Choreography Sketches as a Representational System of Dance Recording : From M . Petipa to M . Fokine

In the long history of ballet, one can particularly emphasise two epochs 19th and early 20th centuries, when the greatest number of works appeared, demonstrating the desire to solve the problems of staged choreography, as well as developing a serious targeted approach to them. We are not talking about the volume and comprehensiveness of works created in the field of ballet, but about the depth of penetration into selected issues of graphic presentation of dance directing, since the total number of such works in these times became more diverse. To date, many important areas of the theory of choreography still survive intact. These include choreography graphics, often associated with the ups and downs of practical experiments, which was not described and conceptualised from beginning to end by the creators. Nevertheless, certain provisions are stipulated in the works of famous choreographers and their successors who were engaged in graphical artwork. Only a few memories survived in the memoirs of contemporaries who witnessed the creativity of the dance masters. Thus, the work of ballet dancers and choreographers in the field of visual fixation of the dance is less studied; it has never been the subject of a special study. There is nothing written about it in the literature. Remaining as optional, this phenomenon is usually excluded from art reasoning. Meanwhile, this work is relevant to a particular art world. It is a special creative element that lives by its own regulations. The use of fine materials by choreographers is rightful, as well as material taken from life, which is of interest to the choreographer. Do not forget that the choreographer’ will thought develops and enriches with the development and enrichment of fine forms. In ballet, there is a certain artistic system, which can be fully revealed only by means of the image. That is why, ultimately, dance is doomed by its functional, purely professional purpose to be attributed to a shoppy director’s development. The greatest interest in this regard is the study of imaging techniques in the course of creating choreographic character. The artistic creativity of the choreographer is seen as a process similar to designing items. However, the problem of expressive figurative and plastic form is not removed, though it is contingent primarily on function. The creative process of Abstract

Introduction
In the long history of ballet, one can particularly emphasise two epochs -19th and early 20th centuries, when the greatest number of works appeared, demonstrating the desire to solve the problems of staged choreography, as well as developing a serious targeted approach to them. We are not talking about the volume and comprehensiveness of works created in the field of ballet, but about the depth of penetration into selected issues of graphic presentation of dance directing, since the total number of such works in these times became more diverse. To date, many important areas of the theory of choreography still survive intact. These include choreography graphics, often associated with the ups and downs of practical experiments, which was not described and conceptualised from beginning to end by the creators. Nevertheless, certain provisions are stipulated in the works of famous choreographers and their successors who were engaged in graphical artwork. Only a few memories survived in the memoirs of contemporaries who witnessed the creativity of the dance masters. Thus, the work of ballet dancers and choreographers in the field of visual fixation of the dance is less studied; it has never been the subject of a special study. There is nothing written about it in the literature. Remaining as optional, this phenomenon is usually excluded from art reasoning. Meanwhile, this work is relevant to a particular art world. It is a special creative element that lives by its own regulations. The use of fine materials by choreographers is rightful, as well as material taken from life, which is of interest to the choreographer. Do not forget that the choreographer' will thought develops and enriches with the development and enrichment of fine forms. In ballet, there is a certain artistic system, which can be fully revealed only by means of the image. That is why, ultimately, dance is doomed by its functional, purely professional purpose to be attributed to a shoppy director's development. The greatest interest in this regard is the study of imaging techniques in the course of creating choreographic character. The artistic creativity of the choreographer is seen as a process similar to designing items. However, the problem of expressive figurative and plastic form is not removed, though it is contingent primarily on function. The creative process of developing choreographic character is viewed as it were "from the inside out". The study of fine art of dance masters is accompanied by in-depth study of compositional graphic tools, based on analysis and experimental study of the properties of the art form.

Materials and Methods
For dance masters, image is a kind of representational system. But just as line and color do not represent the artistic image, the original sketch as well as shooting in this case do not represent artistic creativity. Impartial cribbing from nature is amateurish, though assent to new stages of philosophical and artistic generalisations, expressed in the diversity of aesthetic pursuit, is a lively contradictory form dominant of the choreographic process of the 19th and early 20th centuries. This is the subject of our research.
For the analysis, we chose the artwork of two famous masters of choreography -, whose creative work spans the 19th century, and M. Fokin, reformer of the Russian ballet of the late 19th -early 20th centuries. The study attempts to disclose the specifics of choreographers' fine arts in their ways of functional thinking for specific performance, as well as considering it to be a diagnostic imperative way of mastering the world of dance. The field of our study includes mostly graphic images in which the depth of thought and high visual culture of directing choreographer are interrelated inseparably [1][2][3][4][5][6][7][8][9] . They represent sketches, diagrams and episode pictures of the performance, which are significant in themselves, because we are talking about the images of famous choreographers, each of whom is associated with one or another conception, formulation or period of creativity. They are arranged in the sole sequence, regular for a given case, which is dictated by the general intent of the performance, its main defining idea. Sometimes they are based on a kind of directing choreographer's diary that collects their thoughts, reasoning, stories about their own performances; sometimes they are arranged based on the "hard" director's script. This is everything that occurs daily, away from the theatre audience's eyes, in all its complex dialectics, the ideas, which are yet difficult to generalise; however, the presence of historical distance helps to detect intrinsic, natural essence hidden under the wrapper of accidental. did his work step by step. One of the important periods is creation and work on the libretto, which was written either by himself, or processed according to the tastes, understanding, and time requirements. The study of the era, costumes, and accessories allowed him to give clear assignment (oral or written) to performance artists. Finally, we would like to note that his famous mass dances, rich plastic staging, and even the whole music score were composed after pre-designing complex dancing schemes and fixing them in the drawings. Thus, the clarity of "plastic" and emotional thoughts were achieved, as well as the ability to count on the merger, exceptional simplicity of ideas in choreographic images of the most challenging dance combinations 4.
Schematic drawings of have a variety of advantages, even compared to experienced dance photographers. First of all, they are much deeper in perceiving space in its dance transformation. The organizing principle of each depicted female dancer of the corps de ballet, conventionally indicated in general graphically, are the intuitive features of constant contact with the movement of all other dancers, and this strongly reveals presence of choreographer's thinking. Graphic drawings with lots of figures and objects unfold their narrative in a sequence, characteristic to symbolic writing. Often rows of images include inputted graphical icons resembling prehistoric petroglyphs or Egyptian hieroglyphics. They complement each other in terms of meaning and composition, emphasizing the perception orientation and sequence. However, the temporal sequence of images almost never reflects its temporal length. Simultaneity becomes a time base streak to cover the spatial combination of figures and details, also given in streak. The interrelation of gestures, postures and items, as well as items and human, represent the characters in not so much spatial as topographical context. Sometimes large scale images of human shapes are a sign of their importance, rather than an indication of the geometric magnitudes in the space (much like the Egyptian reliefs and wall paintings). However, these choreographic sketches not only assume the image of space, but the shapes as well -symbols are considered in the space, they look like the combination of front and profile images, made at a right angle or when looking at them from above. The entire internal birth mechanism of the action and the character is often absent. The creative method of. here belongs exactly to those mechanisms, which usually remain "behind the scenes". They do not give comprehensive information about future components of performance, its semantic and figurative-expressive aspect: about the fable, the style, and the manner of performance, though conveying a different thing, namely composition and tempo-rhythm.
Significant relationships between temporal and spatial relationships that are artistically mastered by choreographers in drawings can be called the chronotope (time-space). In choreography, the graphical chronotope represents the adunation of spatial and temporal marks in a meaningful and concrete concept. The time here is stretched, becoming long, and includes rows and chains of figures-characters; the space intensifies, draws into the time movement of the dance. Figures of the dance unfold in space, and the space is comprehended and measured by time. This intersection of rows and merging of marks characterise chorographical chronotope 5.
Petipa and Fokine break down graphic material inside the sketches on rhythmic blocks, set priorities, and determine the compositional drama of each episode of dancing movement. The principle of dynamic equilibrium and correctly found scale of the artwork allows one to combine quite diverse figure patterns and schemes in a single whole, arranged on a plane, and to create on their basis a multifaceted exposure.
Surviving choreography sketches -exposures of to scenes from ballets include "King Kandal" by (available at the Bakhrushin State Central Theatre Museum), "Sleeping Beauty" (1890), the "Nutcracker" (1892) (available at the Bakhrushin State Central Theatre Museum). These are top views and motion schemes to specific acts and scenes. These are search options for a cord groups, their relocation and shape. We see the stage from the height of bird flight. The singularity of the author's opinion gives rise to a geometry that naturally turns into the symbolism. In some drawings performed with graphite pencil and ink, one can see how the right solution comes to mind.
The drawings are already half of the performance. In other words, the ballet is alive not owing to the essence of the movement, but linear building of shapes. From this point of view, the "King Kandal" seems to me typical Baroque. Fairy ballet shows of this kind are long gone. Now the performances are built differently. But the art of creating broad choreographic artworks and large shape compositions must be learnt from Petipa3.

Graphic Images and Choreographic Direction by M. Petipa
Petipa worked not only on the corps de ballet groups' schematics and mass scenes. He used to make sketches of the actual dance movements and shape poses. For example, there exist sketches of several female dancers with tulle (fabric) for the "Mlada" ballet, the sketches of supporting duet movements for the "La Bayadere" and "Mlada" ballets, grotesque drawings of buffoons and characteristic movements of the Slavic dance from the "Mlada" ballet, a sketch of the final scene (Odette and Prince), and the image of a bird of prey for the "Swan Lake" ballet. The abstractive schematic drawings, each resembling petroglyphs of primitive art, give rapid fixation of steps, prisiadkas, moves, pirouettes and other movements, their transitions, repetitions, stops, etc. They are very important for a choreographer since they contain a dynamically technical sense of the future dance. Profile drawings of several female dancers with tulle from the "Mlada" ballet is a foretype, forcing to recollect poetic dancing like aerial scenes of the "La Bayadere" ballet.
In the drawings to "Sleeping Beauty", a significant place is taken by graphical sketches of the waltz to the first act. Here, as nowhere else, one can see digits, crosses, circles, points, arcs, lines, and arrows, showing the number of participants (women, men and children), the structure of the corps de ballet, and the soloists variations. Petipa is looking for the image of the dance, and we see the gradual construction of the waltz figures. Groupings are sometimes spare, not cluttering the area of sheet (stage floor), sometimes solid and compact, shapes are symmetrical, balanced or opposite -asymmetrical and dynamic. Property (baskets, garlands, and flowers), which is so important in this ballet, is also involved in creation of the artistic image.
Choreographer F. Lopukhov notes: "... the image contains the main thing: the idea of garlands. A very beautiful group with the exact lineup of performers came up from this image" 3. Numbers in the circles that are located at different points of the sheets are the characters of the play: Aurora, the Lilac Fairy, Karaboss Fairy, etc. Of course, it is impossible to imagine the appearance of these characters by their schematic drawings, nevertheless they indicate the direction of the culminating points of the plot device, acts and patterns of the play. Intellectually, the drawing hand of M. Petipa is alive with professional idea, frozen on paper.

