Progress 09/15/02 to 09/14/07
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
The objectives of this grant are to complete the genome sequencing of three phytoplasmas: (1) Aster yellows witches' broom phytoplasma (AY-WB), Subgroup A of Candidatus Phytoplasma asteris, NCBI taxonomic ID: 229545. (2) Maize bushy stunt phytoplasma (MBSP), Subgroup B of Candidatus Phytoplasma asteris, NCBI taxonomic ID: 202462. (3) Beet leafhopper transmitted virescence agent (BLTVA), Clover proliferation phytoplasma group (16SrVI), NCBI taxonomic ID: 37694. The sequencing strategy is whole genome shotgun sequencing (WGS) followed by primer walking to close gaps. Because phytoplasmas cannot be cultured, we isolated phytoplasma genomic DNA from the phloem of infected host plants by gel-purification with clamped homogeneous electrical field (CHEF) electrophoresis. A manuscript describing the genome sequence of phytoplasma strain AY-WB and genome comparisons among mollicutes was published: Bai X., Zhang, J., Miller, S.A., Jansco Radek, A., Shevchenko, D.V., Tsukerman,
K., Walunas, T., Lapidus, A., Campbell, J.W., and Hogenhout, S.A. (2006). Living with Genome Stability: the Adaptation of Phytoplasmas to Diverse Environments of Their Insect and Plant Hosts. J. Bact. 188: 3682-3696. Furthermore, the MBSP genome-sequencing project is at the gap closure phase, and annotation of genome sequences has started. Finally, the BLTVA genome is sequenced at 4-fold. Annotation of these sequences is in progress as well. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches' broom (AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phytoplasmas have small repeat-rich genomes. This comparative analysis revealed that the repeated DNAs are organized into large clusters of potential mobile units (PMUs), which contain tra5 insertion sequences (ISs), and specialized sigma factors and membrane proteins. So far, these PMUs appear to be unique to phytoplasmas. Compared to mycoplasmas,
phytoplasmas lack several recombination and DNA modification functions, and therefore phytoplasmas may use different mechanisms of recombination, likely involving PMUs, for the creation of variability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GC skews and presence of ISs and large repeated sequences in the AY-WB and OY-M genomes are indicative of high genomic plasticity. Nevertheless, segments of 250 kb, located between genes lplA and glnQ are syntenic between the two phytoplasmas, and contain the majority of the metabolic genes and no ISs. AY-WB appears to be further along in the reductive evolution process than OY-M. The AY-WB genome is 154 kb smaller than the OY-M genome, primarily as a result of fewer multicopy sequences, including PMUs. Furthermore, AY-WB lacks many genes that are truncated and are part of incomplete pathways in OY-M.
Impacts
Phytoplasmas cause chronic, systemic diseases that affect over 300 species in 38 families of broad-leaf, herbaceous plants and several woody fruit crops. Farmers would benefit from more specific, faster, and cheaper detection methods and from pesticides that can cure trees/plants from phytoplasma diseases as soon as they show symptoms. This would significantly reduce insecticide usage and, hence, production costs. In addition, more knowledge about the pathogen will lead to a better understanding of how phytoplasmas cause the serious disease symptoms in their plant hosts and, hence, this research will aid the development of phytoplasma-resistant crop varieties. US businesses importing and exporting agricultural products will also significantly benefit from better phytoplasma diagnostics methods. Sequence data generated in this project will help to identify phytoplasma-specific membrane proteins that can be used for raising antibodies to establish ELISA-based assays for
the detection of phytoplasma diseases. Further, with the availability of the human, insect, plant and many bacterial genomes, we can now define metabolic pathways and genes specific to phytoplasmas/mollicutes. These pathways or genes can serve as targets for specific control agents that will inhibit phytoplasma/mollicute replication or kill phytoplasmas/mollicutes but will not harm plant or insect hosts, humans and animals, or the environment.
Publications
- Bai X., Zhang, J., Miller, S.A., Jansco Radek, A., Shevchenko, D.V., Tsukerman, K., Walunas, T., Lapidus, A., Campbell, J.W., and Hogenhout, S.A. (2006). Living with Genome Stability: the Adaptation of Phytoplasmas to Diverse Environments of Their Insect and Plant Hosts. J. Bact. 188: 3682-3696.
