Progress 07/01/02 to 07/01/07
Outputs
PI left University, no report given.
Impacts
PI left University, no report given.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
Phytoplasmas are prokaryotes of Gram-positive ancestry and belong to the Class Mollicutes. They are insect-transmitted plant pathogens that cause economically important diseases in hundreds of plant species worldwide including vegetable, ornamental and fruit crops. They are also prevalent in natural forest ecosystems and wild plant species. Phytoplasmas are an enigma among plant pathogens, because they cannot be cultured and are often hard to detect and identify. Serological and molecular detection techniques have been developed to improve detection capabilities for phytoplasmas in plants and insect vectors. The routine use of serological methods, however, has been limited by problems with non-specific background reactivity and lack of appropriate specificity. On the other hand, molecular markers, including the polymerase chain reaction (PCR), have been developed with better specificity and are readily available, but are expensive, require access to an experienced
laboratory, are based on incomplete knowledge, and do not adequately detect the full range of genetic diversity among phytoplasma populations. Therefore, the objective of the research is to develop faster, cheaper and more specific phytoplasma diagnostic assays that can be used in the field, do not require extensive training or access to a specialized lab and allow quick identification of one phytoplasma strain or a group of phytoplasmas. We showed that two strains of Candidatus (Ca.) Phytoplasma asteris (previously known as phytoplasma group 16SrI), Aster yellows phytoplasma strain witches' broom (AY-WB) and onion yellows phytoplasma strain M (OY-M), have repeated sequences that are uniquely organized into clusters, potential mobile units (PMUs), which contain tra5 insertion sequences (ISs), and genes for specialized sigma factors and membrane proteins (Bai et al., 2006. J. Bact. 188: 3682). We now have evidence that these PMUs form episomes and may replicate as extrachromosomal DNAs
(i.e. plasmids). We also found PMUs in the genome of Beet leafhopper transmitted virescence phytoplasma (BLTVP) that belongs to entirely different Candidatus genus, i.e. Ca. Phytoplasma trifolii (previously known as phytoplasma group 16srVI). This suggests that all phytoplasmas may have these PMUs in their genomes. Because PMUs appear to be unique to phytoplasmas and are present as multiple copies, PCRs with PMU primers are likely to increase phytoplasma detection specificity and sensitivity in plant samples and individual insects. Indeed, PCR experiments with various PMU primer demonstrated successful detection of AY-WB in various plant hosts and in individual insects. We aligned PMU sequences of AY-WB, OY-M, Maize bushy stunt phytoplasma (MBSP) and BLTVP, and characterized several regions that are conserved. These conserved regions can be used for development of new primer pairs or generation of specific antibodies for detection of diverse phytoplasmas.
Impacts
Phytoplasmas cause chronic, systemic diseases that affect over 300 species in 38 families of broad-leaf, herbaceous plants and several woody fruit crops. Phytoplasmas are usually detected by amplification of a specific region of the phytoplasma genome by polymerase chain reaction (PCR). However, although effective, PCR is expensive, requires a specialized laboratory, and may generate false positives. The development of more specific, cheaper and easier detection techniques would benefit farmers and would increase the overall likelihood that phytoplasmas are detected in early stages of a potential outbreak. Further, phytoplasmas require transmission by insects to spread, and possibilities of early detection of phytoplasma outbreaks may reduce excessive use of insecticides.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The objective of the research is to develop faster, cheaper and more specific phytoplasma diagnostic assays that can be used in the field, do not require extensive training or access to a specialized lab, and allow quick identification of one phytoplasma strain or a group of phytoplasmas. The availability of genome sequence data of several phytoplasmas allows the identification of membrane proteins specific to a particular phytoplasma or conserved among phytoplasmas. Such proteins are excellent candidates for the development of specific antibodies in diagnostic assays. Further, comparative analysis among phytoplasma genomes will allow the identification of highly variable sequences, which are useful for fingerprinting assays to characterize phytoplasma strains and isolates. The genome of Aster yellows phytoplasma strain witches broom (AY-WB) was sequenced to completion and completely annotated (Bai et al., 2006. Journal of Bacteriology. In press). AY-WB belongs to the
Candidatus (Ca.) Phytoplasma asteris (previously known as phytoplasma group 16SrI). The genome sequences were mined for genes that encode secreted proteins and proteins located on the surface of the phytoplasma cell. This resulted in the identification of 210 AY-WB putative membrane proteins of which 79 proteins were predicted to be secreted to the extracellular space of AY-WB. Subsequent comparative analysis of the 210 AY-WB protein sequences with the completed genome sequence of onion yellows phytoplasma strain M (OY-M), which also belongs to Ca. Phytoplasma asteris, revealed that most of these membrane proteins are unique to AY-WB, whereas only a few are conserved between the two phytoplasmas. This year we obtained genome sequence information from more phytoplasmas in the Ca. Phytoplasma asteris group, including Maize bushy stunt phytoplasma (MBSP) and Aster yellows phytoplasma strain DB2 (AY-DB2). In addition, we have sequence information from Beet leafhopper transmitted
virescence phytoplasma (BLTVP) that belongs to Ca. Phytoplasma trifolii. All phytoplasmas have repeat-rich genomes and the genome comparisons revealed that the repeated DNAs of phytoplasmas are uniquely organized into clusters, potential mobile units (PMUs), which contain tra5 insertion sequences (ISs), and specialized sigma factors and membrane proteins. Some of the membrane proteins encoded by PMUs are conserved among phytoplasmas and are potential candidates for the development of serologically based assays for the detection of all phytoplasmas. Other membrane proteins encoded on PMUs are more specific to each of the phytoplasmas and hence are useful targets for more specific detection assays. We also discovered that PMUs play an important role in phytoplasma genome evolution. Each phytoplasma strain has a unique number of PMUs. For example, the difference in PMU contents between the AY-WB and OY-M genomes largely accounts for the 154 kb smaller genome size of AY-WB. Thus, in
addition to antibody-based assays, fingerprinting using PMU sequences will allow for differentiation of phytoplasma strains or even isolates.
