A 2020 Minor, Mainstream or Situational? Eva Menasse's Tiere Für Fortgeschrittene. Modern Languages Open

Anita Bunyan email@example.com
Gonville and Caius College
University of Cambridge
GB

A 2020 Minor, Mainstream or Situational? Eva Menasse's Tiere Für Fortgeschrittene. Modern Languages Open
2020110.3828/mlo.v0i0.301
This article explores the relationship between minor, mainstream and situational positionality in the work of Eva Menasse. Menasse can be categorized as a secondgeneration Jewish writer, an Austrian writer and a woman writer, and this theme of multiple affiliations or situational positionality lies at the heart of her novels and short stories. The paper will focus on her short story collection Tiere für Fortgeschrittene (2017) to explore the way in which her writing challenges received narratives to move beyond binary and linear concepts of 'Austrian' and 'Jewish' identity, and indeed of the 'minor', towards a more multi-facetted and mobile concept of situational positionality.Tweetable Abstract: Anita Bunyan explores the relationship between minor, mainstream and the more multifaceted and mobile concept of situational 'Austrian' and 'Jewish' positionality in Eva Menasse's Tiere fuer Fortgeschrittene.

This article explores the relationship between minor, mainstream and situational positionality in the work of Eva Menasse. Menasse can be categorized as a secondgeneration Jewish writer, an Austrian writer and a woman writer, and this theme of multiple affiliations or situational positionality lies at the heart of her novels and short stories. The paper will focus on her short story collection Tiere für Fortgeschrittene (2017) to explore the way in which her writing challenges received narratives to move beyond binary and linear concepts of 'Austrian' and 'Jewish' identity, and indeed of the 'minor', towards a more multi-facetted and mobile concept of situational positionality.

Tweetable Abstract: Anita Bunyan explores the relationship between minor, mainstream and the more multifaceted and mobile concept of situational 'Austrian' and 'Jewish' positionality in Eva Menasse's Tiere fuer Fortgeschrittene.

'Power' is a central category in Deleuze and Guattari's concepts of 'major' and 'minor' literature. According to them, while 'major' literature is, for example, connected to the "language of the state, the official tongue" and "the language of power", 'minor' literature is a literature of the relatively powerless -"the literature a minority makes in a major language" (Deleuze and Guattari 1983: 16). It is also political in the sense that "every individual matter is immediately plugged into the political" and communal in that "what the solitary writer says already constitutes a communal action. […] The political field has contaminated all statement, especially literature which finds itself positively charged with the role and the function of collective, and even revolutionary utterance" (Deleuze and Guattari 1983: 16, 17).

It is interesting to consider the writing of the novelist Eva Menasse in the light of this socio-political definition of 'minor' literature. She identifies and is frequently marketed as a second-generation Jewish writer who seeks, in her work as an author and a contemporary public intellectual, to break the silence about, in particular, the Austrian past. 1 As a secondgeneration writer, she has sought to challenge what she regards as an official culture of silence and repression of suffering on the part of the postwar Jewish communities, especially in Sammy wäre lieber am Fenster gesessen, aber wenn er sich beschwerte, pries sie ihm den Platz damit, dass er zwischen ihnen hinausschauen konnte, als wäre er vorn mit dabei. Der Statistik zufolge stirbt der Beifahrer als erstes. Daran dachte sie jedes Mal, wenn Ben und sie nach den vom ADAC empfohlenen anderthalb Stunden die Plätze tauschten. Sein Fehler würde sie das Leben kosten. Das galt auch umgekehrt, aber davor hatte sie, solange sie am Steuer saß, keine Angst. Sie war zwar überzeugt, dass man die Schuldgefühle ein Leben lang nicht loswerden würde, was für einen selbst offenkundig viel quälender wäre als tot zu sein. Aber solange sie fuhr, hatte sie es in der Hand. (Tiere 278) [Sammy would have preferred to sit by the window, but when he complained, she praised the middle seat by pointing out that he would be able to look out through the gap between them, as if he were there in the front. According to statistics, the frontseat passenger is the first to die. She thought about that every time when, after every hour and a half, as recommended by the German Automobile Association, she and Ben swapped places. She would pay with her life for his mistake. The reverse was also the case, but as long as she sat at the wheel, she was not afraid of that. Of course, she was convinced that you would never ever get over the feelings of guilt and that would obviously be more agonising than to be dead. But as long as she was in the driving seat, it was in her hands.]

The mounting references to Jenna's anxious disposition culminate in a description of her suffering a severe panic attack when the car's satellite navigation system leads them into a claustrophobic dead end in the streets of Florence, where even the walls of the street take on a threatening intensity:

Das Navi wurde zu etwas halb Lebendigem, Bösem, das sie ins Unglück zog, in immer engere Winkel führte bis in eine Sackgasse, in der man nicht mehr wenden konnte. Und dort würde etwas Grauenvolles passieren. Außer Jenna ahnte das niemand. Ihre Vorahnungen waren das einzige, was sie am Ende retten würde. Die rostrot gekalkten Wände schoben sich links und rechts näher, als hätten die Häuser verborgene Füßchen. (Tiere 305) [The satnav almost acquired a life of its own, turning into something evil that led her into misfortune, driving her into increasingly tight corners and finally into a cul-de-sac where you couldn't turn around anymore. And there, something horrific would happen. No one, apart from Jenna, suspected that. In the end, her premonitions were the only thing that would save her. The rust-red limewashed walls pushed ever closer from left and right, as if the houses had little hidden feet.] By this point, though, the narrative has revealed that Jenna's anxieties and shame-inducing panic attacks are rooted in her Jewish family's experience of persecution. Although she has no first-hand experience of persecution, her postmemorial trauma focuses on vividly imagined scenes of departure and familial separation, which are brought into especially sharp relief by her own experience of parenthood (Tiere 276,286 [When she tried to imagine her father's departure at that time, the images grew dark. As if one's own thoughts were visually impaired. Dark and cold, masses of people, hysterical in a way that was ghostly and silent. In the middle of the picture, her family in historical dress. Grandparents and teenage uncle frozen in motion. Only her small father, who had no understanding of things, wriggled about. He had these oversized child's eyes which didn't belong to him alone, but to all poor children of the world and of history. Everything in black and white, an old silent film. When Jenna was in an auto-aggressive mood, she imagined piano accompaniment, as was common in the age of silent films. Waltzes, the polka, marching music. Hitler sends the Jews off in the trains. In all the decades that she had spent with this scene, it had scarcely changed.] "Umgeben" [Surrounded] or "durchdrungen" [permeated] (Tiere 277) by this family history of fear, separation, deterritorialisation and death, Jenna struggles increasingly with its farreaching psychological effects. A Jewishness experienced as an early tendency to parsimony and as "unappetitliche kleine Angstreste, eines Tages doch enttarnt und verhaftet zu werden" [unsavoury little remnants of fear that one day you would indeed be discovered and arrested] at border crossings has now given way to acute anxiety: "Geiz war bisher ihre schlechte Eigenschaft gewesen, die verkrampfte Sorge um eine Zukunft, die einen vergessen ließ, die Gegenwart zu genießen" [Miserliness had been her bad habit up until now, a tense concern about the future which prevented you from enjoying the present] (Tiere 290). The motif of anxiety is compounded by that of the shame she experiences when she contrasts her quotidian problems to those faced by her grandparents: "Alltag? Das war ein Luxusproblem, das gab es damals nicht" [The problems of everyday life? That was a luxury. It didn't exist then] (Tiere 300).

As she grapples with her own marital problems, Jenna is conscious of the potentially fatal dangers of failed relationships for her Jewish grandparents' generation, especially for Jews in mixed marriages: "Mit dem Geld und dem Ansehen verspielt man in Wien auch die Beziehungen. Und das wurde lebensgefährlich. Was der Großvater nicht hatte wissen können" [In Vienna, along with your money and reputation, you could also squander relationships. And that became highly dangerous. Which Grandfather couldn't have known] (Tiere 285). Her anxieties also feel shameful in comparison with the suffering endured by her parents who were separated from their own parents as children and survived the Holocaust: "Mit minderen Problemen als dem Tod wollten Jennas Eltern nicht belangt werden" [Jenna's parents didn't want to concern themselves with problems less pressing than death] (Tiere 309). Jenna is convinced that her father, a 'Kindertransport' child, has suppressed the "lebenslanges" [life-long] trauma (Tiere 301) of childhood separation by presenting it stoically as a youthful adventure (Tiere 294). This unintended deprecation of her problems, indeed of her whole existence, is a psychological burden, relieved only by Jenna's hopes for the third generation. Her almost rebellious reflection "dass Überleben nicht ohne weiteres als höchstes Gut anzusehen war" [that survival was not necessarily the greatest good] (Tiere 302) is counterbalanced by her ability to rejoice in her young son's comparatively carefree existence: "Er schaffte, was sie nicht gekonnt hatte, er ging von klein auf angstfrei und neugierig in die Welt" [He managed what she could not. From a young age he approached the world without fear and with curiosity] (Tiere 315).