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Progress 01/01/05 to 12/31/05
Outputs
The objectives of this grant are to complete the genome sequencing of three phytoplasmas, and to organize workshops to educate students and to involve the scientific community and public in this project. The 706,569-bp chromosome and four plasmids of AY-WB were sequenced and fully annotated, and compared to the onion yellows phytoplasma strain M (OY-M) genome (Oshima et al., 2004. Nat. Genet. 36: 27-29). We also completed a comparative genome analysis of phytoplasmas and other mollicutes. A manuscript reporting the AY-WB genome sequence and results of the comparative analyses has been accepted for publication, and will be printed in 2006. We developed pipelines for the identification of candidate phytoplasma virulence factors, and secured additional funding to conduct functional characterizations of 58 AY-WB candidate virulence proteins. We also sequenced the MBSP genome at 10-fold and assembled the sequences into contigs. The MBSP genome appears to contain more
repeats than that of AY-WB. We are in the process of constructing a large-insert library, and we expect that sequences derived from this large-insert library will resolve the assembly of repeat-rich areas and lead to closure of gaps. We have a 4-fold coverage of the BLTVA genome. The annotation of the AY-WB genome sequences and the comparative genome analysis of AY-WB and OY-M resulted in several important findings. First, whereas the phytoplasma genomes are small, they are extremely repeat rich. We discovered that the repeated DNAs are composed of multicopy genes, which are organized into clusters that have similar gene orders and contain tra5 insertion sequences (ISs) that belong to group IS150, family IS3. We named these gene clusters potential mobile units (PMUs). At least one AY-WB PMU has striking features characteristic of a replicative composite transposon. In addition, we discovered that the PMUs contain genes for specialized sigma factors and membrane proteins suggesting
that PMUs are important for phytoplasma interactions with insects and plants. Hence, the PMUs are candidate pathogenicity islands. Further, we have evidence that expression of PMU genes might occur through a process that involves circularization and replicative transposition. Finally, we discovered that the AY-WB genome is 154 kb smaller than the OY-M genome primarily as a result of fewer multicopy sequences. Thus, expansions or reductions of PMUs play a major role in phytoplasma genome evolution. Interestingly, despite the high genomic plasticity of phytoplasmas, segments of 250 kb, located between genes lplA and glnQ, are syntenic between phytoplasmas, and contain the majority of the metabolic genes and no ISs. This is the first comparative phytoplasma genome analysis and report of the existence of PMUs in phytoplasma genomes. We did experiments to determine the plant host range of AY-WB. So far, results have shown that AY-WB can efficiently infect lettuce, China aster, Nicotiana
benthamiana and Arabidopsis thaliana, and occasionally tomato.
Impacts
Phytoplasmas cause chronic, systemic diseases that affect over 300 species in 38 families of broad-leaf, herbaceous plants and several woody fruit crops. Farmers would benefit from more specific, faster, and cheaper detection methods and from pesticides that can cure trees/plants from phytoplasma diseases as soon as they show symptoms. This would significantly reduce insecticide usage and, hence, production costs. In addition, more knowledge about the pathogen will lead to a better understanding of how phytoplasmas cause the serious disease symptoms in their plant hosts and, hence, this research will aid the development of phytoplasma-resistant crop varieties. US businesses importing and exporting agricultural products will also significantly benefit from better phytoplasma diagnostics methods. Sequence data generated in this project will help to identify phytoplasma-specific membrane proteins that can be used for raising antibodies to establish ELISA-based assays for
