Mikhail Kolot

Progressive aggregation of Tomato yellow leaf curl virus coat protein in systemically infected tomato plants, susceptible and resistant to the virus

Tomato yellow leaf curl virus (TYLCV) coat protein (CP) accumulated in tomato leaves during infection. The CP was immuno-detected in the phloem associated cells. At the early stages of infection, punctate signals were detected in the cytoplasm, while in the later stages aggregates of increasing size were localized in cytoplasm and nuclei. Sedimentation of protein extracts through sucrose gradients confirmed that progress of infection was accompanied by the formation of CP aggregates of increasing size. Genomic ssDNA was found in the cytoplasm and in the nucleus, while the dsDNA replicative form was exclusively associated with the nucleus. CP-DNA complexes were detected by immuno-capture PCR in nuclear and cytoplasmic large aggregates. Nuclear aggregates contained infectious particles transmissible to test plants by whiteflies. In contrast to susceptible tomatoes, the formation of large CP aggregates in resistant plants was delayed. By experimentally changing the level of resistance/susceptibility of plants, we showed that maintenance of midsized CP aggregates was associated with resistance, while large aggregates where characteristic of susceptibility. We propose that sequestering of virus CP into midsized aggregates and retarding the formation of large insoluble aggregates containing infectious particles is part of the response of resistant plants to TYLCV.

Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co‐expression of affinity‐tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in‐vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non‐ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9‐GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.

Phosphorylation of the integrase protein of coliphage HK022.

The integrase (Int) proteins of coliphages HK022 and lambda, are phosphorylated in one or more of their tyrosine residues. In Int of HK022 the phosphorylated residue(s) belong to its core-binding/catalytic domains. Wzc, a protein tyrosine kinase of Escherichia coli, is not required for Int phosphorylation in vivo, however, it can transphosphorylate the conserved Tyr(342) catalytic residue of Int in vitro. Int purified from cells that overexpress Wzc has a reduced activity in vitro. In vivo, the lysogenization of wild type HK022 as well as of lambda is not affected by the overexpression of Wzc. However, the nin5 mutant of lambda, which lacks a protein-tyrosine phosphatase gene, shows a significantly reduced lysogenization. It is suggested that phosphorylation of Int by Wzc down regulates the activity of Int.

Site-specific recombination in Arabidopsis plants promoted by the Integrase protein of coliphage HK022.

The gene encoding the wild type Integrase protein of coliphage HK022 was integrated chromosomally and expressed in Arabidopsis thaliana plants. Double-transgenic plants cloned with the int gene as well as with a T-DNA fragment carrying the proper att sites in a tandem orientation showed that Int catalyzed a site-specific integration reaction (attP × attB) as well as a site-specific excision reaction (attL × attR). The reactions took place without the need to provide any of the accessory proteins that are required by Int in the bacterial host. When expressed in tobacco plants a GFP-Int fusion exhibits a predominant nuclear localization.

Core-binding specificity of bacteriophage integrases.

Abstract The site-specific recombination systems of bacteriophages λ and HK022 share the same mechanism and their integrase proteins show strong homology. Nevertheless the integrase protein of each phage can only catalyze recombination between its own att sites. Previous work has shown that the specificity determinants in the att sites are located within the sequences that bind the integrase to the core of att. DNA fragments that carry attL and attR sites of each phage were challenged with each of the two integrases and the DNA-protein complexes were examined by the gel- retardation technique. The results show that each integrase can form higher-order DNA-protein complexes only with its cognate att sites, suggesting that differences in the mode of binding to the core are responsible for the specificity difference between the two integrases.

Position and direction of strand exchange in bacteriophage HK022 integration.

The positions of the endonucleolytic cleavages promoted by the integrase protein (Int) of coliphage HK022 within its attB site were determined. The protein catalyses a staggered cut, which defines an overlap sequence of 7 by within the core site. The overlap region is at the center of symmetry of a palindromic sequence which appears in all four putative att core binding sites for Int. We confirm that the order of strand exchange is similar to that in phage λ.

Determinants that target the integrase of phage HK022 into the mammalian nucleus.

The integrase (Int) protein of coliphage HK022 catalyzes the site-specific integration and excision of the phage into and from its Escherichia coli host chromosome. Int expressed from a plasmid in COS1 monkey cells is localized in the nucleus, as is a fusion protein between Int and the green fluorescent protein (GFP). Mutation analysis of the GFP-Int fusion has revealed in Int two regions of positively charged amino acid residues that cooperate in the nuclear localization. One region harbors residues Arg90 and Arg93. The other, which spans residues 307-340 belongs to the catalytic domain of Int, is rich in basic residues and is strongly conserved within the Int protein family. Being localized in the nucleus renders Int of HK022 as a potential recombinase for site-specific gene manipulations in mammals.

Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates.

Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted by the whitefly Bemisia tabaci to tomato and other crops. TYLCV proteins are endangered by the host defenses. We have analyzed the capacity of the tomato plant and of the whitefly insect vector to degrade the six proteins encoded by the TYLCV genome. Tomato and whitefly demonstrated the highest proteolytic activity in the fractions containing soluble proteins, less-in large protein aggregates; a significant decrease of TYLCV proteolysis was detected in the intermediate-sized aggregates. All the six TYLCV proteins were differently targeted by the cytoplasmic and nuclear degradation machineries (proteases, ubiquitin 26S proteasome, autophagy). TYLCV could confront host degradation by sheltering in small/midsized aggregates, where viral proteins are less exposed to proteolysis. Indeed, TYLCV proteins were localized in aggregates of various sizes in both host organisms. This is the first study comparing degradation machinery in plant and insect hosts targeting all TYLCV proteins.

Efficient Flp-Int HK022 dual RMCE in mammalian cells

Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with different target specificities (dual RMCE). Recently, using the Flp–Cre recombinase pair, dual RMCE proved to be efficient, provided the relative ratio of the enzymes during the reaction is optimal. In the present report, we analyzed how the efficiency of dual RMCE mediated by the Flp–Int (HK022) pair depends on the variable input of the recombinases—the amount of the recombinase expression vectors added at transfection—and on the order of the addition of these vectors: sequential or simultaneous. We found that both in the sequential and the simultaneous modes, the efficiency of dual RMCE was critically dependent on the absolute and the relative concentrations of the Flp and Int expression vectors. Under optimal conditions, the efficiency of ‘simultaneous’ dual RMCE reached ∼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE.

The effect of mutations in the Xis-binding sites on site-specific recombination in coliphage HK022.

Abstract. Excisionase (Xis) is an accessory protein that is required for the excision of the related prophages λ and HK022. Xis binds to two tandemly arranged binding sites (X1 and X2) on the P arm of the recombination sites attP and attR. Gel-retardation analyses and site-specific recombination assays were conducted on derivatives bearing site-directed mutations in the X1 and X2 sites of phage HK022. The results confirm the cooperative binding of Xis to its sites, showing that binding to X1 stimulates further binding to X2. The results also show that mutants affected in a single site are inactive in excision, whereas mutants affected in both sites, which show a complete absence of Xis binding, display significant excision activity. This restored activity is attributed to the interaction of Xis with Integrase, the protein that catalyzes the site-specific recombination reaction.