Accessible Unlicensed Requires Authentication Published by De Gruyter December 1, 2018

Characterization of Non-Target-Site Mechanisms in Imidazolinone-Resistant Sunflower by RNA-seq

Mercedes Gil ORCID logo, Tatiana Vega, Silvina Felitti, Liliana Picardi, Sandrine Balzergue and Graciela Nestares
From the journal Helia

Abstract

Imisun sunflowers (Helianthus annuus L.) are imidazolinone-resistant cultivars in which the two mechanisms of herbicide resistance coexist: (i) mutation in herbicide target-site (target-site resistance) and (ii) non-target-site resistance (NTSR). In Imisun technology, NTSR could be related to herbicide metabolism and might occur as a result of a constitutive up-regulation of resistance genes, or it can appear only after herbicide treatment. The objective of this study was to characterize NTSR in Imisun sunflower in response to imazethapyr using RNA-Seq and to determine whether these mechanisms are constitutive or herbicide-induced. Cypsels were germinated in plastic pots, watered by capillarity and growth in chamber under controlled conditions. Seven-day-old plants were treated with 0 (control) and 1 μM imazethapyr for 12 h. After leaf RNA purification, stranded, paired-end cDNA libraries were constructed. Sequencing was performed with Illumina HiSeq2000. Local mapping, with and without multihits, was carried out over the reference transcriptome HaT13l and differential expression was analysed. Sixty one and 47 contigs (according to mapping strategy) related to xenobiotic metabolism were found: cytochromes P450s, ABC transporters, glycosyltransferases, UDPglucuronosyl/glucosyltransferases and glutathione S-transferases. None of these contigs showed differential expression between control and imazethapyr-treated plants. Seventeen interesting contigs were verified by qRT-PCR. These results suggest that constitutive NTSR mechanisms may account for imidazolinone resistance in Imisun sunflower.

Resumen

Caracterización de mecanismos de resistencia no relacionados al sitio de acción en girasol mediante RNA-seq

Los girasoles Imisun son cultivares resistentes a imidazolinonas en los cuales coexisten dos mecanismos de resistencia: (i) una mutación puntual en el sitio blanco del herbicida (resistencia relacionada al sitio de acción) y (ii) resistencia no relacionada al sitio de acción (NTSR). En la tecnología Imisun, NTSR podría estar relacionada con el metabolismo del herbicida y podría resultar de una sobreexpresión constitutiva de los genes detoxificadores, o inducida luego del tratamiento con herbicida. El objetivo de este trabajo fue caracterizar NTSR en girasoles Imisun en respuesta al tratamiento de imazetapir mediante RNA-seq, y determinar si estos mecanismos son constitutivo o inducidos por el herbicida. Las cipselas fueron germinadas en multimacetas, regadas por capilaridad y se mantuvieron en una cámara bajo condiciones controladas. Plantas de 7 días fueron tratadas con imazetapir 0 (control) y 1 μM por 12 h. Luego de la purificación del RNA de hoja, se construyeron bibliotecas de cDNA stranded y paired-end. La secuenciación se llevó a cabo en Illumina HiSeq2000. Se realizaron dos tipos de estrategias de mapeo local contra el transcriptoma de referencia HaT13l, incluyendo y filtrando multihits, respectivamente y se realizó el análisis de la expresión diferencial. Se identificaron 61 y 47 contigs (de acuerdo a la estrategia de mapeo) relacionados a metabolismo de xenobióticos: citocromos P450, transportadores ABC, glicosiltransferasas, UDPglucuronosil/glucosiltransferasas y glutatión S-transferasas. No se encontró expresión diferencial entre las plantas tratadas y sin tratar con imazetapir para ninguno de los contigs. La expresión de 17 contigs de interés fue validada mediante qRT-PCR. Estos resultados sugieren que mecanismos NTSR constitutivos estarían involucrados con la resistencia a imidazolinonas en girasol.

Résumé

Caractérisation de la réponse NTSR à l’herbicide imazethapyr chez le tournesol Imisun en utilisant la technologie RNA-seq

Les tournesols (génotype Imisun) sont des cultivars imidazolidone-résistants au sein desquels cohabitent 2 mécanismes distints de résistance à cet herbicide: (1) mutation de type « gène cible » et (2) une résistance non géniquement ciblé (NTSR: Non-Target-Site-Resistance). Dans le génotype Imisun, la résistance de type NTSR pourraient être soit reliée au métabolisme de l’herbicide en lui-même, conduisant ainsi à une régulation positive et constitutive des gènes de résistance, soit apparaitrait seulement après chaque traitement herbicide. L’objectif de ce projet était de caractériser la réponse NTSR à l’herbicide « imazethapyr » chez le tournesol Imisun en utilisant la technologie RNA-seq et déterminer ainsi si ce mécanisme était constitif ou induit par l’herbicide seulement. Pour ce faire, des graines ont été mis à germer dans des pôts en plastiques arrosés par capilarité et mis en culture dans des chambres climatiques en conditions environnementales controlées. Des plants de 7 jours ont été traités avec 0 µM d’imazethapyr (condition « controle ») et 1 µM d’imazethapyr (condition « traitement ») pendant 12 h. Après extraction des ARN totaux des feuilles, des banques cDNA orientées et pairées ont été construites. Le séquençage a été réalisé sur des machines Illumina HiSeq2000. Un mapping « local », avec ou sans prise en compte des hits multiples, a été réalisé contre le transcriptome de référence HaT13l. L’analyse différentielle de l’expression génique entre les 2 conditions (« contrôle » vs « traitement ») a été réalisée. Respectivement 61 et 47 contigs ont été trouvés relatif au métabolisme des xenobiotiques selon à méthode de mapping: cytochromes P450, ABC transporters, glycotransferases, UDPglucuronosyl/glucosyltransferases et des glutathione-S-transferases. Aucun de ces contigs n’a été trouvés comme différentiellement exprimés entre la condition controle et le traitement à l’imazethapyr. Dix-sept contigs d’intérets ont été vérifiés par qRT-PCR. Ces résultats suggèrent qu’une réponse constitutive des mécanismes NTSR pourrait expliquer la résistance à l’imidazolinone chez le tournesol (génotype Imisun).

Acknowledgements

Thanks are due to Dr Jerry Miller and to Dr José María Bruniard for kindly providing seed materials. This work was supported by grants from FONCyT (PICT 2013-1010) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Proyecto P-UE 22920160100043). Funding was received from Fundación Tecnológica Argentina para el Desarrollo Social y Ambiental. M. Gil has been awarded a Postgraduate Research Scholarship by Jefatura de Gabinete de la Nación Argentina, BEC.AR 2013. The authors are grateful to Gabriela Venturi for English editing.

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/helia-2018-0012).

Received: 2018-08-22
Accepted: 2018-10-11
Published Online: 2018-12-01

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