the detection of phytoplasma diseases. Further, with the availability of the human, insect, plant and many bacterial genomes, we can now define metabolic pathways and genes specific to phytoplasmas/mollicutes. These pathways or genes can serve as targets for specific control agents that will inhibit phytoplasma/mollicute replication or kill phytoplasmas/mollicutes but will not harm plant or insect hosts, humans and animals, or the environment.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Milestones: Genome sequencing and sequence analysis: (i) Sequenced the genome of phytoplasma strain AY-WB to completion (publication in preparation); (ii) Constructed libraries with isolated MBSP and BLTVA DNA; (iii) Sequenced the genome of MBSP at 8-fold and that of BLTVA at 4-fold; (iv) Completed a comparative analysis of mollicute genomes (Bai et al., 2004. FEMS Microbiol. Lett. 235: 249-258); (v) Mined the AY-WB genome sequences for genes encoding secreted proteins potentially involved in insect transmission and plant pathogenesis (disease effectors); Functional analysis of phytoplasma proteins: (vi) Secured additional funding to conduct functional analyses of phytoplasma proteins; (vii) Designed and conducted high-throughput functional genomic experiments to characterize the functions of candidate phytoplasma effector genes. Other: (viii) Organized a symposium Life Styles and Genomics of Fastidious and Gram-positive Bacteria at the Annual Meeting of the American
Phytopathological Society (APS), July 31 to August 4, 2004, in Anaheim, California. Organizers: Saskia Hogenhout (PI) and Carol Ishimaru. Notable Findings: Genome sequencing and sequence analysis: (i) Comparative analysis of the completed AY-WB (724 kb) and OY genomes (862 kb) revealed that the number of genes for metabolism and transport are similar between the two phytoplasmas consistent with the observation that phytoplasmas rely on a minimal genome complement. However, these phytoplasma genomes have striking differences in numbers of repeated sequence clusters. (ii) SignalP analysis of all predicted AY-WB proteins resulted in the identification of 58 secreted proteins. Eight of the 58 proteins contain nuclear localization signals (NLS) and might target host nuclei. Most of the secreted protein genes are located in the repeated sequence clusters. Functional analysis of phytoplasma proteins: (iii) For validation of the NLS predictions of phytoplasma protein A11 and A30, cellular
localization studies of yellow fluorescent protein (YFP) fusions of these proteins were conducted and showed that A11 targeted the plant cell nucleus and A30 the plant cell nucleolus. Subsequent virus-induced gene silencing (VIGS) experiments showed that the transport of A11 into plant nuclei of N. benthamiana cells is dependent on importina; (iv) The A11 protein ORF is located in an operon-like region that contain other ORFs of putative secreted proteins, and ORFs that are frequently observed in pathogenicity related gene clusters of pathogenic bacteria, such as those of DNA-binding and modification proteins, including transposons, membrane proteins and a sigma factor; (v) We conducted microarray hybridization experiments to monitor A11-induced changes in tomato gene expression. We inoculated tomato plants with PVX expressing the A11 gene of AY-WB and included mock inoculation and the empty PVX vector. Results showed that the PVX expression system is useful for assessment of changes
in tomato gene expression in response to heterologous effectors.
Impacts
Phytoplasmas cause chronic, systemic diseases that affect over 300 species in 38 families of broad-leaf, herbaceous plants and several woody fruit crops. Farmers would benefit from more specific, faster, and cheaper detection methods and from pesticides that can cure trees/plants from phytoplasma diseases as soon as they show symptoms. This would significantly reduce insecticide usage and, hence, production costs. In addition, more knowledge about the pathogen will lead to a better understanding of how phytoplasmas cause the serious disease symptoms in their plant hosts and, hence, this research will aid the development of phytoplasma-resistant crop varieties. US businesses that import/export agricultural products will also significantly benefit from better phytoplasma diagnostics methods. Sequence data generated in this project will help to identify phytoplasma-specific membrane proteins that can be used for raising antibodies to establish ELISA-based assays for the
detection of phytoplasma diseases. Further, with the availability of the human, insect, plant and many bacterial genomes, we can now define metabolic pathways and genes specific to phytoplasmas/mollicutes. These pathways or genes can serve as targets for specific control agents that will inhibit phytoplasma/mollicute replication or kill phytoplasmas/mollicutes but will not harm plant or insect hosts, humans and animals, or the environment.
Publications
- Bai X., Zhang J., Holford I. R., and Hogenhout S.A. (corresponding author) (2004). Comparative genomics identifies genes shared by distantly related insect-transmitted plant pathogenic mollicutes. FEMS Microbiology Letters 235: 249-258.
- Zhang J., Hogenhout S. A., Nault L.R., Hoy C.W. and Miller S.A. (corresponding author) (2004). Molecular and symptom analyses of phytoplasma strains from lettuce reveal a diverse population. Phytopathology 94: 842-849.
