Phytoplasmas are phloem-limited plant pathogens transmitted by phloem-sucking Hemipteran insects. They are wall-less bacteria causing severe economic damage on many crops worldwide. Molecular mechanisms involved in the interaction between phytoplasmas and their hosts are still poorly understood, mainly because phytoplasma cultivation in purity is not yet feasible. This work aimed to set up a laser microdissection (LMD) protocol that could be useful for further studies to elucidate the signaling pathway in phytoplasma colonized host cells, both in plant- and insect-phytoplasma interactions. Two unrelated phytoplasmas (Flavescence dorée, FD, 16SrV-C and Chrysanthemum yellows, CY 'Candidatus Phytoplasma asteris', 16SrI-B), the model plant Arabidopsis thaliana and the leafhopper Euscelidius variegatus, vector of both pathogens, were studied in this work. Protocols were successfully optimized to isolate vascular tissues of A. thaliana from leaves and stems through LMD, and also to extract RNA from collected cells. To verify the optimized procedure, the presence of three plant transcripts (one ubiquitarious and two tissue specific) and of four phytoplasma transcripts (one abundant and one rare for each pathogen) was evaluated by RT-PCR on RNA extracted from isolated phloem cells. In situ hybridization experiments were performed on E. variegatus, by using specific DIG-conjugated probes to localize insect transcripts known to be expressed either in gut or in salivary glands, as well as phytoplasma cells. These experiments were performed to identify specific tissues inside an insect section, which is an essential step for the application of an LMD protocol to dissect them.
Phytoplasma-host interactions: a closer look through laser microdissection and in situ hybridization.
Pesando M;Balestrini R;Bosco D;Galetto L
2016
Abstract
Phytoplasmas are phloem-limited plant pathogens transmitted by phloem-sucking Hemipteran insects. They are wall-less bacteria causing severe economic damage on many crops worldwide. Molecular mechanisms involved in the interaction between phytoplasmas and their hosts are still poorly understood, mainly because phytoplasma cultivation in purity is not yet feasible. This work aimed to set up a laser microdissection (LMD) protocol that could be useful for further studies to elucidate the signaling pathway in phytoplasma colonized host cells, both in plant- and insect-phytoplasma interactions. Two unrelated phytoplasmas (Flavescence dorée, FD, 16SrV-C and Chrysanthemum yellows, CY 'Candidatus Phytoplasma asteris', 16SrI-B), the model plant Arabidopsis thaliana and the leafhopper Euscelidius variegatus, vector of both pathogens, were studied in this work. Protocols were successfully optimized to isolate vascular tissues of A. thaliana from leaves and stems through LMD, and also to extract RNA from collected cells. To verify the optimized procedure, the presence of three plant transcripts (one ubiquitarious and two tissue specific) and of four phytoplasma transcripts (one abundant and one rare for each pathogen) was evaluated by RT-PCR on RNA extracted from isolated phloem cells. In situ hybridization experiments were performed on E. variegatus, by using specific DIG-conjugated probes to localize insect transcripts known to be expressed either in gut or in salivary glands, as well as phytoplasma cells. These experiments were performed to identify specific tissues inside an insect section, which is an essential step for the application of an LMD protocol to dissect them.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