The motif of the persecuted child appears in many of Menasse's narratives and gives vivid expression to the powerlessness experienced by both first-and second-generation Jewish survivors of the Holocaust. It could be regarded as a recurrent feature of Menasse's 'minor' politics and also of a 'minor' aesthetic which allows her narratives to explore concepts of powerlessness and power, victimhood and perpetration, innocence and guilt. The narrative of "Enten" establishes tragicomic parallels between Jenna's father's perilous journey as a 'Kindertransport' child, waiting anxiously in his best suit on the platform of the Westbahnhof in Vienna, and her absurdly overprotected son who travels "beschützt von seinen Akademiker-Eltern, in einem mit grinsenden Schlangen bedruckten, TÜV-geprüften Kindersitz in den Süden" [southwards, protected by his educated parents, in a grinning-snake-print safety seat which has passed its MOT] (Tiere 301). In Jenna's imagination, both boys, with their unnaturally large "Kulleraugen" [big round eyes], are conflated into "eine einzige, zu beschützende Kinderfigur" [the single figure of a child in need of protection] (Tiere 310) for which her generation of "Überlebenstrophäen" [trophy survivors] must assume responsibility (Tiere 293).

The story "Haie" [Sharks] reworks the motif of the persecuted child and explores the possibility of creating new forms of minoritarian community and solidarity. Here, the central protagonist's second-generation Jewishness leads her to forge a bond of solidarity with an ostracised migrant child in contemporary Berlin. Observing proud family groups on their way to celebrate their children's first day at school, "Nora empfand es als einen Morgen voller Einigkeit und völkerverbindender Harmonie" [Nora felt it to be a morning full of unity and a harmony which brought nations together] (Tiere 212). But the motif of the sharks in the title of the story alerts the reader to the fact that Nora's idealism, and that of Berliners who take such pride in their multicultural local neighbourhoods, is wishful thinking. The narrative of "Haie" focuses on the suspicious and mistrustful, almost febrile, atmosphere of Nora's daughter's primary school community in Berlin. Nora's instinctive solidarity with immigrants (Tiere 243) is tested when, on the first day of school, her daughter Clara develops an intense dislike of a somewhat overbearing little boy in her new class. As the school year wears on, Nora finds herself empathising with the allegedly ' difficult' six-year-old Muslim boy, Frederic, the child of wealthy Middle Eastern immigrant parents rumoured to belong to a mafia-like criminal clan: "Und sie fand es nicht richtig, zufällige Untergruppen mit Eltern zu bilden, nur weil sie so ähnlich waren wie sie, weiß, gebildet, deutschsprachig" [And she didn't think it was right to form random subgroups with parents just because they were so similar to her; white, educated, German speaking] (Tiere 221). A number of incidents, such as the deliberate cutting of a child's satchel strap and reports of aggressive behaviour, lead parents and children to ostracise Frederic and his family, despite Nora's increasingly energetic attempts to rationalise the events, stick to the facts and defend the small boy's actions. Something about Frederic's plight, that of the ostracised child, awakens her sympathy (Tiere 225). Even Nora is conflicted, though, and cannot prevent herself from suspecting that it is indeed Frederic who cut the straps of Clara's satchel (Tiere 222).

A link is established in "Haie" between the rumours of mafioso threats and intimidation circulating among Nora's friends and the increasingly exclusionary and scapegoating behaviour of the parents and school authorities towards the little boy. Almost involuntarily, Nora draws a connection between such conduct, which lies beyond the grasp of the law, and scapegoating by the Nazi regime, only to find that the sense of shame she associates with her Jewishness resurfaces: "Es ist genau wie mit den Arisierungen, sagte Nora plötzlich laut, und alle schauten sie an. Sie wurde rot" [It's exactly like the Aryanisations, Nora suddenly said out loud, and everyone looked at her. She blushed] (Tiere 229). Similarly, when she speaks out in defence of Frederic at a parents' evening, she once again finds herself the object of the dominant parental group's gaze: "Mehrere Augenpaare starrten sie an. Warum geht dir das so nahe, fragte eine, die bisher wenig gesagt hatte. Nora schüttelte verzweifelt den Kopf. Ich habe das Gefühl, hier wird einer zum Sündenbock gemacht, begann sie" [Several pairs of eyes stared at her. Why is that such a big deal for you, asked one woman who had said little up to now. Nora shook her head in despair. I have the feeling that someone's being made into a scapegoat here, she began] (Tiere 240).

By the end of the story, Nora is alone in her empathy for Frederic, isolated from the mainstream of "Mittelstands-Eltern" [middle-class parents] who are glad to see the back of the boy when he is withdrawn by his concerned and "zu sichtbar" [too visibly] wealthy (Tiere 234) parents from the state primary and sent to a private school where, they convince themselves, he will get the attention he clearly requires. Meanwhile, other immigrants with children in the class, such as a Turkish family who conform to what they consider to be mainstream middleclass values, customs and self-image, are celebrated: "Diese säkulare, kopftuchfreie Familie mit dem hart arbeitenden Handwerker-Vater galt als Musterbeispiel aufrichtigen Bemühens, fähig zur echten Integration. Bei allen Schulbuffets pries man ihr Couscous" [This secular, headscarfless family whose father was a hardworking skilled tradesman was regarded as the perfect example of honest effort, capable of real integration. At every school fete people praised their couscous] (Tiere 236). But as soon as there is conflict, it is implied, the relaxed and innocent appearances and consciences of the primary school parental group belie the fact that, as the title of the story suggests, in reality, like sharks, they constitute a threat. "Langsam bewegte sich die Gruppe von ihr weg, plaudernd, lachend, entspannt, vollkommen unschuldig. Nora ging allein davon" [Slowly, the group moved away from her, chatting, laughing, relaxed, completely innocent. Nora left on her own] (Tiere 243). Worst of all, by the end of the narrative she is alienated from her partner Paul who, in a chilling moment, is revealed to be the source of the rumour regarding the immigrant family's alleged criminality.

In the vulnerable figure of Frederic, in whom Nora perceives something "Schlaues und Verletztes, das sich nicht erwischen lassen wollte" [sly and wounded, that could not be pinned down] (Tiere 237), she seems to see something that invokes involuntary memories of her own childhood as a second-generation Jewish daughter in postwar Austria: "Kinder in diesem Alter hatten doch längst Geheimnisse, sie schämten sich für so viele unverständliche Dinge. Sie hatten begriffen, dass ihnen verschiedene Rollen nützen konnten, je nach Gesprächspartner. Obwohl sie sich Sorgen um Clara machte, blieb sie einen Moment stehen" [Children at that age had indeed long had secrets, they were ashamed of so many things they didn't understand. They had realised that it could be useful to play different roles, depending on the interlocutor. Although she was concerned about [her daughter] Clara, she stopped for a moment] (Tiere 237). In this situation, which can be seen as constituting a brief moment of revolt, when the adult Nora encounters little Frederic outside the school principal's office, it seems that her Jewish identity asserts itself over the maternal side of herself and she expresses solidarity with the powerless migrant child. Nora's Jewish and maternal identities prove to be situational: There are multiple facets to her minoritarian identity, but in particular situations of encounter or conflict, different aspects of this identity come to the fore.

Menasse's 'minor' politics could be said to find expression through a tragicomic aesthetic. The serious themes and the melancholy, even pessimistic, everyday situations in her work are presented from a humorously ironic perspective. The narratives make extensive use of visual and situational comedy which draws on the aesthetic of the comic strip. In the plot of "Enten", Jenna's desire for autonomy and self-realisation expresses itself in the bathetic and furtive purchase of a garish plastic toy "mit karikaturhaften Augen" [with caricature eyes] for her son (Tiere 311) and in "Haie" Nora attends a school parents' evening where "Die Lehrerin dagegen blieb stehen, oder sie lehnte an ihrem Erwachsenentisch, während sie alle da hockten wie auf dem Topf, die Knie bei den Ohren" [The teacher, by contrast, stayed standing or she leaned against her desk for grown-ups while they all crouched there, as if on potties, their knees up to their ears] (Tiere 220). The tragicomedy is consistent, however, with the cultural practice of a Jewish community that employs humour as a conscious strategy for coping with events and memories that threaten to overwhelm it. Silence and humour, Jenna ruminates, are family strategies: "Erkrankungen wurden grundsätzlich verschwiegen.

[…] Das Thema Krankheiten nur in Form von barschen Witzen" [Illnesses were absolutely not discussed. (…) The theme of illness only in the form of pointed jokes] (Tiere 309). Humour is combined with a degree of emotional distance, which enables these individuals to avoid the "Sog in Richtung der uralten Katastrophen" [undertow pulling them in the direction of the catastrophes of long ago] from which they are not confident they would be able to re-emerge: "Und vielleicht war genau das die Technik, die man in ihrer Familie seit Generationen beherrschte" [And perhaps that was precisely the technique which her family had perfected generations ago] (Tiere 316).

