Cauliflower mosaic virus as a gene expression vector for plants

It is possible to replace the CaMV (cauliflower mosaic virus) ORF (open reading frame) II with foreign sequences without interfering with virus viability. Such recom-binants can induce the synthesis of substantial amounts of a foreign protein in infected plants and confer new properties to these plants. However, so far only three genes have been successfully cloned and expressed in this way. The expression mechanism of CaMV demands precise replacement of ORF II and probably certain structural features of the viral 35S RNA, which should not be disturbed by inserted sequences. Since these features are largely unknown, it cannot at present be pre-dicted whether an insert will be tolerated. It is more likely that larger inserts will disturb the viral gene expression mechanism than smaller ones.     

Characterization of Turnip mosaic virus from the Asian‐BR Population in Iran

Brassicaceae crops in eight provinces of the North‐west Iran were surveyed for Turnip mosaic virus (TuMV) infection during 2011 and 2012. Many symptomatic plants (38%; 226 of 598) were found to be infected with TuMV. The highest frequency was in turnip (61%), followed by radish (55%), oilseed rape (38%), and brassica weeds including annual bastard cabbage (42%), small tumbleweed‐mustard (50%) and wild radish (45%), but not Brassica oleracea and Lepidium sativum. Using biological assays, Iranian TuMV isolates grouped in three [B], [B(R)] and [BR] host‐infecting types. Phylogenetic analysis using complete coat protein (CP) gene nucleotide sequences showed that the Iranian isolates belonged to the Basal‐B and Asian‐BR populations. No evidence of recombination was found in these isolates using different recombination‐detecting programmes. To our knowledge, our study shows for the first time the occurrence of TuMV Asian‐BR subpopulation in the mid Eurasian region of Iran. The data suggest that the Asian‐BR subtype population is found across southern Eurasia and might be a continuous population in East Asia (mostly Japan and China) and Minor Asia (Turkey), the places considered to be one of the origins of TuMV populations.     

Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells

Summary An experimental system to study cell cycle specific gene expression in plant cells was developed using protoplasts from tobacco cells synchronized by aphidicolin treatment. Chimeric plasmids consisting either of the chloramphenicol acetyltransferase (CAT) gene downstream of the cauliflower mosaic virus (CaMV) 35 S promoter or the nopaline synthase (nos) promoter were introduced into synchronized protoplasts of four cell cycle stages by electroporation. In the case of the CaMV 35 S promoter cyclic oscillation of CAT activity was observed which paralleled the cell cycle of the recipient cells. The peak of CAT activity was found in the S phase, while no such cyclic change was observed in the case of the nos promoter. This system clearly shows that it is feasible to search for a cell cycle specific promoter. The significance of these observations is discussed in relation to the study of plant cells.     

A role for plant microtubules in the formation of transmission-specific inclusion bodies of Cauliflower mosaic virus

Interactions between microtubules and viruses play important roles in viral infection. The best-characterized examples involve transport of animal viruses by microtubules to the nucleus or other intracellular destinations. In plant viruses, most work to date has focused on interaction between viral movement proteins and the cytoskeleton, which is thought to be involved in viral cell-to-cell spread. We show here, in Cauliflower mosaic virus (CaMV)-infected plant cells, that viral electron-lucent inclusion bodies (ELIBs), whose only known function is vector transmission, require intact microtubules for their efficient formation. The kinetics of the formation of CaMV-related inclusion bodies in transfected protoplasts showed that ELIBs represent newly emerging structures, appearing at late stages of the intracellular viral life cycle. Viral proteins P2 and P3 are first produced in multiple electron-dense inclusion bodies, and are later specifically exported to transiently co-localize with microtubules, before concentrating in a single, massive ELIB in each infected cell. Treatments with cytoskeleton-affecting drugs suggested that P2 and P3 might be actively transported on microtubules, by as yet unknown motors. In addition to providing information on the intracellular life cycle of CaMV, our results show that specific interactions between host cell and virus may be dedicated to a later role in vector transmission. More generally, they indicate a new unexpected function for plant cell microtubules in the virus life cycle, demonstrating that microtubules act not only on immediate intracellular or intra-host phenomena, but also on processes ultimately controlling inter-host transmission.     