Graphic Images and Choreographic Direction by M. Fokine
Exposure drawings of choreographer are similar to the sketches by, though from an artistic point of view, they are more professional as they are drawn more in detail1. Artistic talent of, the author of the first edition about the choreographer, which today is a rarity: "... he was a regular visitor to the Hermitage Museum and art galleries of the Russian Museum, where he mastered the brush and copied paintings of Russian and foreign artists. Later, his choreographic works so clearly represented the favor of the promising artistic background, which have developed in him under the impression of catholically aesthetic inclinations" 2. Graphical drawings of choreographer can be clearly divided into two groups. First group represents plans (top views) of choreographic breakdown. Fokine's sketches are not a monumental Baroque clutter like those drawn by Petipa. Sketches are of a multi-part composite character. The sheet is divided into parts (small formats) like a ballet performance is isolated to the acts, pictures and scenes. Sketches of groups of actors with their compositional linear, circular, oval, diagonal, grouped or multiple breakdown follow one by one in chronological order under the numbers of 1, 2, 3, etc. 6 We see not the end result, but the observation process, the search process that shows one of the unsolved mysteries of creativity that is especially attractive. The exposition of the dance's order for the "Fire Bird" ballet to Igor Stravinsky's music is solved exactly in this manner (1910, Museum of Theatrical and Musical Culture in St. Petersburg).
Another group of drawings consists of the profile sketches of single, two or three figure compositions. The drawings are specific, each showing a certain movement of dynamic or static poses. It's not only the scheme, but also the motion manner -sketches to the "Blue God" (1911) and "Egyptian Nights" (1908) ballets. Even at the initial stage, these ballets are distinguished by a deep sense of style of the choreographic text, the originality of its perception.
In the chronology of the sketches to the "Blue God" ballet to music, we see the evolution of the movement from a single figure to two-figures, and further three-figures breakdowns. The movement develops from dynamics to statics. The graphic line of Fokine's pencil picks up almost without corrections the proportions and gestures of male and female figures; we feel the hand of the artist 7 .
In the sketches of dance groups to "Egyptian Nights" by A. Arensky, particularly attractive is the stylised central shape of a female, standing on the podium, looking like she descended from sculpted originals of Hermitage or the Cairo Museum collections. This indicates that M. Fokine deeply studied monuments of Egyptian art 8 .
The image of magnificent Baroque dance is depicted in the drawings for the "Awnings Dreams" ballet to the music of Mozart (1921). Three shapes of King, Queen and the little Page, who supports the tail of the Queen, vary the shapes of circular dance, moving towards the viewer in a horizontally-linear manner. "These drawings, masterfully performed with black ink, create an image of a lush and pompous Baroque style, so clearly manifested in costumes of the 18th century. Heavy frame skirts, tight bodice, high hair, powdered wigs, ceremonial movements and slow demonstrative poses -all of this can be seen in such a small, though expressive choreographic sketches by. They contain the narrative and figurative seance, stay on the verge of choreographers' exposures and mounted conceptions" 8 .
Thus, the immediate world of ballet-master, transferred to the plane, required a different study, different measure of the reality. And because of the effort to keep the whole "grand style" of ancient Egypt, ancient Greece or the French Baroque, follows their stylistic logic and at the same time follows the logic of the libretto. The categories of space and time here have their own specifics. It seems like the artist -ballet-master exists simultaneously in two time layers -then and now. On the one hand, poetics set by dramaturgy adjusts to styling, offers accordingly painted visuals, and on the other hand, recorded fragment of the play has a historical projection.

Conclusion
Thus, we reviewed the existing rare material that raises a deep layer of hidden and secret knowledge that lurks behind the outer shell of choreographic images. The basis of our analysis consists in graphical sources that cannot be directly available to non-specialists. This causes certain duality in our attitude to this knowledge. On the one hand, there is a certain elitism of the subject, and on the other hand -an objective need to make it known to a wide audience. And not by accident, though quite natural is that a purely theoretical issues of the ballet theater development, including the preservation, restoration and reconstruction of the performances, are associated with practical aspects of visual creativity and the symbolic system of dance recording. This relationship is in line with fruitful traditions of Russian ballet-master ideas,


s2fieldsofstudy: Art Linguistics

text:
The Convergence between Prose and Poetry in Virginia Woolf’s “Blue & Green”

This paper sheds light on the point of intersection and the moment of cross-fertilization between prose and poetry in Virginia Woolf’s experimental sketch, – “Blue & Green”. It provides an in-depth analysis of Woolf’s poetic technique in her short story in which she utilizes most of the features of poetry such as imagery, anaphora, alliteration, repetition, rhythm, and rhyme. Although classified as a short story, “Blue & Green” defies the definition of short stories as it transcends the traditional literary parameters such as plot, characterisation, and setting. Thus, this paper aims to show that “Blue & Green” can be read as a highly descriptive and sensational prose poem. Using rhythmic and poetical prose, the masterpiece which is narrated in the present tense, is carefully measured by many semi-colons and commas that hold the words and narrative together. This can be seen to herald Woolf’s more liberated style of prose writing that followed.

a visual statement and might described as passing observations rather than stories shaped by a clear narrative vision. They are linked to the post-impressionist view of art as 'subject less' and use a 'language of form and colour' to bring form in close alliance with the chaos of sense perception (p. 50).
Skrbic's argument implies that "Blue & Green" seems to be a visual painting using the language of form and colour rather than to be a story with a clear subject and clear narration. So the understanding of Woolf's "Blue & Green" depends on the reader's imagination to transform the words into a pictorial image. Similarly, Kathryn Benzel (2004) emphasises that Woolf's sketch can be seen reacting against the conventional narrative sense of plot and characterization, and replacing it by a visual and painting-like structure. Benzel (2004) confirms that some of [Woolf's] lesser-known short stories are labelled as sketches or caricatures rather than short stories. "A Haunted House," "Blue & Green," "Monday or Tuesday," and "The String Quartet" are stories that seem to defy definition as sketches or caricatures or short stories. To read these pieces is not to accumulate details in the conventional narrative sense of plot and characterisation, but to experience a visionary moment (p. 157).
While most of the writers studied Woolf's "Blue & Green" as an impressionist or post-impressionist sketch focusing on the way that Woolf uses light, colour and form to create a visual moment, this paper sheds light on the point of convergence and the moment of intersection between prose and poetry in Woolf's prose poem -"Blue & Green". It provides an in-depth analysis of Woolf's poetic technique in her text in which she utilizes most of the features of poetry such as imagery, anaphora, alliteration, repetition, rhythm, and rhyme. This paper aims to show that "Blue & Green" can be read as a highly descriptive and sensational prose poem. Using rhythmic and poetical prose, the masterpiece which is narrated in the present tense, is carefully measured by many semi-colons and commas that hold the words and narrative together. This can be seen to herald Woolf's more liberated style of prose writing that followed.
Flourishing Creativity & Literacy "Blue & Green" was first published in 1921 in Monday or Tuesday, a collection remarkable for "reformulating the definition of 'story' to eliminate its orientation toward an event" (Skrbic, 2004, p. 49). Although classified as a short story, "Blue & Green" defies the definition of short stories as it is liberated from the traditional literary parameters such as plot, characterization, and setting. Woolf asserts in "Modern Fiction" (1919) that "if [the writer] could base his work upon his own feeling and not upon convention, there would be no plot, no comedy, no tragedy, no love interest or catastrophe in the accepted style" (1966, p. 106). In regard to "Blue & Green" and "Monday or Tuesday", Valerie Shaw (1983) observes that both sketches transcend the traditional short story parameters: Dissatisfaction with the medium in which stories are told, made Virginia Woolf cultivate a lyrical style in an attempt to bring words closer to the effects of line and colour which she admired in painting. The result was to dislodge the narrative element of prose fiction so radically in favour of sensations and atmospheres that in Woolf's case it becomes more appropriate to talk of "sketches" than of "stories" (p. 16).
In one of her earliest essays, "The Decay of Essay-writing" (1905), Woolf realises that modern consciousness needs to "liven the faded colours of bygone ages" and, hence, replace the old commodities with "fresh and amusing shapes" (Bowlby,p. 5), affirming that any new "peculiar substance" requires a new narrative form: "you can say in this shape what you cannot with equal fitness say in any other" (Ibid., p. 6). As early as 1908, Woolf reveals to her brother-in-law, the art critic Clive Bell, her aim to approach a new way of writing fiction, as she confides her ambitions to refashion and "reform the novel and capture multitudes of things at present fugitive, enclose the whole, and shape infinite strange shape" (1975, p. 356). Again in 1916 in her essay "Hours in a Library", she asserts the need for a new form of writing to reverse her dissatisfaction with the prevailing form of fiction, insisting that literature was in need of "new forms for our new sensations" (1966, p. 39).
This new narrative form that Woolf seeks to originate is the fusion of prose and poetry -through the arrangement of words, syntax, and figurative language -to create a newly intersected writing style. It is clear that Woolf's desire to experiment with the new modern style does not include only the novel, but extends to embrace other forms of short fiction. In 1917 she expresses her frustration and dissatisfaction with "the frightfully" and "overpowering" novel, adding "I dare say one ought to invent a completely new form. Anyhow its very amusing to try with these short things" (1976, p. 167). Only four years after this letter, her experimental short story collection, Monday or Tuesday, was published.
John Lehmann argues that after the publication of her notable collection of short stories in Monday or Tuesday, Woolf succeeded in merging elements of poetry into her prose: "from that moment onwards Virginia Woolf was to remain more poet than novelist, forever searching for new means of dissolving prose into poetry, of refining away all but the husk of action in works which still went under the name of novels, and irradiating them with this strange new light" (1944, p. 150).
Before undertaking the analysis of the "Blue & Green" as a prose poem, it is appropriate to reflect on the definition and the different features of prose poem, the term which recurs throughout the paper.
In his introduction The Prose Poem: An International Anthology (1976), Michael Benedikt describes the formal characteristics of this literary form, which he perceives to be "a genre of poetry, self-consciously written in prose, and characterized by the intense use of virtually all the device of poetry, which includes the intense use of devices of verse. The sole exception to access to the possibilities, rather than the set priorities of verse is, we would say, the line break" (p. 47).
According to Stephen Fredman (1990), the prose poem evidences "a fascination with language (through puns, rhyme, repetition, elision, disjunction, excessive troping, and subtle foregrounding of diction) that interferes with the progression of story or idea" (p. 1).
Additionally, David Lehman also defines the prose poem, shedding light on its features as he points out that the prose poem is a poem written in prose rather than verse. On the page it can look like a paragraph or fragmented short story, but it acts like a poem. It works in sentences rather than lines. With the one exception of the line break, it can make use of all the strategies and tactics of poetry. Just as free verse did away with meter and rhyme, the prose poem does away with the line as the unit of composition. It uses the means of prose toward the ends of poetry (2003, p. 13).
In "Blue & Green" Woolf sets out to portray the sensation of colour in prose which has many of the characteristics of poetry. Beginning with the title -"Blue & Green" -it is written in italics with the symbol (&) in between, and is divided into two descriptive paragraphs facing one another on two pages with the subtitles "GREEN." and "BLUE." written in capital letters with a full stop following each subtitle as seen in the original Hogarth edition of Monday or Tuesday (Sim, 2010, p. 73). The way that Woolf writes the title and subtitles gives the reader a hint of what is to come. Woolf touches on an idea that was to be popularized by Clive Bell and Roger Fry and that became widely associated with Post-Impressionism, that of "significant form", where art is viewed as using the "language of form and color" (Fry, 'Post-Impressionism', p. 17, 2006), a language in which form and content are bound together to create a visual language through visual metaphors and the arrangement of syntactic elements.
Woolf places "GREEN." on the left side of the page describing the different colours and shapes that are produced when the lustre dropped on the white marble during daylight. In contrast, "BLUE." stands on the right side of the page, the green light with the sun's rays missing, as it is night time. Again, the various images of colours as perceived by the viewer are depicted.
In order to begin the analysis of "Blue & Green" as a prose poem, line breaks and capital letters are added to make the paragraphs look like poems. However, the words and punctuation are exactly as in the original copy.