Jewishness is not only linked to anxiety, shame and trauma, however. It also manifests itself in the stories as a combative desire for justice and self-expression. Jenna reflects on her second-generation role as a custodian of truth about the past in Austria and Germany and on her obligation to take the revolutionary step of revealing it openly: "Sie war der Platzhalter einer Wahrheit, die sich nicht mehr bergen ließ" [She was the custodian of truths that could no longer be concealed] (Tiere 277). The plight of the migrant child energises the side of Nora that is "so kampfeslustig und kohlhaasisch" [pugilistic and Kohlhaasesque] (Tiere 225), where the intertextual reference to the Kleistian protagonist Michael Kohlhaas, whose quest for justice leads him into criminality, suggests that Menasse's short narrative involves an appropriation and modern rewriting of the German novella tradition. Both stories constitute self-reflexive gestures towards the potentially disruptive role of Menasse's 'minor' narratives within German-speaking literary and political discourse. The strong autobiographical connections between the second-generation Jewish author and the protagonists of the narratives strengthen this impression of 'minor' intervention and disruption.

However, on closer inspection, the categories of social identity and power, so central to Deleuze and Guattari's analysis of, for example, the work of Franz Kafka, are revealed in Menasse's narratives to be highly situational. From thematic, authorial and narrative perspectives, it is difficult to categorise Menasse's writings unequivocally as examples of 'minor' literature. In her narratives, 'Jewishness' is not always a 'minor' social identity. In addition, the social identity of the protagonists cannot be reduced to that of the second-generation Jew. As Myrto Aspioti argues in relation to the protagonist of Menasse's novel Quasikristalle (2013), minoritarian identity is rarely clear-cut. Jenna and Nora also have gender, familial, professional and national identities that intersect with and complicate their second-generation Jewish experience. The multifaceted and situational character of their social identities is underscored by the fact that these two protagonists appear in more than one story by Menasse. This narrative intersectionality is a characteristic feature of her prose. Jenna makes an appearance as a supporting character in "Schafe" [Sheep], another story in Tiere für Fortgeschrittene, and Nora is also the main protagonist of "Habgier [Greed]" which appears in Menasse's first collection of short stories, Lässliche Todsünden [Venial Mortal Sins], published in 2009. "Habgier" focuses not on Nora as an Austrian mother in Berlin, but on her professional identity as a Jewish-Austrian freelance journalist who causes a political scandal in Vienna when she uncovers compromising historical film footage which destroys the career of a right-wing political celebrity. In "Habgier", Nora's 'minoritarian' situation and her psychology as a freelance female artist and Jewish daughter are defined by her increasingly conflictual relationship with a mainstream and highly paternalistic Jewish-Viennese familial and social circle represented by her father and Richard Bialik, an influential old family friend: Niemals sollte Richard Bialik behaupten können, dass sie aus Eigensinn einen geopfert habe, den er für anständig hielt. Das wog schwerer als alle Schelte ihres Vaters, ihren Geschäftssinn betreffend. (Todsünden,247) [Richard Bialik must never be allowed to claim that her obstinacy had caused her to sacrifice someone he considered to be decent. That weighed more heavily than any complaints her father might have had about her business acumen.] Nora's situation demonstrates that in Menasse's narratives 'Jewishness' and the 'minor' are not necessarily synonymous.

From authorial and thematic perspectives, Menasse's work can be categorised as writing that focuses not just on 'minor' or 'mainstream' Jewish experience, but also on familial and gender identity. 4 Nora's and Jenna's identities as partners and mothers are central to both narratives. The main focus of "Enten" is the strained marital relationship between Jenna and her husband, Ben, undoubtedly exacerbated by the deep-rooted anxieties that have shaped her since childhood but also affected by the gendered expectations that inform the couple's roles and identities as parents. Narrated from Jenna's perspective, as they drive south to Italy, the narrative reflects on whether their marital interactions are "genervt oder liebevollironisch" [irritated or affectionately ironic] (Tiere 276). Theirs is a relationship on a tightrope: "Auf dieser unerforschlichen Schneide balancierte ihre Ehe seit einer Weile, von beiden Seiten aus" [Their marriage had been balancing on this unfathomable tightrope for some time, from the perspective of both sides] (Tiere 276). Outwardly content, they are internally trapped in the "banalen, aber morastigen Winkel, in denen eine an sich glückliche Ehe feststeckte" [banal but constricted swamp in which apparently happy marriages get mired] (Tiere 282). "Haie" foregrounds Nora's identities as a partner and mother who has set up home in a new country: "Ihre kleine Tigerfamilie" [Her little tiger family], Nora ruminates, is "fest zusammengeschmiedet in der Fremde" [has been firmly welded together away from home] (Tiere 213). This familial solidarity is, however, tested by her identity as a second-generation Jewish daughter and the associated affinity she feels for the ostracised immigrant boy, Frederic.

Jenna and Nora could also be seen as deterritorialised Austrians who have migrated to Berlin. Indeed, only two of the central protagonists of Tiere für Fortgeschrittene, both elderly or middle-aged men, are rooted in their Austrian and Brandenburg surroundings. The other narratives, including "Haie" and "Enten", feature protagonists on the move, on road trips, or in transitional spaces such as hotels, holiday resorts and visiting artists' residences. This migration of the characters highlights the situational and often fluid nature of social identity. It is not just that their identities are characterised by multiple affiliations; rather, the relationship between these facets of the protagonists' identities is situational. In Nora's case, for example, it is particularly evident that her identity as a member of a minority group is situational. In Vienna, the setting of "Habgier", she is set apart from what could be called mainstream Austrian society by her Jewishness, but in mainstream Jewish circles it is her gender, generation and precarious economic status that set her apart. In Berlin, the setting of "Haie", by contrast, Nora is not a freelance artist and Viennese Jew, but primarily a mother and an Austrian adjusting to the customs of a city where everyone seems to have minority status: "bei ihr zu Hause maß man dem ersten Schultag nicht dieselbe Bedeutung zu wie hier, in dieser großen Stadt, die in so unglaublich viele Minderheiten zerfallen schien" [At home, they didn't attach as much significance to the first day at school as they do here, in this big city which seemed fragmented into an incredibly large number of minorities] (Tiere 213). In Berlin, language is a marker of the Austrian identity which sets Nora apart from other German parents in the neighbourhood primary school who claim that their children are victimised by the immigrant boy:

Deutschsprachig, du, hänselte Lydia, die regelmäßig behauptete, bestimmte Wendungen aus Noras Heimat noch nie, aber im Leben nicht gehört zu haben. Das muss ich mir merken, rief Lydia dann immer ein kleines bisschen zu laut. Aber sie merkte sie sich nie. Und außerdem, sagte Lydia, bilde ich keine Weißengruppe, sondern eine Opfergruppe, ist das denn Zufall, deiner Meinung nach, dass nur unsere Kinder. (Tiere 221) [You, a German-speaker, teased Lydia who regularly claimed never before, indeed never before in her life, to have heard certain expressions from Nora's homeland. I must remember that, Lydia then exclaimed, always just a little too loudly. But she never did. And besides, Lydia said, I'm not forming a group of whites, rather a group of victims, do you think it's a coincidence then that it's only our children.] Similarly, Jenna is conscious that, as an Austrian, she cannot identify with the German historical experience that her husband tries to transmit to their son, "die normalerweise mit dem Satz früher gab es, wie ihr wisst, zwei Deutschlands anfingen. Damals, im Wiener Lachkabinett, wusste sie wenig von Deutschland, und dass es in zwei Teile gehackt worden war, hätte sie vermutlich gerecht gefunden" [which normally began with the sentence As you know, in the past there were two Germanys. At that time, in the Viennese comedy booth, she knew little about Germany and she would probably have found it quite fair that it had been chopped into two pieces] (Tiere 303). This consciousness of, even quite subtle, linguistic and historical differences links Austrian and migrant identities and complicates the basis on which Menasse's narratives can be defined as 'minor' literature.

The interpretation of Menasse's narratives as second-generation Jewish 'minor' literature is further complicated by the author's biography as the child of a Catholic mother of Moravian descent. Indeed, several stories in Menasse's collections, most notably "Hochmut" [Pride] in Lässliche Todsünden, concern themselves with the second-generation perpetrator experience. "Zorn" [Anger], another story from this earlier collection, focuses on the strained relationship between a mother, Ilka, and her young son, Joshi, both of whom appear to be consumed by an ill-defined rage whose source remains unexplained. Ilka and a grown-up Joshi reappear in Tiere für Fortgeschrittene as supporting characters in "Raupen" [Caterpillars], which tells the story of Konrad, an elderly Austrian man, and his tragicomic attempts to care for his demented wife, Grete, in the family home. The narrative gradually reveals the fact that they are the parents and grandparents of Ilka (also known as Helena) and Joshi respectively, and also the parents of a now deceased daughter, Fiona -a fragile and damaged woman whose troubled relationships featured in another story, "Gefräßigkeit" [Gluttony]. In "Raupen", the Austrian family emerges as a source of love and support, but there are also strong implications that the source of the family tensions, resentments and deep-rooted anger lies in the nation's fascist past.

