Transformation of Madagascar periwinkle (Catharanthus roseus) for studying infection with ʼCandidatus Phytoplasma solaniʼ and enzymatic activities in infected plants

Anžič, Barbara (author), Dermastia, Marina (mentor), Roitsch, Thomas (comentor)

'Candidatus Phytoplasma solani' is an intracellular plant pathogenic bacterium that lives in phloem tissues and is transmitted from plant to plant by insects through ingestion of the infected phloem sap. This phytoplasma can cause various diseases of important crop plants, presumably through effectors. Previous studies have indicated that phytoplasma infections affect the metabolism of host plant carbohydrates and phenolic compounds, which can be associated with reactive oxygen species and antioxidant enzymes. As it is still not possible to routinely cultivate phytoplasmas under in-vitro conditions, we use phytoplasma-infected Catharanthus roseus as the model plant for this research. In this thesis, we attempted to establish a protocol for stable transformation of C. roseus, and tried to transform this with constructs of six effectors of ‘Ca. P. solani’, which were discovered through bioinformatics tools. In the second part of this thesis, the activities of the key enzymes involved in carbohydrate and antioxidant metabolism were assayed in: (i) grapevine leaf samples from a vineyard naturally infected with ‘Ca. P. solani’; (ii) leaf samples of greenhouse grown C. roseus infected with two different strains of ‘Ca. P. solani’; and (iii) leaf samples of Nicotiana benthamiana after transient transformation with the ‘Ca. P. solani’ effector constructs. The results confirm our hypothesis that infection with ‘Ca. P. solani’ significantly affects carbohydrate metabolism. However, the influence of infection was less obvious for antioxidant metabolism.

• Language: English
• Keywords: phytoplasmas, ‘Candidatus Phytoplasma solani', bois noir, Vitis vinifera, Catharanthus roseus, Nicotiana benthamiana, plant transformation, enzymatic activity, effector proteins
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [B. Anžič]
• Year: 2019
• PID: 20.500.12556/RUL-111641
• UDC: 632.35:602.6:577.15
• COBISS.SI-ID: 5108088
• Publication date in RUL: 06.10.2019

Secondary language

• Language: Slovenian
• Title: Transformacija madagaskarskega zimzelena (Catharanthus roseus) za raziskave okužbe s ‘Candidatus Phytoplasma solani’ in encimske aktivnosti v okuženih rastlinah

Abstract

'Candidatus Phytoplasma solani' je rastlinski znotrajcelični parazit, ki živi znotraj floema in ki ga prenašajo žuželke s sesanjem okuženega floemskega soka. Je povzročiteljica različnih bolezni na nekaterih gospodarsko pomembnih rastlinah, domnevno s pomočjo efektorjev. Dosedanje študije nakazujejo, da okužbe s fitoplazmami vplivajo na metabolizem ogljikovih hidratov in fenolnih komponent v gostiteljski rastlini, ki so lahko povezane z reaktivnimi kisikovimi vrstami in antioksidantnimi encimi. Ker rutinska kultivacija fitoplazem v in vitro pogojih še vedno ni mogoča, uporabljamo s fitoplazmo okuženo rastlino Catharanthus roseus kot modelno rastlino za raziskave. V tej raziskavi, smo poskušali vzpostaviti protokol za stabilno transformacijo C. roseus in jo poskusili transformirati s konstrukti ali šestimi efektorji ‘Ca. P. solani’, ki so bili odkriti s pomočjo bioinformatskih orodij. V drugem delu magistrske naloge smo raziskovali aktivnost ključnih encimov, ki sodelujejo v metabolizmu ogljikovih hidratov in antioksidantov. Raziskovani so bili v: (i) listih vinske trte iz vinogradov, ki so bili naravno okuženi s ‘Ca. P. solani’; (ii) listih C. roseus, ki so bili gojeni v rastlinjaku in okuženi z dvema različnima sevoma ‘Ca. P. solani’; in v (iii) listih Nicotiana benthamiana po prehodni transformaciji s ‘Ca. P. solani’ efektorskimi konstrukti. Rezultati so potrdili našo hipotezo, da okužba s ‘Ca. P. solani’ bistveno vpliva na metabolizem ogljikovih hidratov. Vpliv okužbe je bil manj očiten pri metabolizmu antioksidantov.

• Keywords: fitoplazme, Candidatus Phytoplasma solani, bolezen trsnih rumenic, Vitis vinifera, Catharanthus roseus, Nicotiana benthamiana, transformacija rastlin, encimska aktivnost, efektorski proteini