From Bacteriophage to Plant Genetics.

When first asked to write a review of my life as a scientist, I doubted anyone would be interested in reading it. In addition, I did not really want to compose my own memorial. However, after discussing the idea with other scientists who have written autobiographies, I realized that it might be fun to dig into my past and to reflect on what has been important for me, my life, my family, my friends and colleagues, and my career. My life and research has taken me from bacteriophage to Agrobacterium tumefaciens-mediated DNA transfer to plants to the plant genome and its environmentally induced changes. I went from being a naïve, young student to a postdoc and married mother of two to the leader of an ever-changing group of fantastic coworkers-a journey made rich by many interesting scientific milestones, fascinating exploration of all corners of the world, and marvelous friendships.

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.

In planta somatic homologous recombination assay revisited: a successful and versatile, but delicate tool.

Marker-transgene-dependent lines of Arabidopsis thaliana measuring somatic homologous recombination (SHR) have been available for almost two decades. Here we discuss mechanisms of marker-gene restoration, comment on results obtained using the reporter lines, and stress how caution must be applied to avoid experimental problems or false interpretation in the use of SHR reporter lines. Although theoretically possible, we conclude that explanations other than SHR are unlikely to account for restoration of marker gene expression in the SHR lines when used with appropriate controls. We provide an overview of some of the most important achievements obtained with the SHR lines, give our view of the limitations of the system, and supply the reader with suggestions on the proper handling of the SHR lines. We are convinced that SHR lines are and will remain in the near future a valuable tool to explore the mechanism and influence of external and internal factors on genome stability and DNA repair in plants.

Descendants of primed Arabidopsis plants exhibit resistance to biotic stress.

An attack of plants by pathogens or treatment with certain resistance-inducing compounds can lead to the establishment of a unique primed state of defense. Primed plants show enhanced defense reactions upon further challenge with biotic or abiotic stress. Here, we report that the primed state in Arabidopsis (Arabidopsis thaliana) is still functional in the next generation without additional treatment. We compared the reactions of Arabidopsis plants that had been either primed with ß-amino-butyric acid (BABA) or with an avirulent isolate of the bacteria Pseudomonas syringae pv tomato (PstavrRpt2). The descendants of primed plants showed a faster and higher accumulation of transcripts of defense-related genes in the salicylic acid signaling pathway and enhanced disease resistance upon challenge inoculation with a virulent isolate of P. syringae. In addition, the progeny of primed plants was also more resistant against the oomycete pathogen Hyaloperonospora arabidopsidis. When transgenerationally primed plants were subjected to an additional priming treatment, their descendants displayed an even stronger primed phenotype, suggesting that plants can inherit a sensitization for the priming phenomenon. Interestingly, this primed to be primed phenotype was much reduced in the Arabidopsis ß-amino-butyric acid priming mutant ibs1 (induced BABA sterility1). Our results demonstrate that the primed state of plants is transferred to their progeny and confers improved protection from pathogen attack as compared to the descendants of unprimed plants.

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.

Insertion DNA Promotes Ectopic Recombination during Meiosis in Arabidopsis.

Nucleotide insertion/deletions are common polymorphisms in living organisms; however, little is known about their genetic behavior during meiosis. Here, the recombination frequency (RF) of isogenic strains of transgenic Arabidopsis thaliana, that differ in the presence or absence of an insertion, was compared. We screened over 6 million seedlings and found that during meiosis the unpaired DNA insertions paired with ectopic homologues demonstrated a 13.8 times higher RF than that of noninsertion DNA. The direct measurement of recombination events provided the first evidence that a large piece of insertion DNA had a unique genetic behavior during meiosis. This pattern was consistently observed in different lines varying in overlapping sequence, construct orientation, chromosome location, and crossing direction. We suggest that higher ectopic recombination is promoted by DNA insertions and that this mechanism exists commonly in plants. Therefore, insertion DNA plays a nontrivial role in shaping genetic variation, chromosome instability, and genome evolution.

Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants.

Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) in planta approximately 40-fold. The frequency of transferred DNA (T-DNA) integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a G2-phase retardation that accelerates endoreduplication, and elevated levels of mRNAs coding for proteins involved in HR-all factors that could also contribute to upregulation of HR frequency in fas mutants.

Transgeneration memory of stress in plants.

Owing to their sessile nature, plants are constantly exposed to a multitude of environmental stresses to which they react with a battery of responses. The result is plant tolerance to conditions such as excessive or inadequate light, water, salt and temperature, and resistance to pathogens. Not only is plant physiology known to change under abiotic or biotic stress, but changes in the genome have also been identified. However, it was not determined whether plants from successive generations of the original, stressed plants inherited the capacity for genomic change. Here we show that in Arabidopsis thaliana plants treated with short-wavelength radiation (ultraviolet-C) or flagellin (an elicitor of plant defences), somatic homologous recombination of a transgenic reporter is increased in the treated population and these increased levels of homologous recombination persist in the subsequent, untreated generations. The epigenetic trait of enhanced homologous recombination could be transmitted through both the maternal and the paternal crossing partner, and proved to be dominant. The increase of the hyper-recombination state in generations subsequent to the treated generation was independent of the presence of the transgenic allele (the recombination substrate under consideration) in the treated plant. We conclude that environmental factors lead to increased genomic flexibility even in successive, untreated generations, and may increase the potential for adaptation.