Serological detection of Cucumber mosaic virus on some important host crops in the north region of Iran

Plant virus diseases cause major losses in agricultural and horticultural products, especially in tropical and subtropical regions. The first step to manage these diseases is detecting, identifying and determining the pathogen characteristics. Cucumber mosaic disease is one of the most prevalent and damaging plant diseases in the world which is caused by Cucumber mosaic virus (CMV). Each year, this virus causes yield decreasing and substantial economic damages in its host plants worldwide including the north of Iran. In order to study and identify CMV, 935 leaf samples were collected based on typical symptoms from 10 crops (tomato, pea, tobacco, soybean, watermelon, broad bean, squash, cucumber, eggplant and lettuce) in different regions of Golestan and Mazandaran provinces (north of Iran) during 2009–2010. Suspicious samples were analysed by DAS-ELISA and polyclonal antibodies. The results showed that 275 samples (29.4%) were infected by CMV. Between these hosts, the highest and the lowest CMV infection was associated to watermelon (62.44%) and lettuce (0%), respectively. Among sampling locations, Behshahr (100%) and Minoodasht (3.47%) showed the maximum and minimum infection, respectively.     

Cauliflower mosaic virus gene VI causes growth suppression, development of necrotic spots and expression of defence-related genes in transgenic tobacco plants

Summary In order to study possible functions of the inclusion body matrix protein (IBMP) encoded by gene VI of cauliflower mosaic virus (CaMV), the XbaI fragment containing the gene VI of a Japanese strain of CaMV (CaMV S-Japan) was transferred to tobacco plants by Ti mediated transformation. Eight out of 18 kanamycin resistant plants (40%) expressed detectable levels of IBMP. Those transgenic plants expressing IBMP produced leaves with light green color, and their growth was suppressed as compared with control plants. Symptom-like necrotic spots also appeared on the leaves and stems of the mature transgenic plants. Furthermore, in these transgenic plants, pathogenesis-related proteins 1a, 1b and 1c were highly expressed and the activity of 1,3--glucanase was increased up to eightfold. From these results, we concluded that expression of the IBMP is associated with symptom development.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

Cauliflower mosaic virus promoters direct efficient expression of a bacterial G418 resistance gene in Schizosaccharomyces pombe

We have isolated and sequenced a cDNA clone which contains the entire coding sequence of the precursor to a subunit of wheat phosphoribulokinase (PRKase). (The enzyme is a homodimer). The cDNA contains 1533 bp and has an open reading frame of 1212 nucleotides. This encodes a protein with an amino-terminal transit sequence of 53 amino acids, while the part that forms the mature protein contains 351 amino acids and has a molecular weight of 39,200 daltons. A comparison of the wheat amino acid sequence with that already known for the mature protein of spinach reveals that there are identical residues in 86% of the positions but their transit peptides differ substantially from one another. The mature wheat and spinach proteins are identical in a segment of over 50 amino acids near the amino-terminus which is the region believed to be involved in ATP binding and in regulation by light of the catalytic activity of the enzyme. We further demonstrate that the expression of PRKase mRNA in wheat leaves is regulated in a developmental, tissue-specific and light dependent manner. We also show that the light-induced increase in the steady-state levels of this mRNA is dependent on the developmental stage of the leaf.     

Recombination with coat protein transgene in a complementation system based on Cucumber mosaic virus (CMV)

In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an Nsi I site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants expressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination be     

Variation between Turnip mosaic virus Isolates in Zhejiang Province,China and Evidence for Recombination

The 3′‐terminal sequences (c. 1700 nt) of the RNA genome of 10 Turnip mosaic virus (TuMV) isolates from different hosts in Zhejiang province, China, were determined. Phylogenetic analysis of the coat protein nucleotide sequences revealed that most TuMV sequences fell into two distinct clusters. The Chinese isolates B1‐B4 (from Brassica spp.) were similar and placed in the largest group (Group 1), while the isolates R1‐R6 (from Raphanus) were usually placed in a distinct but smaller group (Group 2). There were only approximately 90% identical nucleotides between the two groups. However, one isolate (R5) showed evidence of recombination in that the region between nucleotides 430 and 450, from the start of the coat protein gene and its 3′‐terminus, was a Group 1 type.     