"Raupen" elicits a degree of sympathy for Konrad's point of view (Tiere 90). Narrated primarily from Konrad's free indirect perspective, it lays bare the domestic challenges and indignities of living with a family member who has dementia. He is "verwitwet, obwohl sie noch da ist" [widowed, although she is still there] (Tiere 63) but works tirelessly to keep his wife clean, safe and at home. He resists his daughters' attempts to secure state help for their mother, reflecting on the seductive nature of words such as the "Dienstleistungsgesellschaft" [service economy] in which his daughters have faith but which, in his view, mask a transactional relationship that can never replace the quality of familial care (Tiere 62). A degree of narrative irony also reveals him to be a stubborn man, prone to bouts of self-pity, who imagines himself alternately as a medieval knight, defending his wife against what he regards as his cold-hearted daughters (Tiere 61) or, in an implied reference to his Austrian Catholic background, as a version of the stoic martyr, Saint Sebastian: "Endlich einmal waren sie sich einig, diese beiden, und gewannen damit eine ungeahnte Macht. Wie der Heilige Sebastian ließ er die Pfeile in sich eindringen, stand er wieder bis zum Knöchel in seinem Seelenblut, doch aufrecht, ohne zu wanken" [For once they were in agreement, these two, and as a result gained an unexpected degree of power. Like Saint Sebastian, he let the arrows pierce him, standing once again up to his ankles in the blood of his soul, but upright, without wavering] (Tiere 58). Konrad is fully aware that his daughters view him as authoritarian and worse: "Beratungsresistent, sagte Helenas Stimme. Dienstleistungsgesellschaft, sagte Katharina. Gefängniswärter, Tyrann, Nazi, Arschloch, sagte Fiona" [Impervious to advice, said Helena's voice. Service economy, said Katharina. Prison guard, tyrant, Nazi, arsehole, said Fiona] (Tiere 76). The possibility that Fiona's accusatory reference to him as a 'Nazi' should be read literally rather than figuratively provides a clue to the source of the family's troubling anger and anxiety. Helena's choice of names for her own family could be read as attempts to distance herself from German culture by adopting the name Ilka, the Hungarian version of Helena, and by giving her son a Hebrew name, Joshua. The troubled behaviour of the sensitive and wayward Joshi, "dieser unberechenbare, halbkriminelle Bursche" [this unpredictable, semicriminal lad] (Tiere 56) who can be calmed only by his grandmother, suggests that the perpetrator community's legacy of repression and anger has been passed on to the innocent third generation. There is a clear parallel, and perhaps even an implied sense of community or solidarity, between Ilka's second-generation anger and the anxiety experienced by the Jewish daughter figures, Nora and Jenna; together they give narrative expression to the complicated legacy of the Austrian past in which, as in the biography of Eva Menasse, victimhood and perpetration, powerlessness and power, the 'minor' and 'major' can often be found in combination.

On a formal and not just thematic level, the multi-perspectival narrative patterns of Menasse's writing also challenge the binary and overly schematic categories of 'minor' and 'major' literature. Menasse has written about her interest in "Muster, Lücken, Strukturen" [patterns, gaps, structures] and the representation of themes, motifs, characters and spaces from a variety of spatial and temporal perspectives is a central aspect of her narrative approach. 5 The multifaceted situational identities of Menasse's protagonists resist reductive categorisation. Narrative perspectives are relativised, especially when the protagonists feature in more than one story. In "Schafe", for example, Jenna and her family are supporting characters and the objects of the main protagonist's observations about his sojourn in a visiting German scholars' residence in Italy. A psychologist, he is trained to analyse the characters and produce "Porträtskizzen" [portrait sketches] of those around him. Jenna is first introduced to the reader through his probing perspective: "Zu ihr fällt einem das Wort intensiv ein, wobei schwer zu sagen ist, ob sich diese Intensität eher in die positive oder negative Richtung entlädt. Heftige Empathie und kühler Hochmut, Lebenslust und Verzweiflung, sie scheint aus Gegensätzen zusammengeklebt" [Intense was the word that struck you about her, although it was hard to say whether this intensity would unfold more in a positive or a negative direction. Powerful empathy and cool arrogance, love of life and despair, she seemed crafted out of contradictions] (Tiere 135). "Enten", by contrast, is narrated mainly from Jenna's free indirect perspective as she surveys her marriage with a new degree of detachment brought about by the family's road trip southwards: "Auch in ihrer Ehe saß sie im Brückenrestaurant und sah die Bewegungen aus kühler Höhe" [In her marriage too she was sitting in a restaurant on a motorway bridge, observing the movements below from a cool distance] (Tiere 290).

The narratives emphasise the fact that multiple 'versions' of reality are possible and that the truth about relationships is always a matter of perspective. Jenna, for instance, notes wryly that she had originally interpreted her husband's "grenzenlose Unaufmerksamkeit" [boundless inattentiveness] (Tiere 281) as an endearing form of "intellektuelle Zerstreutheit" [intellectual absentmindedness] (Tiere 290). She is conscious that her parents' narrative about her marriage is quite different from the one she would construct: "Deshalb hielten sie an jenem kränkenden Mythos fest, der aus Ben geradezu den Dompteur eines Wildtiers machte. Und aus Jenna die Hauptgewinnerin im Männer-Lotto" [They clung therefore to the hurtful myth that cast Ben as a virtual lion-tamer. And Jenna as someone who had won first prize in the national husband lottery] (Tiere 281). Indeed, all the stories in Tiere für Fortgeschrittene call into question the narratives that the central protagonists construct for themselves. The narrator of "Schafe" reflects explicitly on this issue as he mulls over how to present himself and his work to the group: "Schließlich gibt es verschiedene Möglichkeiten dessen, was ich über mich sagen könnte" [In the end there are different ways of presenting myself] (Tiere 137).

This exploration of the protagonists' situational identities over time and space is reinforced by the intriguing narrative connections established between places in the short story collections Lässliche Todsünden and Tiere für Fortgeschrittene. The semi-private, reassuring space of the local 'Beisl', or bistro pub, the Blaubichler -a quintessentially Viennese space which perhaps functions as a microcosm of Viennese middle-class society -features as a more or less important setting in many of the narratives in Lässliche Todsünden and Tiere für Fortgeschrittene, establishing intriguing spatial and personal interconnections between the narratives. These interconnections blur genre boundaries by hinting at the idea that the collections of stories actually constitute a social novel, exploring aspects of contemporary middle-class Viennese and Berlin society from a variety of perspectives.

Menasse's narratives of situational identity reveal the category of the 'major' to be just as problematic as that of the 'minor'. From an authorial and thematic perspective, Menasse and her Jewish-Austrian protagonists, whose families are rooted in pre-war Vienna, could be seen as representatives of a 'major' literature or culture, especially when contrasted to the migration narratives of transnational authors such as Katja Petrowskaja and Sasha Marianna Salzmann. Moreover, the white, middle-class, affluent protagonists can also be considered as representatives of a global cultural or intellectual elite which possesses great cultural power and capital, but whose impotence in the face of contemporary political, social and environmental challenges is satirised by the narratives. This is a description that could apply to Menasse herself, who embraces the role of the public intellectual and is not afraid to intervene publicly in political and social debates. 6 At the same time, she is keenly aware of the limits of her influence as a writer in relation to the overwhelming power of the internet:

Dramatisch aber ist, dass sich die Öffentlichkeit als solche, die sich damals erst gebildet hat als eine Gegenöffentlichkeit zum Staat, gerade komplett auflöst. Ihre Bestandteile sind zwar noch da, aber so fragmentiert wie das Mikroplastik in den Ozeanen. Wen wollen wir denn heute noch erreichen, wenn wir in der Paulskirche sprechen, wenn wir in der NZZ oder in der FAZ schreiben? 7

[What is dramatic though is the way that the public sphere, which emerged originally as a counterweight to the state, is right now completely breaking down. Its component parts still exist, but they are as fragmented as pieces of microplastic in the ocean. Who exactly are we trying to reach when we speak in Frankfurt's St Paul's Church or when we write in the Neue Zürcher Zeitung or in the Frankfurter Allgemeine newspapers?]

Menasse's narratives also reveal that the putative power of those who possess cultural capital can be deceptive. "Schafe", in which Jenna features as a supporting character, is a coruscating satire on the ineffectual attempts of contemporary German intellectuals to rise to the global challenges of their age. The plot revolves around the exploits of a group of creatives from Germany, one of whom is Jenna, who find themselves figuratively and then literally imprisoned in a supposedly paradisiacal artists' residential centre in Italy. The nature and location of the institution are only gradually revealed by the narrator, a German psychologist who is recovering from a breakdown. The artists and intellectuals, "die ausgewählten Künstler und Denker der Nation" [the select artists and thinkers of the nation] (Tiere 179) as the narrator

