PVY) infection by replacing five native siRNAs of TAS1c with 210-bp fragments from the coat protein (CP) region of the PVY genome. The results showed that the transient expression of syn-tasiR-CPpvy2 in Nicotiana benthamiana (N. benthamiana) plants conferred antiviral resistance, supported by the absence of PVY infection symptoms and viral accumulation. This indicated that syn-tasiR-CPpvy2 successfully targeted and silenced the PVY CP gene, effectively inhibiting viral infection. syn-tasiR-CPpvy1 displayed attenuated symptoms and decreased viral accumulation in these plants However, severe symptoms of PVY infection and a similar amount of viral accumulation as the control were observed in plants expressing syn-tasiR-CPpvy3. syn-tasiR-CPpvy/pvx, which targets both PVY and potato virus X (PVX), was engineered using a single precursor. After the transient expression of syn-tasiR-CPpvy/pvx3 and syn-tasiR-CPpvy/pvx5 in N. benthamiana, the plants were resistant to both PVY and PVX. These results suggested that engineered syn-tasiRNAs could not only specifically induce antiviral resistance against one target virus but could also be designed for multi-targeted silencing of different viruses, thereby preventing complex virus infection in plants.

Biocontrol of tobacco black shank disease (Phytophthora nicotianae) by Bacillus velezensis Ba168.

Tobacco black shank (TBS) caused by Phytophthora nicotianae is destructive to almost all tobacco cultivars and is widespread in many tobacco-growing countries. Through lab study and field test, we isolated plant growth-promoting rhizobacteria (PGPR) strain Ba168 which is a promising biocontrol strain of TBS. Ba168 was isolated from 168 soil samples and identified as Bacillus velezensis by its genetic and phenotypic characteristics. A susceptibility test indicated that the P. nicotianae antagonistic materials of Ba168 in extracellular metabolites were composed of effective and stable proteins/peptides. P. nicotianae's growth was suppressed by the ammonium sulfate precipitation of Ba168 culture filtrates (ASPBa) at a minimum inhibitory concentration of 5 µg/mL. Extracellular conductivity, pH, and the wet/dry weights of P. nicotianae's mycelia, along with scanning electron microscope analysis, suggested that Ba168-derived proteins/peptides could effectively inhibit P. nicotianae by causing irreversible damage to its cell walls and membranes. Protein identification of ASPBa supported these results and identified many key proteins responsible for various biocontrol-related pathways. Field assays of TBS control efficacy of many PGPRs and agrochemicals showed that all PGPR preparations reduced the disease index of tobacco, but Ba168 was the most effective. These results demonstrated the importance of Bacillus-derived proteins/peptides in the inhibition of P. nicotianae through irreversible damage to its cell wall and membrane; and the effectiveness of PGPR strain B. velezensis Ba168 for biocontrol of the soil-borne disease caused by P. nicotianae.

Inhibitory Effect of Osthole from Cnidium monnieri on Tobacco Mosaic Virus (TMV) Infection in Nicotiana glutinosa.

The coumarin compound of osthole was extracted from Cnidium monnieri and identified by LC-MS and 1H- and 13C-NMR. Osthole was tested for anti-virus activity against tobacco mosaic virus (TMV) using the half-leaf method. The results showed that stronger antiviral activity on TMV infection appeared in Nicotiana glutinosa than that of eugenol and ningnanmycin, with inhibitory, protective, and curative effects of 72.57%, 70.26%, and 61.97%, respectively. Through observation of the TMV particles, we found that osthole could directly affect the viral particles. Correspondingly, the level of coat protein detected by Western blot was significantly reduced when the concentrations of osthole increased in tested plants compared to that of the control. These results suggest that osthole has anti-TMV activity and may be used as a biological reagent to control the plant virus in the half-leaf method.

Antifungal Effect of Magnolol and Honokiol from Magnolia officinalis on Alternaria alternata Causing Tobacco Brown Spot.

In this study, two phenol compounds, magnolol and honokiol, were extracted from Magnolia officinalis and identified by LC-MS, 1H- and 13C-NMR. The magnolol and honokiol were shown to be effective against seven pathogenic fungi, including Alternaria alternata (Fr.) Keissl, Penicillium expansum (Link) Thom, Alternaria dauci f.sp. solani, Fusarium moniliforme J. Sheld, Fusarium oxysporum Schltdl., Valsa mali Miyabe & G. Yamada, and Rhizoctonia solani J.G. Kühn, with growth inhibition of more than 57%. We also investigated the mechanisms underlying the potential antifungal activity of magnolol and honokiol. The results showed that they inhibited the growth of A. alternata in a dose-dependent manner. Moreover, magnolol and honokiol treatment resulted in distorted mycelia and increased the cell membrane permeability of A. alternata, as determined by conductivity measurements. These results suggest that magnolol and honokiol are potential antifungal agents for application against plant fungal diseases.

Molecular Variability and Distribution of Sugarcane Mosaic Virus in Shanxi, China.

BACKGROUND: Sugarcane mosaic virus (SCMV) is responsible for large-scale economic losses in the global production of sugarcane, maize, sorghum, and some other graminaceous species. To understand the evolutionary mechanism of SCMV populations, this virus was studied in Shanxi, China. A total of 86 maize leaf samples (41 samples in 2012 and 45 samples in 2013) were collected from 4 regions of Shanxi. RESULTS: Double-antibody sandwich (DAS)-ELISA and RT-PCR showed 59 samples (30 samples in 2012 and 29 samples in 2013) to be positive for SCMV, from which 10 new isolates of SCMV were isolated and sequenced. The complete genomes of these isolates are 9610 nt long, including the 5' and 3' non-coding regions, and encode a 3063-amino acid polyprotein. Phylogenetic analyses revealed that 24 SCMV isolates could be divided on the basis of the whole genome into 2 divergent evolutionary groups, which were associated with the host species. Among the populations, 15 potential recombination events were identified. The selection pressure on the genes of these SCMV isolates was also calculated. The results confirmed that all the genes were under negative selection. CONCLUSIONS: Negative selection and recombination appear to be important evolutionary factors shaping the genetic structure of these SCMV isolates. SCMV is distributed widely in China and exists as numerous strains with distinct genetic diversity. Our findings will provide a foundation for evaluating the epidemiological characteristics of SCMV in China and will be useful in designing long-term, sustainable management strategies for SCMV.

[Polysaccharides inhibiting cucumber mosaic virus and their influence on tobacco defensive enzyme activities].

OBJECTIVE: Polysaccharides inhibiting cucumber mosaic virus (CMV) on tobacco were screened and their influence on tobacco defensive enzyme activities was explored. METHOD: We detected the deactivating, preventing and treating effect of 21 polysaccharides on CMV by half leaf method on Nitcotiana tabacum var. Samsun NN. We detected the variation of enzyme activity of Nitcotianatabacum var. NC89 handled by antiviral polysaccharide. RESULT: Results show that Marasmiu sandrosaceus polysaccharide had good deactivating and preventing effect on CMV. The preventing rate could reach 83.41% when the tobacco was dealt with the admixture of its 200-fold dilution and equivalent virus liquid for 30 min. The inhibition rate could reach 93.15% when the tobacco was inoculated after spraying with M. androsaceus polysaccharide for 24h. Moreover, the diversification of enzyme activity of tobacco-related was detected. Results show that peroxidase (POD) , polyphenol oxidase (PPO) andphenylalanine ammonia lyase (PAL) activities significantly enhanced. M. androsaceus polysaccharide sprayed on tobacco after inoculating CMV for 24h. Its enzyme activities increased. The peaksof POD, PPO and PAL activities ofthe treatment were 2.74, 3.45 and 2.82 times of the value of comparison, respectively. CONCLUSION: M. androsaceus polysaccharide can increase the resistance of tobacco on CMV by enhancing defense enzyme activity of tobacco.

Mutasynthesis of pyrrole spiroketal compound using calcimycin 3-hydroxy anthranilic acid biosynthetic mutant.

The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.

Characterization of the N-methyltransferase CalM involved in calcimycin biosynthesis by Streptomyces chartreusis NRRL 3882.

Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 µM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.

[Identification and primary application of TSNA degrading bacterial strain AS97 isolated from aging tobacco leaves].

OBJECTIVE: The purpose of this work was to screen strains having tobacco-specific nitrosamines (TSNA) deteriorating activity, isolated from the inner and superficial of tobacco plants. Then strain AS97 was isolated and identified for further application. METHODS: Strain AS97, with the highest conversion ability against both nitrate and nitrite, was screened by enrichment and selective medium. The strain was identified by morphological, physio-biochemical characteristics and 16S rRNA gene sequence analysis. The concentration of 4-(methylnitrosamino)-1-(3-pyridy)-1-butanone (NNK), N-nitrosonicotine (NNN), N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB) were determined by LC-MS/MS. The fermentation broth of strain AS97 was spraied on the leaves of tobacco to define inoculum concentration and fermentation condition. RESULTS: AS97 was identified as Pseudomonas fluorescens (Genbank accession number: JF 449445). Under the optimal growth conditions with inoculum concentration of 5%, at 30 degrees C for 10 d, AS97 had high biological activity against NNK and NNN with a degradation rate of 59.08% and 38.79%, respectively. The correlation analysis displayed a pronounced correlation (p > 0.01) among the concentration of nitrate, nitrite and TSNA. Furthermore, the results also exhibited that nitrate and nitrite were antecedent substance of TSNA. CONCLUSION: These results indicated that Pseudomonas fluorescens AS97 could be a promising microorganism in the practice of non-harmful cigarette production.

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection.

RNA interference (RNAi) silences gene expression by guiding mRNA degradation in a sequence-specific fashion. Small interfering RNA (siRNA), an intermediate of the RNAi pathway, has been shown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells. Here, we report that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) could inhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-associated 126 kDa protein in intact plant tissue. Our results indicate that transiently expressed shRNA efficiently interfered with TMV infection. The interference observed is sequence-specific, and time- and site-dependent. Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumber mosaic virus (CMV), an unrelated tobamovirus. In order to interfere with TMV accumulation in tobacco leaves, it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation. Our results support the view that RNAi opens the door for novel therapeutic procedures against virus diseases. We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expression could be employed as a potent antiviral treatment in plants.nt antiviral treatment in plants.

A viral protein suppresses siRNA-directed interference in tobacco mosaic virus infection.

Plant viruses encode suppressors of post-transcriptional gene silencing (PTGS), an adaptive defense response that limits virus replication and its spread in plants. The helper component proteinase (HC-Pro) of the potato virus A (PVA, genus Potyvirus) suppresses PTGS of silenced transgenes. Here, the effect of HC-Pro on siRNA-directed interference in the tobacco mosaic virus (TMV) was examined by using a transient Agrobacterium tumefaciens-based delivery system in intact tissues. It was shown that the interference effect was completely blocked by co-infiltration with HC-Pro plus siRNA constructs in both systemic and hypersensitive hosts. In the system host, all plants agro-infiltrated with HC-Pro plus siRNA constructs displayed the same symptoms as the negative control. Meanwhile, TMV RNA accumulation was found to be abundant in the upper leaves using reverse transcriptase-PCR (RT-PCR) and Northern blot assays. On the contrary, plants agro-infiltrated with the siRNA construct alone were free of symptoms. Therefore, our study suggests that the transient expression of HC-Pro inhibited the siRNA-directed host defenses against TMV infection.