GREEN.
THE PORTED fingers of glass hang downwards.
The light slides down the glass, and drops a pool of green.
All day long the ten fingers of the lustre drop green upon the marble.
The feathers of parakeets -their harsh cries -sharp blades of palm trees -green, too; Green needles glittering in the sun.
But the hard glass drips on to the marble; The pools hover above the desert sand; The camels lurch through them; The pools settle on the marble; Rushes edge them; Weeds clog them; Here and there a white blossom; The frog flops over; At night the stars are set there unbroken.
Evening comes, And the shadow sweeps the green over the mantelpiece; The ruffled surface of ocean.
No ships come; The aimless waves sway beneath the empty sky. It's night; The needles drip blots of blue.

BLUE.
The snub-nosed monster rises to the surface and spouts through his blunt nostrils two columns of water, Which, fiery-white in the centre, Spray off into a fringe of blue beads.
Strokes of blue line the black tarpaulin of his hide.
Slushing the water through mouth and nostrils he sings, Heavy with water, And the blue closes over him dowsing the polished pebbles of his eyes.
Thrown upon the beach he lies, Blunt, obtuse, shedding dry blue scales.
Their metallic blue stains the rusty iron on the beach.
Blue are the ribs of the wrecked rowing boat.
A wave rolls beneath the blue bells.
"Green" begins with observations during the daytime where the green light is being personified as having "ported fingers" from which drops of green colour slide down the glass. This personification embodies a metaphor around an artist who is while pouring paints on his/her canvas, the dots of colour dropping unintentionally upon the marble. As the sketch progresses another metaphor to portray the green colour is used; the "feathers of parakeets" is being compared with "sharp blades of palm trees" which look like "green needles glittering in the sun". To connect these phrases together, Woolf does not use linking words; instead she draws line form, or in other words, parenthetic dashes: "The feathers of parakeets -their harsh cries -sharp blades of palm trees -green". Again, her style is compared with the post-impressionist idea of "significant form", as Fry points out that "[t]he artist plays upon us by the rhythm of line, by colour, by abstract form, and by the quality of the matter he employs" (Fry, 2006, p. 14). Additionally, it is clearly apparent that as a poet, Woolf indulges herself in the poetic style through the high preciseness of figures of speech. As noticed above, in five short lines, three different figures of speech are being used; personification, metaphor, and simile.
The second group of lines, which extends from line 6 to line 13, is characterized by the succession of semicolons, which shows the variant elements that Woolf uses to achieve the poetic effect: But the hard glass drips on to the marble; The pools hover above the dessert sand; The camels lurch through them; The pools settle on the marble; Rushes edge them; Weeds clog them; Here and there a white blossom; The frog flops over; It is clearly evident that if each line is read individually, with a pause after each one, the narrative looks like a fragmented short story. However, the sketch acts like a poem with the use of semicolons which hold the narrative together and allow the reader to move from one line to the next without pause, as if moving from one line to another in a stanza. Concerning the sequence of semicolons, Nena Skrbic points out that their succession, with no end-stops, which holds the words together and looks like a consequence of fragmented sentences, sketches a think narrative stretched thin. Added to this, Skrbic argues that "the series of semicolons act as a sort of visual tagline, forcing the reader to break up the reading experience into a series of visual frames" (Skrbic, 2004, p. 54).
Moreover, the poetic coherence is achieved through Woolf's skilful employment of anaphora by the repetition of "the" at the beginning of successive clauses, which is repeated twenty four times throughout the sketch.
In addition to the anaphora that Woolf employs in "Green", the repetition of certain words serves to give emphasis; as for instance, each of "glass", "marble", and "pool(s)" is repeated three times while "green" is repeated six times in the text and twice in the title and subtitle. That is to say that the elements which are very important in depicting the reflection of the green colour on the marble are repeated to emphasise this pictorial image. Also the pronoun "them" is repeated three times at the middle of the sketch and in clauses that are equal in length, portioned by semicolons, and act as an end rhyme: But the hard glass drips on to the marble; The pools hover above the dessert sand; The camels lurch through them; The pools settle on the marble;

Rushes edge them;
Weeds clog them; According to Skrbic, "[t]he repetition of 'them' compels the reader to read spatially, 'to look for similarities,' producing an experience that is almost the antithesis of reading in time" (2004, p. 54).
Added to this, the alliterative patterns and the initial rhyme as shown in the italicised and underlined consonants below also work to create the poetic effect:

THE PORTED fingers of glass hang downwards. a
The light slides down the glass, and drops a pool of green. b All day long the ten fingers of the lustre drop green upon the marble. c The feathers of parakeets -their harsh cries -sharp blades of palm trees -green, too; d The green's out. n To outline the rhyme scheme of this sketch, it would be as follows: abcdecfgchhijklmknopo. The first nine lines form one unit, consisting of two non-rhyming lines followed by a rhyming line (abc, dec, fgc). The next three lines again comprise a distinct unit, in which the first and second lines rhyme while the third is non-rhyming (ggh). In the next two units, the lines are non-rhyming; however, the second line of the first unit rhymes with the second line of the second unit (ijk) and (ljm). The sketch finally ends with the first line rhyming with the third line (non). As a whole, the sketch is therefore, an assortment of different rhyming techniques. Some lines rhyme together while others either fail to rhyme or are completely free of rhyming. The entire sketch can be said to rhyme, but it would be misleading to suggest it as having a rhyme scheme. All of these poetic devices show that Woolf creates a prose poem that is symmetrically arranged.
Furthermore, it is important to shed light on the various types of imagery that Woolf utilises since the aesthetic appreciation and the emotional experience of poetry do not only emerge from rhythm, rhyme, metaphor, simile, and symbols, but also from imagery. "GREEN" is rich in sensuous imagery as now discussed: Woolf begins this sketch with a visual image, depicting a ray of green light dripping onto the marble, thereby encouraging the reader to enter a world of imagination through his/her journey of discovering the sensation of colour. The sense of visual is also reinforced by the idea of green colour. The image is visual but the other sense is clearly tactile shown by the words "slide down" and "drops" which imply contact between the light and the glass, and the marble surface: THE PORTED fingers of glass hang downwards. The light slides down the glass, and drops a pool of green. All day long the ten fingers of the lustre drop green upon the marble.
In the following cluster of clauses, Woolf mixes visual, tactile and auditory imagery; the feathers of the parakeets are glittering like the green needles of palm trees gleaming in the sun. Yet the heat of the sun on the blades of the palm trees will also be felt as a tactile experience. The auditory image recurs with the harsh cries of the parrots: The feathers of parakeets -their harsh cries -sharp blades of palm trees -green, too; green needles glittering in the sun.
Woolf once again combines visual imagery with tactile imagery. Here, she creates a visual image of the pools settling upon the desert land and a tactile image created by the meeting of the camel's legs with the sand.
The pools hover above the desert sand; the camels lurch through them.
"GREEN" ends with a visual image when the green light begins to fade away as evening falls; the viewer feels the atmosphere of the coming of the night; the shadow touches the surface of the ocean; no ships; the waves moves forth and back; the green light is out and new blue light starts to emerge, evoking the image of the sea: At night the stars are set there unbroken. Evening comes, and the shadow sweeps the green over the mantelpiece; the ruffled surface of ocean. No ships come; the aimless waves sway beneath the empty sky. It's night; the needles drip blots of blue. The green's out.
With the vanishing of the green light, the second sketch, "BLUE" has beautifully controlled the narrative and begun by portraying the perception of blue at night when the sunlight has completely disappeared and the colour is inconspicuous.
In comparison to "GREEN", "BLUE" is syntactically smoother; long sentences including no breaks, nor dashes or semicolons: The snub-nosed monster rises to the surface and spouts through his blunt nostrils two columns of water, which, fiery-white in the centre, spray off into a fringe of blue beads.
[…] Slushing the water through mouth and nostrils he sings, heavy with water, and the blue closes over him dowsing the polished pebbles of his eyes.
Thrown upon the beach he lies, blunt, obtuse, shedding dry blue scales.
Their metallic blue stains the rusty iron on the beach. Blue are the ribs of the wrecked rowing boat.
[…] A wave rolls beneath the blue bells. But the cathedral's different, cold, incense laden, faint blue with the veils of madonnas.
As Woolf continues to use imagery to strengthen the reader's engagement with the text and to reconstruct the development of the narrative in "GREEN", she follows the same technique as in "BLUE". Everything in her sketch is translated into imagery. She begins her narrative with a visual image: The snub-nosed monster rises to the surface and spouts through his blunt nostrils two columns of water, which, fiery-white in the centre, spray off into a fringe of blue beads.
[…] Slushing the water through mouth and nostrils he sings, heavy with water, and the blue closes over him dowsing the polished pebbles of his eyes.
While the image is visual, it is clear that the primary sense here is the auditory one, represented in the sound of the splashing of water through the monster's mouth and nostrils. There is also the idea of auditory evocation in the action of singing.
Multiple sensory clustering in "BLUE" helps the reader to imagine the transformation of blue colour in addition to its different shades that have been depicted. It is clearly seen in the following lines: Thrown upon the beach he lies, blunt, obtuse, shedding dry blue scales. Their metallic blue stains the rusty iron on the beach. Blue are the ribs of the wrecked rowing boat.
[…] A wave rolls beneath the blue bells. But the cathedral's different, cold, incense laden, faint blue with the veils of madonnas.
The overall image starts out visually with the monster lying upon the beach and "shedding" his "scale", then the tactile sense is inferred when his body touches the beach. This is followed by the auditory sense of the rowing of the boat and the rolling of the waves, culminating again with the visual reference to colour in the metallic blue stain. One could say that Woolf combines the three senses repeatedly to further intensify this image.
Beyond the imagery techniques already mentioned, Woolf has skilfully mastered the sound techniques which are certainly important elements of her craft. To enhance the integrity of her prose poem, to create a musical quality, and to establish rhythm within it, she uses a good deal of alliteration. John Strachan and Richard Terry clarify the impact of alliteration on the structure of the poem as creating both integrity and formal connections between the lines of the poem: The effect of alliteration is to create a formal connection between the first and second halves of the line (or between the first half-line and second half line as it would originally have been). In this sense, it gives a structural integrity to the poem and also, as all sound patterns do, lends it a sort of musicality. Yet, the soundpatterning overall serves a functional rather than a descriptive purpose (Strachan and Terry, 2011, p. 50).
Repetition of certain words is another technique that Woolf uses to create sound effect and to provide her prose poem with a regular pattern; for example, each of the words "blunt" and "nostrils" is repeated twice, while "water" is repeated three times, and the word "blue" is repeated nine times in the text, and twice in the title and subtitle.
The overall poetic technique that Woolf applies in "Blue & Green" shows that her experimental sketch can be considered as a prose poem rather than a short story.  (Woolf, 1978, p. 310).
In her essay "The Narrow Bridge of Art" (1927), which is also known as "Poetry, Fiction and the Future", Woolf proclaims her dissatisfaction of the traditional way of using prose as it is the common and ordinary form used by all types of people to achieve the common purposes of life; "[p]rose has taken all the dirty work on to her own shoulders; has answered letters, paid bills, written articles, made speeches, served the needs of businessmen, shopkeepers, lawyers, soldiers, peasants". Therefore, she looks forward to creating a new shape of writing, which she proposes will resemble poetry in the sense that it will have 'much of the ordinariness of prose' but many of the features of poetry: [It] will be written in prose, but in prose which has many of the characteristics of poetry. It will have something of the exaltation of poetry, but much of the ordinariness of prose […] it will express the ideas of the characters closely and vividly, but from a different angle. It will resemble poetry in this that it will give not only or mainly people's relations to each other and their activities together, as the novel has hitherto done, but it will give the relation of the mind to general ideas and its soliloquy in solitude (1966, pp. 224-5).
By blurring the lines between prose and poetry, Woolf aims to illuminate the role played by poetry when used together with prose. As quoted above, poetry "will give the relation of the mind to general ideas and its soliloquy in solitude", in addition to the emotions that are aroused and the beauty that is produced, as she says: "poetry has always been overwhelmingly on the side of beauty. She has always insisted on certain rights, such as rhyme, metre, poetic diction" (1966, pp. 223).
"Blue & Green" crystallizes Woolf's aspiration to develop a new way of writing the novel. In fact, the sketch anticipates Mrs Dalloway (1925), To The Lighthouse (1927), Orlando: A Biography (1928) and The Waves (1931) in which the form of writing definitely embraces poetical attributes. Hence "Blue & Green" can be seen as spearheading Woolf's new and alternative way of representing fiction, that being one in which genre, prose and poetry intertwine to create a different and more appealing aesthetic.


Infographics as Eye Candy: Review of World War II in Numbers: An Infographic Guide to the Conflict, Its Conduct, and Its Casualties by Peter Doyle (2013)

Peter Doyle. World War II in Numbers: An Infographic Guide to the Conflict, Its Conduct, and Its Casualties, illustrated by Lindsey Johns (Buffalo NY: Firefly Books, 2013). 224 pp. ISBN: 177085195X. Doyle’s book contains dozens of graphs of statistical data dealing with World War II. Many of these graphs are visually striking. However, they often violate fundamental graphing principles, in that they distort quantitative relationships, use unidentified scales, and often make it difficult to compare quantities. Graphic software makes it easy to create imaginative images, but these can fail to communicate the very information that is the graph’s purpose.

The computer revolution has dramatically changed how graphs are produced and consumed. Software packages have made it vastly easier not just to translate numbers into visual images, but to devise all manner of arresting ways of presenting information. Moreover, the Internet favors short pieces, preferably accompanied by dramatic illustrations. Both the ease of creating graphs and the Internet's seemingly endless appetite for visual displays means that we are encountering a lot more graphs, maps, and other pictorial representations of quantitative information.
However, many of those displays aren't very good. USA Today became famous for its innovative weather maps that summarized temperatures across the country (Monmonier 1999), even as it achieved notoriety for its frequent use of graphs that embodied all manner of bad practices. It became increasingly easy to spot poor graphs in newsmagazines trying to use innovative computer graphics to make their looks more compelling.
There has also been a booming literature on best-and worst-practices. There are guides for those who might want to make graphs (Harris 1999;Robbins 2005), inquiries into graphing's history and practice (Wainer 2005(Wainer , 2009, and of course Edward Tufte's books on theory and principles, especially The Visual Display of Quantitative Information (1983). One can also find magnificent volumes using graphics to convey substantive information (e.g., Shaw et al. 2008), or to simply celebrate an expanding array of possibilities for making information visible (e.g., Klanten et al. 2008). It has probably never been easier to get excellent guidance on the pathways to good graphing, and warnings about the pitfalls that endanger those who stray.
Which brings us to the book at hand. I bought World War II in Numbers because I'm interested in both history and graphs. The back cover promises that the book "brings the statistics of the conflict to life in an innovative, visual way, with graphs, charts, map overlays and high impact infographics." When I opened the book and took one look at the first graph, I realized there were going to be problems. The two-page spread features tables listing the populations in the Axis and Allied nations, and accompanies each table with silhouette figures of a woman and a man in 1940s garb (her dress has distinctive shoulder pads, and he's wearing a fedora). The total population of the Axis countries was 222.1 million, about one-sixth the total for the Allies (1,342.8 million) (p. 12). (Why, you might ask, is the Allied total so large? Because it includes both China and India.) And so-you can probably see this coming-the silhouette figures for the Allies are six times as tall and six times as wide as the Axis figures. Which is to say that a six-fold difference in populations is represented by figures whose area is about 36 times as large.
This is, of course, not an unfamiliar error. It was addressed more than sixty years ago in Darrell Huff's classic How to Lie with Statistics (1954). It is routinely covered in guides to good graphing practices. There is really no excuse for educated people making this mistake. People who make graphs should know better. The people who design graphing software packages ought to make it hard rather than easy to produce bad graphs. And editors who review the finished graphs should apply more critical eyes.
And yet, graph after graph in this book features differences in the sizes of icons-human figures, tanks, airplanes, circles, whatever-displayed by proportionally increasing both height and width. On page 27, There is a circle with a diameter of about 14 mm. (i.e., quite a bit smaller than a dime) that represents the just over 1 million Japanese troops killed in the Second Sino-Japanese War. This is accompanied by a caption: "The number of civilians who died in the struggle was staggering: 22,000,000. The corresponding representative circle is so large it won't fit on this page." Actually, of course, there is plenty of room for a circle that has 22 times as much area, just not for a circle that has a diameter that is 22 times as long.
While that familiar error is repeated many times, what is particularly striking is the novel ways this book finds to translate simple tables of figures into visually exciting yet incomprehensible displays. For example, consider the starburst-shaped graph on page 99 (illustrating Japanese aircraft losses at the battle of Kohima). At the graph's center is a dime-sized circle. The circle is divided into thirds, each devoted to a month between March and May, 1944. For each month there are six wedges emerging from the circle representing different sorts of aircraft losses. The smallest number represented is 5 (denoted by a wedge that is roughly 16 mm. long), the largest number is 100 (a 110-mm. wedge) (see Figure 1 for examples of three different wedges from this graph). It is impossible to figure out how the various numbers translate into these shapes. Is it the area each wedge covers, or is it the length of the wedges (and, if so, should we imagine that the wedge begins at the edge or the center of the circle)? Is there perhaps a logarithmic scale at work? And of course there is a larger question: why exactly should we care about month-to-month categorizations of Japanese aircraft losses? The reader suspects that the choice of graph topics was driven less by their importance than by the availability of a data set that could be graphed. The sources for the numbers are not given. Or consider the graph on page 43, illustrating German, Polish, and Soviet forces deployed during the invasion of Poland through the use of crystal-like wedges (see Figure 2 for the Russian data). In this graph, the leftmost reddish wedge denotes the number of Russian infantry divisions (33), the rightmost gray wedge portrays the number of Russian troops (466,516). It is impossible to guess how these numbers were translated into these shapes.
A seemingly more conventional display appears on pages 116-7, which illustrates the specifications of nine rifles carried by Allied troops. Figure 3 depicts the American's Springfield rifle. At first glance, this seems to be a conventional bar graph, but on closer inspection, we realize that the top bar denotes weight in pounds, the second the rifle's length in feet, the third muzzle velocity in feet per second, and the bottom bar is effective range (again in feet, but obviously using a different scale than the second bar). Therefore, there can be no meaningful comparisons among the lengths of what seem to be bars in a bar graph. In fact, both Figures 2 and 3 illustrate a common approach: separating the graphic elements to be compared. Figure 2's graph has separate clusters of crystal shapes for German, Polish, and Russian forces. If one wants to know, say, whether the Germans had more infantry divisions in the field than the Poles, one needs to compare the sizes of the reddish wedges in the different clusters. Alternatively, one can simply glance at the small table on page 42, which neatly summarizes all the numbers that become those bewildering shapes on the facing page. Similarly, if you're wondering whether the Springfield rifle was unusually light or heavy, you must go to each of the other eight graphs on pages 116-17 (and perhaps the six graphs showing Axis rifles on page 115) to learn that it was, in fact, lighter than some and heavier than others. In other words, the decision to separate comparable data into different displays actually makes it harder to make sense of what is being conveyed. Worse than merely producing unnecessary "chartjunk" (Tufte 1983), these choices add "extraneous cognitive load" that makes it harder for readers to comprehend the information being conveyed (Chandler and Sweller 1991). I could go on, but the point should be clear. Graphs are supposed to present information in a way that makes it easier to understand the data, but this book features a large collection of visual displays that leave the viewer confused. Data that can be presented in simple tables are turned into eye candy-perhaps engaging, but ultimately just empty calories.
The computer revolution has given us ready access to vast amounts of information, but of course there is no guarantee that the information we locate is actually valuable, or that it will be translated into displays that illuminate, rather than confuse.


