Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage.

Condensin, an SMC (structural maintenance of chromosomes) protein complex, extrudes DNA loops using an ATP-dependent mechanism that remains to be elucidated. Here, we show how condensin activity alters the topology of the interacting DNA. High condensin concentrations restrain positive DNA supercoils. However, in experimental conditions of DNA loop extrusion, condensin restrains negative supercoils. Namely, following ATP-mediated loading onto DNA, each condensin complex constrains a DNA linking number difference (∆Lk) of -0.4. This ∆Lk increases to -0.8 during ATP binding and resets to -0.4 upon ATP hydrolysis. These changes in DNA topology do not involve DNA unwinding, do not spread outside the condensin-DNA complex and can occur in the absence of the condensin subunit Ycg1. These findings indicate that during ATP binding, a short DNA domain delimited by condensin is pinched into a negatively supercoiled loop. We propose that this loop is the feeding segment of DNA that is subsequently merged to enlarge an extruding loop. Such a "pinch and merge" mechanism implies that two DNA-binding sites produce the feeding loop, while a third site, plausibly involving Ycg1, might anchor the extruding loop.

Keeping intracellular DNA untangled: A new role for condensin?

The DNA-passage activity of topoisomerase II accidentally produces DNA knots and interlinks within and between chromatin fibers. Fortunately, these unwanted DNA entanglements are actively removed by some mechanism. Here we present an outline on DNA knot formation and discuss recent studies that have investigated how intracellular DNA knots are removed. First, although topoisomerase II is able to minimize DNA entanglements in vitro to below equilibrium values, it is unclear whether such capacity performs equally in vivo in chromatinized DNA. Second, DNA supercoiling could bias topoisomerase II to untangle the DNA. However, experimental evidence indicates that transcriptional supercoiling of intracellular DNA boosts knot formation. Last, cohesin and condensin could tighten DNA entanglements via DNA loop extrusion (LE) and force their dissolution by topoisomerase II. Recent observations indicate that condensin activity promotes the removal of DNA knots during interphase and mitosis. This activity might facilitate the spatial organization and dynamics of chromatin.

Condensin minimizes topoisomerase II-mediated entanglements of DNA in vivo.

The juxtaposition of intracellular DNA segments, together with the DNA-passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.

Transient expression of homologous hairpin RNA interferes with PVY transmission by aphids.

Hairpin RNAs have been used to confer resistance to viruses in plants through RNA silencing. However, it has not been demonstrated that RNA silencing was effective against inoculation by aphids of non-persistently transmitted viruses, the major route of plant virus spread in nature. As a proof-of-principle strategy, we made use of Agrobacterium tumefaciens to transiently express a hairpin RNA homologous to Potato virus Y (PVY) in plant tissues. A complete and specific interference with aphid transmission of PVY was achieved by inducers of RNA silencing, as demonstrated by specific siRNAs accumulation in agroinfiltrated tissues. To our knowledge, this is the first report of successful interference with non-persistent transmission of a plant virus using RNA interference.

Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs.

In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco rattle virus (TRV)-derived siRNAs. TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3, or DCL2 targets, alone or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNAs reveals differential contributions throughout the TRV genome to siRNA formation. Indirect evidence suggests that DCL2 is responsible for production of a subset of siRNAs derived from the 3' end region of TRV. TRV siRNA biogenesis and antiviral silencing are strongly dependent on the combined activity of the host-encoded RNA-dependent RNA polymerases RDR1, RDR2, and RDR6, thus providing evidence that perfectly complementary double-stranded RNA serves as a substrate for siRNA production. We conclude that the overall composition of viral siRNAs in TRV-infected plants reflects the combined action of several interconnected pathways involving different DCL and RDR activities.

Production of plum pox virus HC-Pro functionally active for aphid transmission in a transient-expression system.

Potyviruses are non-persistently transmitted by aphid vectors with the assistance of a viral accessory factor known as helper component (HC-Pro), a multifunctional protein that is also involved in many other essential processes during the virus infection cycle. A transient Agrobacterium-mediated expression system was used to produce Plum pox virus (PPV) HC-Pro in Nicotiana benthamiana leaves from constructs that incorporated the 5' region of the genome, yielding high levels of HC-Pro in agroinfiltrated leaves. The expressed PPV HC-Pro was able to assist aphid transmission of purified virus particles in a sequential feeding assay, and to complement transmission-defective variants of the virus. Also, HC-Pro of a second potyvirus, Tobacco etch virus (TEV), was expressed and found to be functional for aphid transmission. These results show that this transient system can be useful for production of functionally active HC-Pro in potyviruses, and the possible uses of this approach to study the mechanism of transmission are discussed.

A Single Amino Acid Mutation in the Plum pox virus Helper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities.

ABSTRACT The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation ( HCL(134)H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL(134)H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro.

Expression of functionally active helper component protein of Tobacco etch potyvirus in the yeast Pichia pastoris.

Tobacco etch potyvirus (TEV) is transmitted by aphids in a non-persistent manner with the assistance of a virus-encoded protein known as helper component (HC-Pro). To produce a biologically active form of recombinant TEV HC-Pro protein, heterologous expression in the methylotrophic yeast Pichia pastoris was used. A cDNA encoding the TEV HC-Pro region, fused to a Saccharomyces cerevisiae alpha-mating factor secretory peptide coding region, was inserted into the P. pastoris genome using a modified version of the pPIC9 vector. The expressed TEV HC-Pro protein was obtained directly from culture medium of recombinant yeast colonies; it was able to interact with TEV particles in a protein overlay binding assay, and also to assist aphid transmission of purified TEV particles to plants using the aphid Myzus persicae as vector. Our results indicate that P. pastoris provides a rapid and low-cost heterologous expression system that can be used to obtain biologically active potyvirus HC-Pro protein for in vitro transmission assays.