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Progress 01/01/03 to 12/31/03
Outputs
(i) We isolated genomic DNA (800 kb) of the phytoplasma strain AY-WB (previously aster yellows phytoplasma (AYP) OH Bolt) from the phloem of infected lettuce plants by gel-purification with clamped homogeneous electrical field (CHEF) electrophoresis. The genome was sequenced using whole genome shotgun sequencing (WGS). We constructed libraries using 5 micrograms of gel isolated AY-WB chromosomal DNA. Following random sequencing and assembly, we used primer walking to close the gaps. The phytoplasma genome is AT-rich and contains many repeats. We are constructing a large-insert library to connect repeat-rich regions flanking the 9 contigs. (ii) A comparative genomics project resulted in the identification of four genes shared by the insect-transmitted plant-pathogens AY-WB and Spiroplasma kunkelii, but absent from the human and animal pathogenic Mycoplasma and Ureaplasma spp. This is interesting because spiroplasmas are more related to mycoplasmas and ureaplasmas than
to phytoplasmas. We hypothesize that these four genes are important for the persistent infection of insects and plants by AY-WB and S. kunkelii. A publication reporting this work is in preparation. (iii) The available AY-WB sequence data were mined for genes involved in insect transmission and plant pathogenesis. This research is based on the hypothesis that secreted phytoplasma proteins are potential virulence factors (effector proteins). Hence, computer algorithms were employed to predict whether proteins have a N-terminal signal peptide required for secretion by the Sec-dependent or type II secretion systems. Additional criteria, including length, annotation, and lack of transmembrane domain, were used to select 56 putative soluble secreted proteins for functional analysis. PI Hogenhout and PhD student Xiaodong Bai have secured additional funding to conduct the functional analyses. A publication reporting this work is in preparation. (iv) We have identified 11 insertion sequences
(ISs) in the genome of aster yellows phytoplasma strain AY-WB. These 11 ISs share high nucleotide sequence similarity with those of IS150 group in IS3 family. The presence of the ISs among various phytoplasmas was investigated by PCR using primers designed on sequences internally and externally to inverted repeats of AY-WB ISs. Results showed that ISs can be used as markers to distinguish phytoplasma isolates. A publication reporting this work is in preparation. (v) Other accomplishments: (a) We have isolated DNA of MBSP and BLTVA. DNA will be send to Integrated Genomics for library construction and shotgun sequencing. (b) PI Hogenhout, Postdoc Jianhua Zhang, and PhD student Xiaodong Bai visited Integrated Genomics Inc., Chicago to receive training for using the genome sequence annotation database, ERGO. (c) A proposal to organize a symposium on Life Styles and Genomics of Fastidious and Gram-positive Bacteria at the Annual Meeting of the American Phytopathological Society (APS), July
31 to August 4, 2004, in Anaheim, California was approved by the APS Program Committee. Drs Saskia Hogenhout and Carol Ishimaru co-organize this symposium and will submit final plans by March 22, 2004.
Impacts
Phytoplasmas cause chronic, systemic diseases that affect over 300 species in 38 families of broad-leaf, herbaceous plants and several woody fruit crops. Farmers would benefit from more specific, faster, and cheaper detection methods and from pesticides that can cure trees/plants from phytoplasma diseases as soon as they show symptoms. This would significantly reduce insecticide usage and, hence, production costs. In addition, more knowledge about the pathogen will lead to a better understanding of how phytoplasmas cause the serious disease symptoms in their plant hosts and, hence, this research will aid the development of phytoplasma-resistant crop varieties. US businesses that import/export agricultural products will also significantly benefit from better phytoplasma diagnostics methods. Sequence data generated in this project will help to identify phytoplasma-specific membrane proteins that can be used for raising antibodies to establish ELISA-based assays for the
detection of phytoplasma diseases. Further, with the availability of the human, insect, plant and many bacterial genomes, we can now define metabolic pathways and genes specific to phytoplasmas/mollicutes. These pathways or genes can serve as targets for specific control agents that will inhibit phytoplasma/mollicute replication or kill phytoplasmas/mollicutes but will not harm plant or insect hosts, humans and animals, or the environment.
Publications
- No publications reported this period
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