) . 3
.Wenn sie versuchte, sich die Abreise ihres Vaters damals vorzustellen, verdunkelten sich die Bilder. Als hätte man in den eigenen Gedanken eine Sehstörung. Finster und kalt, Menschenmassen, die auf gespenstisch-lautlose Weise hysterisch waren. In der Bildmitte ihre Familie, in historischen Kostümen. Die Großeltern und der halbwüchsige Onkel in der Bewegung eingefroren, nur ihr kleiner Vater, der gar nichts verstand, zappelte. Er hatte diese übergroßen Kinderaugen, die ihm nicht allein gehörten, sondern allen armen Kindern der Welt und der Geschichte. Alles in Schwarzweiß, ein alter Film ohne Ton. Wenn Jenna autoaggressiv aufgelegt war, stellte sie sich als Untermalung Klaviermusik vor, wie zur Stummfilmzeit üblich. Walzer, Polka, Marschmusik. Der Führer schickt die Juden auf den Zug. In all den Jahrzehnten, die sie mit dieser Szene verbrachte hatte, hatte sie sich kaum verändert. (Tiere 276-7)
See, for example, Schmitz. On the theme of breaking the silence in Austria, see Reiter 6, 9. On Austria's 'belated culture of remembrance', see Krylova. On Menasse as a Jewish-Austrian writer, see Reiter 122-9.
Translations into English are my own.
On the concept of 'postmemory', see Hirsch.
On Menasse as a 'woman writer', see Seemann 35-51.
See her biographical entry on the website of the Villa Massimo, the Deutsche Akademie in Rome, where she held a visiting Fellowship in 2015: https://www.villamassimo.de/de/stipendiaten/eva-menasse.
Art. 14, page 11 of 14
See her collection of essays and public speeches, Lieber aufgeregt als abgeklärt.7  Menasse 2019.
For more reflections on situational positionality, see. See also the conclusion that writing by Jews in Germany up to 2005 also seeks "to find a way out of its ' either-or' dilemma" in McGlothlin 232. 9 See Deleuze 1994.
Art. 14, page 12 of 14 notes sardonically, are given absurd little tasks to accomplish and riddles to solve by the shadowy director and bureaucrats who run the institution. They spend days working as a collective to unlock the deeper meaning behind a series of picture postcards distributed by one of their number until he admits that the images were completely random, thus rendering their efforts utterly futile (Tiere 176). Gradually worn down by the oppressive and increasingly threatening Italian heat, the cowardly, submissive, but now increasingly hysterical group of Germans summons the courage to rise up against the bureaucrats who deny them air-conditioning (Tiere 169). They storm the pavilion that contains the air-conditioning apparatus, only to get locked in for several excruciating days and nights (Tiere 171). This comically absurd course of events culminates in a narrative climax when the narrator is revealed to have hidden the key to the pavilion and suffers another psychological collapse (Tiere 179). The powerlessness and utterly ineffectual activity of the arrogant collective is then again vividly emphasised by the revelation that the key has been found by the intelligent guesswork of a child -Jenna's son, Sammy (Tiere 178). All this comedy satirises a tragic reality. The intellectual "Geplänkel" [squabbles] and the self-serving ideological self-positioning and "Geweihschau" [posturing] (Tiere 146) of the nation's best and brightest are totally unequal to the social, global and environmental challenges of the age. Paralysed by anxiety, inertia and a cowardly submissiveness to authority, this pathetic "ängstliche Gesellschaft" [anxious society] cannot even sort out the air-conditioning, let alone save the planet which carries on revolving "obwohl sie ununterbrochen schmutziger, voller und gewalttätiger wird" [although it continues to get dirtier, more crowded and more violent]. Belief in the power of the cultural elite to effect progress is replaced by the end of the narrative with this philosophy of eternal recurrence, which Europe's cultural and social elites are powerless to alter (Tiere 180). At the very least, this conclusion suggests that Deleuze and Guattari's concepts of the ' official tongue' and the 'language of power' might need to be rethought, because the location of the 'major' or 'mainstream' is increasingly difficult to establish in a world where, in some sense, ' all becoming is minoritarian'(Deleuze and Guattari 2013, 123).The powerlessness of mainstream society in the face of overwhelming global challenges is reinforced by Tiere für Fortgeschrittene's series of self-absorbed, inflexible and self-pitying male protagonists who together constitute a satire on contemporary concepts of masculinity defined by work, tempo, competition and transactional relationships with women. These human flaws are not the preserve of the male protagonists, however; they reflect a wider society characterised by lack of personal commitments to other humans, to nature and to the wider world (Tiere 205). The story "Igel" [Hedgehog], for example, whose main protagonist finds a hedgehog in the grounds of a luxury hotel sporting a plastic collar from a discarded fast-food beaker, exposes the polluting activities of fast-food companies and satirises the ineffectual forms of empathy and virtue-signalling on the part of a wealthy middle class that profits from such businesses (Tiere 123). Yet, as with Nora's brief but powerful connection with Frederic, these fleeting, if apparently ineffectual, moments of solidarity provide some respite from the melancholy underlying many of Menasse's narratives. Moreover, their rhizomatic character holds out the possibility of fruitful connections. To quote from Deleuze and Guattari's later publication, A Thousand Plateaus:[U]nlike trees or their roots, the rhizome connects any point to any other point, and its traits are not necessarily linked to traits of the same nature; […] It has neither beginning nor end, but always a middle (milieu) from which it grows and which it overspills.[…]In contrast to centred (even polycentric) systems with hierarchical modes of communication and preestablished paths, the rhizome is an acentred, nonhierarchical, nonsignifying system without a General and without an organizing memory or central automaton, defined solely by a circulation of states.(Deleuze and Guattari 2013, 21-2)Art. 14, page 13 of 14The narratives of Tiere für Fortgeschrittene are alert to the fact that power is often situational and decentred. This diffuse nature of power is encapsulated briefly in "Haie" in the image of the city of Berlin as a decentred space where there is no centre of power but multiple forms of power relationships. One of the achievements of Tiere für Fortgeschrittene is to demonstrate through narrative the situational or positional nature of power. When read together, these stories reinforce the situational character of Jenna's and Nora's power and of their intersectional identities. They challenge received narratives to move beyond the binary concepts of 'German' (or 'Austrian') and 'Jewish' identity, perpetration and victimhood, mainstream and minor, towards a more multifaceted and mobile concept of situational positionality. 8 This has perhaps less in common with Deleuze and Guattari's concept of the 'minor' and more with their concept of the new, eternally 'becoming' subject. 9Art. 14, page 14 of 14
Unpacking Europe: Towards a Critical Reading. Leslie Adelson, Salah Hassan and Iftikhar DadiNaiRotterdamAgainst Between: A ManifestoAdelson, Leslie. "Against Between: A Manifesto". In Salah Hassan and Iftikhar Dadi (eds.), Unpacking Europe: Towards a Critical Reading. Rotterdam: Nai, 2001, pp. 244-55.

Difference and Repetition. Gilles Deleuze, New York: Columbia U PDeleuze, Gilles. Difference and Repetition. New York: Columbia U P, 1994.

. Gilles Deleuze, Félix Guattari, London: BloomsburyA Thousand PlateausFirst editionDeleuze, Gilles and Félix Guattari. A Thousand Plateaus. London: Bloomsbury, 2013. First edition, 1987.

What is a Minor Literature. Gilles Deleuze, Félix Guattari, Mississippi Review. 113Deleuze, Gilles and Félix Guattari. "What is a Minor Literature?". Mississippi Review, Vol. 11, No. 3, 1983, pp. 13-33.

The Generation of Postmemory: Writing and Visual Culture after the Holocaust. Marianne Hirsch, New York; Columbia U PHirsch, Marianne. The Generation of Postmemory: Writing and Visual Culture after the Holocaust. New York: Columbia U P, 2012.

The Long Shadow of the Past: Contemporary Austrian Literature, Film and Culture. Katya Krylova, Camden HouseWoodbridgeKrylova, Katya. The Long Shadow of the Past: Contemporary Austrian Literature, Film and Culture. Woodbridge: Camden House, 2017.

Writing by Germany's Jewish Minority. Erin Mcglothlin, Contemporary German Fiction: Writing in the Berlin Republic. Stuart TabernerCambridge; Cambridge U PMcGlothlin, Erin. "Writing by Germany's Jewish Minority". In Stuart Taberner (ed.), Contem- porary German Fiction: Writing in the Berlin Republic. Cambridge: Cambridge U P, 2007, pp. 230-46.

Alles geht in Trümmer -und das, was Öffentlichkeit war, wird bald nicht einmal mehr eine Erinnerung gewesen sein. Eva Menasse, Neue Zürcher Zeitung. Accessed 03.05.2020Menasse, Eva. "Alles geht in Trümmer -und das, was Öffentlichkeit war, wird bald nicht ein- mal mehr eine Erinnerung gewesen sein". Neue Zürcher Zeitung, 27 May 2019. https:// www.nzz.ch/feuilleton/eva-menasse-sieht-die-oeffentlichkeit-vor-dem-zerfall-ld.1484079 [Accessed 03.05.2020].

. Eva Menasse, Todsünden, Kiepenheuer & WitschCologneMenasse, Eva. Lässliche Todsünden. Cologne: Kiepenheuer & Witsch, 2009.

Eva Menasse, 10.4324/9780203074671Lieber aufgeregt als abgeklärt. Essays. Cologne: Kiepenheuer & Witsch, 2015. Menasse, Eva. Tiere für Fortgeschrittene. Cologne: Kiepenheuer & Witsch, 2017. Reiter, Andrea. Contemporary Jewish Writing: Austria after Waldheim. London/New York: Routledge. Menasse, Eva. Lieber aufgeregt als abgeklärt. Essays. Cologne: Kiepenheuer & Witsch, 2015. Menasse, Eva. Tiere für Fortgeschrittene. Cologne: Kiepenheuer & Witsch, 2017. Reiter, Andrea. Contemporary Jewish Writing: Austria after Waldheim. London/New York: Routledge, 2013. DOI: https://doi.org/10.4324/9780203074671

Für Juden ist es befremdlich, wie Porzellanelefanten behandelt zu werden', Eva Menasse im Interview. Thorsten Schmitz, Süddeutsche Zeitung. Accessed 03.15.2019Schmitz, Thorsten. "'Für Juden ist es befremdlich, wie Porzellanelefanten behandelt zu werden', Eva Menasse im Interview". Süddeutsche Zeitung, 20 May 2017. https://www. sueddeutsche.de/kultur/eva-menasse-im-interview-fuer-juden-ist-es-befremdlich-wie- porzellanelefanted-behandelt zu werden-1.3512912 [Accessed 03.15.2019].