extfieldsofstudy: Biology Medicine

text:
Corrigendum: Tomato yellow leaf curl virus infection mitigates the heat stress response of plants grown at high temperatures

Scientific Reports 6: Article number: 19715; Published online: 21 January 2016; Updated: 11 May 2016 The original version of this Article contained errors in the spelling of the authors Ghandi Anfoka, Adi Moshe, Lilia Fridman, Linoy Amrani, Or Rotem, Mikhail Kolot, Mouhammad Zeidan, Henryk Czosnek and Rena Gorovits which were incorrectly given as Anfoka Ghandi, Moshe Adi, Fridman Lilia, Amrani Linoy, Rotem Or, Kolot Mikhail, Zeidan Mouhammad, Czosnek Henryk and Gorovits Rena respectively.

In the Middle East and in other tropical and sub-tropical countries, tomatoes grown in the field in the spring and summer, are exposed to high temperatures (40 o C and higher), often in combination with Tomato yellow leaf curl virus (TYLCV) infection. TYLCV englobes a number of viruses (family Geminiviridae, genus Begomovirus) infecting tomato cultures (Solanum lycopersicum) worldwide 1 . Following the inoculation of virions in the plant vascular system by the whitefly Bemisia tabaci, the virus reaches the phloem-associated cells. The viral ssDNA genome is shuttled by the coat protein into the nucleus where it replicates via a dsDNA intermediate encoding into six multifunctional proteins. Newly synthesized coat protein (CP) and viral genomes are assembled into viral particles, which move to the cytoplasm and translocate to neighboring cells and long-distance via the vascular system, from where they are acquired by the whitefly vector during feeding 2 . Geminiviruses reprogram the cell cycle of mature plant cells, interacting with host factors to create a permissive environment for viral replication 3 . TYLCV CP recruits HSP70, one of the main cellular chaperones, to promote CP translocation into the nuclei of infected cells 4 .
To ensure a successful long-term infection cycle, geminiviruses must restrain their destructive effect on the host cells and prevent drastic plant responses. Recently, we described the suppression of host cell death, induced by inhibition of HSP90 and its co-chaperone SGT1, in tomato plants infected by TYLCV 5 . The accumulation of damaged ubiquitinated proteins and inhibition of 26S proteasome were significantly relieved under TYLCV infection. Moreover, HSP90-dependent activation of the heat shock transcription factors (HSFs: HsfA2 and Scientific RepoRts | 6:19715 | DOI: 10.1038/srep19715 HsfB1) and of the downstream genes Hsp17 and Apx1/2 (ascorbate peroxidase) under heat stress (HS) conditions were suppressed in TYLCV-infected tomatoes. Following suppression of the plant stress response, TYLCV can replicate and accumulate in a permissive environment.
One of the broadly used host protection mechanisms in preventing viral replication and spread involves the destruction of viral components, frequently CP, by cellular proteolytic degradation systems such as the 26S proteasome and autophagy. The degradation of TYLCV CP was recently described 6 . TYLCV counters proteolysis by inducing the aggregation of viral proteins such as the CP 7 and V2 proteins 8 , thereby limiting the accessibility of the viral proteins to cellular degradation. Indeed, the progress of TYLCV infection was shown to be accompanied with the development of CP aggregates of increasing size, first in the cytoplasm and then in the nucleus of infected tomato cells 7 . Large nuclear aggregates contain infectious virus particles that are transmissible to test tomato plants by B. tabaci. In contrast to TYLCV-susceptible (S) tomatoes, TYLCV-resistant (R) plants respond to TYLCV by a significant delay in the formation of large aggregates 7 . Accordingly, we proposed that the accumulation of large TYLCV aggregates is an indicator of a successful viral infection.
Elevated temperatures interfere with plant-pathogen interactions, often compromising R gene-mediated disease responses, including the hypersensitive response (HR) 9 . At high temperatures, the collapse of the Nicotiana tabacum N gene-mediated Tobacco mosaic virus (TMV) resistance was caused by heat-induced conformational changes of the plant R protein and was associated with downregulation of NADPH oxidase and superoxide and stimulation of dehydroascorbate reductase 10 . Mild increases in temperature also compromised the R gene-mediated HR following expression in N. benthamiana of Potato virus X (PVX) CP or of TMV helicase 11 . At high temperatures, Tomato spotted wilt virus (TSWV) suppressed the TSWV-mediated HR in pepper plants (Capsicum annuum) 12 . The combination of HS, drought and Turnip mosaic virus (TuMV) infection was investigated in Arabidopsis 13 . A significant reduction in biomass was found in all single stress conditions, which was further exaggerated when the different stresses were applied together, especially, under a combination of virus and heat stresses. Moreover, heat or heat and drought increased the susceptibility of Arabidopsis plants to virus infections. The cause for this increased susceptibility was claimed to reside in an altered expression of components of the signal transduction pathway and/or in a modified metabolite signaling.
Increasing temperatures involve reprogramming of signal transduction components, transcription factors and proteins associated with the metabolism of stress-generated reactive oxygen species (ROS) (reviewed in 14 and references therein). Transcript profiling of tomato plants showed that genes affected by high temperatures included those encoding for heat shock proteins (HSPs), osmolytes, enzymes that affect the membrane fluidity and enzymes involved in ROS homeostasis 15 . Further analysis suggests that high temperature response requires a coalition of pathways that culminate in the activation/synthesis of HSFs and accumulation of HSPs. Plants possess a larger number of Hsf genes than animals, leading to the hypothesis that HSFs have gained additional functions in plants 16 . Support for this hypothesis comes from the overexpression of HSFA2 in transgenic Arabidopsis, which resulted in an increased tolerance to combined light and HS 17 . In addition, different biotic stresses induce HSF expression indicating that they may also play a role in pathogen defense 16 . Since biotic-and abiotic stress-dependent signaling pathways may act antagonistically 18 , the control of these pathways is essential for an effective plant response.
In the current study, we examined whether the tomato defenses against TYLCV infection were influenced by HS or whether the virus affected HS response. In general, plant immunity occurs at different levels and can be divided into basal and R gene-mediated resistance 19 . However, we have shown before that TYLCV infection does not induce R gene-mediated resistance 20 . Furthermore, only some key markers of basal resistance, such as pathogen related proteins (PR), were up-regulated by TYLCV, and only at the late stages of infection 21,22 . These data allowed us to omit here the analysis of R genes and PR genes patterns in our experiments and to emphasis the changes in the patterns of several key HSPs, HSFs and HSF-regulated genes. In tomato cell cultures subjected to heat stress, the constitutively expressed HS transcription factor HsfA1 is complemented by two HS-inducible forms, HsfA2 and HsfB1. Because of its stability, HsfA2 accumulates to high levels during prolonged HS and recovery. It tightly regulates the expression of the APX gene family, which may play a major role in removing intercellular H 2 O 2 and preventing ROS overproduction 23 . Therefore, we have initiated the investigation of the combined effect on tomato plants of TYLCV infection and heat stress by studying the dominant transcription factor HSFA2 and its interaction with viral proteins. Particular attention was paid to the role of protein aggregation in the processes of tomato adaptation to heat and viral stresses. TYLCV-S and R tomatoes grown at different temperature regimes and the plant responses to combination of HS and virus infection were analyzed.

