Bianchedi, P.

Janick, K.

Malnoy, M.

Moser, M.

Tabarelli, M.

Trenti, M.

English

Archivio istituzionale della ricerca - Fondazione Edmund Mach

INTRODUCTION  OVEREXPRESSION OF THE MOLECULAR TARGET OF APPLE  PROLIFERATION PHYTOPLASMA EFFECTOR IN APPLE  Mattia Tabarelli123, Katrin Janik2, Pier Luigi Bianchedi3, Massimiliano Trenti2, Mirko Moser3, Mickael Malnoy3  1 - Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Italy   2 - Research Centre for Agriculture and Forestry Laimburg, Bolzano, Italy   3 - Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Trento, Italy.  MATERIALS AND METHODS Apple Proliferation (AP) is a severe          disease widespread in apple-growing        areas within Europe. Typical symptoms comprise shoot proliferation, small leaves  with altered shape and undersized, tasteless and colorless fruits. Causal agent of the disease is the phytoplasma ‘Candidatus phytoplasma mali’  (‘Ca. P. mali’). The genome analysis of different The SAP11-TCP binding leads to the deactivation of the TFs (Sugio et al., 2011), although direct molecular evidences of the symptoms induction by the TFs deactivation are        still lacking. strains of ‘Ca. P. mali’ revealed the presence of one effector, SAP11CaPM (Siewert et al., 2014), which has been observed to target at least two apple class II TCP transcription factors (TF), namely MdTCP24 and MdTCP25 (Janik et al., 2017) Goal of this work was to induce a stable overexpression of MdTCP25 in apple to shed light on the role of this gene in the apple physiology and in the development      of the disease. Thank you very much for reading my poster! For any comment or question you are very welcome to contact me at: mattia.tabarelli@fmach.it or mattia.tabarelli@laimburg.it Have a nice day  Mattia Tabarelli, 3d year PhD student in Agricultural Sciences and Biotechnology @ UniUD, Italy was extracted and amplified      using Agrobacterium-specific primers to exclude an ongoing  infection and vector’s backbone/ TCP-specific to confirm the successful transformation. Plantlets emerged from transformed calluses were propagated and chosen for further analyses: DNA  Confirmed transgenic lines were resampled and DNA was extracted from leaves. The number of copies of               35S::MdTCP25 integrated in the genome was determined as described by Dalla Costa and collaborators (2019) using the marker gene nptII as target.  Results are displayed in the  graph below. The full length MdTCP25     gene was subcloned in the destination vector pK7WG2.D for in planta expression (35s promoter, KanR as selection marker) used  for A. Tumefaciens-mediated transformation of apple, Cv. Gala. After the incubation with Agrobacterium, the leaves were  put in regeneration on  Kanamycin-containing  media for several months.  RNA was extracted   from the leaves of 6 independent lines,  retrotranscripted and used for MdTCP25 expression profiling. Ub and EF1 were chosen as housekeeping genes. Results, normalized such that the Wild 00,511,522,533,5WT In the bar chart is plotted the  expression of MdTCP25 in transgenic lines calculated via qPCR, compared to the wild type (white bar). Error bars are calculated on biological and technical triplicates. The expression was normalized on two housekeeping genes (Ubiquitin and Elongation Factor 1). The  purple numbers inside the bars represent the copy number for each line.  In the picture below  the transgenic acclimatized lines are shown compared to the Wild Type Gala (first plant on the left). 2,1 2,7 3,5 3,3 2,4 1,9 Copy number determined using nptII as target MdTCP25 expression relative to Gala determined via qPCR N CONCLUSIONS AND FUTURE PERSPECTIVES Type expression is equal to 1, are displayed in the graph below The expression of TCP25 in transgenic lines is lower than expected and, in some cases, even lower than the basal WT expression. Given that the TCP family is comprised of more than 50 genes regulated at a very fine level, we suppose that only individuals with a low level of overexpression were able to survive. Nonetheless, a slight change in the TCP25 expression seems to be sufficient to induce some phenotype effects: the MdTCP25 overexpressing plants tend to display loss of apical dominance, smaller leaves and shorter stem compared to the Wild Type.  Some selected transgenic lines are currently being infected with Ca. P. Mali via micrografting and an extensive analysis of their expression will follow.  Dalla Costa L, Bozzoli M, Pompili V, Piazza S, Broggini G, Patocchi A, Malnoy M (2019). Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification. Eur Food Res Technol 245, 643–652. https://doi.org/10.1007/s00217-018-3187-0 Janik K, Mithöfer A, Raffeiner M, Stellmach H, Hause B, Schlink K (2017). An effector of apple proliferation phytoplasma targets TCP transcription factors—a generalized virulence strategy of phytoplasma? Molecular Plant Pathology, 18(3), 435–442. https://doi.org/10.1111/mpp.12409 Siewert C, Luge T, Duduk B, Seemüller E, Büttner C, Sauer S, Kube M (2014).  Analysis of expressed genes of the bacterium “candidatus phytoplasma mali” highlights key features of virulence and metabolism. PLoS ONE, 9(4). https://doi.org/10.1371/journal.pone.0094391 Sugio SA, Kingdom HN, MacLean AM, Grieve VM, Hogenhout SA (2011).  Phytoplasma protein effector SAP11 enhances insect vector reproduction by manipulating plant development and defence hormone biosynthesis. Proceedings of the National Academy of Sciences, USA 108: E1254–E1263. doi: 10.1073/pnas.1105664108 REFERENCES: Photo by Biologische Bundesanstalt für Land- und Forstwirtschaft Ca. P mali (AP-MLO) symptoms, where the typical ‘’witches brooms’’ and enlarged stipules are clearly visible.  7th International Horticulture Research Conference  -  July 1st - 30th, 2020  Online  BAR CHART – COPY NUMBER AND MDTCP25 EXPRESSION  

Overexpression of the molecular target of Apple Proliferation Phytoplasma in Apple

https://openpub.fmach.it/bitstream/10449/64082/1/Overexpression%20of%20the%20molecular%20target%20of%20Apple%20Proliferation%20Phytoplasma%20in%20Apple.pdf

Overexpression of the molecular target of Apple Proliferation Phytoplasma in Apple

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