vstispСадоводство и виноградарствоHorticulture and viticulture0235-25912618-9003Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»10.31676/0235-2591-2022-3-15-29vstisp-876Research ArticleГЕНЕТИКА, СЕЛЕКЦИЯ, СЕМЕНОВОДСТВОGENETICS, BREEDING, SEED PRODUCTIONОценка влияния трансгенного компонента привойно-подвойной комбинации на устойчивость к вирусу шарки сливыEvaluation of the eff ect of the transgenic component of the graft-twin combination on resistance to the Plum pox virusСидороваТ. Н.SidorovaT. N.

Сидорова Т. Н. — младший научный сотрудник

проспект Науки, 6, г. Пущино, Московская область, 142290

Sidorova T. N., Junior Researcher

6, Prospekt Nauki, Pushchino, Moscow region, 142290

МирошниченкоД. Н.MiroshnichenkoD. N.

Мирошниченко Д. Н. — кандидат биологических наук, старший научный сотрудник

проспект Науки, 6, г. Пущино, Московская область, 142290

Miroshnichenko D. N., PhD (Biol.), Senior Researcher, Chief Researcher

6, Prospekt Nauki, Pushchino, Moscow region, 142290

КировИ. В.KirovI. V.

Киров И. В. — кандидат биологических наук, старший научный сотрудник

Москва

Kirov I. V., PhD (Biol.), Senior Researcher

Moscow

ПушинА. С.PushinA. S.

Пушин А. С. — младший научный сотрудник

проспект Науки, 6, г. Пущино, Московская область, 142290

Pushin A. S., Junior Researcher

6, Prospekt Nauki, Pushchino, Moscow region, 142290

ДолговС. В.DolgovS. V.

Долгов Сергей Владимирович — доктор биологических наук, главный научный сотрудник

проспект Науки, 6, г. Пущино, Московская область, 142290

Sergey V. Dolgov, Dr. Sci. (Biol.), Chief Researcher

6, Prospekt Nauki, Pushchino, Moscow region, 142290

dolgov@bibch.ruФилиал Института биоорганической химии им. М. М. Шемякина и Ю. А. Овчинникова Российской академии наукРоссияBranch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of ScienceRussian FederationФилиал Института биоорганической химии им. М. М. Шемякина и Ю. А. Овчинникова Российской академии наук; Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии Российской академии наукРоссияBranch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science; All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of ScienceRussian FederationВсероссийский научно-исследовательский институт сельскохозяйственной биотехнологии Российской академии наукРоссияAll-Russia Research Institute of Agricultural Biotechnology, Russian Academy of ScienceRussian FederationФилиал Института биоорганической химии им. М. М. Шемякина и Ю. А. Овчинникова Российской академии наук; Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии Российской академии наукРоссияBranch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science; All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of ScienceRussian FederationФилиал Института биоорганической химии им. М. М. Шемякина и Ю. А. Овчинникова Российской академии наук; Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии Российской академии наук; Федеральный научный селекционно-технологический центр садоводства и питомниководстваРоссияBranch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science; All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Science; Federal Horticultural Center for Breeding, Agrotechnology and NurseryRussian Federation202215062022031529Copyright © Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture», 20222022Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»https://www.sadivin.com/jour/about/submissions#copyrightNoticehttps://www.sadivin.com/jour/article/view/876