Molecular Characterization of the 3'-Terminal Region of Turnip mosaic virus Isolates from Eastern China

Turnip mosaic virus (TuMV; genus Potyvirus, family Potyviridae) causes great losses to cruciferous crop production worldwide. The 3′‐terminal genomic sequences of eight TuMV isolates from eastern China were compared with those of 74 other Chinese TuMV isolates of known host origin in the GenBank and isolated during the past 25 years. The reported sequences of the eight TuMV isolates are 1125 or 1126‐nucleotides (nt) long excluding the poly(A) tail. They all contain one partial open reading frame of 912 nt, encoding 304 amino acids, followed by a stop codon and a non‐translated region of 209–210 nt. Results of phylogenetic analyses showed that Chinese TuMV isolates clustered into three groups: basal‐BR, Asian‐BR and world‐B. The ratios of non‐synonymous and synonymous substitutions and results of amino acid alignment provided evidence for purifying or negative selection in TuMV populations of China.     

Effect of deletions in the cauliflower mosaic virus polyadenylation sequence on the choice of the polyadenylation sites in tobacco protoplasts

Summary Deletions were made in the cauliflower mosaic virus polyadenylation sequence which was cloned downstream of the -glucuronidase gene (gus). The populations of mRNAs generated in tobacco mesophyll protoplasts by transient expression with the various constructs were analysed using a polymerase chain reaction procedure. When no deletion was present in the sequence, the mRNA appeared to be polyadenylated at two major polyadenylation sites. A deletion upstream from the AATAAA sequence made the population of polyadenylated mRNAs very heterogenous at their 3 ends. A deletion downstream of the AATAAA sequence had no effect on the choice of the site. Alternative polyadenylation sites were used when the native polyadenylation site was deleted. These results are discussed in relation to data obtained with other polyadenylation sequences from both plants and animals.     

A putative replicative form of the abutilon mosaic virus (gemini group) in a chromatin-like structure

Summary The putative replicative form of the abutilon mosaic virus (AbMV), a geminivirus, was purified from infected plants. It was shown to consist of a bipartite genome of 2660 and 2640 bp. This double-stranded DNA has a closed or relaxed circular conformation and part of it is packed in nucleoprotein complexes with a chromatin-like structure. Similarities between the geminiviruses and the animal simian virus 40 are discussed against this back-ground.Cloning was performed under L2/B1 conditions according to the licence of the ZKBS 1526/1This article is based on a doctoral study by A.A. and T.F. in the Faculty of Biology, University of Hamburg     

Identification of Arabidopsis proteins that interact with the cauliflower mosaic virus (CaMV) movement protein

Gene I of cauliflower mosaic virus (CaMV) encodes a protein that is required for virus movement. The CaMV movement protein (MP) was used in a yeast 2-hybrid system to screen an Arabidopsis cDNA library for cDNAs encoding MP-interacting (MPI) proteins. Three different clones were found encoding proteins (MPI1, -2 and -7) that interact with the N-terminal third of the CaMV MP. The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants. A non-infectious MP mutant, ER2A, with two amino acid changes in the N-terminal third of the MP failed to interact with MPI7, while an infectious second-site mutant, that differed from ER2A by only a single amino acid change, interacted in the 2-hybrid system. MPI7 is encoded by a member of a large, but diverse gene family in Arabidopsis. MPI7 is related in sequence, size and hydropathy profile to mammalian proteins (such as rat PRA1) described as a rab acceptor. The gene encoding MPI7 is expressed widely is Arabidopsis plants, and in transgenic plants the MPI7:GFP fusion protein is localized in the cytoplasm, concentrated in punctate spots. In protoplasts transfected with CFP:MP and MPI7:YFP, CFP:MP colocalized to some of the sites where MPI7:YFP is expressed. At these sites, fluorescence resonance energy transfer (FRET) between fluorophores was observed indicating an interaction in planta between the CaMV MP and MPI7.