Yoshiko Ishii

Dramatic Transcriptional Changes in an Intracellular Parasite Enable Host Switching between Plant and Insect

Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the “host switching” between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to “host switching” between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of “host switching” mechanism may contribute to the development of novel pest controls.

Presence of two glycolytic gene clusters in a severe pathogenic line of Candidatus Phytoplasma asteris.

SUMMARY Phytoplasmas are plant-pathogenic bacteria that are associated with numerous plant diseases. We have previously reported the complete genomic sequence of Candidatus Phytoplasma asteris, OY strain, OY-M line, which causes mild symptoms. The phytoplasma genome lacks several important metabolic genes, implying that the consumption of metabolites by phytoplasmas in plants may cause disease symptoms. Here we show that the approximately 30-kb region including the glycolytic genes was tandemly duplicated in the genome of OY-W phytoplasma, which causes severe symptoms. Almost duplicated genes became pseudogenes by frameshift and stop-codon mutations, probably because of their functional redundancy. However, five kinds of genes, including two glycolytic genes, remained full-length ORFs, suggesting that it is advantageous for the phytoplasma to retain these genes in its lifestyle. In particular, 6-phosphofructokinase is known as a rate-limiting enzyme of glycolysis, implying that the different number of glycolytic genes between OY-W and OY-M may influence their respective glycolysis activities. We previously reported that the phytoplasma population of OY-W was higher than that of OY-M in their infected plants. Taking this result into account, the higher consumption of the carbon source may affect the growth rate of phytoplasmas and also may directly or indirectly cause more severe symptoms.

Cloning and Characterization of the Antigenic Membrane Protein (Amp) Gene and In Situ Detection of Amp from Malformed Flowers Infected with Japanese Hydrangea Phyllody Phytoplasma

A Japanese hydrangea phyllody (JHP) disease found throughout Japan causes economic damage to the horticultural industry. JHP phytoplasma-infected Japanese hydrangea plants show several disease symptoms involved in floral malformations, such as virescence, phyllody and proliferation. Here, we cloned and characterized the antigenic membrane protein (Amp) gene homolog from the JHP phytoplasma (JHP-amp), expressed the JHP-Amp protein in Escherichia coli cells, and then obtained an antibody against JHP-Amp. The antibody against JHP-Amp had no cross-reactions with the antibody against the Amp protein from a closely related onion yellows phytoplasma. This serologic specificity is probably due to the high diversity of the hydrophilic domains in the Amp proteins. The in situ detection of the JHP-Amp protein revealed that the JHP phytoplasma was localized to the phloem tissues in the malformed flower. This study shows that the JHP-Amp protein is indeed a membrane protein, which is expressed at detectable level in the JHP phytoplasma-infected hydrangea.

Unique morphological changes in plant pathogenic phytoplasma-infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ-specific manner.

Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, onion yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.

Process of reductive evolution during 10 years in plasmids of a non-insect-transmissible phytoplasma.

A non-insect-transmissible phytoplasma strain (OY-NIM) was obtained from insect-transmissible strain OY-M by plant grafting using no insect vectors. In this study, we analyzed for the gene structure of plasmids during its maintenance in plant tissue culture for 10 years. OY-M strain has one plasmid encoding orf3 gene which is thought to be involved in insect transmissibility. The gradual loss of OY-NIM plasmid sequence was observed in subsequent steps: first, the promoter region of orf3 was lost, followed by the loss of then a large region including orf3, and finally the entire plasmid was disappeared. In contrast, no mutation was found in a pseudogene on OY-NIM chromosome in the same period, indicating that OY-NIM plasmid evolved more rapidly than the chromosome-encoded gene tested. Results revealed an actual evolutionary process of OY plasmid, and provide a model for the stepwise process in reductive evolution of plasmids by environmental adaptation. Furthermore, this study indicates the great plasticity of plasmids throughout the evolution of phytoplasma.

New ex vivo reporter assay system reveals that σ factors of an unculturable pathogen control gene regulation involved in the host switching between insects and plants

Analysis of the environmental regulation of bacterial gene expression is important for understanding the nature, pathogenicity, and infection route of many pathogens. “Candidatus Phytoplasma asteris”, onion yellows strain M (OY‐M), is a phytopathogenic bacterium that is able to adapt to quite different host environments, including plants and insects, with a relatively small ~850 kb genome. The OY‐M genome encodes two sigma (σ) factors, RpoD and FliA, that are homologous to Escherichia coli σ70 and σ28, respectively. Previous studies show that gene expression of OY‐M dramatically changes upon the response to insect and plant hosts. However, very little is known about the relationship between the two σ factors and gene regulatory systems in OY‐M, because phytoplasma cannot currently be cultured in vitro. Here, we developed an Escherichia coli‐based ex vivo reporter assay (EcERA) system to evaluate the transcriptional induction of phytoplasmal genes by the OY‐M‐derived σ factors. EcERA revealed that highly expressed genes in insect and plant hosts were regulated by RpoD and FliA, respectively. We also demonstrated that rpoD expression was significantly higher in insect than in plant hosts and fliA expression was similar between the hosts. These data indicate that phytoplasma‐derived RpoD and FliA play key roles in the transcriptional switching mechanism during host switching between insects and plants. Our study will be invaluable to understand phytoplasmal transmission, virulence expression in plants, and the effect of infection on insect fitness. In addition, the novel EcERA system could be broadly applied to reveal transcriptional regulation mechanisms in other unculturable bacteria.