У косточковых плодовых культур устойчивость к вирусу шарки сливы (PPV) может быть достигнута за счет специфической деградации вирусной РНК по механизму РНК-интерференции (RNAi). Однако трансгенные устойчивые к вирусам растения вызывают серьезные опасения относительно биобезопасности из-за встраивания и экспрессии шпилечных конструкций, которые обычно содержат различные селективные чужеродные гены. Поскольку взрослое дерево плодовых культур представляет собой комбинацию привоя и подвоя, прививка коммерческих сортов на трансгенные устойчивые к вирусам подвои является возможным подходом для смягчения проблем биобезопасности. Настоящее исследование направлено на получения ответа на следующий вопрос: в какой степени сигналы сайленсинга малые интерферирующие РНК (siRNA) передаются из трансгенного подвоя в прививку коммерческого сорта и насколько это влияет на его устойчивость к PPV? Были изучены две комбинации, NT:GM и GM:NT (привой:подвой), с акцентом на первую комбинацию. Инокуляцию вирусом проводили либо на привое, либо на подвое. Форму клонового подвоя косточковых культур `Элита` [(Prunus pumila L..P. salicina Lindl.)x(P. cerasifera Ehrh.)] использовали в качестве подвоя для домашней сливы сорта Стартовая (Prunus domestica L.). Трансгенные формы как подвоя, так и привоя экспрессировали конструкцию РНК- шпильки, генерирующую малые интерферирующие РНК (siRNA) комплементарные гену белка оболочки PPV (CP), и демонстрировали высокую вирусную устойчивость. Данные о последовательностях пулов малых РНК (sRNA) показали, что накопление специфичной для конструкции малой интерферирующей (siRNA) в трансгенном подвое сливы достигло более 2 % от общего количества. Повышенный уровень siRNA обеспечивал устойчивость к PPV и блокировал перемещение вируса через GM-ткани к NT-партнеру при инокуляции трансгенных тканей. В то же время сигнал мобильной siRNA не перемещался от GM-подвоя к целевой NT-ткани привоя до уровня, достаточного для запуска сайленсинга транскриптов PPV и обеспечения надежной вирусной устойчивости. Отсутствие подвижности молекул siRNA, полученных из трансгена, сопровождалось переносом различных эндогенных специфичных для подвоя siRNA в NT-привой, что указывает на исключительное нарушение транзитивности исследуемого сигнала РНК интерференции. Представленные здесь результаты показывают, что трансплантация косточковых плодовых культур остается непредсказуемой практикой и требует дальнейшего изучения транспорта siRNA в комбинации подвой/привой на молекулярном уровне.

In stone fruit trees, resistance to Plum pox virus (PPV) can be achieved through the specific degradation of viral RNA by the mechanism of RNA interference (RNAi). Transgenic virus-resistant plants, however, raise serious biosafety concerns due to the insertion and expression of hairpin constructs that usually contain various selective foreign genes. Since a mature stone tree represents a combination of scion and rootstock, grafting commercial varieties onto transgenic virus-tolerant rootstocks is a possible approach to mitigate biosafety problems. The present study was aimed at answering the following question: To what extent are molecular RNAi silencing signals transmitted across graft junctions in transgrafted plum trees and how much does it affect PPV resistance in genetically modified (GM)/non-transgenic (NT) counterparts? Two combinations, NT:GM and GM:NT (scion:rootstock), were studied, with an emphasis on the first transgrafting scenario. Viral inoculation was carried out on either the scion or the rootstock. The interspecific rootstock `Elita` [(Prunus pumila L..P. salicina Lindl.)x(P. cerasifera Ehrh.)] was combined with cv. Startovaya (Prunus domestica L.) as a scion. Transgenic plum lines of both cultivars were transformed with a PPV-coat protein (CP)-derived intron-separate hairpin-RNA construct and displayed substantial viral resistance. High-throughput sequence data of small RNA (sRNA) pools indicated that the accumulation of construct-specific small interfering RNA (siRNA) in transgenic plum rootstock reached over 2 %. The elevated siRNA level enabled the resistance to PPV and blocked the movement of the virus through the GM tissues into the NT partner when the transgenic tissues were inoculated. At the same time, the mobile siRNA signal was not moved from the GM rootstock to the target NT tissue to a level sufficient to trigger silencing of PPV transcripts and provide reliable viral resistance. Th e lack of mobility of transgenederived siRNA molecules was accompanied by the transfer of various endogenous rootstock-specific siRNAs into the NT scion, indicating the exceptional transitivity failure of the studied RNAi signal. The results presented here indicate that transgrafting in woody fruit trees remains an unpredictable practice and needs further in-depth examination to deliver molecular silencing signals.

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The authors declare that there are no conflicts of interest present.