A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize

Plant RNA virus-based guide RNA (gRNA) delivery has substantial advantages compared to that of the conventional constitutive promoter-driven expression due to the rapid and robust amplification of gRNAs during virus replication and movement. To date, virus-induced genome editing tools have not been developed for wheat and maize. In this study, we engineered a barley stripe mosaic virus (BSMV)-based gRNA delivery system for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated targeted mutagenesis in wheat and maize. BSMV-based delivery of single gRNAs for targeted mutagenesis was first validated in Nicotiana benthamiana. To extend this work, we transformed wheat and maize with the Cas9 nuclease gene and selected the wheat TaGASR7 and maize ZmTMS5 genes as targets to assess the feasibility and efficiency of BSMV-mediated mutagenesis. Positive targeted mutagenesis of the TaGASR7 and ZmTMS5 genes was achieved for wheat and maize with efficiencies of up to 78% and 48%. Our results provide a useful tool for fast and efficient delivery of gRNAs into economically important crops.     

Cucumber mosaic virus coat protein-mediated protection

Transgenic tobacco plants (CP +) that express the coat protein gene of cucumber mosaic virus (CMV)-Y strain were highly protected from infection with either CMV virions or CMV RNA, while transgenic protoplasts were also protected from infection with CMV virions but not with CMV RNA. CP + plants showed greater susceptibility to infection with satellite RNA-free CMV-Y than CMV-Y containing satellite RNA. At temperatures above 30°C, CP + plants did not or poorly resist infection with CMV. Elevated temperature affected the accumulation of CP rather than its mRNA, suggesting that CP molecules are mainly involved in virus resistance in CP + plants.     

Age-related Resistance in Bell Pepper to Cucumber mosaic virus

We demonstrated the occurrence of mature plant resistance in Capsicum annuum‘Early Calwonder’ to Cucumber mosaic virus (CMV) under greenhouse conditions. When Early Calwonder plants were sown at 10 day intervals and transplanted to 10‐cm square pots, three distinct plant sizes were identified that were designated small, medium and large. Trials conducted during each season showed that CMV accumulated in inoculated leaves of all plants of each size category. All small plants (with the exception of the winter trial) developed a systemic infection that included accumulation of CMV in uninoculated leaves and severe systemic symptoms. Medium plants had a range of responses that included no systemic infection to detection of CMV in uninoculated leaves with the systemically infected plants being either symptomless or expressing only mild symptoms. None of the large plants contained detectable amounts of CMV in uninoculated leaves or developed symptoms. When plants were challenged by inoculation of leaves positioned at different locations along the stem or different numbers of leaves were inoculated, large plants continued to accumulate CMV in inoculated leaves but no systemic infection was observed. When systemic infection of large plants did occur, e.g. when CMV‐infected pepper was used as a source of inoculum, virus accumulation in uninoculated leaves was relatively low and plants remained symptomless. A time‐course study of CMV accumulation in inoculated leaves revealed no difference between small and large plants. Analyses to examine movement of CMV into the petiole of inoculated leaves and throughout the stem showed a range in the extent of infection. While all large plants contained CMV in inoculated leaves, some had no detectable amounts of virus beyond the leaf blade, whereas others contained virus throughout the length of the stem but with limited accumulation relative to controls.     

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.     

Interaction of Cucumber mosaic virus and Bean yellow mosaic virus in co-infected plants of bean and broad bean

After evaluation of the responses of bean and broad bean common cultivars against an isolate of Cucumber mosaic virus (CMV-K) and Bean yellow mosaic virus (BYMV-K), interaction of isolates was statistically studied on co-infected plants of bean cv. Bountiful and broad bean cv. Lahijan at two trials. Based on viral relative concentration determined by quantitative enzyme-linked immunosorbent assay, BYMV interacts synergistically with CMV in bean at 14 days post inoculation, while in co-infection with BYMV, CMV interacts antagonistically in both host plants at least in one of the two trials. This suggests that CMV/BYMV interaction is dependent on host species and developmental stage of plant. Co-infection like single infection with CMV in bean plants led to significantly decrease in plants’ height and fresh weight than BYMV singly infected and healthy plants, while viral infection of broad bean plants did not significantly affect growth parameters. Decline effect of viral infection (especially co-infection) on chlorophyll and carotenoids value of bean plants was greater than those of broad bean. Viral infection (singly or doubly) caused irregular changes in nutrient elements values of both hosts compared with healthy ones.     