The Re-Construction and Deconstruction of a Family Narrative: Eva Menasse's Vienna. Daphne Seemann, Valerie Heffernan and Gillian PyeAmsterdam/New York: RodopiTransitions: Emerging Women Writers in German-language LiteratureSeemann, Daphne. "The Re-Construction and Deconstruction of a Family Narrative: Eva Menasse's Vienna". In Valerie Heffernan and Gillian Pye (eds.), Transitions: Emerging Women Writers in German-language Literature. Amsterdam/New York: Rodopi, 2013, pp. 35-51.


Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

John Richard Tuttle
Candace H Haigler
Dominique Robertson
Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system
M E T H O D O L O G Y Open Access
Background: We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium(Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns.Results: The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively. Conclusions: These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.

Background

Virus-induced gene silencing (VIGS) is a reverse genetics technique that exploits the plant's post-transcriptional gene silencing (PTGS) machinery to obtain a sequencespecific transcript reduction for a targeted gene [1,2]. The technique employs a virus that has been modified to carry a fragment of a host gene. When the virus infects the plant, it triggers PTGS against the viral genome as well as the included host sequence. This leads to the degradation of RNAs with homology to the viral genome and produces a knockdown phenotype for the targeted gene(s). Because it can rapidly silence genes without the need for stable transformation, VIGS has become an attractive alternative to other reverse genetics strategies, which are timeconsuming and especially difficult in plant species like cotton that are recalcitrant to transformation/regeneration. VIGS vectors have been developed from a variety of virus/host combinations [1][2][3]. Although vectors have been constructed from RNA and DNA viruses as well as viral DNA satellites, we will only focus on vectors used in this research as derived from the bipartite DNA viruses of the family Geminiviridae, genus Begomovirus. These are single stranded DNA viruses with a conserved, well-characterized genome organization. The two genome components are designated DNA A and DNA B. Sequences coding for replication and movement proteins are split between the A and B components, respectively [4]. The A component contains 5 predicted open reading frames that code for the replication related proteins AL1 and AL3, the transactivator/anti-silencing protein AL2, the putative silencing suppressor AL4, and the coat protein AR1. The B component contains two open reading frames that code for the intercellular and intracellular movement proteins, BL1 and BR1 respectively. The two components share a~200-bp region of high homology [4] referred to as the common region, which contains the origin of replication and a consensus sequence that is cleaved and ligated by the AL1 protein during rolling circle replication [5,6]. When a common region is placed on either side of the genome as a direct repeat, the region between the replication origins is released in planta to form a functional viral episome [7,8].

To accommodate targeting sequences, a multiple cloning site is typically inserted either in place of the AR1 sequence or downstream of the BR1 gene [9][10][11][12]. Peele and coworkers reported that the latter approach resulted in more extensive silencing, but insertion of sequences downstream of the CLCrVBR1 gene failed to result in a systemic infection [9], [Tuttle and coworkers, unpublished]. The deletion or replacement of AR1 sequence renders begomoviruses non-transmissable by their whitefly vector [13,14]. Therefore, to achieve VIGS the viral vector must be introduced into the plant cells either mechanically or through the use of Agrobacterium vectors.

Particle bombardment employs a "gene gun" to blast particles coated with viral DNA into the plant. The gene gun can be commercial (BioRad's Helios or PDS-1000) or a homemade particle inflow gun (PIG; [15]). Both are powered by pressurized helium. A solenoid valve on the gas cylinder controls the rapid release of helium, which passes into a vacuum chamber through a filter holding micron or submicron particles of gold or tungsten that carry the nucleic acids. The metal particles are then forced into the samples below the filter [15]. Although there have been several publications detailing the construction of homemade gene guns, some degree of technical skill is required for their construction. This together with the high cost of commercial gene guns means that VIGS vectors that rely on biolistic delivery methods are not useful in all labs [15][16][17].

There are two other relatively easy and inexpensive methods for inoculating begomovirus vectors: DNA abrasion and agroinoculation. DNA abrasion involves the use of an abrasive (carborundum or ground glass) to introduce a viral DNA solution into leaf cells [18]. Although this method of inoculation is reasonably efficient for viruses that are not phloem-limited, it is not effective for all virus/host combinations, including Cotton leaf crumple virus (CLCrV)/cotton, the combination described here. The success of this method depends in part on the tissue specificity of the virus as well as the mechanical properties of the host leaf being inoculated [18,19]. For inoculation using Agrobacterium [20], similar to biolistic vectors, each component of the vector must be flanked by directly repeated common regions. For bipartite begomoviruses, a mixture of Agrobacterium carrying the A and B component plasmids is introduced into the plant through stem inoculation, wounding, or infiltration into intercellular leaf spaces [20][21][22]. In each of these methods, a single unit-length viral genome component is released from plasmid DNA to establish a systemic infection.

Cotton leaf crumple virus (CLCrV) is a cotton-infecting geminivirus endemic to the southwestern United States and Mexico [23]. Because it is a vascular-associated virus [12,24] it is more difficult to inoculate than other begomoviruses that show broader tissue specificity [19]. We developed the A DNA as a vector for foreign DNA by replacing the coat protein gene with a multiple cloning site and demonstrated VIGS in cotton (Gossypium hirsutum) [12]. Co-bombardment of cotyledons with the modified A DNA and wild type B-DNA produced systemic silencing in several G. hirsutum cultivars that persisted throughout the plant for over a year and was most extensive in the cultivars Acala SJ-1 and Deltatype Weber [25]. VIGS was visualized using a 500-bp fragment of the chlorophyll biosynthetic gene, Magnesium Chelatase subunit I (ChlI). Silencing of this gene produced a sectored pattern of chlorophyll loss that was more extensive at lower growth temperatures [12]. We also showed that CLCrV could be used as an expression vector by inserting a full-length GFP in place of the coat protein gene [12].

In this paper, we expand the potential of cotton VIGS by: (a) demonstrating the effectiveness of CLCrV VIGS vectors after moving them into binary vectors for agroinoculation and (b) providing instructions for making two inexpensive gene guns and demonstrating their potential for inoculating VIGS vectors.

Results and discussion

Binary vectors for Agrobacterium-mediated delivery of CLCrV Many labs lack the necessary equipment to inoculate the biolistic form of the CLCrV vector. To address this, we cloned each of the vector's components into the opensource binary plasmid, pCambia1300 (CAMBIA, Canberra, Australia). The A-DNA vector, pJRT.Agro. CLCrVA.008 (referred to as CLCrVA:CP-), contains a multiple cloning site in place of the coat protein gene and produces an episome that is identical to that from the biolistic vector following inoculation. Episomes from the B-DNA binary plasmid, pJRT.Agro.CLCrVB1.3 (referred to as CLCrVB), are also identical to their biolistic counterpart. Two additional A-DNA plasmids, one for silencing ChlI (pJRT.Agro.CLCrVA.009) and one expressing GFP (pJRT.Agro.CLCrVA.010), were made by swapping XbaI/SacI fragments consisting of the 3' region of the AL1 gene, AL2, AL3, the multiple cloning site, and one of the two duplicated common regions with the same region from the biolistic vector ( Figure 1). These plasmids will be referred to as CLCrVA:ChlI and CLCrVA:GFP, respectively. Binary plasmids were transformed into the Agrobacterium strain GV3101:pMP90 [26] using the freeze-thaw method [27]. The plasmid pMP90 was derived from the pTiC58 plasmid pGV2201 and contains a deleted T-DNA region, the necessary virulence functions, and gentamycin resistance [26].

We used CLCrVA:ChlI to compare the efficiency and extent of silencing from the biolistic and Agrobacteriumbased methods of delivery. Cotton seedlings were agroinoculated by infiltration of the bottom surface of the cotyledons with a 1-ml syringe lacking a needle. The inoculum consisted of 1:1 mixtures of Agrobacterium cultures harboring CLCrVA:ChlI or CLCrVB. Following infiltration, theChlI silencing phenotype of leaf yellowing was first observed at 12 to 20 days post inoculation (dpi). Both particle bombardment and agroinoculation methods resulted in similar silencing efficiencies ( Figure 2).We obtained an average inoculation efficiency of 81% (n = 30) over three agroinoculation experiments, which was not significantly different from the 69% efficiency we observed for three biolistic experiments (n = 27; p-value 0.34; two-tailed non-parametric t-test). Both the onset and extent of ChlI silencing after agroinoculation were similar to plants inoculated by particle bombardment (Figure 3). Similarly, the expression of solublemodified red-shifted GFP (smRS-GFP) [28] from the  The efficiency of silencing from agroinoculation and particle bombardment of CLCrVA:ChlI was comparable. Dark and light grey bars represent silencing efficiencies in three agroinoculation or three particle bombardment experiments, respectively. Particle bombardment was conducted as described previously (12) with the homemade gun described by Finer and coworkers (15).

