The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants.

Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases.

Top 10 plant viruses in molecular plant pathology.

Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a 'Top 10' based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Brome mosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus. This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.

Antibegomoviral activity of the agrobacterial virulence protein VirE2.

Mungbean yellow mosaic geminivirus (MYMV) causes severe yellow mosaic disease in blackgram, mungbean, Frenchbean, pigeonpea, soybean and mothbean. We attempted to induce resistance against this virus using the transcriptional activator protein gene deleted in the C-terminal activation domain (TrAP-∆AD) and Agrobacterium tumefaciens virE2. MYMV is known to replicate in agroinoculated tobacco leaf discs. Three transgenic tobacco plants which harboured a truncated MYMV transcriptional activator protein gene and two tobacco plants transformed with the octopine type A. tumefaciens virE2 gene were agroinoculated with an A. tumefaciens strain which harboured the partial dimers of both DNA A and DNA B of MYMV. The level of viral DNA accumulation in leaf discs of transgenic plants correlated inversely to the level of the MYMV TrAP-∆AD transcript. Two VirE2-transgenic plants, which complemented tumorigenesis of a virE2 mutant A. tumefaciens strain, effectively reduced MYMV DNA accumulation in the leaf disc agroinoculation assay.

Replication stress leads to genome instabilities in Arabidopsis DNA polymerase delta mutants.

Impeded DNA replication or a deficiency of its control may critically threaten the genetic information of cells, possibly resulting in genome alterations, such as gross chromosomal translocations, microsatellite instabilities, or increased rates of homologous recombination (HR). We examined an Arabidopsis thaliana line derived from a forward genetic screen, which exhibits an elevated frequency of somatic HR. These HR events originate from replication stress in endoreduplicating cells caused by reduced expression of the gene coding for the catalytic subunit of the DNA polymerase delta (POLdelta1). The analysis of recombination types induced by diverse alleles of poldelta1 and by replication inhibitors allows the conclusion that two not mutually exclusive mechanisms lead to the generation of recombinogenic breaks at replication forks. In plants with weak poldelta1 alleles, we observe genome instabilities predominantly at sites with inverted repeats, suggesting the formation and processing of aberrant secondary DNA structures as a result of the accumulation of unreplicated DNA. Stalled and collapsed replication forks account for the more drastic enhancement of HR in plants with strong poldelta1 mutant alleles. Our data suggest that efficient progression of DNA replication, foremost on the lagging strand, relies on the physiological level of the polymerase delta complex and that even a minor disturbance of the replication process critically threatens genomic integrity of Arabidopsis cells.

Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect.

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.

Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection.

The low efficiency of current microinjection-based animal transgenesis techniques is largely the result of poor embryo survival. We have developed a new, bacterial recombinase-based transgenesis method. Intracytoplasmic sperm injection (ICSI) of single stranded DNA (ssDNA) complexed with E. coli recombinase RecA into mouse metaphaseII (MII) arrested oocytes resulted in RecA-dependent transgenesis. This approach offers significant advantages over pronuclear microinjection and previous ICSI-based transgenesis approaches in terms of improved embryo survival, which translates into greater transgenesis efficiency. It also opens the possibility to attempt experiments, which may affect gene targeting by homologous recombination into DNA of mammalian single celled pre-implantation embryos.

Dynamic response of plant genome to ultraviolet radiation and other genotoxic stresses.

Oligonucleotide microarray technology was used to identify genes, which are responding after exposure to UV-C radiation and to other agents causing genotoxic stress. The effect of these conditions on recombinational DNA repair was monitored in parallel. Global changes in gene expression were investigated in Arabidopsis wild-type plants challenged with UV-C, bleomycin, another abiotic agent and xylanase, a biotic factor, all leading to elevated homologous recombination frequencies. The comparison of the expression profile of each treatment allowed defining genes specifically involved in the dynamic response to UV. In the future, the potential roles of such genes in the different forms of stress recognition, signal transduction, and their roles in DNA repair processes will be assessed by using reverse genetic tools available for Arabidopsis thaliana.

Transcriptome profiling reveals similarities and differences in plant responses to cadmium and lead.