Infection dynamics in viral spread and interference under the synergism between Cucumber mosaic virus and Turnip mosaic virus

Mixed infection of Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV) induced more severe symptoms on Nicotiana benthamiana than single infection. To dissect the relationships between spatial infection patterns and the 2b protein (2b) of CMV in single or mixed infections, the CMV vectors expressing enhanced green fluorescent or Discosoma sp. red fluorescent proteins (EGFP [EG] or DsRed2 [Ds], respectively were constructed from the same wild-type CMV-Y and used for inoculation onto N. benthamiana. CMV2-A1 vector (C2-A1 [A1]) has a functional 2b while CMV-H1 vector (C2-H1 [H1]) is 2b deficient. As we expected from the 2b function as an RNA silencing suppressor (RSS), in a single infection, A1Ds retained a high level of accumulation at initial infection sites and showed extensive fluorescence in upper, noninoculated leaves, whereas H1Ds disappeared rapidly at initial infection sites and could not spread efficiently in upper, noninoculated leaf tissues. In various mixed infections, we found two phenomena providing novel insights into the relationships among RSS, viral synergism, and interference. First, H1Ds could not spread efficiently from vasculature into nonvascular tissues with or without TuMV, suggesting that RNA silencing was not involved in CMV unloading from vasculature. These results indicated that 2b could promote CMV to unload from vasculature into nonvascular tissues, and that this 2b function might be independent of its RSS activity. Second, we detected spatial interference (local interference) between A1Ds and A1EG in mixed infection with TuMV, between A1Ds (or H1Ds) and TuMV, and between H1Ds and H1EG. This observation suggested that local interference between two viruses was established even in the synergism between CMV and TuMV and, again, RNA silencing did not seem to contribute greatly to this phenomenon.     

Cucumber mosaic virus infection affects sugar transport in melon plants

Viral infection often affects carbon assimilation and metabolism in host plants. To better understand the effect of cucumber mosaic virus (CMV) infection on sugar transport, carbohydrate levels and the amounts of the various sugars in the phloem sap were determined in infected melon (Cucumis melo L.) plants. Source leaves infected with CMV were characterized by high concentrations of reducing sugars and relatively low starch levels. The altered level of carbohydrates was accompanied by increased respiration and decreased net photosynthetic rates in the infected leaves. Although stachyose was the predominant sugar in phloem sap collected from petioles of control leaves, sucrose (Suc) was a major sugar in the phloem sap of infected leaves. Moreover, analyses of the newly fixed (14)CO(2) revealed a high proportion of radioactive Suc in the phloem sap of infected leaves 60 min post-labeling. The alteration in phloem sap sugar composition was found in source, but not old, leaves. Moreover, elevations in Suc concentration were also evident in source leaves that did not exhibit symptoms or contain detectable amounts of virus particles. The mode by which CMV infection may cause alterations in sugar transport is discussed in terms of the mechanism by which sugars are loaded into the phloem of cucurbit plants.     

Molecular Characterization and Distribution of Cucumber green mottle mosaic virus in China

Cucumber green mottle mosaic virus (CGMMV) is an emerging virus on watermelon in China. We report here the almost complete nucleotide sequence and the characterization of the genome of a Chinese isolate (CGMMV-LN). Nucleotide sequence comparisons showed CGMMV-LN is closely related to CGMMV-KOM, with 99.4% identity. On the basis of the nucleotide sequence, a digoxigenin-labelled cDNA probe CG, complementary to the replicase gene region of CGMMV was synthesized. The specificity and sensitivity of the probe was tested. The detection limit of the method was equivalent to 0.8 μg fresh tissues infected by CGMMV. Two hundreds and eighteen watermelon samples collected from different regions in China during 2006–2007 were tested by this method. The distribution pattern of CGMMV in China during these years was revealed. The virus has spread in five provinces of China so far, including Liaoning, Hebei, Guangdong, Hubei and Shandong and might be an increasing tendency, which provides important information for CGMMV control in China.     