Impacts
Phytoplasmas cause chronic, systemic diseases that affect over 300 species in 38 families of broad-leaf, herbaceous plants and several woody fruit crops. Phytoplasmas are usually detected by amplification of a specific region of the phytoplasma genome by polymerase chain reaction (PCR). However, although effective, PCR is expensive, requires a specialized laboratory, and may generate false positives. The development of more specific, cheaper and easier detection techniques would benefit farmers and would increase the overall likelihood that phytoplasmas are detected in early stages of a potential outbreak. Further, phytoplasmas require transmission by insects to spread, and possibilities of early detection of phytoplasma outbreaks may reduce excessive use of insecticides.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The objective of the research is to develop faster and cheaper serologically based phytoplasma detection assays that can be used in the field, do not require extensive training or access to a specialized lab and allow quick identification of one phytoplasma strain or a group of phytoplasmas. The availability of genome sequence data of several phytoplasmas allows the identification of membrane proteins specific to a particular phytoplasma or conserved among phytoplasmas. Such proteins are excellent candidates for the development of specific antibodies. The Hogenhout lab recently sequenced the genome of the phytoplasma strain aster yellows witches broom (AY-WB) to completion, and used computer algorithms to identify sequences of membrane proteins. So far, we have identified 210 membrane proteins based on the presence of one or more membrane domains in the protein sequence. Further, a signal peptide sequence was detected in 79 phytoplasma protein sequences indicating
that these 79 proteins are secreted by the phytoplasmas and hence are exposed to the surface. Comparative analysis of these 289 (210 + 79) AY-WB sequences with another phytoplasma, onion yellows phytoplasma (OY) that was sequenced by a Japanese group, showed that some of the 289 appear unique to AY-WB, whereas others were also present in OY and may therefore be conserved among phytoplasmas. To further investigate the presence of these proteins among phytoplasmas, the Hogenhout lab recently started a collaboration with TIGR (The Institute for Genomic Research) to sequence more phytoplasma genomes. Phytoplasma strains considered for genome sequencing are AY-S, AY-BW, AY-SG and AY-DB2 (Zhang J., Hogenhout S. Nault L.R., Hoy C.W. and Miller S.A., 2004. Phytopathology 94: 842-849). These sequences and the genomes of Maize bushy stunt phytoplasma (MBSP) and Beet leafhopper transmitted virescence phytoplasma (BLTVP) that are sequenced as part of another funded project (CSREES
2002-35600-12752) should provide the required information to identify membrane proteins specific to a particular phytoplasma or conserved among phytoplasmas. Two of the 79 proteins were cloned in a plasmid (pFLAG) for expression by the bacterium Escherichia coli. The pFLAG expression system allows quick production and purification of large amounts of proteins. So far, the Hogenhout lab has expressed five phytoplasma genes and purified three proteins, and raised antibodies to one protein (A11). The antibodies could successfully detect A11 in phytoplasma-infected insects by immunofluorescence confocal laser scanning microscopy. However, detection of this protein in protein extracts of phytoplasma-infected plants and insects was not successful. However, this research showed that we can successfully express phytoplasma proteins in heterologous systems and produce antibodies. To make more and faster progress with this project, I and other PIs submitted a proposal to setup a Center that
brings together a multidisciplinary team of OARDC scientists and their unique expertise and perspectives in the diagnosis of pathogens affecting food animals and plants.
