Phytoplasmas: DNA barcoding and host adaptation

Olga Vladimirovna Makarova

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling


Phytoplasmas are plant pathogenic bacteria that are transmitted by leafhopper vectors. These phytopathogens cause disease in over 100 economically important plants, including apple, grapevine, rice, coconut, pear, apricot, cotton and potato. When infected, susceptible plants usually fail to germinate or die. Currently, disease management is limited to control of insect vectors and elimination of infected plants, where correct identification plays a crucial role. As phytoplasmas cannot be cultured in vitro and do not possess a distinct morphology, traditional microbiological identification methods are not applicable, and current identification relies on sequence-based methods. However, only a few methods can be used for universal identification of phytoplasmas from a wide range of species. Phytoplasmas are cell wall-less intracellular pathogens of plants and insects. Their small genomes appear to lack or have many incomplete metabolic pathways. Nevertheless, phytoplasmas display an ability to not only survive in very different cell environments of their plant and insect hosts, but also to manipulate them. Phytoplasmas are known to change the morphology and physiology of plants to their own advantage by making plants more attractive to their insect vectors, although the exact mechanisms of how they do it are largely unknown. Thus, phytoplasmas present a unique study object for both basic and applied research, owing to their importance as pathogens of plants (and associated difficulties with disease management and pathogen identification) and an interesting lifestyle that involves colonisation and manipulation of two highly dissimilar hosts. The present Ph.D. project addresses two main research questions: (i) whether it is possible to develop a universal sequence-based identification of phytoplasmas, and (ii) what are the mechanisms behind their remarkable ability to adapt to cell environments of their plant and insect hosts. To answer the first question, the potential of DNA barcoding for phytoplasma identification has been explored. DNA barcoding is a method of identification that is based on a short conserved molecular marker present in all species, but polymorphic enough to allow species separation. This molecular marker (DNA barcode) is then sequenced with a single primer pair from all species in question and compared with the sequences in the reference database. QBOL, the EUsponsored project, has been launched in order to develop a DNA barcoding identification system for major plant pests and pathogens, including phytoplasmas. As a part of this initiative, this Ph.D. project focused on the identification of DNA regions suitable for DNA-barcoding of phytoplasmas, sequencing of these DNA barcodes and establishment of a reference DNA barcode database, as well as the development and optimisation of suitable protocols. DNA barcodes of two genes (Tuf and 16S rRNA) were obtained from all major phytoplasma species/groups that included both quarantine and non-quarantine strains. The phylogenetic analysis proved their ability to separate main phytoplasma species/groups. Identification procedures have been developed and the DNA barcodes were deposited in the DNA barcode database. This DNA barcoding-based system is expected to be implemented in the EU and aid plant health inspectors. To elucidate the mechanisms employed by phytoplasmas for host adaptation, this Ph.D. project used the AY-WB phytoplasma – leafhopper Macrosteles quadrilineatus - Arabidopsis thaliana model pathosystem and a systems biology approach to identify genes involved in this process. Relative gene expression values were determined for 232 AY-WB phytoplasma genes (ca. 35% 6 of all AY-WB genes), that were likely to be involved in host adaptation. These included genes from fourteen COG categories that are relevant to central metabolism, previously identified genes of predicted secreted proteins (effectors, or SAPs) and secreted proteins with transmembrane domain(s) (SAMPs). The majority of the genes tested (62%) were differentially expressed. Furthermore, host-responsive genes were more likely to exhibit signs of positive and/or negative selection. These included genes involved in direct interaction with the host cell environment, defence-related genes and genes present in multiple copies within the genome. Surprisingly, metabolic adaptation was also reflected in the patterns of gene expression, with genes involved in malate metabolism being upregulated in the insect host, suggesting that phytoplasmas use malate as an additional source of energy in this host. Based on these findings, a list of candidate genes involved in host adaptation was proposed for experimental validation. Furthermore, two effector proteins identified as candidate host adaptation genes were characterised during this Ph.D. study. Phenotypic analysis of the transgenic plants expressing SAP54 and EP phytoplasma secreted proteins showed that the effectors alter plant morphology and development. The preliminary results on characterisation of EP showed that it interacts with transcription factors of the plant host. Taken together, these findings show that phytoplasmas readily respond to host shifts by changing their gene expression and employ a number of mechanisms to adapt and manipulate their hosts, which include (but are not limited to) secretion of effector molecules and changes in central metabolic pathways. The list of identified candidate genes will help prioritise further studies on host adaptation of these bacteria. The present Ph.D. project contributed to phytoplasma research in two ways. The development of a universal identification system will improve phytoplasma diagnostics. Identification of genes involved in host adaptation and further validation of two phytoplasma effectors have increased the current understanding of pathogenicity mechanisms employed by this bacterium and this will form the basis for further development of disease control strategies.
ForlagAarhus Universitet, Institut for Agroøkologi
Antal sider211
ISBN (Trykt)978-87-92869-23-4
StatusUdgivet - feb. 2012


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