Efficient high-throughput discovery of large peptidic hormones and biomarkers

A novel approach is presented for the simultaneous identification and relative quantification of secreted peptides, particularly those that have been historically difficult to analyze in a concerted manner. Peptides exceeding 60 residues with various degrees of post-translational modification were identified on a liquid chromatographic time scale. The approach demonstrates high efficiency pattern-based recognition analysis of complex neuroendocrine peptide sets and enables rapid identification of biomarkers from biological material.     

Cucumber mosaic virus: viral genes as virulence determinants

TAXONOMIC RELATIONSHIPS: Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus in the family Bromoviridae, which also encompasses the Peanut stunt virus (PSV) and the Tomato aspermy virus (TAV). Nucleotide sequence similarity among these three cucumoviruses is 60%-65%. CMV strains are divided into three subgroups, IA, IB and II, based on the sequence of the 5' untranslated region of the genomic RNA 3. Overall nucleotide sequence similarity among CMV strains is approximately 70%-98%. GEOGRAPHICAL DISTRIBUTION, HOST RANGE AND SYMPTOMATOLOGY: CMV is distributed worldwide, primarily in temperate to tropical climate zones. CMV infects more than 1200 species of 100 plant families, including monocot and dicot plants. Symptoms caused by CMV infection vary with the host species and/or CMV strain, and include mosaic, stunt, chlorosis, dwarfing, leaf malformation and systemic necrosis. CMV disease is spread primarily by aphid transmission in a nonpersistent manner. PHYSICAL PROPERTIES: In tobacco sap, the thermal inactivation point of the viral infectivity is approximately 70 °C (10 min), the dilution end-point is approximately 10(-4) and viral infectivity is lost after a few days of exposure to 20 °C. Viral infectivity can be retained in freeze-dried tissues and in the form of virions purified using 5 mm sodium borate, 0.5 mm ethylenediaminetetraacetic acid and 50% glycerol (pH 9.0) at -20 °C. CMV particles are isometric, approximately 28-30 nm in diameter and are composed of 180 capsid subunits arranged in pentamer-hexamer clusters with T= 3 symmetry. The sedimentation coefficient (s(20) ,(w) ) is c. 98 S and the particle weight is (5.8-6.7) × 10(6) Da. The virions contain 18% RNA. The RNA-protein interactions that stabilize the CMV virions are readily disrupted by sodium dodecylsulphate or neutral chloride salts. GENOMIC PROPERTIES: The genomic RNAs are single-stranded messenger sense RNAs with 5' cap and 3' tRNA-like structures containing at least five open reading frames. The viral RNA consists of three genomic RNAs, RNA 1 (c. 3.3 kb), RNA 2 (c. 3.0 kb) and RNA 3 (c. 2.2 kb), and two subgenomic RNAs, RNA 4 (c. 1.0 kb) and RNA 4A (c. 0.7 kb). The 3' untranslated regions are conserved across all viral RNAs. CMV is often accompanied by satellite, noncoding, small, linear RNA that is nonhomologous to the helper CMV.     

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.     

Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants

The expression of viral coat protein (CP) in transgenic plants has been shown to be very effective in virus plant protection. However, the introduction of CP genes into plants presents the potential risk of the encapsidation of a superinfecting viral genome in the transgenic protein, an event which could change the epidemiology of the disease. To detect the potential heterologous encapsidation of the cucumber mosaic virus (CMV) genome by alfalfa mosaic virus (AIMV) CP expressed in transgenic tobacco plants, a system of immunocapture (IC) and amplification by polymerase chain reaction (PCR) was optimized. This provided high sensitivity and reliable selection of the heterologously encapsidated CMV genome in the presence of natural CMV particles. As little as 2 pg of virus could be detected by immunocapture/polymerase chain reaction (IC/PCR) technique. Evidence for heterologous encapsidation of the CMV genome was found in 11 of the 33 transgenic plants tested two weeks after CMV inoculation. This demonstrates a significant rate of heterologous encapsidation events between two unrelated viruses in transgenic plants. Since CP is involved in the interactions of the virus particle with its vector, the release in the field of such transgenic plants could alter the transmission properties of some important viruses.     