Impacts
Phytoplasmas cause chronic, systemic diseases that affect over 300 species in 38 families of broad-leaf, herbaceous plants and several woody fruit crops. Phytoplasmas are usually detected by amplification of a specific region of the phytoplasma genome by polymerase chain reaction (PCR). However, although effective, PCR is expensive, requires a specialized laboratory, and may generate false positives. The development of more specific, cheaper and easier detection techniques would benefit farmers and would increase the overall likelihood that phytoplasmas are detected in early stages of a potential outbreak. Further, phytoplasmas require transmission by insects to spread, and possibilities of early detection of phytoplasma outbreaks may reduce excessive use of insecticides.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
We have utilized the available genome sequence data of the phytoplasma strain aster yellows witches broom (AY-WB) for the development of ELISA-based detection methods. ELISA-based detection of pathogens is generally quick, and it is cheaper than PCR-based detections currently used for detection and characterization of phytoplasmas. ELISA-based detection methods require specific antibodies that react with one phytoplasma, a group of related phytoplasmas, or all phytoplasmas. However, because phytoplasmas cannot be cultured in cell-free medium, the development of specific antibodies that do not cross-react with proteins from the plant host is difficult. The availability of genome sequence data of several phytoplasmas allows the identification of membrane proteins specific to a particular phytoplasma or conserved among phytoplasmas. Such proteins are excellent candidates for the development of specific antibodies. Membrane proteins often have a transmembrane domain that
can be recognized based on the presence of a particular amino acid sequence. Further, membrane proteins that are secreted can be recognized by the presence of a N-terminal signal peptide sequence. We have used various bioinformatics tools to identify proteins that are potentially secreted. Each gene corresponding to such protein was cloned in a plasmid (pFLAG) for expression by the bacterium Escherichia coli. The pFLAG expression system allows quick production and purification of large amounts of proteins. So far we have expressed five phytoplasma genes, and purified three proteins. We will raise antibodies to purified proteins and test the antibodies for detection of AY-WB in an ELISA-based detection assays. Genome sequencing of a related phytoplasma to AY-WB, Onion yellows phytoplasma (OY), has been completed, and those of phytoplasmas distantly related to AY-WB are in progress. A preliminary comparison of the genome sequence data of these phytoplasmas resulted in the identification
of membrane proteins specific to AY-WB, and membrane proteins conserved among phytoplasmas. We plan to expand our comparative genome analysis, and select candidate genes for expression and subsequent antibody production.
Impacts
Phytoplasmas cause chronic, systemic diseases that affect over 300 species in 38 families of broad-leaf, herbaceous plants and several woody fruit crops. Phytoplasmas are usually detected by amplification of a specific region of the phytoplasma genome by polymerase chain reaction (PCR). However, although effective, PCR is expensive, requires a specialized laboratory, and may generate false positives. The development of more specific, cheaper and easier detection techniques would benefit farmers and would increase the overall likelihood that phytoplasmas are detected in early stages of a potential outbreak. Further, phytoplasmas require transmission by insects to spread, and possibilities of early detection of phytoplasma outbreaks may reduce excessive use of insecticides.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
We accomplished the following goals: 1) Characterization of the AYP isolate. Mixed infections of different aster yellows phytoplasma strains can occur in field-grown plants (Zhang and Miller, unpublished). Therefore, single strains were purified from field-collected lettuce plants (Celeryville, OH area in late summer, 2000) showing typical `bolt' and `severe' symptoms. Ph.D. student Jianhua Zhang (JZ) determined that the AYP `bolt' strain belongs to the aster yellows subgroup A cluster (16SrIA). We selected AYP `bolt' for sequencing because of its small genome. 2) Isolation of the AYP 850 kb chromosome from pulse-field gels. Phytoplasmas cannot be cultured and consequently phytoplasma chromosomal DNA has to be purified from plants or insects. A protocol to purify AYP `bolt' DNA from pulse field gels was established to collect sufficient purified AYP DNA for construction of shotgun and cosmid libraries. We isolated 20 microgram of aster yellows phytoplasma DNA within a
period of three months. 3) Library construction, and sequencing and assembly of AYP'bolt' genome sequences. The shotgun library was constructed at Integrated Genomics Inc., Chicago (IG) with 5 microgram of pulse-field gel isolated AYP chromosomal DNA. After random sequencing and primer walking, sequences were assembled into 17 contigs totaling 765 kb of the AYP `OH bolt' chromosome. The average GC content of the sequences is 30%, which is similar to that of other mollicute genomes. 4) Analysis of AYP `bolt' sequences. The contigs were submitted to ERGO for automated annotation. The annotation results revealed that 16 contigs were derived from the AYP and 1 contig from the lettuce chloroplast genome. The contaminating plant sequences were easily identified because they assembled into separate contigs, and the entire mitochondria and chloroplast genomes of several plant species have been sequenced. The genome size of plant chloroplasts are approximately 150 kb. Phylogenetic analysis
using gene sequences of the elongation factor TU (tuf) gene confirmed that AYP `OH bolt' belongs to the 16SrIA cluster and is most closely related to the phytoplasma causing virescence of Plantago coronopus (PVM) with a predicted chromosome size of 810 kb. These results demonstrate that despite the fact that phytoplasmas cannot be cultured, sufficient phytoplasma DNA can be purified for high-throughput genome sequencing. The next step is to find phytoplasma-specific sequences that can be used for detection and targets for control agents.
Impacts
This project will provide us with information on how phytoplasmas differ from other mollicutes (e.g. spiroplasmas) and reveal genes specific for phytoplasmas that then can serve as targets for novel pesticides and detection methods. We expect that the generated data will provide ample opportunities for the development of PCR-based detection assays for phytoplasmas in general and, more specifically, for aster yellows phytoplasma.
Publications
- No publications reported this period
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