We analyzed the influence of salts of two heavy metals--lead and cadmium (Pb2+ and Cd2+) on plants, including plant and root size, plant genome stability as well as global genome expression. Measurement of the metal uptake showed that there was a significantly higher incorporation of Cd than of Pb, 0.6 and 0.15 uM per gram of dry weight, respectively. The analysis of the root length and plant size showed a dose dependent decrease in plants exposed to cadmium. In contrast there was little difference in the size of plants exposed to lead, although there was nearly four-fold increase of the root length. Analysis of the genome stability revealed that cadmium led to a dose dependent increase of homologous recombination whereas lead had no effect. Analysis of the global genome expression of plants chronically exposed to 50 uM of Cd and Pb revealed 65 and 338 up- and down-regulated genes by Cd and 19 and 76 by Pb, respectively. Interestingly, half of the genes that changed their expression in Pb-treated plants also changed their expression in Cd-treated ones. The greater number of genes regulated by Cd reflects generally higher genome instability of plants as well as higher uptake as compared to Pb.

Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair.

The budding yeast INO80 complex is a conserved ATP-dependent nucleosome remodeler containing actin-related proteins Arp5 and Arp8. Strains lacking INO80, ARP5, or ARP8 have defects in transcription. Here we show that these mutants are hypersensitive to DNA damaging agents and to double-strand breaks (DSBs) induced by the HO endonuclease. The checkpoint response and most transcriptional modulation associated with induction of DNA damage are unaffected by these mutations. Using chromatin immunoprecipitation we show that Ino80, Arp5, and Arp8 are recruited to an HO-induced DSB, where a phosphorylated form of H2A accumulates. Recruitment of Ino80 is compromised in cells lacking the H2A phosphoacceptor S129. Finally, we demonstrate that conversion of the DSB into ssDNA is compromised in arp8 and H2A mutants, which are both deficient for INO80 activity at the site of damage. These results implicate INO80-mediated chromatin remodeling directly at DSBs, where it appears to facilitate processing of the lesion.

Pathogen-induced systemic plant signal triggers DNA rearrangements.

Plant genome stability is known to be affected by various abiotic environmental conditions, but little is known about the effect of pathogens. For example, exposure of maize plants to barley stripe mosaic virus seems to activate transposable elements and to cause mutations in the non-infected progeny of infected plants. The induction by barley stripe mosaic virus of an inherited effect may mean that the virus has a non-cell-autonomous influence on genome stability. Infection with Peronospora parasitica results in an increase in the frequency of somatic recombination in Arabidopsis thaliana; however, it is unclear whether effects on recombination require the presence of the pathogen or represent a systemic plant response. It is also not clear whether the changes in the frequency of somatic recombination can be inherited. Here we report a threefold increase in homologous recombination frequency in both infected and non-infected tissue of tobacco plants infected with either tobacco mosaic virus or oilseed rape mosaic virus. These results indicate the existence of a systemic recombination signal that also results in an increased frequency of meiotic and/or inherited late somatic recombination.

Pathogen stress increases somatic recombination frequency in Arabidopsis.

Evolution is based on genetic variability and subsequent phenotypic selection. Mechanisms that modulate the rate of mutation according to environmental cues, and thus control the balance between genetic stability and flexibility, might provide a distinct evolutionary advantage. Stress-induced mutations stimulated by unfavorable environments, and possible mechanisms for their induction, have been described for several organisms, but research in this area has mainly focused on microorganisms. We have analyzed the influence of adverse environmental conditions on the genetic stability of the higher plant Arabidopsis thaliana. Here we show that a biotic stress factor-attack by the oomycete pathogen Peronospora parasitica-can stimulate somatic recombination in Arabidopsis. The same effect was observed when plant pathogen-defense mechanisms were activated by the chemicals 2,6-dichloroisonicotinic acid (INA) or benzothiadiazole (BTH), or by a mutation (cim3). Together with previous studies of recombination induced by abiotic factors, these findings suggest that increased somatic recombination is a general stress response in plants. The increased genetic flexibility might facilitate evolutionary adaptation of plant populations to stressful environments.