Materials and Methods
Viruses, insects and plants. TYLCV from Israel 24 and from Jordan 25 were used in location to inoculate tomato plants (these two isolates are stains from the same virus since their sequences show more than 95% identity). Whiteflies (Bemisia tabaci B biotype, termed also MEAM1) were used to inoculate tomato plants. Plantlets (3 weeks after sowing) were caged with adult viruliferous whiteflies for 48 h (approximately 50 whiteflies per plant) as described 26

Extraction of proteins from tomato leaves for SDS-PAGE and sucrose gradient analyses. For
SDS polyacrylamide gel electrophoresis (SDS-PAGE) analyses, proteins were prepared as follows. Leaves (about 50 mg pooled from three plants) were minced, frozen in liquid nitrogen and drill-homogenized in a standard SDS-PAGE loading buffer supplemented with 2% SDS. Samples were boiled for 10 min and centrifuged for 40 min at 10,000 × g; about 30 μ g protein from the supernatants were subjected to SDS-PAGE.
Native total proteins for sucrose gradient analyses were extracted as follows. Leaves (about 500 mg pooled from three plants) were drill-homogenized in 50 mM Tris-HCl pH 7.5, 80 mM KCl, 10 mM MgCl 2 , 0.2 mM EDTA, 0.5% Nonidet P40, 1 mM dithiothreitol and Complete Protease Inhibitor Mixture (Roche, Mannheim, Germany). Homogenates were incubated on ice for 45 min, vortexed and centrifuged at 1,200 × g for 10 min at 4 o C. The supernatant (0.5 ml) was loaded on top of a 10 ml linear 10-50% sucrose gradient. After centrifugation at 104,000 g for 20 h at 4 o C (Beckman SW27 rotor), the content of each gradient tube was divided into 10 fractions as previously described 7 . Aliquots of 50 μ l were subjected to 12% SDS PAGE and Western blot analyzed. Each gradient immunodetection was repeated at least five times. Cytoplasmic and nuclear proteins were prepared as previously described 7 . In vitro immunodetection of viral and plant proteins. Antibodies were prepared against TYLCV CP (Hadar Biotech, Rehovot, Israel) expressed in E. coli 7 . Antibodies against HSP70, HSP90, HSP100/ClpB, BiP, Histone H3 and cytoplasmic HSP70 were purchased from Agrisera (Sweden). Anti-HSFA2 was a gift from Prof. K.D. Scharf (Goethe University Frankfurt am Main, Germany). Anti-OE33, a 33-kDa subunit of photosystem II oxygen-evolving complex, was a gift from Prof. Z. Adam (The Hebrew University of Jerusalem, Israel). Incubation with primary antibodies was followed by exposure to secondary goat peroxidase coupled antibodies (Agrisera, Sweden) and by ECL detection (Amersham, UK). Each immunodetection was repeated at least three times for each set of plants (pooled tissues from the upper-most fully developed leaves of three plants).

Visualization in situ of TYLCV CP and plant HSFA2 in tomato leaves. For histological analyses,
cross sections of tomato leaves (cut into 0.5 cm squares) were processed as described 7,8 . Histological samples were incubated for 12 h at 4 o C with anti-HSFA2 diluted 1:1000 in 2% BSA/MTSB, followed by exposure to Cy3-conjugated anti-rabbit secondary antibody (Jackson Immunoresearch, USA) diluted 1:200. After HSFA2 visualization, slides were incubated again with 2% BSA/MTSB for 2 h and exposed to anti-TYLCV-CP diluted 1:500, followed by Cy3 antibodies (Jackson Immunoresearch, USA) labeling. The samples were inspected using a stereoscopic fluorescence zoom microscope (SMZ1500, Nikon, Japan) and fluorescence microscope (Eclipse 80i, Nikon, Japan); CP was detected as a green fluorescent signal, HSFA2 was detected as a red fluorescent signal. Plant nuclei were stained with DAPI (Thermo Scientific, USA), at 1 μ g/ml for 20 min at 25 °C, and detected as a blue fluorescent signal.
Purification of TYLCV proteins expressed in Escherichia coli and in vitro protein-protein interaction assay. The six full-length TYLCV open reading frames were PCR-amplified using specific primers containing an additional NdeI (5′ end) and BamHI (3′ end) restriction site. The amplicons were cloned into the NdeI and BamHI sites of plasmid pET-14B (Novagen, USA). The recombinant plasmids were used to transform BL21 E. coli cells. Following IPTG induction, the His-tagged TYLCV proteins were purified from recombinant bacteria by Ni-affinity chromatography in the presence of 6 M urea using HisPur Ni-NTA Resin (Thermo Scientific, USA). E. coli protein extracts were mixed with equal volume of equilibration buffer (PBS with 20 mM imidazole),incubated for 1 h at RT, washed with two resin-bed volumes of wash buffer (PBS with 25 mM imidazole) and eluted with elution buffer (PBS with 250 mM imidazole). The purified proteins were dialyzed for 16 h at 4 o C against 50 mM Tris-HCl pH 8.6, 5 mM EDTA, 500 mM arginine, 500 mM NaCl, 12.5% glycerol, and centrifuged at 12,000 g for 20 min. The TYLCV protein-containing supernatants were stored at − 80 o C in 12.5% glycerol and consequently used for protein-protein interaction assays.
For in vitro interaction assay, TYLCV His-tagged proteins were diluted four times with equilibration buffer and re-applied to Ni-NTA resin. After 3 h incubation at 4 o C, the resin was washed with wash buffer. About 200 μ g of total proteins, extracted from TYLCV-infected and HS-treated tomatoes were loaded on the column for a 12 h incubation at 4 o C. After washing with equilibration buffer and wash buffer, bound proteins were eluted with the elution buffer (this fraction was named "elute 1"), mixed with SDS-PAGE sample buffer and boiled. The remaining proteins were extracted by boiling the resin with sample buffer ("elute 2"). Both kinds of elutes were analyzed by western blotting using anti-HSFA2 antibodies.
DNA, RNA extractions and qPCR detection of TYLCV DNA. DNA was prepared from 100 mg leaf tissues as described 7 . RNA was prepared from 100 mg leaf tissues using the Tri-Reagent method (Sigma-Aldrich, USA); cDNA was prepared using the EZ-first strand cDNA synthesis kit (Biological Industries, Israel) according to the manufacturer. PCR and reverse transcription PCR (RT-PCR) were conducted in triplicate with three five batches of DNA or RNA. DNA and cDNA were analyzed by qPCR in the presence of SYBR Green I (Takara, Japan) in a Corbett Research Rotor-Gene 6000 cycler. The reaction was as follows: 30 s at 94 o C followed by 40 cycles consisting of 10 s at 94 o C, 30 s at 59 o C, and 20 s at 72 o C. The primers used to amplify a 182 bp fragment of TYLCV (X15656) were sense TCTGTTCAAGGATTTCGTTG and reverse sense GCTGTCGAAGTTCAG CCTTC. As an internal reference for DNA detection, a 183 bp fragment of the tomato "Expressed" housekeeping gene (SGN-U346908) was amplified using the sense CTAAGAACGCTGGACCTAATG and reverse

Results
Tomato plants subjected to elevated temperatures contain increased TYLCV amounts, whether susceptible or resistant to the virus. We have used two tomato lines to analyze the interplay between virus (TYLCV) and heat stress (HS). Line 967 is highly susceptible to TYLCV; line GF967 is resistant to TYLCV. GF967 originated from line 906-7 previously used to study the molecular basis of TYLCV resistance 21,28 . Upon infection, 967 presents typical disease symptoms of leaf curling and stunting and contains high levels of virus, while GF967 remains symptomless and contains smaller amounts of virus.
GF967 and 967 tomatoes were grown in two separated greenhouses in the Jordan Valley (Jordan), at two temperature regimen: regime 1 was 22-25 o C/18-20 o C, day/night, regime 2 was 40-45 o C/20-25 o C, day/night. In each greenhouse, two groups of plants were grown, TYLCV-infected and uninfected tomatoes. Growing infected virus-susceptible 967 plants at high temperature exacerbated the magnitude of the disease symptoms already observed at normal temperatures, as shown 28 days after infection (dpi) (Fig. 1A,1). On the contrary, virus-resistant GF967 plants infected the same day as the 967 plants and subsequently grown at high temperature did not show significant disease symptoms (Figs 1A and 2). Samples of young leaves, which developed during the course of TYLCV infection, were collected every 7 days for 28 days. In parallel, samples of uninfected tomatoes were collected the same days. DNA, RNA and proteins were extracted from the leaf samples.
When the virus-susceptible plants (967) were grown at normal temperatures (regime 1), the amounts of viral DNA in leaves (estimated by qPCR) continuously increased during infection. However, when these plants were grown at high temperature (regime 2), the amount of viral DNA accumulating during infection nearly doubled (Fig. 1B,1). On the other hand, when the virus-resistant plants (GF967) were grown at normal temperature (regime 1), the amounts of virus increased significantly only after the third week post-inoculation, albeit to very low levels. However, when these plants were grown at high temperature (regime 2), significant amounts of viral DNA started to accumulate two weeks earlier, increasing steadily, but not reaching the amounts found in the susceptible plants (Figs 1B and 2).
All along the infection, the time-course of TYLCV coat protein (CP) accumulation, estimated by western blot immuno-detection, paralleled that of the TYLCV DNA (Fig. 1C). In line 967, virus CP was detected already at 14 dpi and its amount steadily increased with the development of the infection, a feature shared with other TYLCV susceptible lines 20 . Long-term HS treatment led to a significant increase in the CP amounts all along the infection (Fig. 1C). By comparison, in leaves of GF967 plants grown at temperature regime 1, CP was barely detectable even at 28 dpi; however, when these plants were subjected to high temperatures, the virus CP was conspicuous already at 14 dpi. These results, together with the estimation of viral DNA amounts, indicated that long term HS caused increased TYLCV accumulation in the virus-susceptible and even in the virus-resistant tomatoes.
Heat stress induces an increase in size of TYLCV CP aggregates. We have previously shown that, with the progress of TYLCV infection, TYLCV CP aggregated in inclusions of increasing size. The appearance of the large CP aggregates was a feature of susceptibility to TYLCV 7 . Here we have analyzed the degree of CP aggregation in lines 967 and GF967, subjected to normal and to high temperatures.
Native proteins, extracted from leaves of the susceptible tomato line 967 at 21 and 28 dpi, were subjected to ultracentrifugation in sucrose gradients. This method has been successfully applied for the detection of plant virus-induced protein aggregates and their separation according to size 7,30 . In samples from tomatoes grown at normal temperatures (regime 1), the viral CP at 21 dpi was detected in small/midsized and large aggregates (fractions 6-10) (Fig. 2). Although CP amounts increased during infection, the CP at 28 dpi was present in the same gradient fractions as at 21 dpi. Tomatoes grown at high temperature (regime 2) showed a different CP pattern: whether at 21 or 28 dpi, CP concentrated almost exclusively in the bottom of the gradient (fraction N10), where large aggregates/inclusion bodies are confined. Hence, exposure of TYLCV infected tomatoes to constant HS caused the development of large aggregates/inclusions.