Agrobacterium vector CLCrVA:GFP was also comparable to particle bombardment. As described previously [12], GFP fluorescence was confined to vascular associated cells, reflecting the known tissue specificity of CLCrV (data not shown).

The biolistic vector has 6 different unique restriction sites in its MCS but, due to its larger size, only two of these sites are unique in the Agrobacterium vector. This makes cloning silencing fragments into the Agrobacterium vector more difficult. To overcome this, silencing fragments can be introduced into the biolistic CLCrV A vector and then sub-cloned into the Agrobacterium vector using SacI and XbaI (Figure 1). Although not in the MCS, the SacI and XbaI sites occur only once in both of the CLCrV A vectors.

One minor drawback of agroinoculation was that initial seedling growth was slightly delayed compared to their biolistic counterparts for both ChlI silencing and GFP expression constructs. This difference occurred despite the production of identical viral episomes in both cases. The transient slower growth of agroinoculated plants was consistent with native Agrobacterium acting as a plant pathogen [29] and causing systemic changes in inoculated plants [30].

Development of low-cost particle inflow guns

As an alternative to commercial particle bombardment systems, a 5-dollar pump-action plastic water gun can serve as a gene gun ( Figure 4A,B). The only modifications required were the removal of a small plastic tip covering the outlet nozzle, and cutting of the outlet nozzle to match the outer diameter of a Millipore swinex syringe filter, used to hold the DNA-coated particles ( Figure 4B). The gun employed a small hand-powered pump to charge a cylinder with compressed air, which was then released to travel through the filter tip so that the 1-micron gold particles carrying the VIGS vectors were propelled into the target cells. At least one comparatively simple gene gun has been described previously [17], but required the use of a separate compressed air supply.

This gun was used to inoculate cotton with the biolistic CLCrV-based VIGS vector carrying a 500-bp fragment of the ChlI gene. The number of pump strokes was used to gauge pressure in the gun, and 40 to 60 strokes resulted in successful inoculation of the CLCrV silencing vector. Inoculation efficiency was visually assessed by counting the number of plants showing photobleaching. In a preliminary experiment, silencing was observed in 1 of 3 inoculated cotton plants, but in a subsequent experiment silencing was observed in 3 of 6 inoculated cotton plants ( Figure 5A). More plants can be inoculated to compensate for the relatively low inoculation efficiency with this simple gene gun. The extent of silencing (yellow leaf area) was similar to plants inoculated by other methods.

Although it was useful, the modified water gun did not withstand the rigorous cleaning needed to conduct sterile bombardments. Therefore, we developed a more durable version of the gun from off-the-shelf parts purchased at a hardware store for approximately 50 dollars. Most of the cost was attributable to the brass components. The metal gun was used to introduce principles of gene silencing to students in a biochemistry lab. In these experiments both wild type and Tomato golden mosaic virus (TGMV)-derived VIGS vectors were inoculated into Nicotian abenthamiana. Efficiency was assessed with either photobleaching from the TGMV silencing vector carrying a 156-bp ChlI fragment (TGMVB:ChlI [9]) or symptom development (contorted leaves) from the wildtype virus. The built-in gauge on the bicycle pump was used to measure pressure in the gun. A pressure of 80 lb/ in 2 was sufficient to obtain silencing in 3 of 5 inoculated plants and to produce TGMV infection in 5 of 5 inoculated plants ( Figure 5B,C).

Conclusions

Tools for several affordable methods for the delivery of the CLCrV VIGS vector have been developed and demonstrated. The new molecular tools were binary plasmids for agroinoculation of the CLCrV cotton VIGS vector by syringe infiltration. Agroinoculation resulted in essentially equivalent efficiency and extent of silencing compared to biolistic inoculation. The Agrobacteriumvectors were larger than the biolistic VIGS vectors and thus contain fewer unique restriction sites for cloning in silencing fragments, but the ability to sub-clone fragments from the biolistic vectors circumvents this problem. The new hardware consisted of two low-cost Figure 4 Examples of two homemade gene guns. A, B) A plastic gene gun made from a slightly modified 5-dollar water gun. A) The only modification required was to connect a Millipore swinex filter tip to the nozzle for compressed air. This was achieved by removing three screws in the plastic body, followed by the plastic nozzle cover (arrow). B) The $5 gene gun with the filter tip screwed into the cut-off nozzle (arrow). C) An example of a metal gene gun built for approximately $50 from parts at a local hardware store (see Materials and Methods for construction details).

Figure 5

Chl Isilencing from two different VIGS vectors inoculated using two low-cost particle inflow guns. A) ChlI silencing in cotton (64 dpi) co-bombarded with the CLCrVA:ChlI and CLCrVB plasmids using the modified plastic water gun shown in Figure 4A,B. B) N. benthamiana (37 dpi) showing the viral symptom of contorted upper leaves (arrows) after bombardment with the wild type TGMV virus using the homemade metal gene gun. C) N. benthamiana (37 dpi) showing ChlI silencing after co-bombardment with TGMVA and TGMVB:ChlI using the metal gene gun ( Figure 4C).

($5 -$50) particle inflow gunsmade exclusively from offthe-shelf components that can be used instead of expensive commercial devices for biolistic inoculation. The $50 gene gun is durable and withstands rigorous cleaning while also being easy to construct and use. These simple and affordable biolistic guns will extend the potential for research and teaching based on VIGS.

Methods

Vector availability

All CLCrV vectors described here are available for nonprofit research from Addgene.org following a standard material transfer agreement.

Cloning the vector into pCambia

To generate pJRT.Agro.CLCrVA.008, the biolistic empty vector plasmid pJRTCLCrVA.008 [12] was digested with XbaI and XhoI. A 1130-bp fragment from pJRTCLCrVA.008 containing one common region, AL4, and the 5' 664-bp of AL1 was ligated into pCambia1300 cut with XbaI and SalI to produce pJRT.Agro. CLCrVA.008.1. In a second digestion and ligation, pJRTCLCrVA.008 was cut with XbaI and SacI and the 1386-bp resulting fragment was cloned into pJRT.Agro. CLCrVA.008.1 cut with XbaI and SacI to generate a 11,443-bp plasmid pJRT.Agro.CLCrVA.008.

To produce the ChlI-silencing and GFP-expressing marker constructs pJRT.Agro.CLCrVA.009 and pJRT.Agro. CLCrVA.010, the biolistic constructs pJRTCLCrVA.009 and pJRTCLCrVA.010 were each digested with SacI and XbaI to release 1,883-bp and 2,062-bp fragments respectively. These fragments were then ligated into SacI, XbaI digested pJRT.Agro.CLCrVA.008 to produce pJRT.Agro. CLCrVA.009 and pJRT.Agro.CLCrVA.010.

Prior to inserting the B component of the viral genome into pCambia1300, it was necessary to reduce the duplication of B component sequence from a complete dimer [31] to a version with only one BR1 gene. This was accomplished by using PCR and the primers BDAgR1new (5' -ACC CAG ACT AGT AAA CGC TAT TAT ATA GG -3') and BDAgF1 (5' -GCG GAG CTC CAG AAC GAT CTC AGT TAG GTC ATG GG -3') to amplify a 906-bp fragment, containing a single common region, from the CLCrV B dimer. The BDAgF1 primer introduced a SacI restriction enzyme site at the 5' end of the PCR product. The additional SacI site was used in conjunction with an internal SpeI site to insert the 896bp fragment into the respective restriction sites of the pBluescript SK + II vector (Agilent Technologies, www. agilent.com) to create pJRTCLCrVB.1. Next, a 2,549-bp fragment was cut from the CLCrV B dimer using the restriction enzyme SpeI. This fragment contained a second viral common region as well as the two full-length open reading frames BL1 and BR1. The fragment was purified by gel extraction and ligated into the SpeI restriction site of pJRTCLCrVB.1. The resulting clones were screened for proper orientation of the insert by XbaI digestion and called pJRTCLCrVB1.3. The plasmid JRTCLCrVB1.3 contains two viral common regions that flank the two fulllength open reading frames BL1 and BR1 in the pBluescript SK+ II vector.

After construction of pJRTCLCrVB1.3, the construct was digested with the restriction enzymes HindIII and SacI. The 3,490-bp fragment produced from this digestion was cloned into the respective sites in the HindIII/SacI digested and dephosphorylated pCambia1300 plasmid to produce the 12,400-bp plasmid pJRT.Agro.CLCrVB1.3.

Agroinoculation of the vector

A. tumefaciens strain GV3101:pMP90 was first made competent by inoculating 50 ml of Luria Bertani medium with 125 μL of an overnight Agrobacterium culture. The 50-ml culture was then grown at 30°C for 12 hours, spun for 10 minutes at 4000 x rpm to pellet cells, and washed in 5 ml of sterile TE buffer; this step was repeated once. The TE buffer was removed and the cells were then resuspended in 5 ml of Luria Bertani medium and 200-μL aliquots were refrozen in liquid nitrogen.