Cucumber mosaic virus D satellite RNA-induced programmed cell death in tomato

D satellite RNA (satRNA) with its helper virus, namely, cucumber mosaic virus, causes systemic necrosis in tomato. The infected plant exhibits a distinct spatial and temporal cell death pattern. The distinct features of chromatin condensation and nuclear DNA fragmentation indicate that programmed cell death is involved. In addition, satRNA localization and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling show that cell death is initiated from the infected phloem or cambium cells and spreads to other nearby infected cells. Timing of the onset of necrosis after inoculation implicates the involvement of cell developmental processes in initiating tomato cell death. Analysis of the accumulation of minus- and plus-strand satRNAs in the infected plants indicates a correlation between high amounts of minus-strand satRNA and tomato cell death.     

First Report of Cucumber mosaic virus Infecting Chinese Mallow in China

The virus in naturally infected, stunted Chinese mallow plants and mosaic leaves was identified as Cucumber mosaic virus (CMV). Six symptomatic plants and one symptomless plant were collected in Chongqing, China. DAS‐ELISA suggested CMV was likely associated with the diseased Chinese mallow. Double‐stranded RNA was extracted from the samples, analysed by RT‐PCR, and the coding sequences of their coat proteins (CPs) were sequenced. The results further confirmed CMV was the pathogen causing Chinese mallow stunted, mosaic disease. The isolate was named CMV‐DXC. The full sequence of CMV‐DXC CP was determined, and it had the highest nucleotide identity (99.4%) of those of CMV‐lily, CMV‐WSJ and CMV‐Hnt, respectively. Phylogenetic analysis shows that CMV‐DXC belongs to CMV subgroup II. To our knowledge, this is the first report of CMV infecting Chinese mallow in China.     

黄瓜花叶病毒cDNA载体在烟草细胞中表达外源基因

为了探讨利用黄瓜花叶病毒(CMV)构建表达载体的可行性,分离了山东株(SD) CMV RNA 3的全长cDNA。测定其全序列后,采用定点突变的方法在衣壳蛋白(CP)基因起始密码子处改造出一个NsiⅠ位点,可将外源基因引入NsiⅠ位点和CP基因终止密码子上游附近的XhoⅠ或SalⅠ位点而置换掉CP基因。分别用绿色荧光蛋白(GFP)基因、β-葡糖醛酸酶(GUS)基因以及小鼠二氢叶酸还原酶(DHFR)基因3种报告基因置换CP基因。将Fny株CMV RNA 1、RNA 2和SDCMV嵌合RNA 3的cDNA分别插入pCass载体的35S启动子和终止子之间,将构建的置换型载体直接以质粒的方式转染烟草原生质体,表达了3种报告基因。     

RNA interference-mediated resistance to maize dwarf mosaic virus

Maize dwarf mosaic virus (MDMV) is a widespread pathogen that causes serious yield loss to maize crops. A hairpin RNA expression vector was constructed herein to overcome the low efficiency of cultural protection against MDMV and to improve the MDMV resistance mediated by a shorter transgenic inverted-repeat sequence. This expression vector contained a 451 bp inverted-repeat sequence, homologous to the protease gene (P1) of MDMV. It was used for the Agrobacterium tumefaciens-mediated transformation of maize calli induced from a susceptible inbred line. A total of 17 T₂ transgenic lines were identified by both specific PCR amplification and Southern blot hybridization. Of these lines, 15 were evaluated for MDMV resistance in inoculation field trials under two environments. The relative replication levels of the P1 gene were analyzed by quantitative real-time (qRT)-PCR. Results demonstrated that all of the 15 T₂ lines showed an enhanced resistance to MDMV in comparison with that of the non-transformed parent line. Six lines were deemed to be ‘resistant’ with an average disease index below 25 %, which was not significantly different from that of the resistant control. The relative replication levels of the virus gene were significantly reduced in these resistant T₂ transgenic lines. The efficiency of virus gene silencing was directly related to the transgene copy numbers presented in these transgenic lines.