Single-stranded DNA plant pathogens in Eilat.

An international conference on "Inter- and Intracellular Dynamics of ssDNA Plant Pathogens: Implications for Improving Resistance'' was sponsored by the United States-Israel Binational Agricultural Research and Development Fund (BARD) and organized in Eilat, Israel in November 2005. The topic of this meeting was single-stranded plant pathogens, their inter- as well as intra-cellular dynamics and their implications for improving resistance. Most of the talks concentrated on new and very new findings on principles of virus and bacterium-host interactions, studies that no doubt will lead eventually to the establishment of plants resistant to viral and bacterial infections.

CENTRIN2 interacts with the Arabidopsis homolog of the human XPC protein (AtRAD4) and contributes to efficient synthesis-dependent repair of bulky DNA lesions.

Arabidopsis thaliana CENTRIN2 (AtCEN2) has been shown to modulate Nucleotide Excision Repair (NER) and Homologous Recombination (HR). The present study provides evidence that AtCEN2 interacts with the Arabidopsis homolog of human XPC, AtRAD4 and that the distal EF-hand Ca(2+) binding domain is essential for this interaction. In addition, the synthesis-dependent repair efficiency of bulky DNA lesions was enhanced in cell extracts prepared from Arabidopsis plants overexpressing the full length AtCEN2 but not in those overexpressing a truncated AtCEN2 form, suggesting a role for the distal EF-hand Ca(2+) binding domain in the early step of the NER process. Upon UV-C treatment the AtCEN2 protein was shown to be increased in concentration and to be localised in the nucleus rapidly. Taken together these data suggest that AtCEN2 is a part of the AtRAD4 recognition complex and that this interaction is required for efficient NER. In addition, NER and HR appear to be differentially modulated upon exposure of plants to DNA damaging agents. This suggests in plants, that processing of bulky DNA lesions highly depends on the excision repair efficiency, especially the recognition step, thus influencing the recombinational repair pathway.

Isolation and characterization of the RAD54 gene from Arabidopsis thaliana.

Homologous recombination (HR) is an essential process in maintaining genome integrity and variability. In eukaryotes, the Rad52 epistasis group proteins are involved in meiotic recombination and/or HR repair. One member of this group, Rad54, belongs to the SWI2/SNF2 family of DNA-stimulated ATPases. Recent studies indicate that Rad54 has important functions in HR, both as a chromatin remodelling factor and as a mediator of the Rad51 nucleoprotein filament. Despite the importance of Rad54 in HR, no study of Rad54 from plants has yet been performed. Here, we cloned the full-length AtRAD54 cDNA sequence; an open reading frame of 910 amino acids encodes a protein with a predicted molecular mass of 101.9 kDa. Western blotting analysis showed that the AtRad54 protein was indeed expressed as a protein of approximately 110 kDa in Arabidopsis. The predicted protein sequence of AtRAD54 contains seven helicase domains, which are conserved in all other Rad54s. Yeast two-hybrid analysis revealed an interaction between Arabidopsis Rad51 and Rad54. AtRAD54 transcripts were found in all tissues examined, with the highest levels of expression in flower buds. Expression of AtRAD54 was induced by gamma-irradiation. A T-DNA insertion mutant of AtRAD54 devoid of full-length AtRAD54 expression was viable and fertile; however, it showed increased sensitivity to gamma-irradiation and the cross-linking reagent cisplatin. In addition, the efficiency of somatic HR in the mutant plants was reduced relative to that in wild-type plants. Our findings point to an important role for Rad54 in HR repair in higher plants.

Tn5 transposase-mediated mouse transgenesis.

We have developed a novel method for mouse transgenesis. The procedure relies on a hyperactive Tn5 transposase to insert a transgene into mouse chromosomes during intracytoplasmic sperm injection. This procedure integrates foreign DNA into the mouse genome with dramatically increased effectiveness as compared to conventional methods such as pronuclear microinjection and traditional sperm injection-mediated transgenesis. Our data indicate that with this method, transgenic mice, both hybrids and inbreds, can be produced more consistently and with lower numbers of manipulated oocytes required for traditional microinjection methods. The transposase-mediated transgenesis technique is also effective with round spermatids, offering the potential for rescuing the fertility of azoospermic animals using sperm precursor cells.

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.

The dual nature of homologous recombination in plants.

Homologous recombination creates covalent linkages between DNA in regions of highly similar or identical sequence. Recent results from several laboratories, many of them based on forward and reverse genetics in Arabidopsis, give insights into the mechanisms of the enzymatic machinery and the involvement of chromatin in somatic and meiotic DNA recombination. Also, signaling pathways and interconnections between repair pathways are being discovered. In addition, recent work shows that biotic and abiotic influences from the environment can dramatically affect plant genomes. The resulting changes in the DNA sequence, exerted at the level of somatic or meiotic tissue, might contribute to evolution.