For transformation, cell aliquots were thawed on ice, and 1 μg of plasmid DNA was added to the cells on ice for 5 minutes. Next, the cells and DNA were transferred to liquid nitrogen for 5 minutes, then a 37°C water bath for 5 minutes. 1 ml of Luria Bertani medium was added to the tube, and the cells were incubated at room temperature for 4 hours with agitation. The culture was spun down for 10 minutes at 4000 rpm and all but approximately 100 μL of the supernatant was removed. Cells were resuspended and then plated on Petri plates with 25 μg/ml each of kanamycin, rifampicin, and gentamycin and allowed to grow for 2 days at 30°C.

The vector was introduced into cotton seedlings by infiltration. Cultures of each component were prepared as previously described [32]. After adjusting the density of each culture (OD 600 of 1.5) and allowing them to incubate at room temperature in the dark for 4 hours without shaking, A and B component cultures were mixed in a 1:1 ratio and drawn up into a needleless 5-cc syringe. The mixture was introduced into cotton seedlings at the cotyledon stage by gently forcing the solution into the spongy mesophyll on the bottom surface of the cotyledon. As the infiltration solution moved through the cotyledon, it became noticeably darker, and infiltration was continued until the entire cotyledon was darkened.

Construction of a low-cost particle delivery device

To construct an extremely low cost particle delivery device, we first purchased a pump-action water gun; the MAX D 2000 (Hasbro Inc., email@example.com) from a local store for approximately 4 dollars. A small Phillips head screwdriver was used to remove three screws from the plastic housing on the front of the gun. This allowed the plastic housing to be pried open just enough for the plastic nozzle cover to be removed. The exposed conical nozzle of the gun was then modified to hold a 13 mm Millipore swinex filter tip (Millipore Inc., www.millipore.com). The plastic nozzle was cut back (~18 mm) to the outer housing so that its inner diameter was large enough to allow for the insertion of the threaded filter tip. For inoculation, the built-in hand pump was pumped 40-60 times immediately prior to bombardment. No vacuum was used and the tip of the Millipore filter was placed less than a centimeter from the bottom surface of the cotyledon.

A second more durable gun was constructed consisting of a bicycle pump connected to one end of a brass cylinder via a section of 6-ft fuel line with a ¼-in inner diameter. A blowgun was adapted to the other end of the brass cylinder and fitted with a 13-mm Millipore swinex tip. A bicycle pump with a pressure gauge rated up to 100 lb/in 2 (Schwinn Inc., www.schwinn.com) was adapted to the fuel line using a ¼-in inflation nozzle (provided in a Kobalt 5-PC blowgun kit; Lowes Inc., www.lowes.com, Item #001174).This was essentially a ¼in threaded male fitting on one end and a barb on the other. The fuel line was secured to the barb using a #4 pipe clamp (King Seal Fastener Technology Co., Ltd., Anhui, China, Item #62508). The other end of the fuel line was connected to a brass ¼-in male barb adapter (Watts Industries Inc., www.watts.com, Item #A-192) and was secured with a #4 pipe clamp. The ¼-in male barb adapter was screwed into a female ¼-in to ½-in brass coupling (Watts Industries Inc., Item #A-813) that was threaded onto a ½-in X 6-in brass pipe nipple (Watts Industries Inc., Item #LA-845). Another ¼-in to ½-in female coupling was put in place at the other end of the brass pipe. This coupling was attached to a Kobalt blowgun via a ¼-in male brass nipple (Watts Industries Inc., Item #A-875). The end of the blowgun was fitted with a blowgun adapter included in the above mentioned kit, and the Millipore swinex filter tip was threaded directly into this adapter. For inoculation of N. benthamiana with TGMV, the bicycle pump was pumped to approximately 80 lb/in 2 and the trigger of the blowgun was quickly compressed with the tip of the Millipore syringe filter held less than 1 cm from the surface of the leaf.

For a schematic image of this gene gun see Additional file 1: Figure S1.

Microcarrier preparation

For particle bombardment, aliquots of 1 μm gold particles (INBIO Gold, Victoria, AU, Catalog #BD061) were prepared by suspending 60 mg of gold powder in 1 ml of 100% ethanol. Particles were pelleted by centrifugation for 10 seconds at 13,000 rpm. The supernatant was discarded and the particles were resuspended in 1 ml of sterile water. The particles were pelleted by centrifugation and resuspended in sterile water two additional times. Following the final resuspension, 50-μl aliquots were transferred to 1.5 ml microfuge tubes and stored at −20°C.

To precipitate viral DNA onto the gold particles, 5 μg of each viral component was added to a 50-μl aliquot and vortexed for 30 seconds. 50 μl of 2.5 M CaCl 2 was added and the tube was vortexed for 30 seconds. 20 μl of 0.1 M spermidine was added to the tube and the mixture was vortexed for 3 minutes. The particles were pelleted by centrifugation at 10,000 rpm for 10 seconds. The supernatant was removed and discarded. The pellet was resuspended in 250 μl of 100% ethanol by vortexing. The particles were again pelleted by centrifugation at 10,000 rpm for 10 seconds and the supernatant was discarded. The pellet was resuspended in 65 μl of 100% ethanol. 12 μl of the particle suspension was loaded onto the center of the filter tip for bombardment (see Additional file 2: Figure S2).

Additional files

Additional file 1: Schematic image of metal gene gun. Image of a disassembled metal gene gun labeled with corresponding part numbers.

Competing interests

The authors declare that they have no competing interests.

Figure 1
1Details of the CLCrV-derived VIGS vectors described in this paper. A, B) Maps of CLCrV plasmids for biolistic inoculation. C,D) Maps of the CLCrV:CP-(C) and CLCrVB (D) binary vectors for agroinoculation. The pBluescript SKII + (A,B) and the pCambia1300 (C,D) plasmid backbones are shown by a thin black line and geminivirus DNA is blue or red. Unique SacI and XbaI sites that may be used to sub-clone silencing fragments from the biolistic vector into the Agrobacterium vector are marked with asterisks, and the resulting fragment is shown in red. MCSmultiple cloning site (light blue), CRcommon region (green), LB and RBleft and right T-DNA borders respectively, HygRhygromycin resistance, AmpRampicillin resistance, KanRkanamycin resistance. AL1, AL2, AL3, and AL4 are genes encoded by the A-DNA and BL1 and BR1 by the B-DNA of CLCrV.

Figure 2
2Figure 2 The efficiency of silencing from agroinoculation and particle bombardment of CLCrVA:ChlI was comparable. Dark and light grey bars represent silencing efficiencies in three agroinoculation or three particle bombardment experiments, respectively. Particle bombardment was conducted as described previously (12) with the homemade gun described by Finer and coworkers (15).

Figure 3
3Silencing and expression from the CLCrV Agrobacterium vector was comparable to the biolistic vector. A) New growth on cotton plants agro-inoculated with the CLCrVB and CLCrV:CP-lacked visible symptoms at 35 dpi. B) New growth on cotton plants agro-inoculated with CLCrVB and CLCrVA:ChlI showed a sectored loss of chlorophyll throughout the leaf at 35 dpi. C) Plant bombarded with the biolistic form of the vector used in (B) showed a similar pattern of silencing as the plant shown in (B) at 38 dpi. D) Overlay of GFP and brightfield images showed vein-delimited GFP expression (white arrowhead) in new growth of plants agro-inoculated with CLCrVB and CLCrVA:GFP at 28 dpi.

plants (G. hirsutum cv Deltapine 4515) were grown in a 25°C/23°C (day/night) chamber at approximately 60% relative humidity under a bank of 16 VHO T5 fluorescent lamps fixed at a height of 27 inches. Lighting (135 μmol s -1 m -2 at the shelf level) was longday: 16 hours on, 8 hours off. Cotton plants were grown in 6-in diameter pots in Metromix 360 potting mix (Wyatt Quarles Seed Company, www.wqseeds.com), watered daily, and fertilized once per week with Miracle Grow W (The Scotts Company LLC, www.scotts.com).

Additional file 2 :
2Close-up image of Millipore swinex filter tip. Close-up image of a disassembled Millipore swinex filter tip. The red arrow marks the placement of the microcarrier suspension.

Abbreviations VIGS: Virus-induced gene silencing; CLCrV: Cotton leaf crumple virus; TGMV: Tomato golden mosaic virus; PTGS: Post-transcriptional gene silencing; PIG: Particle inflow gun; ChlI: Magnesium chelatase subunit I; dpi: Days post infection; smRS-GFP: Soluble-modified red-shifted green fluorescent protein; CR: Common region; LB: Left border; RB: Right border.
http://www.plantmethods.com/content/8/1/27
AcknowledgementsFor research support, we thank Cotton Inc. (Cary, NC) and the National Science Foundation (grant number IOS1025947).AuthorAuthors' contributions JRT carried out the molecular cloning, test inoculations, developed the gene guns, and drafted the manuscript. CHH and DR helped to plan research and write the manuscript. All authors read and approved the final manuscript.
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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. Tuttle, Plant Methods. 827Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system. Submit your manuscript at www.biomedcentral.com/submitCite this article as: Tuttle et al.: Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system. Plant Methods 2012 8:27. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit