An improved cucumber mosaic virus-based vector for efficient decoying of plant microRNAs.

We previously devised a cucumber mosaic virus (CMV)-based vector system carrying microRNA target mimic sequences for analysis of microRNA function in Arabidopsis thaliana. We describe an improved version in which target mimic cloning is achieved by annealing two partly-overlapping complementary DNA oligonucleotides for insertion into an infectious clone of CMV RNA3 (LS strain) fused to the cauliflower mosaic virus-derived 35S promoter. LS-CMV variants carrying mimic sequences were generated by co-infiltrating plants with Agrobacterium tumefaciens cells harboring engineered RNA3 with cells carrying RNA1 and RNA2 infectious clones. The utility of using agroinfection to deliver LS-CMV-derived microRNA target mimic sequences was demonstrated using a miR165/166 target mimic and three solanaceous hosts: Nicotiana benthamiana, tobacco (N. tabacum), and tomato (Solanum lycopersicum). In all three hosts the miR165/166 target mimic induced marked changes in developmental phenotype. Inhibition of miRNA accumulation and increased target mRNA (HD-ZIP III) accumulation was demonstrated in tomato. Thus, a CMV-derived target mimic delivered via agroinfection is a simple, cheap and powerful means of launching virus-based miRNA mimics and is likely to be useful for high-throughput investigation of miRNA function in a wide range of plants.This work was supported by the National Natural Science Foundation of China (grants 31170141 and 31470007), a Marie Curie International Incoming Fellowship (PIIF-GA-2009-236443), the 521 Talents Development Project (grant no.11610032521303) to ZD, the Leverhulme Trust (F/09741/F and RPG-2012-667) and the UK Biotechnology and Biological Sciences Research Council (BB/D014376/1 and BB/J011762/1) to JPC.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep1317

Movement Protein Mediates Systemic Necrosis in Tomato Plants with Infection of Tomato Mosaic Virus

The necrogenic strain N5 of tomato mosaic virus (ToMV-N5) causes systemic necrosis in tomato cultivar Hezuo903. In this work, we mapped the viral determinant responsible for the induction of systemic necrosis. By exchanging viral genes between N5 and a non-necrogenic strain S1, we found that movement protein (MP) was the determinant for the differential symptoms caused by both strains. Compared with S1 MP, N5 MP had an additional ability to increase virus accumulation, which was not due to its functions in viral cell-to-cell movement. Actually, N5 MP, but not S1 MP, was a weak RNA silencing suppressor, which assisted viral accumulation. Sequence alignment showed that both MPs differed by only three amino acid residues. Experiments with viruses having mutated MPs indicated that the residue isoleucine at position 170 in MP was the key site for MP to increase virus accumulation, but also was required for MP to induce systemic necrosis in virus-infected tomato plants. Collectively, the lethal necrosis caused by N5 is dependent on its MP protein that enhances virus accumulation via its RNA silencing suppressor activity, probably leading to systemic necrosis responses in tomato plants

Movement Protein Mediates Systemic Necrosis in Tomato Plants with Infection of Tomato Mosaic Virus

The necrogenic strain N5 of tomato mosaic virus (ToMV-N5) causes systemic necrosis in tomato cultivar Hezuo903. In this work, we mapped the viral determinant responsible for the induction of systemic necrosis. By exchanging viral genes between N5 and a non-necrogenic strain S1, we found that movement protein (MP) was the determinant for the differential symptoms caused by both strains. Compared with S1 MP, N5 MP had an additional ability to increase virus accumulation, which was not due to its functions in viral cell-to-cell movement. Actually, N5 MP, but not S1 MP, was a weak RNA silencing suppressor, which assisted viral accumulation. Sequence alignment showed that both MPs differed by only three amino acid residues. Experiments with viruses having mutated MPs indicated that the residue isoleucine at position 170 in MP was the key site for MP to increase virus accumulation, but also was required for MP to induce systemic necrosis in virus-infected tomato plants. Collectively, the lethal necrosis caused by N5 is dependent on its MP protein that enhances virus accumulation via its RNA silencing suppressor activity, probably leading to systemic necrosis responses in tomato plants

Heterologous Replicase from Cucumoviruses can Replicate Viral RNAs, but is Defective in Transcribing Subgenomic RNA4A or Facilitating Viral Movement

Interspecific exchange of RNA1 or RNA2 between the cucumoviruses cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) was reported to be non-viable in plants previously. Here we investigated viability of the reassortants between CMV and TAV in Nicotiana benthamiana plants by Agrobacterium-mediated viral inoculation. The reassortants were composed of CMV RNA1 and TAV RNA2 plus RNA3 replicated in the inoculated leaves, while they were defective in viral systemic movement at the early stage of infection. Interestingly, the reassortant containing TAV RNA1 and CMV RNA2 and RNA3 infected plants systemically, but produced RNA4A (the RNA2 subgenome) at an undetectable level. The defect in production of RNA4A was due to the 1a protein encoded by TAV RNA1, and partially restored by replacing the C-terminus (helicase domain) in TAV 1a with that of CMV 1a. Collectively, exchange of the replicase components between CMV and TAV was acceptable for viral replication, but was defective in either directing transcription of subgenomic RNA4A or facilitating viral long-distance movement. Our finding may shed some light on evolution of subgenomic RNA4A in the family Bromoviridae

Upregulated Palmitoleate and Oleate Production in <i>Escherichia coli</i> Promotes Gentamicin Resistance

Metabolic reprogramming mediates antibiotic efficacy. However, metabolic adaptation of microbes evolving from antibiotic sensitivity to resistance remains undefined. Therefore, untargeted metabolomics was conducted to unveil relevant metabolic reprogramming and potential intervention targets involved in gentamicin resistance. In total, 61 metabolites and 52 metabolic pathways were significantly altered in gentamicin-resistant E. coli. Notably, the metabolic reprogramming was characterized by decreases in most metabolites involved in carbohydrate and amino acid metabolism, and accumulation of building blocks for nucleotide synthesis in gentamicin-resistant E. coli. Meanwhile, fatty acid metabolism and glycerolipid metabolism were also significantly altered in gentamicin-resistant E. coli. Additionally, glycerol, glycerol-3-phosphate, palmitoleate, and oleate were separately defined as the potential biomarkers for identifying gentamicin resistance in E. coli. Moreover, palmitoleate and oleate could attenuate or even abolished killing effects of gentamicin on E. coli, and separately increased the minimum inhibitory concentration of gentamicin against E. coli by 2 and 4 times. Furthermore, palmitoleate and oleate separately decreased intracellular gentamicin contents, and abolished gentamicin-induced accumulation of reactive oxygen species, indicating involvement of gentamicin metabolism and redox homeostasis in palmitoleate/oleate-promoted gentamicin resistance in E. coli. This study identifies the metabolic reprogramming, potential biomarkers and intervention targets related to gentamicin resistance in bacteria

Primers used for constructing plasmids and strand-specific reverse transcription-polymerase chain reactions.

Primers used for constructing plasmids and strand-specific reverse transcription-polymerase chain reactions.</p

The TLS element is essential for viral RNAs to enhance satRNA replication in <i>trans</i>-replication assays.

(A) Schematic diagrams of L3, ncL3 and their derivatives. The 3′ UTR of CMV RNAs is divided into three regions: a variable region (VR) at the 5′ end, a conserved TLS at the 3′ end, and a highly conserved region (CR) separating them. Deleted sequences in the constructed mutants were indicated by dashed lines. The TLS in ncL3 was substituted with the TLS of BMV, PSV, TAV, TMV, or TYMV, to generate six chimeric ncL3 mutants. (B-D, F) Northern blotting analyses of the accumulation of sat-T1, L3 and its mutants in the 5th true leaves of Nicotiana benthamiana transiently expressing LS replication proteins (L1a+L2a) and the RNA silencing suppressor P19. The mutants of L3 or ncL3 tested in these experiments are shown above. (E) Northern blotting analyses of TLS, RNA4 (L4) and its noncoding version (ncL4) of L3, as well as both polarities of sat-T1 in the trans-replication assays. In this replication assay, the TLS, L4 and ncL4 were provided separately in trans via agroinfiltration. It is worth mentioning that the probe used to detect RNA3 and its subgenomic RNA4 in (C) & (D) is the digoxin-labeled oligonucleotide complementary to the sequence spanning from nt 1200 to 1333 of L3. The digoxin-labeled oligonucleotide probe used to detect these RNAs in (E) is complementary to the sequence positioning at nt 2128–2167 of L3. Mock plants were treated by infiltration solution alone. The relative accumulation levels of RNA3, and positive-sense or negative-sense RNA of sat-T1 in (B-D, F) are shown below. Ethidium bromide-stained ribosomal RNAs served as the loading control.</p

The replication proteins of Fny-CMV and LS-CMV exhibit significant differences in their ability to recruit positive-sense RNA of sat-T1.

(A) The hairpin structures containing the Box-B sequence in blue and the mutated Box-B (mBox-B) in red. mF3 denotes the F3 mutant, in which the Box-B was substituted with mBox-B. mF3-T1(+) and mF3-T1(-) are mF3 derivatives with positive-sense or negative-sense RNA of sat-T1 inserted between the CP and 3′ UTR in mF3, respectively. A fragment of the GUS gene (337 nt), equivalent in size of sat-T1, was introduced into F3 or mF3, resulting to the creation of F3-gus or mF3-gus, respectively. (B) The accumulation levels of F3 and mF3 in the trans-replication assay. Either F3 or mF3 was co-expressed with LS replication proteins and P19 in the 5th true leaves of N. benthamiana plants. Mock plants were treated with infiltration solution. At 3 days post-infiltration, total RNAs were extracted separately from three infiltrated leaves for each treatment and subjected to northern blotting analyses. The relative accumulation levels of F3 and mF3 are shown below as the mean values with standard errors from three independent biological samples. (C) Determination of the replication activities of mF3-T1(+), mF3-T1(-), and the controls F3-gus and mF3-gus. These four F3 derivatives was co-expressed with the P19 suppressor and the replication proteins of LS-CMV (upper panel) or Fny-CMV (the lower panel) in the 5th true leaves of Nicotiana benthamiana plants. Mock plants were treated with infiltration solution. At 3 days post-infiltration, total RNAs were extracted separately from three infiltrated leaves for each treatment, and analyzed by northern blot hybridization. The relative accumulation levels with standard errors shown below were calculated from three independent biological samples. “UD” denotes the undetectable level. Ethidium bromide-stained ribosomal RNAs were used as a loading control for normalization of the relative accumulation levels.</p

Subcellular distribution of F1a-mCherry when co-expressed with 6×MS2-satT1 in the leaf tissues of <i>Nicotiana benthamiana</i>.

The lower epidermis of the leaves was infiltrated with Agrobacterium cells harboring the binary plasmids to express F1a-mCherry, 6×MS2-satT1, and p19. At 2 days post-agroinfiltration, the infiltrated leaves were subjected to Laser confocal microscopy for visualizing red fluorescence omitted from F1a-mCherry. (TIF)</p

Substitution of the Box-B motif in RNA3 with sat-T1 inactivated the replication of the RNA3 variants by the replicase of LS-CMV.

(A) Schematic diagrams of RNA3 and its derivative (R3-ΔBox-T1), in which the Box-B sequence was substituted with the (+) strand of sat-T1. (B) Northern blotting analyses of the accumulation of RNA3 and its variants. RNA3 from Fny-CMV (F3) or LS-CMV (L3), as well as their mutants or vector pCB301 was separately co-expressed with the replicase of LS-CMV, together with the RNA silencing suppressor P19. At 3 days post-agroinfiltration, total RNAs were extracted from the infiltrated leaves and subjected to northern blot hybridization. Mock plants were treated by infiltration solution alone. Ethidium bromide-stained ribosomal RNAs were used to assess the loading amounts of all RNA samples. (TIF)</p