The principles by which cortical microtubules self-organize into a global template hold important implications for cell wall patterning. Microtubules move along bundles of microtubules, and neighboring bundles tend to form mobile domains that flow in a common direction. The bundles themselves move slowly and for longer than the individual microtubules, with domains describing slow rotary patterns. Despite this tendency for colinearity, microtubules have been seen to branch off extant microtubules at ;458. To examine this paradoxical behavior, we investigated whether some microtubules may be born on and grow along extant microtubule(s). The plus-end markers Arabidopsis thaliana end binding protein 1a, AtEB1a-GFP, and Arabidopsis SPIRAL1, SPR1-GFP, allowed microtubules of known polarity to be distinguished from underlying microtubules. This showed that the majority of microtubules do branch but in a direction heavily biased toward the plus end of the mother microtubule: few grow backward, consistent with the common polarity of domains. However, we also found that a significant proportion of emergent comets do follow the axes of extant microtubules, both at sites of apparent microtubule nucleation and at cross-over points. These phenomena help explain the persistence of bundles and counterbalance the tendency to branch.

The cell-to-cell movement of Tobacco mosaic virus through plasmodesmata (PD) requires virus-encoded movement protein (MP). The MP targets PD through the endoplasmic reticulum (ER)/actin network, whereas the intercellular movement of the viral RNA genome has been correlated with the association of the MP with mobile, microtubule-proximal particles in cells at the leading front of infection as well as the accumulation of the protein on the microtubule network during later infection stages. To understand how the associations of MP with ER and microtubules are functionally connected, we applied multiple marker three-dimensional confocal and time-lapse video microscopies to Nicotiana benthamiana cells expressing fluorescent MP, fluorescent RNA and fluorescent cellular markers. We report the reconstitution of MP-dependent RNA transport to PD in a transient assay. We show that transiently expressed MP occurs in association with small particles as observed during infection. The same MP accumulates in PD and mediates the transport of its messenger RNA transcript to the pore. In the cellular cortex, the particles occur at microtubuleproximal sites and can undergo ER-associated and latrunculin-sensitive movements between such sites. These and other observations suggest that the microtubule network performs anchorage and release functions for controlling the assembly and intracellular movement of MP-containing RNA transport particles in association with the ER.

Light and dark have antagonistic effects on shoot elongation, but little is known about how these effects are translated into changes of shape. Here we provide genetic evidence that the light/gibberellin-signaling pathway affects the properties of microtubules required to reorient growth. To follow microtubule dynamics for hours without triggering photomorphogenic inhibition of growth, we used Arabidopsis thaliana light mutants in the gibberellic acid/DELLA pathway. Particle velocimetry was used to map the mass movement of microtubule plus ends, providing new insight into the way that microtubules switch between orthogonal axes upon the onset of growth. Longitudinal microtubules are known to signal growth cessation, but we observed that cells also self-organize a strikingly bipolarized longitudinal array before bursts of growth. This gives way to a radial microtubule star that, far from being a random array, seems to be a key transitional step to the transverse array, forecasting the faster elongation that follows. Computational modeling provides mechanistic insight into these transitions. In the faster-growing mutants, the microtubules were found to have faster polymerization rates and to undergo faster reorientations. This suggests a mechanism in which the light-signaling pathway modifies the dynamics of microtubules and their ability to switch between orthogonal axes.

The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

No abstract

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA.

Summary• Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division.• Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Doublelabelling in interphase and mitosis enabled localization studies.• We found that the transient transformation efficiency was highest when BY-2 ⁄ Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle.• Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle.

The genetic variation of Citrus tristeza virus (CTV) was analysed by comparing the predominant sequence variants in seven genomic regions (p33, p65, p61, p18, p13, p20 and p23) of 18 pathogenically distinct isolates from seven different countries. Analyses of the selective constraints acting on each codon suggest that most regions were under purifying selection. Phylogenetic analysis shows diverse patterns of molecular evolution for different genomic regions. A first clade composed of isolates that are genetically close to the reference mild isolates T385 or T30 was inferred from all genomic regions. A second clade, mostly comprising virulent isolates, was defined from regions p33, p65, p13 and p23. For regions p65, p61, p18, p13 and p23, a third clade that mostly included South American isolates could not be related to any reference genotype. Phylogenetic relationships among isolates did not reflect their geographical origin, suggesting significant gene flow between geographically distant areas. Incongruent phylogenetic trees for different genomic regions suggested recombination events, an extreme that was supported by several recombination-detecting methods. A phylogenetic network incorporating the effect of recombination showed an explosive radiation pattern for the evolution of some isolates and also grouped isolates by virulence. Taken together, the above results suggest that negative selection, gene flow, sequence recombination and virulence may be important factors driving CTV evolution.