Heat stress induces different responses in infected TYLCV susceptible (967) and resistant (GF967) tomatoes.
In parallel to CP accumulation, we examined the patterns of several HS response proteins in infected 967 and GF967 tomatoes. Compared to plants grown at normal temperatures, whether susceptible or resistant to the virus, the application of constant heat during infection usually caused an increase in the abundances of HSFA2, HSP90, HSP100/ClpB, HSP70 and BiP (an ER HSP70 resident) (Fig. 3A). This heat-induced increase in the amounts of HS-dependent proteins was also observed in non-infected 967 and GF967 tomatoes (Supplementary Fig. 1).
The patterns of HSFA2, HSP90, HSP100/ClpB (but not of HSP70 and BiP) were different in infected 967 and GF967 tomato lines. The activation of HSFA2, HSP90, HSP100/ClpB was less pronounced in line 967, which contained large amounts of virus, than in line GF967 (Fig. 3A). However, even in GF967 plants, HS-induced protein expression was diminished at the late stages of infection, when virus started to be detectable. Hence, there is correlation between TYLCV accumulation and a decline of plant HS response efficiency. In parallel to the HS-induced proteins, the amounts of HsfA2, HsfB1, Hsp17, Apx1, Apx2, Hsp90 transcript were estimated two weeks after the onset of TYLCV infection (14 dpi) in the leaves of 967 and GF967 tomatoes grown at normal and at high temperatures. The results showed that heat enhanced the accumulation of HsfA2, HsfB1, Hsp17, Apx1, Apx2, Hsp90 transcripts in both lines (Fig. 3B). However in line GF967, the increase was higher than in the 967 line. For example, HS induced a 2.5 fold increase of HsfA2 transcript amounts in 967 tomatoes compared to a 5 fold increase in GF967; Hsp17 -was increased 2.5 fold in 967 and 10 fold in GF967. The expression of Apx genes was increased only in 0.5-2 fold in 967 and 5-15 fold in GF967. Such differences might The results were normalized using the tomato expressed gene as an internal marker. Results were analyzed using student's t test. One asterisk denotes p < 0.05. Two asterisks denotes p < 0.01. Bars represent the average and standard deviation of the relative expression from five independent biological repeats. (C) Western blot analyses of TYLCV CP in tomatoes grown at normal temperatures (− , regime 1) and high temperatures (+ , regime 2). Samples were taken every seven days after the onset of viral infection (0, 7, 14, 21, 28 dpi). The OE33 chloroplast protein (OE33) was used as unrelated control.

TYLCV accumulation correlates with changed efficiency of HS response. To better understand
how TYLCV influences the host plant HS response, we established assays in the laboratory where TYLCV susceptible tomatoes were subjected to HS before and after TYLCV infection. A recovery step was added, where plant samples were returned from high to room temperature (23-25 o C).
Tomato leaf samples were incubated in Petri dishes with water and subjected to a HS (43 o C for 30 min, 1or 2 h) and to a recovery period when mentioned (HS followed by incubation at 23-25 o C for 2 h). Leaf samples incubated with water at room temperature (23-25 o C) were used as controls. HS treatment induced an increase of the amounts of HSFA2, HSP90 and HSP70 proteins in control samples (Fig. 4A). When TYLCV-infected leaves were heat treated, the increase in the amounts of HSFA2 and HSP90 was less pronounced. Conversely, the behavior of HSP70 upon heat shock and recovery was similar in infected and in non-infected plants. The stage of viral infection, but not virus amounts, influenced HS-protein expressions. The efficiency of down-regulation of plant HS response was strong at the beginning of TYLCV infection (14 dpi), reached maximal levels at 21-28 dpi (shown for 21 dpi) and declined at 49 dpi (Fig. 4A). These results corroborated the pattern of HsfA2 transcription, which was more downregulated at the beginning than at the end of viral infection (14 dpi vs. 49 dpi) (Fig. 4B), even though the viral amounts were maximal levels at the late infectious stages 14 . The transcription levels of the other HS transcription factor HsfB1 and HSF regulated genes, such as Hsp17, Apx1, Apx2, Hsp90, were at the lowest levels at 14 dpi, while some up-regulation could be observed at 49 dpi (Fig. 4B).
Special interest aroused during the recovery period, when tomato leaf samples were returned to room temperature (23-25 o C) after heat shock. HsfA2, HsfB1, Hsp17, Apx1, Apx2, Hsp90 genes were still less expressed in leaves of TYLCV-infected plants than in uninfected tomatoes (Fig. 4B). The decreased amounts of HSFA2 coincided with a significant reduction in the expression of several heat-inducible genes, as demonstrated for Hsp17 and Apx1/2 (Fig. 3A). During the recovery period, infected tomatoes did not show the same levels of stress gene expression as those observed in uninfected plants (Fig. 4).

TYLCV infection changes the intracellular distribution of HSFA2. HSFs exist as inactive proteins
mostly found in the cytoplasm. HS causes HSFA2 activation with translocation to the nucleus, where it binds to its target sequences (HSEs) present in the promoter regions of HS genes (30, and references therein).
HSFA2 nuclear translocation was compared in tomato cells of uninfected vs. infected 967 plants (14, 21 dpi). Detached leaves were exposed to heat shock for 1 h, then protein extracts were separated into cytoplasmic and nuclear fractions, where HSFA2 was immuno-detected (Fig. 5A). The heat shock caused the re-localization of the transcription factor from cytoplasm to nucleus in uninfected tomato cells. In the infected leaves, HSFA2 abundance in the nucleus did not exceed that in the cytoplasm; sometimes HSFA2 levels were even less pronounced in the nucleus (Fig. 5A). Hence, in the presence of TYLCV, a decreased translocation of HSFA2 into the nucleus was observed. HSFA2 is able to form complexes with TYLCV proteins. Interaction between HSFA2 in tomato extracts and purified TYLCV proteins was studied by using purified His-tagged viral proteins over-expressed in E. coli and bound to His-Bind Resin. Plant total proteins, extracted from infected tomatoes (14 dpi) and heat shock-treated for 1 h, were passed through a Ni-column with bound viral His-tagged CP, V2, C1, C2, C3, and C4. Following elution, the two kinds of bound proteins (described in Materials and Methods) were immuno-detected with anti-HSFA2 antibodies (Fig. 5B). Complexes between HSFA2 and all the six TYLCV proteins were detected. E. coli extracts, passed through the same resins, were used as negative controls to confirm the absence of any background originating from the protein purification procedure. By comparison, HSP 70 interacted with only three viral proteins (data not shown, except for CP, described in 7 . To identify HSFA2-TYLCV complexes in the tomato leaf cellular compartments, proteins from separated cytoplasmic and nuclear fractions were passed through Ni resin columns bound to the viral proteins. Pull down of plant HSFA2 with viral CP was demonstrated in nuclear, but not cytoplasmic protein extracts (Fig. 5C). HSFA2-CP binding was not found in E. coli protein extracts (not shown). These results indicated that TYLCV CP was able to bind cellular HSFA2, restricting the capacity of free transcription factor to promote HS-genes transcription in plant nuclei.
The presence of HSFA2-CP complexes was confirmed by in situ immuno-fluorescent visualization of plant HSFA2 (red staining) and viral CP (green staining) in leaf sections of symptomatic tomato plants at 21 dpi, heat shock treated in vitro 1 h (Fig. 5D). Double in situ immuno-detection of HSFA2 and CP demonstrated their co-localized in cytoplasm (yellow staining) and nuclei (pink staining).
Combined heat and viral stresses induce the aggregation of the cellular chaperones HSP90 and HSP70. As in the case of TYLCV CP (Fig. 2), we investigated whether heat shock alone and heat shock together with TYLCV infection influence the degree of aggregation of two key cellular chaperones, HSP90 and HSP70. After 2 h of heat treatment, tomato leaves were incubated for 2 h at room temperature to recover. We expected that at this time the chaperones returned to their previous soluble state to regulate the protein quality control systems.

Figure 3. Differences in protein amounts and expression of HS-inducible genes in TYLCV-susceptible (967) and resistant (GF967) tomatoes grown at normal and high temperatures. (A) Western blot analyses
of plant transcription factor HSFA2 and HSPs in tomatoes grown at normal temperature (− , regime 1) and at high temperatures (+ , regime 2). The OE33 chloroplast protein was used as protein loading control. (B) qRT-PCR analyses of Hsf, Hsp and Apx gene transcripts of the two lines grown for 21 days at normal temperature (− , regime 1) and at high temperatures (+ , regime 2) at 14 dpi. The expression level of each gene was calculated in relation to leaves of susceptible line grown at high temperature. The results were normalized using the β-actin gene as an internal marker. Results were analyzed using student's t test. One asterisk denotes p < 0.05. Two asterisks denotes p < 0.01. Bars represent the average and standard deviation of the relative expression from five independent biological repeats; pooled leaves of three different plants were taken for each sample.
Scientific RepoRts | 6:19715 | DOI: 10.1038/srep19715 Ultracentrifugation through sucrose gradients showed that in extracts of leaves incubated at normal temperatures, most HSP90 was detected in the gradient fractions containing soluble proteins (fractions 1-2) and small protein complexes (fractions 3-4). TYLCV infection induced the aggregation of some of the HSP90, which appeared in fraction 10 of the gradient, where large protein aggregates are present (Fig. 6, upper panel). Similar results have been shown previously for HSP70 ((4) and Fig. 6, lower panel). Two hours of heat shock (42-43 o C) led to increased amounts of HSPs and their appearance in large aggregates. Heat shock of TYLCV-infected leaves caused an increase in the amounts of chaperone present in gradient fraction 10 (Fig. 6). Recovery from heat shock (2 h at 23-25 o C) led to a decrease in the amount of aggregated HSP90/HSP70 in uninfected tomatoes; the pattern resembled that of leaves incubated at room temperature (Fig. 6). By comparison, in TYLCV-infected leaves that recovered from heat shock, some amounts of aggregated HSPs remained in large aggregates (fraction 10) (Fig. 6). It has to be noted that after recovery there is more HSPs in fraction 10 of infected plants than before heat shock. These results showed that TYLCV enhances heat shock-induced protein aggregation. These aggregates contained viral CP together with at least two cellular chaperones. Restoration of initial chaperone patterns during recovery was impaired in viral infected plants.

Discussion
The combination of extreme temperature and viral infection occurs quite often in the field in the Middle East and in many tropical regions, impairing crop productivity. It is well known that increasing temperatures facilitate pathogen spread in major food crops 31 . Temperature was identified as the dominant abiotic factor directly affecting herbivorous insects by changing their development, survival, range and abundance 32 . Moreover, many abiotic The results were normalized using the β-actin gene as an internal marker. Results were analyzed using analysis of variance (ANOVA). One asterisk denotes p < 0.05. Two asterisks denotes p < 0.01. Bars represent the average and standard deviation of the relative expression from five independent biological repeats. Bars represent the average and standard deviation of the relative expression from five independent biological repeats. Pooled leaves of three different plants were taken for each sample. Pull-down assay with purified TYLCV proteins (CP, V2, C1, C2, C3, C4) used as baits for binding with HSFA2 present in leaf total protein extracts. HSFA2 immunodetection revealed potential complexes in plant elutes 1 and/or 2 (E1, E2), but not in E. coli protein extracts (Ec). The immunodetection in crude extracts was used as direct control (D). (C) Immunodetection of HSFA2 in pull down assays, where TYLCV CP preferentially binds HSFA2 in the nuclear protein fraction of uninfected and infected leaves. (D) Co-localization of CP and HSFA2 in cytoplasm and nuclei of infected (14 dpi) tomato leaf (cross leaf section and leaf midrib). Fluorescent microscopy using primary anti-CP antisera and Cy2-labeled secondary antibody, primary anti-HSFA2 antisera and Cy3-labeled secondary antibody; nuclei were DAPI stained. Viral CP appears as green, cellular HSFA2 as red, nuclei as blue; CP co-localizing with HSFA2 as yellow (yellow arrow), CP co-localizing with HSFA2 in nuclei as pink (pink arrow). Bar: 50 μ m. stresses were shown to weaken the defense mechanisms of plants and enhance their susceptibility to pathogen infection 33,34 . Current climate prediction models point to a gradual increase in ambient temperatures in the near future and to an enhancement in the frequency, length and amplitude of heat waves 35,36 . Therefore, there is a need to generate crops with enhanced combined tolerance to heat and pathogens.
In the current study, we have highlighted several mechanisms of interaction between HS and TYLCV infection. The effect of HS in promoting virus accumulation was confirmed by subjecting TYLCV susceptible (line 967) and TYLCV-resistant (line GF967) tomato lines grown in near-field conditions. Plants from line 967 grown at high temperatures showed exacerbated symptoms compared to plants grown at milder temperatures (Fig. 1A). Heat application significantly increased the amounts of viral DNA (Fig. 1B) and CP (Fig. 1C) not only in line 967, but also in line GF967. In GF967 tomatoes grown at normal temperatures (regime 1), TYLCV CP was undetectable during prolonged infection (28 dpi). High temperatures (regime 2) led to an increase in CP amounts. Our previous studies indicated that during the course of infection, increasing amounts of TYLCV DNA and CP were present in aggregates of increasing sizes. Moreover, the size expansion of such aggregates was considered as a marker of a successful infection 7 . Heat shock treatment of TYLCV-infected tomato leaves led to the accumulation of viral CP exclusively in large aggregates (Fig. 2).
Comparing the HS-dependent activation profiles of the main heat response players such as HSFs and HSPs in TYLCV-infected and uninfected tomatoes, provides some clues on how tomato plants act under TYLCV and HS. It has been shown that in tomato cell cultures subjected to HS, the constitutively expressed HS transcription factor HSFA1 is complemented by two HS-inducible forms, HSFA2 and HSFB1. Because of its stability, HSFA2 accumulates to high levels in the course of prolonged HS and recovery regimes 37 . Since HSFA2 is an abundant transcription factor with a high activation potential, it is the dominant HSF in tomato upon HS treatment 38 . HSFB1 was suggested to act as a coactivator of HSFA1 during HS 39,40 . HSFs may act as molecular sensors to detect the presence of ROS such as H 2 O 2 and activate downstream stress-responsive genes such as Apx1/2, Hsp17 and others, allowing plants to respond to different environmental conditions 41 . In rice, the induction patterns of HSPs and HSFs following stress treatment such as drought, heat, cold, and salt showed some overlap, but also a specific response to each condition 13 One of the important results of the current study is the discovery that TYLCV infection is accompanied by a decreased activation of cellular HS response. The transcriptional and/or protein patterns of HsfA2/HSFA2, HsfB1, Hsp90/HSP90 (but not of HSP70 and BiP), HSP100, Hsp17, Apx1 and Apx2 were significantly decreased in TYLCV-infected tomatoes (Figs 3 and 4). The failure of TYLCV to induce the expression of various plant HSPs has been described previously 20,22 . Here we have shown that TYLCV infection inactivates cellular HS response, by decreasing the expression levels of HSFs, HSPs and HSF-regulated proteins (Figs 3 and 4). The current experiments do not completely exclude the possibility that the observed effect on HS response suppression is a secondary effect of changing cell metabolism in the aging infected cells. However, potential TYLCV capacity to suppress the HS response depended on the stage of infection, not on aging of the infected tomato plants. It was minor at 7 dpi (data not shown), started to be significant at 14 dpi and was maximal at 21 dpi, while it has decreased at 49 dpi (Fig. 4), even though the amounts of TYLCV were higher at the later Infected and uninfected tomato leaves were incubated at room temperature (control), and at 42-43 o C for stage of infection. In uninfected tomatoes, some changes in the patterns of HS-inducible proteins during aging were observed ( Supplementary Fig. 1), but were much less pronounced than in TYLCV infected plants.
TYLCV infection was accompanied by changes in the intracellular distribution of HSFA2. The decrease in the abundance of HSFA2 occurred mostly in nuclei (Fig. 5A). All the six TYLCV purified proteins could bind to HSFA2 in total protein extracts in in vitro assays (Fig. 5B); in addition, CP was shown to be able to interact with HSFA2 in nuclear extracts (Fig. 5C,D). The changes in the HSFA2 patterns point to the reduced presence of free, active nuclear HSFA2, which may be a reason of down-regulated transcriptional levels of HS-genes described through this study (Figs 3 and 4).
In uninfected tomatoes, during the recovery periods that followed heat shock, the key cellular chaperones HSP70 and HSP90 reverted to fractions containing soluble active proteins or protein complexes. In TYLCV-infected plants, this restoration was not complete (Fig. 6). The mobilization of chaperones in large aggregates could weaken the HS response. They could be used by the virus as elements of the protein quality control machinery to reactivate cellular proteins needed for folding and assembly of multimeric protein complexes required for virus replication, transcription and encapsidation. The finding that HSP70 (4), HSP90 (current study), and components of 26S proteasome degradation (6) were recruited into large CP aggregates/inclusion bodies support this hypothesis.
In TYLCV infected tomatoes, a significant reduction in the levels of transcription and translation of the heat-inducible genes can lead to a decreased temperature tolerance, HS response and consequently to reduced cell death. The importance of CD suppression by TYLCV infection was recently described (5). Likely, reduced stress responses supply enough time for successful viral replication. On the other hand, infected tomatoes become much more susceptible to various environmental stresses. HSF signaling is one of the main contributors to resistance against main abiotic stresses and is essential for the maintenance of normal growth and productivity under stress conditions 41 . Stable HSFA2 is expected to confer a stress resistance phenotype while maintaining yield productivity. Indeed, the maintenance of HSFA2 dependent activation of stress response in TYLCV-infected GF967 plants (compared with TYLCV-infected 976 plants) may indicate a better capacity to manage HS and possibly other abiotic stresses. The development of stable multiple stress tolerance traits in important crop plants will improve yields particularly in areas with adverse environmental conditions, and contributing to global food security. One of the successful example of such multiple stress tolerant plants is the TYLCV-resistant GF967 tomato line, which survives under heat and TYLCV stresses. It might be possible to increase the heat tolerance of TYLCV-susceptible plants by pre-inoculating (by agroinfection) seedlings with a TYLCV symptomless mutant lacking 20 amino acids near the N-terminus of the CP 42 , and therefore not transmissible by whiteflies, before planting in the field. We expect that the mutant will have the same capacity to suppress the HS response and to increase heat tolerance as the wild type virus, though this has to be proven experimentally.