Phytoplasmas are insect-transmitted plant pathogens that replicate in both insect and plant hosts and cause severe diseases in over 300 plant species worldwide. Phytoplasma research has been hampered by the fact that they cannot be cultured in cell-free media. However, with the recent advances in genome sequencing, phytoplasmas became an exciting research object for insect and plant pathologists. Symptoms of AY-WB infection of plants includes phyllody and virescence, which led to the hypothesis that phytoplasma proteins affect the developmental regulation of host plants. In collaboration with Integrated Genomics Inc. 700 kb of the predicted 800 kb genome of strain Aster Yellows Witches' Broom (AY-WB) was sequenced, and analysis of the genome sequence data suggest that putative effector proteins of AY-WB are most likely secreted by type I or II systems. With the assistance of computer algorithms, proteins secreted by the bacterial type II secretion system were predicted based on the presence of N-terminal signal peptides. Subsequently, corresponding genes were cloned into the binary potato virus X (PVX) expression vector, pGR106, and expressed in tomato, tobacco and Nicotiana benthamiana via Agrobacterium mediated inoculation to identify genes that induce disease-like symptoms. Further, genes were cloned into vector, pFLAG-ATS, for secreted expression by Escherichia coli. Synthesized proteins will be used for in vitro experiments and antibody synthesis to allow immunolocalization of AY-WB secreted proteins in the insect vector Macrosteles quadrilineatus and AY-WB-infected plant hosts. This is the first functional genomics project of an insect-transmitted plant-pathogenic mollicute.
Species 1: Acholeplasmatales Acholeplasmataceae Phytoplasma
Species 2: Homoptera Cicadellidae Macrosteles quadrilineatus
Keywords: gene expression, genomics
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